UNDERCARBOXYLATED/UNCARBOXYLATED OSTEOCALCIN INCREASES BETA-CELL PROLIFERATION, INSULIN SECRETION, INSULIN SENSITIVITY, GLUCOSE TOLERANCE AND DECREASES FAT MASS

Abstract

ates to methods and compositions for treating and diagnosing disorders related to energy metabolism and the OST-PTP signaling pathway involving gamma-carboxylase, osteocalcin and adiponectin. Such disorders include, but are not limited to, metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity.

Description

[0001]This application claims the benefit of U.S. Provisional Patent Applications Nos. 60/844,203 filed Sep. 13, 2006; 60/870,604 filed Dec. 18, 2006; 60/909,712 filed Apr. 2, 2007; and 60/945,081 filed Jun. 19, 2007; the contents of which are hereby incorporated by reference as if set forth fully herein.

FIELD OF THE INVENTION

[0003]The present invention relates to methods and compositions for treating, preventing and diagnosing disorders related to energy metabolism and the OST-PTP signaling pathway, which involves gamma-carboxylase, osteocalcin and adiponectin. Such disorders include, but are not limited to, metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity.

BACKGROUND OF THE INVENTION

[0004]The prevailing research paradigm in bone biology is that differentiation and functions of the two bone-specific cell types, osteoblasts and osteoclasts, are determined by secreted molecules that can either be cytokines acting locally, or hormones acting systemically (Harada and Rodan, 2003; Takayanagi, 2006; Teitelbaum and Ross, 2003). Applicants have discovered a previously unknown genetic pathway related to energy metabolism and occurring in osteoblasts in which decreased activity of OST-PTP leads to decreased activity of gamma-carboxylase, which in turn leads to increased secretion of undercarboxylated/uncarboxylated osteocalcin from the osteoblasts, with beneficial effects on glucose homeostasis.

[0005]OST-PTP is the protein encoded by the Esp gene. The Esp gene was originally named for embryonic stem (ES) cell phosphatase and it has also been called the Ptprv gene in mice. (Lee et al, 1996, Mech Dev 59: 153-164). OST-PTP is a receptor-like protein osteotesticular protein tyrosine phosphatase as well as fragments and variants thereof. OST-PTP is a large, 1711 amino-acid long protein that includes three distinct domains. OST-PTP has a 1068 amino-acid long extracellular domain containing multiple fibronectin type III repeats.

[0006]Esp expression is restricted to ES cells, the gonads and the skeleton. In the gonads, Esp is specifically expressed in Sertoli cells of the testis and coelomic epithelial cells of the ovaries. During development, Esp is initially expressed in the apical ectodermal ridge of the limbs. Later during embryonic development and after birth, its expression becomes restricted to pre-osteoblasts and osteoblasts (i.e., Runx2-positive cells) of the perichondrium and periosteum. Because of its bone and testicular localization, the gene product of Esp is often referred to as osteoblast testicular protein tyrosine phosphatase (OST-PTP).

[0007]Osteocalcin, one of the very few osteoblast-specific proteins, has several features of a hormone. Ducy et. al. demonstrated that mineralized bone from aging osteocalcin-deficient mice was two times thicker than that of wild-type. It was shown that the absence of osteocalcin led to an increase in bone formation without impairing bone resorption and did not affect mineralization. Multiple immunoreactive forms of human osteocalcin have been discovered in circulation (Garnero et al. J Bone Miner Res 1994; 9:255-4) and also in urine (Taylor et al. J. Clin. Endocrin. Metab. 1990; 70:467-72). Fragments of human osteocalcin can be produced either during osteoclastic degradation of bone matrix or as the result of the catabolic breakdown of the circulating protein after synthesis by osteoblasts.

[0008]Metabolic syndrome is a combination of medical disorders that increase the risk of cardiovascular disease and diabetes. Some of the symptoms of metabolic syndrome include: fasting hyperglycemia, high blood pressure, decreased HDL cholesterol, elevated triglycerides, and elevated uric acid levels.

[0009]The experiments described herein provide the first evidence that the skeleton is an endocrine regulator of energy metabolism and thereby determines, in part, the onset and severity of metabolic syndrome or type 2 diabetes, as well as the risk of developing these disorders. The experiments described herein establish that the skeleton makes and secretes undercarboxylated osteocalcin which acts as a hormone regulating energy metabolism. Described herein is a previously unknown genetic pathway relating to energy metabolism and occurring in osteoblasts in which decreased activity of OST-PTP leads to decreased activity of gamma-carboxylase, which in turn leads to increased secretion of undercarboxylated osteocalcin from the osteoblasts, with beneficial effects on glucose homeostasis.

SUMMARY OF THE INVENTION

[0010]The present invention provides pharmaceutical compositions comprising an agent that modulates energy metabolism and the OST-PTP signaling pathway, wherein the agent reduces OST-PTP phosphorylase activity, reduces gamma-carboxylase activity, or increases undercarboxylated/uncarboxylated osteocalcin, wherein the pharmaceutical compositions comprise the agent in an amount that produces an effect selected from the group consisting of an increase in glucose tolerance, an increase in insulin production, an increase insulin sensitivity, an increase in pancreatic beta-cell proliferation, an increase in adiponectin serum level, a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, and a decrease in the thickness of arterial plaque.

[0011]In certain embodiments, the pharmaceutical compositions comprise the agent in an amount effective to treat or prevent a disorder selected from the group consisting of metabolic syndrome, glucose intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and obesity. In certain embodiments, the agent inhibits the expression or activity of OST-PTP or gamma-carboxylase, inhibits phosphorylation of gamma-carboxylase, increases the level of undercarboxylated or uncarboxylated osteocalcin, inhibits carboxylation of osteocalcin, decarboxylates osteocalcin. In certain embodiments, the agent is selected from the group consisting of a small molecule, an antibody, a nucleic acid and a biologically active fragment or variant thereof.

[0012]In certain embodiments, the agent is undercarboxylated/uncarboxylated osteocalcin. In certain embodiments, at least one of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21 and 24 of mature human osteocalcin is not carboxylated. In certain embodiments, all three of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21 and 24 of mature human osteocalcin are not carboxylated.

[0013]In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin is a preparation of undercarboxylated/uncarboxylated osteocalcin in which more than about 20% of the total Glu residues at the positions corresponding to positions 17, 21 and 24 of mature human mature human osteocalcin in the preparation are not carboxylated. In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin shares at least 80% amino acid sequence identity with mature human osteocalcin when the undercarboxylated/uncarboxylated osteocalcin and mature human osteocalcin are aligned for maximum sequence homology.

[0014]In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin is a polypeptide selected from the group consisting of: [0015](a) a fragment comprising mature human osteocalcin missing the last 10 amino acids from the C-terminal end; [0016](b) a fragment comprising mature human osteocalcin missing the first 10 amino acids from the N-terminal end; [0017](c) a fragment comprising amino acids 62-90 of SEQ ID NO:2; [0018](d) a fragment comprising amino acids 1-36 of mature human osteocalcin; and [0019](e) variants of the above.

[0020]In certain embodiments, the pharmaceutical composition comprises a small molecule selected from the group consisting of warfarin, beta-blockers, statins, vitamin K inhibitors and biologically active fragments or variants thereof. In a preferred embodiment, the small molecule is warfarin. In another preferred embodiment, the agent is a small molecule that increases the activity or expression of osteocalcin or adiponectin.

[0021]In certain embodiments, the pharmaceutical composition comprises an antibody or antibody fragment that binds to OST-PTP or gamma-carboxylase. Preferably, the antibody or antibody fragment is a monoclonal antibody. In certain embodiments, the antibody or antibody fragment binds to the extracellular domain of OST-PTP. In preferred embodiments, the OST-PTP is human OST-PTP. In certain embodiments, the OST-PTP is the mouse OST-PTP of SEQ ID NO:19 or an OST-PTP having an amino acid sequence that is substantially homologous or identical to SEQ ID NO:19. In certain embodiments, the OST-PTP is an OST-PTP having an amino acid sequence that is at least 70% homologous or identical to SEQ ID NO:19.

[0022]In certain embodiments, the pharmaceutical composition comprises a nucleic acid that inhibits the expression or activity of OST-PTP or gamma-carboxylase. In certain embodiments, the nucleic acid is an antisense oligonucleotide or a siRNA. In certain embodiments, the nucleic acid is an isolated nucleic acid that is selected from the group consisting of an antisense DNA, antisense RNA, and small interfering RNA, which nucleic acid is sufficiently complementary to SEQ ID NO:18 or a sequence that is substantially homologous or identical to SEQ ID NO:18 to permit specific hybridization to SEQ ID NO:18 or the sequence that is substantially homologous or identical to SEQ ID NO:18, and wherein the hybridization prevents or reduces expression of OST-PTP in osteoblasts.

[0023]In certain embodiments, the pharmaceutical composition comprises an agent that increases the level of serum insulin.

[0024]In certain embodiments, the pharmaceutical composition comprises about 1 mg to about 750 mg of the agent. In certain embodiments, the pharmaceutical composition comprises an agent that is formulated into a controlled release preparation. In certain embodiments, the pharmaceutical composition comprises an agent that is chemically modified to prolong its half life in the human body.

[0025]In certain embodiments, the pharmaceutical composition comprises an anti-coagulant, a vasodilator, a drug used to treat atherosclerosis, a drug used to treat diabetes, a vitamin K inhibitor, a statin, or a beta blocker.

[0026]The present invention also provides a pharmaceutical composition comprising an undercarboxylated osteocalcin polypeptide comprising an amino acid sequence

TABLE-US-00001 (SEQ ID NO: 23) YLYQWLGAPVPYPDPLX₁PRRX₂VCX₃LNPDCDELADHIGFQEAYRRFYG PV

wherein [0027]X₁, X₂ and X₃ are each independently selected from an amino acid or amino acid analog, with the proviso that if X₁, X₂ and X₃ are each glutamic acid, then X₁ is not carboxylated, or less than 50 percent of X₂ is carboxylated, and/or less than 50 percent of X₃ is carboxylated, [0028]or said osteocalcin polypeptide comprises an amino acid sequence that is different from SEQ. ID. NO:23 at 1 to 7 positions other than X₁, X₂ and X₃; and [0029]wherein said amino acid sequence can include one amide backbone substitutions.

[0030]In certain embodiments, the osteocalcin polypeptide of SEQ. ID. NO:23 is a fusion protein. In certain embodiments, the arginine at position 43 of SEQ. ID. NO:23 is replaced with an amino acid or amino acid analog that reduces susceptibility of the osteocalcin polypeptide to proteolytic degradation. In certain embodiments, the arginine at position 44 of SEQ. ID. NO:23 is replaced with f3-dimethyl-arginine. In certain embodiments, the osteocalcin polypeptide is a retroenantiomer of uncarboxylated human osteocalcin (1-49).

[0031]The present invention also provides a method of modulating a pathway related to energy metabolism and the OST-PTP signaling pathway comprising administering an agent that reduces OST-PTP phosphorylase activity, reduces gamma-carboxylase activity, or increases undercarboxylated/uncarboxylated osteocalcin, wherein the agent is administered in an amount that produces an effect selected from the group consisting of an increase in glucose tolerance, an increase in insulin production, an increase insulin sensitivity, an increase in pancreatic beta-cell proliferation, an increase in adiponectin serum level, a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, and a decrease in the thickness of arterial plaque. In certain embodiments, the agent is administered in an amount effective to treat or prevent a disorder selected from the group consisting of metabolic syndrome, glucose intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and obesity.

[0032]In certain embodiments, the method comprises administering the agent in an amount effective to treat or prevent a disorder selected from the group consisting of metabolic syndrome, glucose intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and obesity. In certain embodiments, the agent inhibits the expression or activity of OST-PTP or gamma-carboxylase, inhibits phosphorylation of gamma-carboxylase, increases the level of undercarboxylated or uncarboxylated osteocalcin, inhibits carboxylation of osteocalcin, decarboxylates osteocalcin. In certain embodiments, the agent is selected from the group consisting of a small molecule, an antibody, a nucleic acid and a biologically active fragment or variant thereof.

[0033]In certain embodiments, the agent is undercarboxylated/uncarboxylated osteocalcin. In certain embodiments, at least one of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21 and 24 of mature human osteocalcin is not carboxylated. In certain embodiments, all three of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21 and 24 of mature human osteocalcin are not carboxylated. In certain embodiments, the present invention provides methods of administering undercarboxylated/uncarboxylated osteocalcin to increase insulin production.

[0034]In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin is a preparation of undercarboxylated/uncarboxylated osteocalcin in which more than about 20% of the total Glu residues at the positions corresponding to positions 17, 21 and 24 of mature human mature human osteocalcin in the preparation are not carboxylated. In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin shares at least 80% amino acid sequence identity with mature human osteocalcin when the undercarboxylated/uncarboxylated osteocalcin and mature human osteocalcin are aligned for maximum sequence homology.

[0035]In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin is a polypeptide selected from the group consisting of: [0036](a) a fragment comprising mature human osteocalcin missing the last 10 amino acids from the C-terminal end; [0037](b) a fragment comprising mature human osteocalcin missing the first 10 amino acids from the N-terminal end; [0038](c) a fragment comprising amino acids 62-90 of SEQ ID NO:2; [0039](d) a fragment comprising amino acids 1-36 of mature human osteocalcin; and [0040](e) variants of the above.

[0041]In certain embodiments, the agent is a small molecule that inhibits the expression or activity of OST-PTP or gamma-carboxylase. In certain embodiments, the agent is a small molecule selected from the group consisting of warfarin, beta-blockers, statins, vitamin K inhibitors and biologically active fragments or variants thereof. In a preferred embodiment, the small molecule is warfarin. In another preferred embodiment, the agent is a small molecule that increases the activity or expression of osteocalcin or adiponectin.

[0042]In certain embodiments, agent is an antibody or antibody fragment that binds to OST-PTP or gamma-carboxylase. Preferably, the antibody or antibody fragment is a monoclonal antibody. In certain embodiments, the antibody or antibody fragment binds to the extracellular domain of OST-PTP. In preferred embodiments, the OST-PTP is human OST-PTP. In certain embodiments, the OST-PTP is the mouse OST-PTP of SEQ ID NO:19 or an OST-PTP having an amino acid sequence that is substantially homologous or identical to SEQ ID NO:19. In certain embodiments, the OST-PTP is an OST-PTP having an amino acid sequence that is at least 70% homologous or identical to SEQ ID NO:19.

[0043]In certain embodiments, the agent is a nucleic acid that inhibits the expression or activity of OST-PTP or gamma-carboxylase. In certain embodiments, the nucleic acid is an antisense oligonucleotide or a siRNA. In certain embodiments, the nucleic acid is an isolated nucleic acid that is selected from the group consisting of an antisense DNA, antisense RNA, and small interfering RNA, which nucleic acid is sufficiently complementary to SEQ ID NO:18 or a sequence that is substantially homologous or identical to SEQ ID NO:18 to permit specific hybridization to SEQ ID NO:18 or the sequence that is substantially homologous or identical to SEQ ID NO:18, and wherein the hybridization prevents or reduces expression of OST-PTP in osteoblasts.

[0044]In certain embodiments, the methods of the present invention are carried out by administering about 1 mg to about 750 mg of the agent. In certain embodiments, the agent that is formulated into a controlled release preparation. In certain embodiments, the agent is chemically modified to prolong its half life in the human body. In certain embodiments, the agent is co-administered with an anti-coagulant, a vasodilator, a drug used to treat atherosclerosis, a drug used to treat diabetes, a vitamin K inhibitor, a statin, or a beta blocker.

[0045]The present invention also provides a method of diagnosing a patient at risk of developing a disease related to energy metabolism and the OST-PTP signaling pathway comprising (i) determining the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in a biological sample from the patient; and (ii) comparing the ratio to a standard ratio; wherein, if the patient ratio is lower than the standard ratio, the patient is at risk of developing a disease related to the OST-PTP signaling pathway.

[0046]In certain embodiments, the disease related to the OST-PTP signaling pathway is selected from the group consisting of metabolic syndrome, glucose intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and obesity. In certain embodiments, disease related to the OST-PTP signaling is characterized by decreased insulin production, decreased insulin sensitivity, decreased glucose tolerance and/or increased fat mass.

[0047]In certain embodiments, the biological sample is blood.

[0048]In certain embodiments of the diagnostic method described above, the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35.

[0049]The present invention provides a use of an agent that modulates energy metabolism and the OST-PTP signaling pathway and reduces OST-PTP phosphorylase activity, reduces gamma-carboxylase activity, or increases undercarboxylated/uncarboxylated osteocalcin, in an amount that produces an effect selected from the group consisting of an increase in glucose tolerance, an increase in insulin production, an increase insulin sensitivity, an increase in pancreatic beta-cell proliferation, an increase in adiponectin serum level, a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, and a decrease in the thickness of arterial plaque, as a medicament.

[0050]In certain embodiments, the agent is used to treat or prevent a disorder selected from the group consisting of metabolic syndrome, glucose intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and obesity.

[0051]In certain embodiments, the agent inhibits phosphorylation of gamma-carboxylase. In certain embodiments, the agent increases the level of uncarboxylated osteocalcin. In certain embodiments, the agent inhibits carboxylation of osteocalcin. In certain embodiments, the agent decarboxylates osteocalcin.

[0052]In certain embodiments, the agent is undercarboxylated/uncarboxylated osteocalcin. In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin increases insulin production. In certain embodiments, at least one of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21 and 24 of mature human osteocalcin is not carboxylated. In certain embodiments, all three of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21 and 24 of mature human osteocalcin are not carboxylated. In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin is a preparation of undercarboxylated/uncarboxylated osteocalcin in which more than about 20% of the total Glu residues at the positions corresponding to positions 17, 21 and 24 of mature human mature human osteocalcin in the preparation are not carboxylated. In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin shares at least 80% amino acid sequence identity with mature human osteocalcin when the undercarboxylated/uncarboxylated osteocalcin and mature human osteocalcin are aligned for maximum sequence homology.

[0053]In certain embodiments, the undercarboxylated/uncarboxylated osteocalcin is a polypeptide selected from the group consisting of: [0054](a) a fragment comprising mature human osteocalcin missing the last 10 amino acids from the C-terminal end; [0055](b) a fragment comprising mature human osteocalcin missing the first 10 amino acids from the N-terminal end; [0056](c) a fragment comprising amino acids 62-90 of SEQ ID NO:2; [0057](d) a fragment comprising amino acids 1-36 of mature human osteocalcin; and [0058](e) variants of the above.

[0059]In certain embodiments, the agent is selected from the group consisting of a small molecule, an antibody, a nucleic, acid and a biologically active fragment or variant thereof.

[0060]In certain embodiments, the agent is a small molecule that inhibits the expression or activity of OST-PTP or gamma-carboxylase. In certain embodiments, the agent is a small molecule selected from the group consisting of warfarin, beta-blockers, statins, vitamin K inhibitors and biologically active fragments or variants thereof. In a preferred embodiment, the small molecule is warfarin. In another preferred embodiment, the agent is a small molecule that increases the activity or expression of osteocalcin or adiponectin.

[0061]In certain embodiments, agent is an antibody or antibody fragment that binds to OST-PTP or gamma-carboxylase. Preferably, the antibody or antibody fragment is a monoclonal antibody. In certain embodiments, the antibody or antibody fragment binds to the extracellular domain of OST-PTP. In preferred embodiments, the OST-PTP is human OST-PTP. In certain embodiments, the OST-PTP is the mouse OST-PTP of SEQ ID NO:19 or an OST-PTP having an amino acid sequence that is substantially homologous or identical to SEQ ID NO:19. In certain embodiments, the OST-PTP is an OST-PTP having an amino acid sequence that is at least 70% homologous or identical to SEQ ID NO:19.

[0062]In certain embodiments, the agent is a nucleic acid that inhibits the expression or activity of OST-PTP or gamma-carboxylase. In certain embodiments, the nucleic acid is an antisense oligonucleotide or a siRNA. In certain embodiments, the nucleic acid is an isolated nucleic acid that is selected from the group consisting of an antisense DNA, antisense RNA, and small interfering RNA, which nucleic acid is sufficiently complementary to SEQ ID NO:18 or a sequence that is substantially homologous or identical to SEQ ID NO:18 to permit specific hybridization to SEQ ID NO:18 or the sequence that is substantially homologous or identical to SEQ ID NO:18, and wherein the hybridization prevents or reduces expression of OST-PTP in osteoblasts.

[0063]In certain embodiments, about 750 mg of the agent is used as a medicament. In certain embodiments, the agent is formulated into a controlled release preparation. In certain embodiments, the agent is chemically modified to prolong its half life in the human body. In certain embodiments, the agent is co-administered with an anti-coagulant, a vasodilator, a drug used to treat atherosclerosis, a drug used to treat diabetes, a vitamin K inhibitor, a statin, or a beta blocker.

[0064]The present invention provides the use of an undercarboxylated osteocalcin polypeptide, or mimetic thereof, in the manufacture of a medicament for treatment of a metabolic condition.

[0065]The present invention also provides the use of an agent that modulates energy metabolism and the OST-PTP signaling pathway and reduces OST-PTP phosphorylase activity, reduces gamma-carboxylase activity, or increases undercarboxylated/uncarboxylated osteocalcin, in an amount that produces an effect selected from the group consisting of an increase in glucose tolerance, an increase in insulin production, an increase insulin sensitivity, an increase in pancreatic beta-cell proliferation, an increase in adiponectin serum level, a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, and a decrease in the thickness of arterial plaque, for the manufacture of a medicament for the treatment or prevention of a disorder selected from the group consisting of metabolic syndrome, glucose intolerance, type 1 diabetes, type 2 diabetes, atherosclerosis, and obesity.

BRIEF DESCRIPTION OF THE FIGURES

[0066]FIG. 1. Increased insulin secretion and beta-cell proliferation in Esp-/- mice. (A) LacZ-stained tissues from newborn Esp-/- mice demonstrating Esp locus activity in bone and testis but not in pancreas or fat pads. (B) Expression of Esp in osteoblasts, adipocytes, and pancreatic islets by real-time PCR in 1-month-old mice. (C) Southern blot analysis showing efficient recombination at the Esp locus in osteoblasts of Esposb-/- mice. (D) Using real-time PCR Esp expression is 90% decreased in osteoblasts but not altered in testis of Esposb-/- mice. (E) Decreased percentage at weaning of Esp-/- pups born from crosses between Esp+/- mice. (F) Lower survival at birth and at weaning of Esp-/- pups born from Esp+/- and Esp-/- mothers. (G and H) Blood glucose levels (G) and serum insulin levels (H) in WT and Esp-/- newborn before feeding (P0) or after random feeding at indicated ages. (I-J) GSIS (I) and GTT (J) test in 1-month-old WT and Esp-/- mice. (K) H&E staining, insulin immunostaining, and insulin/Ki67 double immunostaining showing larger islets and increased beta-cell proliferation in pancreas of WT and 1-month-old Esp-/- mice. Arrowheads indicate islets, and arrows point at Ki67-positive cells. Scale bars are 100 mm except in upper panels, where they are 800 mm. Histomorphometric comparisons of islet number, size, and beta-cell mass between 1-month-old WT and Esp-/- mice (lowest panel). (L) Pancreas insulin content in 1-month-old WT and Esp-/- mice. (M) Quantification of the number of Ki67-immunoreactive cells in pancreatic islets of P5 and 1-month-old WT and Esp-/- mice. All panels except (I) and (J), ° p<0.05 and *p<0.01 versus WT (Student's t test). (I and J) ° p<0.05 versus WT and *p % 0.001 versus WT (ANOVA followed by post hoc analysis).

[0067]FIG. 2. Increased Insulin Sensitivity and Adiponectin Expression in Esp-/- Mice. All experiments compare 1-month-old mice WT and Esp-/- unless otherwise indicated. (A) ITT. (B) Glucose infusion rate during hyperinsulinemic-euglycemic clamp. (C) Expression of markers of insulin sensitivity in skeletal muscle measured by real-time PCR. (D) Electron microscopy images (upper panel, 20,0003) and corresponding quantification (lower panel) of mitochondrial area in gastrocnemius muscle. Scale bars are 1 mm. (E) Decreased number of lipid droplets on Oil red O stained liver sections (upper panel) and modified expression of insulin target genes by real-time PCR (lower panel) in Esposb-/- mice. Scale bars are 50 mm. (F) Fat pad mass (fat pad weight over body weight). (G) Energy expenditure. (H) Serum triglyceride levels after an overnight fast. (I) H&E staining of adipose tissues of WT and Esp-/- mice (upper panel) and respective distribution of diameters for 100 measured adipocytes per slide (lower panel). Scale bars are 50 mm. (J) Expression of markers of adipogenesis, lipogenesis, fat uptake, and lipolysis in fat. (K) Serum free fatty acid (FFA) in fed and overnight-fasted mice. (L) Expression of Leptin, Resistin, and Adiponectin in fat. (M) Serum levels of adiponectin in newborn mice before feeding (P0) and after random feeding at other indicated ages. (N) Expression of adiponectin target genes, in tissues of WT and Esp-/- mice. In (A), ° p<0.05 versus WT and *p % 0.001 versus WT (ANOVA followed by post hoc analysis); in (B)-(N), *p<0.01 versus WT (Student's t test).

[0068]FIG. 3. Esp-/- Mice Are Protected from Obesity and Glucose Intolerance A-F) Food intake per day (A), body weight curve (B), fat pad mass (C), serum triglyceride levels (D), GTT (E), and ITT (F) in 4-month-old WT and Esp-/- mice 3 months after GTG or vehicle injection. (G-I) Body weight curve (G), GTT (H), and ITT (I) in 3 month-old WT and Esp-/- mice fed a high fat diet for 6 weeks. (J and K) Serum insulin levels (J) and pancreatic insulin content (K) in 1-month-old WT and Esp-/- mice 8 days after STZ or vehicle injection. (L and M) Survival of mice (L) and change of blood glucose levels (M) in 1-month-old WT and Esp-/- mice during the 8 days following STZ injection. (N) Urinary glucose assays in 1-month-old WT and Esp-/- mice 8 days after STZ injection. In (A)-(F), (J), and (K): a, WT versus Esp-/-; b,WT+GTG (or STZ) versus WT+vehicle; c,WT+GTG (or STZ) versus Esp-/-+GTG (or STZ); d, Esp-/-+GTG (or STZ) versus Esp-/-+vehicle. In (G)-(I) and (M), *p<0.05 WT versus Esp-/-. In (A), (C), (D), (J), and (K), Student's t test, p<0.05 for a-d; in (B), (E)-(I), (L), and (M), ANOVA followed by post hoc analysis when number of groups >2, p % 0.001 for a-d.

[0069]FIG. 4. Osteoblasts Secrete a Factor Regulating Insulin and Adiponectin Expression. A-E) All experiments compare 1-month-oldWT and al(I)-Esp mice. (A) Insulin immunostaining (upper panel) and histomorphometric comparisons of islet number, size, beta-cell mass, and Ki67-immunoreactive cells in pancreas (lower panel). Scale bars are 100 mm. (B) Blood glucose and serum insulin/adiponectin levels. (C) GSIS test. (D) GTT. (E) ITT. (F) Expression of Insulin and Glucagon in WT islets cocultured with fibroblasts or osteoblasts. (G) Expression of Adiponectin and Leptin in WT adipocytes cocultured with fibroblasts or osteoblasts. (H) Expression of Insulin and Adiponectin in Esp-/- indicated cells cocultured with fibroblasts or osteoblasts. (I and J) Expression of Insulin (I) and Adiponectin (J) in WT indicated cells cocultured with or without osteoblasts in presence of a filter preventing cell-cell contact or in presence of conditioned medium (CM) collected from osteoblast cultures. (A, B, and F-J) *p<0.05 versus WT (Student's t test); (C-E) ° p<0.05 versus WT and *p % 0.001 versus WT (ANOVA).

[0070]FIG. 5. Osteocalcin Regulates Beta-Cell Proliferation, Insulin Secretion, and Insulin sensitivity. All experiments compare 3-month-old WT and Oc-/- mice unless otherwise indicated. (A) Blood glucose levels after random feeding. (B) Insulin levels. (C) GSIS test. (D) GTT. (E) ITT. (F) Glucose infusion rate during hyperinsulinemic-euglycemic clamp. (G) Energy expenditure. (H) Expression of insulin target genes by real-time PCR. (I) Histomorphometric comparisons of islet numbers, islet size, beta-cell mass, insulin content in pancreas, and Ki67 immunoreactive cells in pancreatic islets. P5, 5-day-old pups; 3M, 3-month-old mice. (J) Fat pad mass (fat pad weight over body weight). (K) Serum triglyceride levels after an overnight fast. (L and M) serum levels (L) and gene expression (M) of adiponectin. (N) Expression of adiponectin target genes by real time PCR. (O) Expression of Insulin and Glucagon in WT pancreatic islets co-cultured with osteoblasts of indicated genotypes. (P) Expression of Adiponectin and Leptin in WT adipocytes co-cultured with osteoblasts of indicated genotypes. (Q) Expression of Insulin and Adiponectin in WT indicated cells cultured in presence of conditioned media from COS cells transfected with an Osteocalcin expression vector or its empty counterpart. (R) Expression of Insulin and Adiponectin in WT islets and adipocytes co-cultured with fibroblasts in presence of recombinant osteocalcin (3 ng/ml) or vehicle, or with osteoblasts expressing (5d) or not (1d) Osteocalcin. (S and T) Dynamic of glucose (S) and insulin levels (T) in Ocn-/- mice injected simultaneously with glucose and 20 ng of recombinant osteocalcin or vehicle. Panels A, B, F-R: *p<0.05 vs WT (Student's t test); panels C-E, S and T, ° p<0.01 vs WT and *p<0.001 vs WT (ANOVA). Results are given as means±SD except in FIG. 5F where means±SEM are shown.

[0071]FIG. 6. Osteocalcin Regulates Insulin Sensitivity via Adiponectin. (A-E) Comparison between 6-week-old WT, Adiponectin+/- (Adipo+/-), Osteocalcin+/- (Ocn+/-), and Ocn+/-; Adipo+/- mice. (A) ITT. (B) Insulin serum levels. (C) Blood glucose levels. (D) GSIS test. (E) Adiponectin serum levels. In (A) and (D), *p % 0.001 versus WT (ANOVA followed by post hoc analysis); in (B), (C), and (E), *p<0.05 versus WT (Student's t test).

[0072]FIG. 7. Esp-/- Mice Are a Model of Increased Osteocalcin Bioactivity. (A-G) Comparison between 6-week-old WT, Esp-/, Ocn+/-, and Esp-/-; Ocn+/- mice. (A) Blood glucose levels. (B) Serum insulin levels. (C) Serum adiponectin levels. (D) GTT. (E) ITT. (F) GSIS test. (G) Quantification of the number of Ki67-immunoreactive cells in pancreatic islets. (H and I) Quantification of the percentage of osteocalcin bound to hydroxyapatite (HA) resin after a 15min incubation of serum of 1-month-old mice of indicated genotypes (H) or of conditioned medium from osteoblast cultures treated with warfarin or vehicle (I). (J) Expression of Adiponectin in WT adipocytes cocultured with osteoblasts treated with warfarin or vehicle. (K) Expression of Adiponectin in WT adipocytes cultured in presence of vehicle or of 1 ng/ml of commercially available carboxylated osteocalcin (Immunotopics) or bacterially produced uncarboxylated osteocalcin. (L) Expression of Insulin and Cyclin D1 in WT islets cultured in presence of 0.3 ng/ml of bacterially produced uncarboxylated osteocalcin or vehicle. (M) Metabolic parameters and total serum osteocalcin levels in control and obese patients. (N-O) Quantification of carboxylated osteocalcin (HA-bound osteocalcin) in control and obese patients. In (A)-(C) and (G)-(L): *p<0.05 versus WT (Student's t test); in (D)-(F), ° p<0.05 versus WT and *p % 0.001 versus WT (ANOVA followed by post hoc analysis).

[0073]FIG. 8. OST-PTP was mutated in COS cells using site directed mutagenesis using a PCR method and a commercially available kit. Mutated OST-PTP (GST-PTP CA) interacts with insulin receptor (InsR) in COS cells (left upper panel) and ROS cells (right upper panel) (third lane) whereas WT OST-PTP (GST-PTP WT) does not interact (second lane). The same amount of GST fusion proteins were used for substrate trapping (lower panels). GST=Recombinant bacterially produced glutathione S-Transferase protein.

[0074]FIG. 9. Mutant enzyme OST-PTP.sup.D1316A traps gamma-carboxylase, thereby demonstrating that gamma-carboxylase is a substrate of OST-PTP. This, however, does not mean that gamma-carboxylase is the only substrate of OST-PTP. There was no binding in the GST lane because there is no PTP transfected. It is a control to show that if there is trapping, it is not due to the GST part of any GST fusion protein. There was also no trapping with GST-PTP^WT because this form dephosphorylated the substrate gamma-carboxylase, which is then released. A band is clearly seen in the lane having the mutant OST-PTP (GST-PTP.sup.D1316A) because the mutation engineered a defect in OST-PTP phosphatase activity that allowed the substrate to irreversibly bind to and be retained by the enzyme.

[0075]FIG. 10. Osteocalcin subcutaneous infusion decreases glycemia in wt mice. Indicated doses of recombinant osteocalcin or PBS were infused subcutaneously for 28 days in wt mice. Blood glucose was measured at indicated days.

[0076]FIG. 11. Osteocalcin subcutaneous infusion increases glucose tolerance in wt mice. Wt mice were infused subcutaneously with indicated doses of recombinant osteocalcin or PBS for 14 days before receiving a single injection of glucose. Blood glucose was measured thereafter at indicated times.

[0077]FIG. 12. Osteocalcin subcutaneous infusion increases insulin sensitivity in wt mice. Wt mice were infused subcutaneously with indicated doses of recombinant osteocalcin or PBS for 18 days before receiving a single injection of insulin. Blood glucose was measured thereafter at indicated times.

[0078]FIG. 13. Osteocalcin subcutaneous infusion decreases fat mass in wt mice. 1) Indicated doses of recombinant osteocalcin or PBS were infused subcutaneously for 28 days in wt mice. Body weight was recorded at indicated days. (2) Gonadal fat pad mass was measured after 28 days.

[0079]FIG. 14. Osteocalcin subcutaneous infusion prevents GTG-induced obesity in wt mice. Wt mice were injected with gold thioglucose (GTG) or vehicle to induce hyperphagia and obesity. Two weeks later they were implanted with subcutaneous osmotic pumps infusing 1 n/hr of recombinant osteocalcin or PBS for 28 days before. Body weight gain was recorded thereafter at indicated days.

[0080]FIG. 15. A fragment of osteocalcin (1-36) is as potent as native osteocalcin in inducing adiponectin expression in vitro. Wt adipocytes were treated for 4 hours with recombinant full-length osteocalcin (1-46) or a truncated form (1-36) or vehicle. Adiponectin expression was then quantified by real time PCR.

[0081]FIG. 16. Shows the effect of applying isoproterenol (enhancing SNS activity) to wild type osteoblasts on the expression of ptprv=Esp/OST-PTP, Ggcx=gamma-carboxylase, Vkor=recycling of vitamin K/necessary for ggcx activity, and Bgp=osteocalcin. mRNA levels in wt osteoblasts were measured using quantitative PCR.

[0082]FIG. 17. The level of serum insulin in one week old mice having various genotypes: WT mice, ob-/+ mice (heterozygous for obesity), ob/ob mice, Bgp -/+ (heterozygous for osteocalcin), BGP-/- mice, and ob/ob/mice that are also Bgp-/- (Oc deficient).

[0083]FIG. 18. Ostecalcin-deficient mice develop atherogenic lesions after 6 weeks on the western diet. Histological analysis of the entrance aorta at the level of the aortic valves clearly shows that atherogenic lesions are present.

[0084]FIG. 19. Absence of the Esp gene that encodes OST-PTP protects ApoE-/- mice from developing atherosclerotic lesions. ApoE-/- mice develop atherosclerotic lesions after being fed a western diet for 6 weeks, while double mutant ApoE-/-; Esp-/- mice on the same diet do not.

[0085]FIG. 20. The pathway for regulation of osteocalcin production by bone. (Upper panel) OST-PTP, the protein encoded by Esp favors the γ-carboxylation of osteocalcin thus lowering the pool of uncarboxylated osteocalcin, which is the active form of this hormone. As a result β-cell proliferation, Insulin expression and Adiponectin expression are normally stimulated. (Lower panel) In absence of OST-PTP γ-carboxylation of osteocalcin is hampered, there is more uncarboxylated osteocalcin and, as a result, β-cell proliferation, Insulin expression and Adiponectin expression are increased. This results in improved glucose handling and decreased fat mass.

[0086]FIG. 21. A diagram showing the high degree of conserved amino acid sequence homology of osteocalcin among species.

[0087]FIG. 22. Generation of Esposb-/- mice and normal bone formation in Esp-/- animals. (A) Targeting construct for conditional inactivation of Esp. White boxes, exons encoding the phosphatase domain of OST-PTP; grey triangles, LoxP sites; black bars, 5' and 3' external probes; S, Sacl; EV, EcoRV. (B) PCR genotyping of Esposb-/- mice. WT and floxed allele (F1) yield 280-bp and 350-bp products, respectively. 1(I) collagen-Cre (1(I)-Cre)) transgenic mice (TG) harbor a transgene-specific band. (C-D) Similar body weight gain (C) and linear growth (D) in WT and Esp-/- mice. P0, newborn; 1M, 1 month (E) Alizarin red/alcian blue staining of skeletons of newborn WT and Esp-/- mice. Mice were dissected, fixed in 95% ethanol, and stained in alcian blue and alizarin red as previously described (Duey et al., 1996). There is no overt defect in mineralization that could explain the perinatal death of mutant pups. (F) Bone histomorphometry of 2 month-old WT and Esp-/- mice. BV/TV, bone volume per Total tissue volume (%); N.ob/B.Pm, number of osteoblasts per bone perimeter (mm-1); Dpd/Creat. Relative levels of deoxypyridinolone crosslinks, a marker of bone resorption.

[0088]FIG. 23. (A-E) Comparison of 1 month-old WT and Esp-/- mice. Serum levels of C-peptide (A), serum Glucagon level (left) and glucagons content in pancreas (right) (B) and serum levels of IGF-1 (C), PYY (D), and amylin (E) in Esp-/- mice. (F) Ratio of muscle mass over body mass calculated by proton magnetic resonance spectroscopy (1H-MRS) in 10 week-old WT and Esp-/- mice. (G) Representative images of proton 1H-MRS of 10 week-old WT and Esp-/- mice. (H) Food intake per day in 1 month- and 3 month-old Esp-/- and WT mice. (I and J) Comparison of expression level by real time PCR (I) and of serum levels (J) of TNF- (left) and IL-6 (right) in 1 month-old Esp-/- and WT mice. (K) Serum leptin (left) and resistin (right) levels in 1 month-old Esp-/- and WT mice. In all panels data represent the means±SD of experiments. *, P<0.01 (t-test).

[0089]FIG. 24. Destruction of VMH nuclei by GTG. Cresyl violet staining of section from hypothalamus of WT and Esp-/- mice injected with GTG or vehicle. Arcuate nuclei are circled in blue, VMH in red.

[0090]FIG. 25. Absence of cell transdifferentiation during the co-culture assays. (A-D) Analysis of Runx2 (A), Osteocalcin (B), adiponectin (C), and Leptin (D) expression by real time PCR in indicated cells 4h after co-culture of osteoblasts with adipocytes or islets.

[0091]FIG. 26. Bone-specific expression of osteocalcin. (A) In situ hybridization analysis of osteocalcin and Esp expression in pancreas of 18.5 dpc embryos. Neither gene is expressed in pancreas. Insulin expression was used as a positive control. Hematoxylin-eosin staining of adjacent sections was used to assess tissue integrity. (B) Real time PCR analysis of osteocalcin expression in osteoblasts, adipocytes, and pancreatic islets collected from 1 month-old WT mice. Osteocalcin is not expressed in adipocytes or islets.

[0092]FIG. 27. Normal osteocalcin expression and serum level in Esp-/- and alpha1(I) Esp mice. (A-B) Real-time PCR analysis of osteocalcin expression (A) and osteocalcin serum levels (B) in 1 month-old WT, Esp-/- and alpha1(I) Esp mice. (C) Analysis of the purity of bacterially produced osteocalcin by SDS-PAGE stained with Coomassie blue.

[0093]FIG. 28. Overexpression of adiponectin in transgenic mice decreases fat mass and increases insulin sensitivity.(A) Schematic representation of the transgene used to overexpress adiponectin (Adipo) in mice. (B) PCR genotyping of SAP-Adipo transgenic mice. (C) Adiponectin serum levels in 3 distinct SAP-Adipo transgenic lines at 1 month of age. (D) Fat pad mass in WT and SAP-Adipo transgenic mice at 3 month of age. (E) Serum insulin levels in WT and SAP-Adipo transgenic mice at 3 month of age. (F) Insulin tolerance test in WT and SAP-Adipo transgenic mice at 3 month of age.

[0094]FIG. 29: Overexpression of insulin in transgenic mice decreases fat mass and increases glucose tolerance. (A-B) Blood glucose levels after fasting (A) or random feeding (B) in SAP-insulin transgenic and wt mice. (C) Serum levels of triglycerides in WT and SAP-insulin transgenic mice. (D) Serum levels of free fatty acids in WT and SAP-insulin transgenic mice. (E) Fat pad mass in WT and SAP-insulin transgenic mice. (F) Glucose tolerance test in WT and SAP-insulin transgenic mice. (G) Food intake in

[0095]WT and SAP-insulin transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

[0096]The present invention is based in part on the discovery that undercarboxylated/uncarboxylated osteocalcin secreted by osteoblasts in bone is responsible for regulating various aspects of energy metabolism. For example, it increases pancreatic beta-cell proliferation, insulin secretion, insulin sensitivity, glucose tolerance, and serum adiponectin and decreases weight gain and fat mass. It also reduces the pathological effects of atherosclerosis. Therefore, certain aspects of the invention are directed to the therapeutic use of undercarboxylated/uncarboxylated osteocalcin, fragments and variants thereof, to treat or prevent metabolic syndrome, type 1 and type 2 diabetes, atherosclerosis and obesity.

[0097]The present invention is also based on the discovery that gamma-carboxylase carboxylates osteocalcin, thereby inactivating osteocalcin. Such inactivation of osteocalcin decreases pancreatic beta-cell proliferation, insulin secretion, insulin sensitivity, glucose tolerance, and serum adiponectin and increases weight gain and fat mass. It also increases the pathological effects of atherosclerosis. Therefore, certain aspects of the invention are directed to the therapeutic use of agents that inhibit the activity of gamma-carboxylase, to treat or prevent metabolic syndrome, type 1 and type 2 diabetes, atherosclerosis and obesity.

[0098]The present invention is further based on the discovery that OST-PTP activates gamma-carboxylase through dephosphorylation. As indicated above, activation of gamma-carboxylase leads to inactivation of osteocalcin. Such inactivation of osteocalcin decreases pancreatic beta-cell proliferation, insulin secretion, insulin sensitivity, glucose tolerance, and serum adiponectin and increases weight gain and fat mass. It also it increases the pathological effects of atherosclerosis. Therefore certain aspects of the invention are directed to the therapeutic use of agents that inhibit the activity of OST-PTP, to treat or prevent metabolic syndrome, type 1 and type 2 diabetes, atherosclerosis and obesity.

[0099]The present invention is also based on the discovery that undercarboxylated/uncarboxylated osteocalcin increases the level of adiponectin expression, resulting in increased insulin sensitivity, glucose tolerance, and decreased weight gain and fat mass. It also reduces the pathological effects of atherosclerosis. Therefore, certain aspects of the invention are directed to the therapeutic use of undercarboxylated/uncarboxylated osteocalcin to regulate the expression of adiponectin, to treat or prevent metabolic syndrome, type 1 and type 2 diabetes, atherosclerosis and obesity.

[0100]The present invention thus relates to methods and compositions for treating and diagnosing disorders related to the OST-PTP signaling pathway involving gamma-carboxylase, osteocalcin and adiponectin. Such disorders include, but are not limited to, metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity. The invention is based on the discovery that OST-PTP dephosphorylates gamma-carboxylase, thereby leading to activation of gamma-carboxylase. Activation of gamma-carboxylase results in carboxylation of osteocalcin, which, as demonstrated herein, leads to symptoms associated with metabolic syndrome, diabetes types 1 and 2, atherosclerosis and obesity.

[0101]Other aspects of the invention are directed to diagnostic methods based on detection of decreased levels of undercarboxylated/uncarboxylated osteocalcin, which is found to be associated with metabolic syndrome, type 1 and type 2 diabetes, atherosclerosis and obesity. In one aspect, the method of diagnosing a disease, including metabolic syndrome, diabetes types 1 and 2, atherosclerosis and obesity, in a patient comprises (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient (ii) comparing the patient level of undercarboxylated/uncarboxylated osteocalcin and a control level of undercarboxylated/uncarboxylated osteocalcin, and (iii) if the patient level is significantly lower than the control level, then the patient is diagnosed as having, or being at risk for, the disease, including metabolic syndrome, diabetes types 1 and 2, atherosclerosis and obesity.

[0102]Other aspects of the invention are directed to diagnostic methods based on detection of decreased ratios of undercarboxylated/uncarboxylated vs carboxylated osteocalcin. Such ratios are found to be associated with metabolic syndrome, type 1 and type 2 diabetes, atherosclerosis and obesity. In one aspect, the method of diagnosing a disease, including metabolic syndrome, diabetes types 1 and 2, atherosclerosis and obesity, in a patient comprises (i) determining a patient ratio of undercarboxylated/uncarboxylated vs carboxylated osteocalcin in a biological sample taken from the patient (ii) comparing the patient ratio of undercarboxylated/uncarboxylated vs carboxylated osteocalcin and a control ratio of undercarboxylated/uncarboxylated vs carboxylated osteocalcin, and (iii) if the patient ratio is significantly lower than the control ratio, then the patient is diagnosed has having, or being at risk for, the disease, including metabolic syndrome, diabetes types 1 and 2, atherosclerosis and obesity.

[0103]Other aspects of the invention are directed to methods for diagnosing a patient at risk of developing a disease, including metabolic syndrome, glucose intolerance, impaired pancreatic beta-cell proliferation, impaired insulin secretion, impaired insulin sensitivity, atherosclerosis and obesity, by determining the patient level of, or level of activity of, OST-PTP and/or gamma-carboxylase, wherein increases in said levels, as compared to controls, indicates a patient is at risk of developing the disease.

PHARMACEUTICAL COMPOSITIONS OF THE INVENTION

[0104]The present invention provides pharmaceutical compositions comprising an agent for modulating the OST-PTP signaling pathway, which as disclosed herein involves gamma-carboxylase and osteocalcin, or for treating or preventing disorders related to the OST-PTP signaling pathway. In particular embodiments, the agent inhibits OST-PTP phosphorylase activity, reduces gamma-carboxylase activity, and/or increases undercarboxylated/uncarboxylated osteocalcin. In particular embodiments, the agent decarboxylates osteocalcin. The agent may be selected from the group consisting of small molecules, polypeptides, antibodies, and nucleic acids. The pharmaceutical compositions of the invention provide an amount of the agent effective to treat or prevent a disorder associated with the OST-PTP signaling pathway. In certain embodiments, a pharmaceutical composition provides an amount of the agent effective to treat or prevent metabolic syndrome or a component thereof, diabetes type 1, diabetes type 2, atherosclerosis, or obesity in a subject. In other embodiments, the composition provides an amount of the agent effective to treat or prevent a disease characterized by decreased insulin production, decreased insulin sensitivity, and decreased glucose tolerance or increased fat mass

[0105]The pharmaceutical compositions of the invention may function to increase serum osteocalcin levels (preferably undercarboxylated or uncarboxylated osteocalcin), serum adiponectin levels and/or serum insulin levels. The pharmaceutical compositions may also increase glucose tolerance, increase insulin sensitivity, and/or increase pancreatic beta-cell proliferation. Other beneficial effects may include a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, and a decrease in hypertension.

[0106]In particular embodiments of the invention, therapeutic agents that may be administered include undercarboxylated osteocalcin; uncarboxylated osteocalcin; or inhibitors that reduce the expression or activity of gamma-carboxylase or OST-PTP (e.g., antibodies, small molecules, antisense nucleic acids or siRNA). The pharmaceutical agents may also include agents that decarboxylate osteocalcin.

[0107]The therapeutic agents are generally administered in an amount sufficient to treat or prevent metabolic syndrome, obesity, diabetes type 1 and 2 and atherosclerosis in a subject. The therapeutic agents may also be administered to reduce fat mass in a subject.

[0108]Biologically active fragments or variants of the therapeutic agents are also within the scope of the present invention. By "biologically active" is meant capable of modulating the OST-PTP signaling pathway involving gamma-carboxylase, osteocalcin and adiponectin. As described herein, "biologically active" means reducing the expression of OST-PTP or its ability to dephosphorylate gamma-carboxylase and reducing the expression of gamma-carboxylase or its ability to carboxylate osteocalcin, or decarboxylating carboxylated osteocalcin thereby leading to increased levels of undercarboxylated/uncarboxylated osteocalcin, insulin and adiponectin. "Biologically active" also means capable of causing at least one effect selected from the group consisting of increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, decreasing fat mass, weight loss, increasing serum adiponectin, a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, and a decrease in hypertension. Fragments and variants are described below. The screens or assays described below may be used to identify, or assay, biologically active fragments and variants of the therapeutic agents of the invention, as well as other agents.

Compositions Comprising Undercarboxylated Osteocalan

[0109]In a specific embodiment of the invention, pharmaceutical compositions comprising osteocalcin, particularly undercarboxylated or uncarboxylated osteocalcin, are provided.

[0110]Osteocalcin, one of the very few osteoblast-specific proteins, has several features of a hormone. For instance, it is synthesized as a pre-pro-molecule and is secreted in the general circulation (Hauschka et al., 1989; Price, 1989). Because of their exquisite cell-specific expression, the osteocalcin genes have been intensively studied to identify osteoblast-specific transcription factors and to define the molecular bases of bone physiology (Ducy et al., 2000b; Harada and Rodan, 2003).

[0111]Osteocalcin is the most abundant non-collagenous protein found associated with the mineralized bone matrix and it is currently being used as a biological marker for clinical assessment of bone turnover. Osteocalcin is a small (46-50 residue) bone specific protein that contains 3 gamma-carboxylated glutamic acid residues in its primary structure. The name osteocalcin (osteo, Greek for bone; Calc, Latin for lime salts; in, protein) derives from the protein's ability to bind Ca²+ and its abundance in bone. Osteocalcin undergoes a peculiar post-translational modification whereby glutamic acid residues are carboxylated to form gamma-carboxyglutamic acid (Gla) residues; hence osteocalcin's other name, bone Gla protein (Hauschka et al., 1989). Gla residues usually confer on proteins high affinity for mineral ions, yet loss- and gain--of function experiments until now have failed to identify a function for osteocalcin in extracellular matrix mineralization in vivo (Ducy et al., 1996; Murshed et al., 2004).

[0112]Osteocalcin is a vitamin K-dependent calcium binding protein (Price et al. (1976) Proc. Natl. Acad. Sci. 73:3373-375). Mature human osteocalcin contains 49 amino acids with a predicted molecular mass of 5,800 kDa (Poser et al. (1980) The Journal of Biological Chemistry, Vol 255, No. 18, pp. 8685-8691). Osteocalcin is synthesized primarily by osteoblasts and ondontoblasts and comprises 15 to 20% of the non-collagenous protein of bone. Poser et al. (1980) J. Biol. Chem. 255:8685-8691 showed that mature osteocalcin contains three carboxyglutamic acid residues which are formed by post-translational vitamin K-dependent modification of glutamic acid residues. The carboxylated Gla residues are at positions 17, 21 and 24 of mouse and human mature osteocalcin. Some human osteocalcin has been shown to contain only 2 Gla residues. Poser, J. W. & Price, P. A. (1979) A Method for Decarboxylation of γ-Carboxyglutamic Acid in Proteins. J. Biol. Chem. 254, 431-436.

[0113]The conformation of decarboxylated (or uncarboxylated or undercarboxylated) osteocalcin lies somewhere between the random coil and helical form. Thus, in solution the peptide occurs as a flexible structure and a single conformation cannot be defined for it (Atkinson et al. Eur. J. Biochem. 1995; 232:515-21). Peptide bonds between arginine residues 19 and 20 and between residues 43 and 44 are susceptible to tryptic hydrolysis, leading to peptides 1-19, 20-43, 45-49, 1-43, and 20-49 which may be the main products of human osteocalcin breakdown in the circulation (Farrugia and Melick, Calcif Tissue Int 1986; 39:234-8, Hellman et al. J Bone Miner Res 1996; 11:1165-75 and Garnero et al. J Bone Miner Res 1994; 9:255-4).

[0114]Conformational study of osteocalcin by circular dichroism (CD) has shown the existence of alpha-helical conformation in osteocalcin and that addition of Ca²+ induces higher helical content. Two-dimensional nuclear magnetic resonance (NMR) studies of osteocalcin in solution, while structurally inconclusive, revealed that the calcium-free protein was effectively unstructured except for the turn required by the disulfide bridge between Cys23 and Cys29. All the proline residues (Hyp9, Pro11, Pro13, Pro15, and Pro27) were in the trans conformation. Beta-turns are present in the region of Tyr12, Asp14 and Asn26. The hydrophobic core of the molecule is composed of the side chains of Leu2, Leu32, Va136 and Tyr42. The calcium-induced helix is extremely rigid due to, in part, the hydrophobic stabilization of the helical domain by the C-terminal domain.

[0115]Osteocalcin in solution binds Ca²+ with a dissociation constant ranging from 0.5 to 3 mM, with a stoichiometry of between 2 and 5 mol Ca²+/mol protein, and to hydroxyapatite (Kd. approximately equal to 10^-7 M). It appears that the Gla residues in osteocalcin are important for its affinity toward Ca²+. Binding of Ca²+ induces normal osteocalcin to adopt the alpha-helical conformation; however, thermally decarboxylated osteocalcin showed higher alpha-helical content than normal osteocalcin and the calcium induced alpha-helical formation was lost. Decarboxylated osteocalcin also lost its specific binding to hydroxyapatite, which is consistent with the results showing that uncarboxylated osteocalcin is the secreted bone hormone. When bound to hydroxyapatite, the Gla residues are protected from thermal decarboxylation. Furthermore, osteocalcin synthesized in animals treated with warfarin, which inhibits the formation of Gla, failed to bind to bone. Furthermore, hydroxyapatite competition studies demonstrated that prothrombin (10 Gla/molecule) and decarboxylated osteocalcin fail to compete with ¹²⁵I-labeled osteocalcin bound to hydroxyapatite. Combining all the information discussed above, a structural model has been constructed. This model consists of two antiparallel alpha-helical domains. The Gla residues are spaced about 5.4 angstroms apart on one of the helices, which is similar to the interatomic lattice spacing of Ca²+ in the x-y plane of hydroxyapatite. It was therefore predicted that the Gla residues in osteocalcin bind to the (001) plane of the hydroxyapatite lattice.

[0116]"Osteocalcin" also known as Bone Gla Protein or BGP, refers to a small vitamin K-dependent calcium binding protein (Price et al. (1976) Proc. Natl. Acad. Sci. 73:3373-5) which is highly conserved among animal species. "Osteocalcin" includes both carboxylated, uncarboxylated and undercarboxylated forms as well as fragments and variants thereof as described herein.

[0117]"Undercarboxylated osteocalcin" means osteocalcin in which one or more of the Glu residues at positions Glu17, Glu21 and Glu24 of the amino acid sequence of the mature human osteocalcin having 49 amino acids, or at the positions corresponding to Glu17, Glu21 and Glu24 in other forms of osteocalcin, are not carboxylated. Undercarboxylated osteocalcin includes uncarboxylated osteocalcin, i.e., osteocalcin in which all three of the glutamic acid residues at positions 17, 21, and 24 are not carboxylated. Recombinant osteocalcin expressed in bacteria is uncarboxylated because bacteria do not have gamma-carboxylase. Preparations of osteocalcin are considered to be "undercarboxylated osteocalcin" if more than about 10% of the total Glu residues at positions Glu17, Glu21 and Glu24 (taken together) in mature osteocalcin (or the corresponding Glu residues in other forms) of the preparation are not carboxylated. In particular preparations of undercarboxylated osteocalcin, more than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the total Glu residues at positions Glu17, Glu21 and Glu24 in mature osteocalcin (or the corresponding Glu residues in other forms) of the preparation are not carboxylated. In particularly preferred embodiments, essentially all of the Glu residues at positions Glu17, Glu21 and Glu24 in mature osteocalcin (or the corresponding Glu residues in other forms) of the preparation are not carboxylated.

[0118]Human osteocalcin cDNA (SEQ ID NO:1) encodes a mature osteocalcin protein represented by the last 49 amino acids of SEQ ID NO:2 (i.e., positions 52-100) with a predicted molecular mass of 5,800 kDa (Poser et al., (1980) The Journal of Biological Chemistry, Vol 255, No. 18, pp. 8685-8691). SEQ ID NO:2 is the pre-pro-sequence of human osteocalcin and mature human osteocalcin is the processed product of SEQ ID NO:2. In this application, the amino acid positions of mature human osteocalcin are referred to. It will be understood that the amino acid positions of mature human osteocalcin correspond to those of SEQ ID NO:2 as follows: position 1 of mature human osteocalcin corresponds to position 52 of SEQ ID NO:2; position 2 of mature human osteocalcin corresponds to position 53 of SEQ ID NO:2, etc. In particular, positions 17, 21, and 24 of mature human osteocalcin correspond to positions 68, 72, and 75, respectively, of SEQ ID NO:2.

[0119]When positions in two amino acid sequences correspond, it is meant that the two positions align with each other when the two amino acid sequences are aligned with one another to provide maximum homology between them. This same concept of correspondence also applies to nucleic acids.

[0120]For example, in the two amino acid sequences AGLYSTVLMGRPS and GLVSTVLMGN, positions 2-11 of the first sequence correspond to positions 1-10 of the second sequence, respectively. Thus, position 2 of the first sequence corresponds to position 1 of the second sequence; position 4 of the first sequence corresponds to position 3 of the second sequence; etc. It should be noted that a position in one sequence may correspond to a position in another sequence, even if the positions in the two sequence are not occupied by the same amino acid.

[0121]Osteocalcin is synthesized primarily by osteoblasts and ondontoblasts. "Osteocalcin" includes the mature protein and further includes biologically active fragments derived from full-length osteocalcin (SEQ ID NO:2) or the mature protein, including various domains, as well as variants as described herein.

[0122]In an embodiment of the invention, the pharmaceutical compositions of the invention comprise a mammalian uncarboxylated osteocalcin. In a preferred embodiment of the invention, the compositions of the invention comprise human osteocalcin having the amino acid sequence of SEQ ID NO:2, or portions thereof, and encoded for by the nucleic acid of SEQ ID NO:1, or portions thereof, or the compositions of the invention may comprise one or more of the human osteocalcin fragments described herein.

[0123]In a specific embodiment, the present invention provides pharmaceutical compositions comprising human undercarboxylated osteocalcin which does not contain a carboxylated glutamic acid at one or more of positions corresponding to positions 17, 21 and 24 of mature human osteocalcin. A preferred form of osteocalcin for use in the present invention is mature human osteocalcin wherein at least one of the glutamic acid residues at positions 17, 21, and 24 is not carboxylated. Preferably, all three of the glutamic acid residues at positions 17, 21, and 24 are not carboxylated. The amino acid sequence of mature human osteocalcin is shown in SEQ. ID. NO: 25.

[0124]The invention also includes the use of polypeptide fragments of osteocalcin. Fragments can be derived from the full-length, naturally occurring amino acid sequence of osteocalcin (e.g., SEQ. ID. NO:2). Fragments may also be derived from mature osteocalcin. The invention also encompasses fragments of the variants of osteocalcin described herein. A fragment can comprise an amino acid sequence of any length that is biologically active.

[0125]Preferred fragments of osteocalcin include fragments containing Glu17, Glu21 and Glu24 of the mature protein. Also preferred are fragments of the mature protein missing the last 10 amino acids from the C-terminal end of the mature protein. Also preferred are fragments missing the first 10 amino acids from the N-terminal end of the mature protein. Also preferred is a fragment of the mature protein missing both the last 10 amino acids from the C-terminal end and the first 10 amino acids from the N-terminal end. Such a fragment comprises amino acids 62-90 of SEQ ID NO:2.

[0126]Other preferred fragments of osteocalcin for the pharmaceutical compositions of the invention described herein include polypeptides comprising, consisting of, or consisting essentially of, the following sequences of amino acids:

[0127]positions 1-19 of mature human osteocalcin

[0128]positions 20-43 of mature human osteocalcin

[0129]positions 20-49 of mature human osteocalcin

[0130]positions 1-43 of mature human osteocalcin

[0131]positions 1-42 of mature human osteocalcin

[0132]positions 1-41 of mature human osteocalcin

[0133]positions 1-40 of mature human osteocalcin

[0134]positions 1-39 of mature human osteocalcin

[0135]positions 1-38 of mature human osteocalcin

[0136]positions 1-37 of mature human osteocalcin

[0137]positions 1-36 of mature human osteocalcin

[0138]positions 1-35 of mature human osteocalcin

[0139]positions 1-34 of mature human osteocalcin

[0140]positions 1-33 of mature human osteocalcin

[0141]positions 1-32 of mature human osteocalcin

[0142]positions 1-31 of mature human osteocalcin

[0143]positions 1-30 of mature human osteocalcin

[0144]positions 1-29 of mature human osteocalcin

[0145]positions 2-49 of mature human osteocalcin

[0146]positions 2-45 of mature human osteocalcin

[0147]positions 2-40 of mature human osteocalcin

[0148]positions 2-35 of mature human osteocalcin

[0149]positions 2-30 of mature human osteocalcin

[0150]positions 2-25 of mature human osteocalcin

[0151]positions 2-20 of mature human osteocalcin

[0152]positions 4-49 of mature human osteocalcin

[0153]positions 4-45 of mature human osteocalcin

[0154]positions 4-40 of mature human osteocalcin

[0155]positions 4-35 of mature human osteocalcin

[0156]positions 4-30 of mature human osteocalcin

[0157]positions 4-25 of mature human osteocalcin

[0158]positions 4-20 of mature human osteocalcin

[0159]positions 8-49 of mature human osteocalcin

[0160]positions 8-45 of mature human osteocalcin

[0161]positions 8-40 of mature human osteocalcin

[0162]positions 8-35 of mature human osteocalcin

[0163]positions 8-30 of mature human osteocalcin

[0164]positions 8-25 of mature human osteocalcin

[0165]positions 8-20 of mature human osteocalcin

[0166]positions 10-49 of mature human osteocalcin

[0167]positions 10-45 of mature human osteocalcin

[0168]positions 10-40 of mature human osteocalcin

[0169]positions 10-35 of mature human osteocalcin

[0170]positions 10-30 of mature human osteocalcin

[0171]positions 10-25 of mature human osteocalcin

[0172]positions 10-20 of mature human osteocalcin

[0173]positions 7-30 of mature human osteocalcin

[0174]positions 7-25 of mature human osteocalcin

[0175]positions 7-23 of mature human osteocalcin

[0176]positions 7-21 of mature human osteocalcin

[0177]positions 7-19 of mature human osteocalcin

[0178]positions 7-17 of mature human osteocalcin

[0179]positions 8-30 of mature human osteocalcin

[0180]positions 8-25 of mature human osteocalcin

[0181]positions 8-23 of mature human osteocalcin

[0182]positions 8-21 of mature human osteocalcin

[0183]positions 8-19 of mature human osteocalcin

[0184]positions 8-17 of mature human osteocalcin

[0185]positions 9-30 of mature human osteocalcin

[0186]positions 9-25 of mature human osteocalcin

[0187]positions 9-23 of mature human osteocalcin

[0188]positions 9-21 of mature human osteocalcin

[0189]positions 9-19 of mature human osteocalcin

[0190]positions 9-17 of mature human osteocalcin

[0191]Especially preferred is a fragment comprising positions 1-36 of mature human osteocalcin. Another preferred fragment is a fragment comprising positions 20-49 of mature human osteocalcin. Other fragments can be designed to contain Pro13 to Tyr76 or Pro 13 to Asn26 of mature human osteocalcin. Additionally, fragments containing the cysteine residues at positions 23 and 29 of mature human osteocalcin, and capable of forming a disulfide bond between those two cysteines, are useful.

[0192]Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment, a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the osteocalcin fragment and/or an additional region fused to the carboxyl terminus of the fragment.

[0193]Also provided for use in the compositions and methods of the present invention are variants of the osteocalcin and osteocalcin fragments described above. "Variants" refers to osteocalcin peptides that contain modifications in their amino acid sequences such as one or more amino acid substitutions, additions, deletions and/or insertions but that are still biologically active. In some instances, the antigenic and/or immunogenic properties of the variants are not substantially altered, relative to the corresponding peptide from which the variant was derived. Such modifications may be readily introduced using standard mutagenesis techniques, such as oligonucleotide directed site-specific mutagenesis as taught, for example, by Adelman et al. (DNA, 2:183, 1983) or by chemical synthesis. Variants and fragments are not mutally exclusive terms. Fragments also include peptides that may contain one or more amino acid substitutions, additions, deletions and/or insertions such that the fragments are still biologically active.

[0194]One particular type of variant that is within the scope of the present invention is a variant in which one of more of the positions corresponding to positions 17, 21 and 24 of mature human osteocalcin is occupied by an amino acid that is not glutamic acid. In some embodiments, the amino acid that is not glutamic acid is also not aspartic acid. Such variants are versions of undercarboxylated osteocalcin because at least one of the three positions corresponding to positions 17, 21 and 24 of mature human osteocalcin is not carboxylated glutamic acid, since at least one of those positions is not occupied by glutamic acid.

[0195]In particular embodiments, the present invention provides osteocalcin variants comprising the amino acid sequence

TABLE-US-00002 (SEQ. ID. NO: 23) YLYQWLGAPV PYPDPLX₁PRR X₂VCX₃LNPDCD ELADHIGFQE AYRRFYGPV

wherein [0196]X₁, X₂ and X₃ are each independently selected from an amino acid or amino acid analog, with the proviso that if X₁, X₂ and X₃ are each glutamic acid, then X₁ is not carboxylated, or less than 50 percent of X₂ is carboxylated, and/or less than 50 percent of X₃ is carboxylated.

[0197]In certain embodiments, the osteocalcin variants comprise an amino acid sequence that is different from SEQ. ID. NO: 23 at 1 to 7 positions other than X₁, X₂ and X₃.

[0198]In other embodiments, the osteocalcin variants comprise an amino acid sequence that includes one or more amide backbone substitutions.

[0199]Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitutions of similar amino acids, which results in no change, or an insignificant change, in function. Alternatively, such substitutions may positively or negatively affect function to some degree. The activity of such functional osteocalcin variants can be determined using assays such as those described herein.

[0200]Variants can be naturally-occurring or can be made by recombinant means, or chemical synthesis, to provide useful and novel characteristics for undercarboxylated/uncarboxylated osteocalcin. For example, the variant osteocalcin polypeptides may have reduced immunogenicity, increased serum half-life, increase bioavailability and/or increased potency. In particular embodiments, serum half-life is increased by substituting one or more of the native Arg residues at positions 19, 20, 43, and 44 of mature osteocalcin with another amino acid or an amino acid analog, e.g., β-dimethyl-arginine. Such substitutions can be combined with the other changes in the native amino acid sequence of osteocalcin described herein.

[0201]Provided for use in the pharmaceutical compositions and methods of the present invention are variants that are also derivatives of the osteocalcin and osteocalcin fragments described above. Derivatization is a technique used in chemistry which transforms a chemical compound into a product of similar chemical structure, called derivative. Generally, a specific functional group of the compound participates in the derivatization reaction and transforms the educt to a derivate of deviating reactivity, solubility, boiling point, melting point, aggregate state, functional activity, or chemical composition. Resulting new chemical properties can be used for quantification or separation of the educt or can be used to optimize the compound as a therapeutic agent. The well-known techniques for derivatization can be applied to the above-described osteocalcin and osteocalcin fragments. Thus, derivatives of the osteocalcin and osteocalcin fragments described above will contain amino acids that have been chemically modified in some way so that they differ from the natural amino acids.

[0202]Provided also are osteocalcin mimetics. "Mimetic" refers to a synthetic chemical compound that has substantially the same structural and functional characteristics of a naturally or non-naturally occurring polypeptide, and includes, for instance, polypeptide- and polynucleotide-like polymers having modified backbones, side chains, and/or bases. Peptide mimetics are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. Generally, mimetics are structurally similar (i.e., have the same shape) to a paradigm polypeptide that has a biological or pharmacological activity, but one or more polypeptide linkages are replaced. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity.

[0203]By way of example, example, Cho et al., 1993, Science 261:1303-5 discloses an "unnatural biopolymer" consisting of chiral aminocarbonate monomers substituted with a variety of side chains, synthesis of a library of such polymers, and screening for binding affinity to a monoclonal antibody. Similarly, Cho et al, 1998, J. Am. Chem. Soc. discloses libraries of linear and cyclic oligocarbamate libraries and screening for binding to the integrin GPIIb/IIIa. Simon et al., Proc. Natl. Acad. Sci. 89:9367-71 discloses a polymer consisting of N-substituted glycines ("peptoids") with diverse side chains. Schumacher et al, 1996, Science 271:1854-7 discloses D-peptide ligands specific for Src homology domain 3 (SH3 domain) by screening phage libraries of L-peptides against a proteins (SH3) synthesized with D-amino acids and then synthesizing a selected L-peptide using D-amino acids. Brody et al., 1999, Mol. Diagn. 4: 381-8 describes generation and screeing of hundreds to thousands of aptamers.

[0204]A particular type of osteocalcin variant within the scope of the invention is an osteocalcin mimetic in which one or more backbone amides is replaced by a different chemical structure or in which one or more amino acids are replaced by an amino acid analog. In a particular embodiment, the osteocalcin mimetic is a retroenantiomer of uncarboxylated human osteocalcin.

[0205]Osteocalcin, as well as its fragments and variants, is optionally produced by chemical synthesis or recombinant methods and may be produced as a modified osteocalcin molecule (i.e., osteocalcin fragments or variants) as described herein. Osteocalcin polypeptides can be produced by any conventional means (Houghten, R. A. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135). Simultaneous multiple peptide synthesis is described in U.S. Pat. No. 4,631,211 and can also be used. When produced recombinantly, osteocalcin may be produced as a fusion protein, e.g., a GST-osteocalcin fusion protein.

[0206]Undercarboxylated/uncarboxylated osteocalcin molecules that fall within the scope of the invention include proteins substantially homologous to human osteocalcin including proteins derived from another organism, i.e., an ortholog. One particular ortholog is mouse osteocalcin. Mouse osteocalcin gene 1 cDNA is SEQ ID NO:3; mouse osteocalcin gene 2 cDNA is SEQ ID NO:4; the amino acid sequence of mouse osteocalcin gene 1 and gene 2 is SEQ ID NO:5.

[0207]As used herein, two proteins are substantially homologous, or identical, when their amino acid sequences are at least about 70-75%, typically at least about 80-85%, and most typically at least about 90-95%, 97%, 98% or 99% or more homologous. "Homology" between two amino acid sequences or nucleic acid sequences can be determined by using the alogorithms disclosed herein. These algorithms can also be used to determine percent identity between two amino acid sequences or nucleic acid sequences.

[0208]In a specific embodiment of the invention, the undercarboxylated/uncarboxylated osteocalcin is a osteocalcin molecule sharing at least 80% homology with the human osteocalcin of SEQ ID:2 or a portion of SEQ ID:2 that is at least 8 amino acids long. In another embodiment of the invention, the undercarboxylated/uncarboxylated osteocalcin is a osteocalcin molecule sharing at least 80% amino acid sequence identity with the human osteocalcin of SEQ ID:2 or a portion of SEQ ID:2 that is at least 8 amino acids long. Homologous sequences include those sequences that are substantially identical. In preferred embodiments, the homology or identity is over the entire length of mature human osteocalcin.

[0209]To determine the percent homology or percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Preferably, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% or more of the length of the sequence that the reference sequence is compared to. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0210]The invention also encompasses polypeptides having a lower degree of identity but which have sufficient similarity so as to perform one or more of the same functions performed by undercarboxylated/uncarboxylated osteocalcin. Similarity is determined by considering conserved amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., Science 247:1306-1310 (1990).

[0211]Examples of conservative substitutions are the replacements, one for another, among the hydrophobic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys, His and Arg; replacements among the aromatic residues Phe, Trp and Tyr; exchange of the polar residues Gln and Asn; and exchange of the small residues Ala, Ser, Thr, Met, and Gly.

[0212]The comparison of sequences and determination of percent identity and homology between two osteocalcin polypeptides can be accomplished using a mathematical algorithm. For example, Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, van Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991. A non-limiting example of such a mathematical algorithm is described in Karlin et al. (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0213]The percent identity or homology between two osteocalcin amino acid sequences may be determined using the Needleman et al. (1970) (.I Mol. Biol. 48:444-453) algorithm. Another non-limiting example of a mathematical algorithm that may be utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).

[0214]A substantially homologous osteocalcin, according to the present invention, may also be a polypeptide encoded by a nucleic acid sequence capable of hybridizing to the human osteocalcin nucleic acid sequence under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encoding a functionally equivalent gene product; or under less stringent conditions, such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989 supra), yet which still encodes a biologically active undercarboxylated/uncarboxylated osteocalcin.

[0215]A substantially homologous osteocalcin, according to the present invention, may also be a polypeptide encoded by a nucleic acid sequence capable of hybridizing to a sequence having at least 70-75%, typically at least about 80-85%, and most typically at least about 90-95%, 97%, 98% or 99% identity to the human osteocalcin nucleic acid sequence, under stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encoding a functionally equivalent gene product; or under less stringent conditions, such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989 supra), yet which still encodes a biologically active undercarboxylated/uncarboxylated osteocalcin.

[0216]It will be understood that a biologically active fragment or variant of human osteocalcin may contain a different number of amino acids than native human osteocalcin. Accordingly, the position number of the amino acid residues corresponding to positions 17, 21 and 24 of mature human osteocalcin may differ in the fragment or variant. One skilled in the art would easily recognize such corresponding positions from a comparison of the amino acid sequence of the fragment or variant with the amino acid sequence of mature human osteocalcin.

[0217]Peptides corresponding to fusion proteins in which full length osteocalcin, mature osteocalcin, or an osteocalcin fragment or variant is fused to an unrelated protein or polypeptide are also within the scope of the invention and can be designed on the basis of the osteocalcin nucleotide and amino acid sequences disclosed herein. Such fusion proteins include fusions to an enzyme, fluorescent protein, or luminescent protein which provides a marker function. In a preferred embodiment of the invention, the fusion protein comprises fusion to a polypeptide capable of targeting the osteocalcin to a particular target cell or location in the body. For example, osteocalcin polypeptide sequences may be fused to a ligand molecule capable of targeting the fusion protein to a cell expressing the receptor for said ligand. Osteocalcin can also be made as part of a chimeric protein for drug screening or use in making recombinant protein. These comprise an osteocalcin peptide sequence operatively linked to a heterologous peptide having an amino acid sequence not substantially homologous to the osteocalcin. "Operatively linked" in this context indicates that the osteocalcin peptide and the heterologous peptide are fused in-frame. The heterologous peptide can be fused to the N-terminus or C-terminus of osteocalcin or can be internally located. In one embodiment, the fusion protein does not affect osteocalcin function. For example, the fusion protein can be a GST-fusion protein in which the osteocalcin sequences are fused to the N- or C-terminus of the GST sequences. Other types of fusion proteins include, but are not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL-4 fusions, poly-His fusions and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant osteocalcin. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence. Therefore, the fusion protein may contain a heterologous signal sequence at its N-terminus.

[0218]EP-A 0 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions (Fc regions). The Fc region is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0 232 262). In drug discovery, for example, human proteins have been fused with Fc regions for the purpose of high-throughput screening assays to identify antagonists (Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson et al. J. Biol. Chem. 270:9459-9471). Thus, various embodiments of this invention also utilize soluble fusion proteins containing an osteocalcin polypeptide and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (e.g., IgG, IgM, IgA, IgE, IgB). Preferred as immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgG1, where fusion takes place at the hinge region. For some uses, it is desirable to remove the Fc region after the fusion protein has been used for its intended purpose, e.g., when the fusion protein is to be used as antigen for immunizations. In a particular embodiment, the Fc part can be removed in a simple way by a cleavage sequence, which is also incorporated and can be cleaved, e.g., with factor Xa.

[0219]A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences can be ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al. (1992) Current Protocols in Molecular Biology). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). An osteocalcin-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to osteocalcin.

[0220]Chimeric osteocalcin proteins can be produced in which one or more functional sites are derived from a different isoform, or from another osteocalcin molecule from another species. Sites also could be derived from osteocalcin-related proteins that occur in the mammalian genome but which have not yet been discovered or characterized.

[0221]Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally-occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in polypeptides are described below.

[0222]Accordingly, the osteocalcin polypeptides of the present invention also encompass derivatives which contain a substituted amino acid residue that is not one encoded by the genetic code, in which a substituent group is included, in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the osteocalcin polypeptide, such as a leader or secretory sequence or a sequence for purification of the osteocalcin polypeptide or a pro-protein sequence.

[0223]Undercarboxylated/uncarboxylated osteocalcin can be modified according to known methods in medicinal chemistry to increase its stability, half-life, uptake or efficacy. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0224]In a specific embodiment of the invention, modifications may be made to the osteocalcin to reduce susceptibilty to proteolysis at residue ARG43 as a means for increasing serum half life. Such modifications include, for example, the use of retroenantio isomers, D-amino acids, or other amino acid analogs.

[0225]Acylation of the N-terminal amino group can be accomplished using a hydrophilic compound, such as hydroorotic acid or the like, or by reaction with a suitable isocyanate, such as methylisocyanate or isopropylisocyanate, to create a urea moiety at the N-terminus. Other agents can also be N-terminally linked that will increase the duration of action of the osteocalcin derivative as known in this art.

[0226]Reductive amination is the process by which ammonia is condensed with aldehydes or ketones to form imines which are subsequently reduced to amines. Reductive amination is a useful method for conjugating undercarboxylated/uncarboxylated osteocalcin and its fragments or variants to PEG. Covalent linkage of poly(ethylene glycol) (PEG) to undercarboxylated/uncarboxylated osteocalcin and its fragments and variants may result in conjugates with increased water solubility, altered bioavailability, pharmacokinetics, immunogenic properties, and biological activities. See, e.g., Bentley et al., J. Pharm. Sci. 1998 November; 87(11):1446-9.

[0227]Several particularly common modifications that may be applied to undercarboxylated/uncarboxylated osteocalcin and its fragments and variants such as glycosylation, lipid attachment, sulfation, hydroxylation and ADP-ribosylation are described in most basic texts, such as Proteins--Structure and Molecular Properties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (1990) Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann. NY: Acad. Sci. 663:48-62.

[0228]As is also well known, polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing events and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translational natural processes and by synthetic methods.

[0229]Modifications can occur anywhere in the undercarboxylated/uncarboxylated osteocalcin and its fragments and variants, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. Blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally-occurring and synthetic polypeptides and may be applied to the undercarboxylated/uncarboxylated osteocalcin or its fragments and variants of the present invention. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine. Thus, undercarboxylated/uncarboxylated osteocalcin and its fragments and variants with N-formylmethionine as the amino terminal residue are within the scope of the present invention.

[0230]A brief description of various protein modifications that come within the scope of this invention are set forth in the table below:

TABLE-US-00003 TABLE 1 Protein Modification Description Acetylation Acetylation of N-terminus or ε-lysines. Introducing an acetyl group into a protein, specifically, the substitution of an acetyl group for an active hydrogen atom. A reaction involving the replacement of the hydrogen atom of a hydroxyl group with an acetyl group (CH₃CO) yields a specific ester, the acetate. Acetic anhydride is commonly used as an acetylating agent, which reacts with free hydroxyl groups. Acylation may facilitate addition of other functional groups. A common reaction is acylation of e.g., conserved lysine residues with a biotin appendage. ADP-ribosylation Covalently linking proteins or other compounds via an arginine-specific reaction. Alkylation Alkylation is the transfer of an alkyl group from one molecule to another. The alkyl group may be transferred as an alkyl carbocation, a free radical or a carbanion (or their equivalents). Alkylation is accomplished by using certain functional groups such as alkyl electrophiles, alkyl nucleophiles or sometimes alkyl radicals or carbene acceptors. A common example is methylation (usually at a lysine or arginine residue). Amidation Reductive animation of the N-terminus. Methods for amidation of insulin are described in U.S. Pat. No. 4,489,159. Carbamylation Nigen et al. describes a method of carbamylating hemoglobin. Citrullination Citrullination involves the addition of citrulline amino acids to the arginine residues of a protein, which is catalyzed by peptidylarginine deaminase enzymes (PADs). This generally converts a positively charged arginine into a neutral citrulline residue, which may affect the hydrophobicity of the protein (and can lead to unfolding). Condensation of amines Such reactions, may be used, e.g., to attach a peptide to other with aspartate or glutamate proteins labels. Covalent attachment of Flavin mononucleotide (FAD) may be covalently attached to flavin serine and/or threonine residues. May be used, e.g., as a light-activated tag. Covalent attachment of A heme moiety is generally a prosthetic group that consists heme moiety of an iron atom contained in the center of a large heterocyclic organic ring, which is referred to as a porphyrin. The heme moiety may be used, e.g., as a tag for the peptide. Attachment of a nucleotide May be used as a tag or as a basis for further derivatising a or nucleotide derivative peptide. Cross-linking Cross-linking is a method of covalently joining two proteins. Cross-linkers contain reactive ends to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules. Several chemical groups may be targets for reactions in proteins and peptides. For example, Ethylene glycol bis[succinimidylsuccinate, Bis[2- (succinimidooxycarbonyloxy)ethyl]sulfone, and Bis[sulfosuccinimidyl]suberate link amines to amines. Cyclization For example, cyclization of amino acids to create optimized delivery forms that are resistant to, e.g., aminopeptidases (e.g., formation of pyroglutamate, a cyclized form of glutamic acid). Disulfide bond formation Disulfide bonds in proteins are formed by thiol-disulfide exchange reactions, particularly between cysteine residues (e.g., formation of cystine). Demethylation See, e.g., U.S. Pat. No. 4,250,088 (Process for demethylating lignin). Formylation The addition of a formyl group to, e.g., the N-terminus of a protein. See, e.g., U.S. Pat. Nos. 4,059,589, 4,801,742, and 6,350,902. Glycylation The covalent linkage of one to more than 40 glycine residues to the tubulin C-terminal tail. Glycosylation Glycosylation may be used to add saccharides (or polysaccharides) to the hydroxy oxygen atoms of serine and threonine side chains (which is also known as O-linked Glycosylation). Glycosylation may also be used to add saccharides (or polysaccharides) to the amide nitrogen of asparagine side chains (which is also known as N-linked Glycosylation), e.g., via oligosaccharyl transferase. GPI anchor formation The addition of glycosylphosphatidylinositol to the C- terminus of a protein. GPI anchor formation involves the addition of a hydrophobic phosphatidylinositol group - linked through a carbohydrate containing linker (e.g., glucosamine and mannose linked to phosphoryl ethanolamine residue) - to the C-terminal amino acid of a protein. Hydroxylation Chemical process that introduces one or more hydroxyl groups (--OH) into a protein (or radical). Hydroxylation reactions are typically catalyzed by hydroxylases. Proline is the principal residue to be hydroxylated in proteins, which occurs at the C.sup.γ atom, forming hydroxyproline (Hyp). In some cases, proline may be hydroxylated at its C.sup.β atom. Lysine may also be hydroxylated on its C.sup.δ atom, forming hydroxylysine (Hyl). These three reactions are catalyzed by large, multi-subunit enzymes known as prolyl 4-hydroxylase, prolyl 3-hydroxylase and lysyl 5-hydroxylase, respectively. These reactions require iron (as well as molecular oxygen and α-ketoglutarate) to carry out the oxidation, and use ascorbic acid to return the iron to its reduced state. Iodination See, e.g., U.S. Pat. No. 6,303,326 for a disclosure of an enzyme that is capable of iodinating proteins. U.S. Pat. No. 4,448,764 discloses, e.g., a reagent that may be used to iodinate proteins. ISGylation Covalently linking a peptide to the ISG15 (Interferon- Stimulated Gene 15) protein, for, e.g., modulating immune response. Methylation Reductive methylation of protein amino acids with formaldehyde and sodium cyanoborohydride has been shown to provide up to 25% yield of N-cyanomethyl (--CH₂CN) product. The addition of metal ions, such as Ni²+, which complex with free cyanide ions, improves reductive methylation yields by suppressing by-product formation. The N-cyanomethyl group itself, produced in good yield when cyanide ion replaces cyanoborohydride, may have some value as a reversible modifier of amino groups in proteins. (Gidley et al.) Methylation may occur at the arginine and lysine residues of a protein, as well as the N- and C-terminus thereof. Myristoylation Myristoylation involves the covalent attachment of a myristoyl group (a derivative of myristic acid), via an amide bond, to the alpha-amino group of an N-terminal glycine residue. This addition is catalyzed by the N- myristoyltransferase enzyme. Oxidation Oxidation of cysteines. Oxidation of N-terminal Serine or Threonine residues (followed by hydrazine or aminooxy condensations). Oxidation of glycosylations (followed by hydrazine or aminooxy condensations). Palmitoylation Palmitoylation is the attachment of fatty acids, such as palmitic acid, to cysteine residues of proteins. Palmitoylation increases the hydrophobicity of a protein. (Poly)glutamylation Polyglutamylation occurs at the glutamate residues of a protein. Specifically, the gamma-carboxy group of a glutamate will form a peptide-like bond with the amino group of a free glutamate whose alpha-carboxy group may be extended into a polyglutamate chain. The glutamylation reaction is catalyzed by a glutamylase enzyme (or removed by a deglutamylase enzyme). Polyglutamylation has been carried out at the C-terminus of proteins to add up to about six glutamate residues. Using such a reaction, Tubulin and other proteins can be covalently linked to glutamic acid residues. Phosphopantetheinylation The addition of a 4'-phosphopantetheinyl group. Phosphorylation A process for phosphorylation of a protein or peptide by contacting a protein or peptide with phosphoric acid in the presence of a non-aqueous apolar organic solvent and contacting the resultant solution with a dehydrating agent is disclosed e.g., in U.S. Pat. No. 4,534,894. Insulin products are described to be amenable to this process. See, e.g., U.S. Pat. No. 4,534,894. Typically, phosphorylation occurs at the serine, threonine, and tyrosine residues of a protein. Prenylation Prenylation (or isoprenylation or lipidation) is the addition of hydrophobic molecules to a protein. Protein prenylation involves the transfer of either a farnesyl (linear grouping of three isoprene units) or a geranyl-geranyl moiety to C- terminal cysteine(s) of the target protein. Proteolytic Processing Processing, e.g., cleavage of a protein at a peptide bond. Selenoylation The exchange of, e.g., a sulfur atom in the peptide for selenium, using a selenium donor, such as selenophosphate. Sulfation Processes for sulfating hydroxyl moieties, particularly tertiary amines, are described in, e.g., U.S. Pat. No. 6,452,035. A process for sulphation of a protein or peptide by contacting the protein or peptide with sulphuric acid in the presence of a non-aqueous apolar organic solvent and contacting the resultant solution with a dehydrating agent is disclosed. Insulin products are described to be amenable to this process. See, e.g., U.S. Pat. No. 4,534,894. SUMOylation Covalently linking a peptide a SUMO (small ubiquitin- related Modifier) protein, for, e.g., stabilizing the peptide. Transglutamination Covalently linking other protein(s) or chemical groups (e.g., PEG) via a bndge at glutamine residues tRNA-mediated addition of For example, the site-specific modification (insertion) of an amino acids (e.g., amino acid analog into a peptide. arginylation) Ubiquitination The small peptide ubiquitin is covalently linked to, e.g., lysine residues of a protein. The ubiquitin-proteasome system can be used to carryout such reaction. See, e.g., U.S. 2007-0059731.

[0231]To practice the methods of the invention, it may be desirable to recombinantly express the osteocalcin protein. The cDNA sequence and deduced amino acid sequence of human osteocalcin is represented in SEQ ID NO:1 and SEQ ID NO:2. Osteocalcin nucleotide sequences may be isolated using a variety of different methods known to those skilled in the art. For example, a cDNA library constructed using RNA from a tissue known to express osteocalcin can be screened using a labeled osteocalcin probe. Alternatively, a genomic library may be screened to derive nucleic acid molecules encoding the osteocalcin protein. Further, osteocalcin nucleic acid sequences may be derived by performing a polymerase chain reaction (PCR) using two oligonucleotide primers designed on the basis of known osteocalcin nucleotide sequences. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from cell lines or tissue known to express osteocalcin.

[0232]While the osteocalcin polypeptides and peptides can be chemically synthesized (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y.), large polypeptides derived from osteocalcin and the full length osteocalcin itself may be advantageously produced by recombinant DNA technology using techniques well known in the art for expressing a nucleic acid. Such methods can be used to construct expression vectors containing the osteocalcin nucleotide sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al., 1989, supra, and Ausubel et al., 1989, supra).

[0233]A variety of host-expression vector systems may be utilized to express the osteocalcin nucleotide sequences. In a preferred embodiment, the osteocalcin peptide or polypeptide is secreted and may be recovered from the culture media.

[0234]Appropriate expression systems can be chosen to ensure that the correct modification, processing and subcellular localization of the osteocalcin protein occurs. To this end, bacterial host cells are preferred for expression of osteocalcin, as such cells are unable to carboxylate osteocalcin.

[0235]The isolated osteocalcin can be purified from cells that naturally express it, e.g., osteoblasts, or purified from cells that naturally express it but have been modified to overproduce osteocalcin, e.g., purified from cells that have been altered to express it (recombinant), synthesized using known protein synthesis methods, or by modifying cells that naturally encode osteocalcin to express it. In a particular embodiment, a recombinant cell has been manipulated to activate expression of the endogenous osteocalcin gene. For example, WO 99/15650 and WO 00/49162 describe a method of expressing endogenous genes termed random activation of gene expression (RAGE), which can be used to activate or increase expression of endogenous osteocalcin. The RAGE methodology involves non-homologous recombination of a regulatory sequence to activate expression of a downstream endogenous gene. Alternatively, WO 94/12650, WO 95/31560, WO 96/29411, U.S. Pat. No. 5,733,761 and U.S. Pat. No. 6,270,985 describe a method of increasing expression of an endogenous gene that involves homologous recombination of a DNA construct that includes a targeting sequence, a regulatory sequence, an exon, and a splice-donor site. Upon homologous recombination, a downstream endogenous gene is expressed. The methods of expressing endogenous genes described in the forgoing patents are hereby expressly incorporated by reference.

[0236]Compositions Comprising Adiponectin and Undercarboxylated/Uncarboxylated Osteocalcin

[0237]In a specific embodiment of the invention, pharmaceutical compositions comprising adiponectin and undercarboxylated/uncarboxylated osteocalcin or biologically active undercarboxylated/uncarboxylated osteocalcin fragments or variants may be administered to a patient in need of such administration. In an embodiment of the invention, the adiponectin is a mammalian adiponectin. In a preferred embodiment of the invention, the pharmaceutical compositions of the invention comprise human adiponectin. The cDNA sequence of human adiponectin is shown in SEQ ID NO:6. The amino acid sequence of human adiponectin is shown in SEQ ID NO:7.

[0238]In another aspect of the present invention, biologically active adiponectin fragments or variants together with undercarboxylated/uncarboxylated osteocalcin or biologically active undercarboxylated/uncarboxylated osteocalcin fragments in a pharmaceutical composition can be used in the treatment methods of the present invention. Adiponectin fragments or variants encompass the kinds of changes in the native sequence of adiponectin that are described above for osteocalcin and can be produced by the same methods that are described above for producing osteocalcin fragments and variants.

[0239]Compositions Comprising Inhibitors of Gamma-Carboxylase and/or OST-PTP

[0240]In another embodiment of the invention, the pharmaceutical compositions of the invention comprise an inhibitor that reduces the expression or activity of gamma-carboxylase or OST-PTP. Preferably, the biological activity of gamma-carboxylase or OST-PTP (as previously described) is inhibited. The inhibitors may be antibodies (monoclonal or polyclonal) or fragments of antibodies, small molecules, polypeptides or proteins, or nucleic acids (e.g., antisense DNA or RNA, siRNA).

[0241]In certain embodiments, the inhibitors reduce the activity of OST-PTP having the amino acid sequene of SEQ ID NO:19. In other embodiments, the inhibitors reduce the activity of an OST-PTP having an amino acid sequence that is substantially homologous or identical, as previouly described to the amino acid sequence of SEQ ID NO:19.

[0242]In certain embodiments, the inhibitors reduce the activity of gamma-carboxylase having the amino acid sequence of SEQ ID NO:11. In other embodiments, the inhibitors reduce the activity of an gamma-carboxylase having an amino acid sequence that is substantially homologous or identical, as previouly described to the amino acid sequence.

Small Molecule Inhibitors of OST-PTP and Gamma-Carboxylase

[0243]In certain embodiments, the agent is a small molecule. By "small molecule" is meant organic compounds of molecular weight of more than 100 and less than about 2,500 daltons, and preferably less than 500 daltons. Such small molecules inhibit the biological activity of OST-PTP or gamma-carboxylase.

[0244]The inhibitors may comprise agents that act as inhibitors of vitamin K, beta blockers, statins, and/or thiol-specific inhibitors that function to increase serum adiponectin, serum insulin and/or serum osteocalcin levels, preferably undercarboxylated/uncarboxylated osteocalcin. The agents may also increase glucose tolerance, increase insulin sensitivity, increase beta-cell proliferation, and/or cause other effects of biologically active agents as previously described.

[0245]Warfarin and other vitamin K inhibitors, including coumadin and other derivatives, beta-blockers, statins, and fragments and modifications thereof, may be administered to patients who would benefit from inhibition of gamma-carboxylase. In a specific embodiment of the invention, the small molecule warfarin may be used to inhibit the activity of gamma-carboxylase. Warfarin derivatives are exemplified by acenocoumarol, phenprocoumon and phenindione. Warfarin and other coumadin derivatives block vitamin K-dependent gamma-carboxylation, thus increasing the level of undercarboxylated/uncarboxylated osteocalcin.

[0246]Beta blockers are used to treat high blood pressure (hypertension), congestive heart failure (CHF), abnormal heart rhythms (arrhythmias), and chest pain (angina). Beta blockers are sometimes used in heart attack patients to prevent future heart attacks. There are 2 main beta receptors: beta 1 and beta 2. Some beta blockers are selective, which means that they block beta 1 receptors more than they block beta 2 receptors. Beta 1 receptors are responsible for heart rate and the strength of the heartbeat. Nonselective beta blockers block both beta 1 and beta 2 receptors. Beta 2 receptors are responsible for the function of smooth muscles; they are also the only beta receptors expressed by osteoblasts. Non-limiting examples of beta blockers include sotalol, timolol, esmolol, carteolol, carvedilol, nadolol, propranolol, betaxolol, penbutolol, metoprolol, labetalol, acebutolol, atenolol, metoprolol, labetalol, pindolol, and bisoprolol.

[0247]Statins are further exemplified by atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Non-limiting examples of beta-blockers include sotalol, carvedilol, metroprolol. Other small molecules can be identified using the screens and assays disclosed herein.

[0248]Other inhibitors include thiol specific inhibitors of gamma-carboxylase. Cys and His residues of gamma carboxylase are implicated in the carboxylase mechanism of gamma carboxylase, and it is observed that the enzyme is inhibited by thiol-specific inhibitors, such as N-ethylmaleimide (NEM) and mercurials such as p-hydroxymurcuribenzoate (pHMB). Additional non-limiting examples of these inhibitors include 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), 2-nitro-5-thiocyanobenzoic acid (NTCB), iodoacetamide (IA), N-phenylmaleimide (PheM), N-(1-pyrenyl) maleimide (PyrM), naphthalene-1,5-dimaleimide (NDM), N,N'-(1,2-phenylene) dimaleimide (oPDM), N,N'-1,4-phenylene dimaleimide (pPDM), N,N'-1,3-phenylene dimaleimide (mPDM), 1,1-(methylenedi-4,1-phenylene)bismaleimide (BM), 4-(N-maleimido)phenyltrimethylammonium (MPTM), N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine (BMP), N-succinimidyl 3-(2-pyridyldithio)propionate, diethyl pyrocarbonate, p-chloromercuribenzene sulphonic acid and thiosulfinates. These inhibitors may also be provided as conjugate or derivative, such as with, e.g., BSA or aminodextran.

Antibody Inhibitors of OST-PTP and Gamma-Carboxylase

[0249]The present invention also provides compositions comprising an antibody or antibodies, as well as biologically active fragments or variants thereof, that are capable of binding to an epitope of OST-PTP or gamma-carboxylase polypeptides. An antibody against OST-PTP that decreases its activity can be used therapeutically. In certain embodiments, the antibody against OST-PTP binds to the extracellular domain of OST-PTP.

[0250]In certain embodiments, the antibody against OST-PTP binds to an epitope in the mouse OST-PTP of SEQ ID NO:19 or an OST-PTP having an amino acid sequence that is substantially homologous or identical to SEQ ID NO:19. In other embodiments, the antibody against OST-PTP binds to an epitope in an OST-PTP having an amino acid sequence that is at least 70% homologous or identical to SEQ ID NO:19.

[0251]Human OST-PTP can be obtained by isolating the human ortholog of mouse OST-PTP (SEQ ID NO:18) (or rat OST-PTP; SEQ ID NO:24) by methods known in the art. For example, one could prepare a cDNA library from human osteoblasts and identify human OST-PTP cDNA by hybridizing the cDNA clones from the library to a mouse probe. The mouse probe could be based on a portion of mouse OST-PTP (SEQ ID NO:18). Alternatively, PCR, using primers based on the mouse sequence, can be used to obtain the human OST-PTP gene.

[0252]Gamma-carboxylase is an intracellular protein, so antibodies or fragments of antibodies against it are preferably used therapeutically when combined with technologies for delivering the antibodies, fragments or variants into the interior of target cells expressing gamma-carboxylase, e.g., osteoblasts. Antibodies, fragments or variants against gamma-carboxylase can also be used diagnostically or in drug screening assays. Antibodies or antibody fragments or variants against osteocalcin and adiponectin similarly can be used with technologies for delivering the antibodies or fragments into the interior of target cells and can also be used in diagnostics and drug screening assays.

[0253]In a particular embodiment, the present invention provides antibodies, fragments or variants of antibodies that recognize an epitope in OST-PTP that includes the amino acid at position 1316 of mouse OST-PTP or the corresponding position of human OST-PTP. In certain embodiments, these antibodies, fragments or variants of antibodies block or inhibit the ability of OST-PTP to activate gamma-carboxylase. In certain embodiments, use of these antibodies or fragments results in OST-PTP losing 50%, 60%, 70%, 80%, 90%, 95%, or essentially all of its ability to activate gamma-carboxylase.

[0254]The term "epitope" refers to an antigenic determinant on an antigen to which an antibody binds. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes generally have at least five contiguous amino acids. The terms "antibody" and "antibodies" include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab')₂ fragments. Polyclonal antibodies are heterogeneous populations of antibody molecules that are specific for a particular antigen, while monoclonal antibodies are homogeneous populations of antibodies to a particular epitope contained within an antigen. Monoclonal antibodies are particularly useful in the present invention.

[0255]Antibody fragments that have specific binding affinity for the polypeptide of interest (e.g., OST-PTP or gamma-carboxylase) can be generated by known techniques. Such antibody fragments include, but are not limited to, F(ab')₂ fragments that can be produced by pepsin digestion of an antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')₂ fragments. Alternatively, Fab expression libraries can be constructed. See, for example, Huse et al. (1989) Science 246:1275-1281. Single chain Fv antibody fragments are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15 to 18 amino acids), resulting in a single chain polypeptide. Single chain Fv antibody fragments can be produced through standard techniques, such as those disclosed in U.S. Pat. No. 4,946,778.

[0256]Once produced, antibodies or fragments thereof can be tested for recognition of the target polypeptide by standard immunoassay methods including, for example, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay assay (RIA). See, Short Protocols in Molecular Biology eds. Ausubel et al., Green Publishing Associates and John Wiley & Sons (1992).

[0257]The immunoassays, immunohistochemistry, RIA, IRMAs used herein are based on the generation of various antibodies, including those that specifically bind to osteocalcin, OST-PTP, gamma-carboxylase, adiponectin, vitamin K, or their fragments or variants. Antibodies and methods of using antibodies to quantitate the amount of osteocalcin, in particular, in a sample are also described in Hosoda et al. (U.S. Pat. No. 5,681,707). Hosoda et al. disclose antibodies that bind to the N-terminal 20 amino acids, or the C-terminal 14 amino acids of osteocalcin. Anti-OST-PTP antibodies are commercially available.

[0258]In one embodiment, antibodies against OST-PTP or gamma-carboxylase that reduce its activity are useful in the treatment of a patient having a disorder related to the OST-PTP pathway. Such disorders include metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity. Such disorders are characterized by decreased insulin production, decreased insulin sensitivity, decreased glucose tolerance and/or increased fat mass.

Nucleic Acid Inhibitors of OST-PTP and Gamma-Carboxylase

[0259]Other embodiments of the present invention are directed to the use of antisense nucleic acids or small interfering RNA (siRNA) to reduce or inhibit expression and hence the biological acitivity of proteins or peptides, particularly OST-PTP and gamma-carboxylase. The cDNA sequences encoding OST-PTP and gamma-carboxylase are set forth below. Based on these known sequences, antisense DNA or RNA that hybridize sufficiently to the respective gene or mRNA encoding OST-PTP and gamma-carboxylase to turn off or reduce expression can be readily designed and engineered, using methods known in the art.

[0260]In a specific embodiment of the invention, antisense or siRNA molecules for use in the present invention are those that bind under stringent conditions to the human gamma-carboxylase nucleic sequence of SEQ ID NO:10. In yet another embodiment of the invention, the antisense or siRNA molecules are those that that bind under stringent conditions to the OST-PTP nucleic acid sequence of SEQ ID NO:18, or sequences that are substantially homologous to SEQ ID NO:18. In other embodiments, the antisense or siRNA molecules bind under stringent conditions to sequences that are substantially homologous or identical to SEQ ID NO:18.

[0261]Antisense-RNA and anti-sense DNA have been used therapeutically in mammals to treat various diseases. See for example Agrawal, S. and Zhao, Q. (1998) Curr. Opin. Chemical Biol. Vol. 2, 519-528; Agrawal, S and Zhang, R. (1997) CIBA Found. Symp. Vol. 209, 60-78; and Zhao, Q, et al., (1998), Antisense Nucleic Acid Drug Dev. Vol 8, 451-458; the entire contents of which are hereby incorporated by reference as if fully set forth herein. Antisense oligodeoxyribonucleotides (antisense-DNA), oligoribonucleotides (antisense-RNA), and other polymeric antisense compounds (e.g., oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside linkages and non-naturally-occurring portions which function similarly) can base pair with a gene or its transcript. An antisense PS-oligodeoxyribonucleotide for treatment of cytomegalovirus retinitis in AIDS patients is the first antisense oligodeoxyribonucleotide approved for human use in the US. Anderson, K. O., et al., (1996) Antimicrobiol. Agents Chemother. Vol. 40, 2004-2011, and U.S. Pat. No. 6,828,151 by Borchers, et al., entitled "Antisense modulation of hematopoietic cell protein tyrosine kinase expression," describe methods for making and using antisense nucleic acids and their formulation, the entire contents of which are hereby incorporated by reference as if fully set forth herein.

[0262]Methods of making antisense nucleic acids are well known in the art. Further provided are methods of modulating the expression of OST-PTP and gamma-carboxylase genes and mRNA in cells or tissues by contacting the cells or tissues with one or more of the antisense compounds or compositions of the invention. As used herein, the terms "target nucleic acid" encompass DNA encoding OST-PTP or gamma-carboxylase and RNA (including pre-mRNA and mRNA) transcribed from such DNA. The specific hybridization of a nucleic acid oligomeric compound with its target nucleic acid interferes with the normal function of the target nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense." The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of the protein encoded by the DNA or RNA. In the context of the present invention, "modulation" means reducing or inhibiting in the expression of the gene or mRNA for OST-PTP and/or gamma-carboxylase. DNA is the preferred antisense nucleic acid.

[0263]The targeting process includes determination of a site or sites within the target DNA or RNA encoding the OST-PTP and/or gamma-carboxylase for the antisense interaction to occur such that the desired inhibitory effect is achieved. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the mRNA for OST-PTP or gamma-carboxylase, preferably human OST-PTP or gamma-carboxylase. Since, as is known in the art, the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon," the "start codon" or the "AUG start codon." A minority of genes have a translation initiation codon having the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been shown to function in vivo. Thus, the terms "translation initiation codon" and "start codon" can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine in eukaryotes. It is also known in the art that eukaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, "start codon" and "translation initiation codon" refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene. Routine experimentation will determine the optimal sequence of the antisense or siRNA.

[0264]It is also known in the art that a translation termination codon (or "stop codon") of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA, respectively).

[0265]The terms "start codon region" and "translation initiation codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon. Similarly, the terms "stop codon region" and "translation termination codon region" refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.

[0266]The open reading frame (ORF) or "coding region," which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5' untranslated region (5'UTR), known in the art to refer to the portion of an mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3' untranslated region (3'UTR), known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene.

[0267]It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.

[0268]Once one or more target sites have been identified, nucleic acids are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect of inhibiting gene expression and transcription or mRNA translation.

[0269]In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of a nucleic acid is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the nucleic acid and the DNA or RNA are considered to be complementary to each other at that position. The nucleic acid and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the nucleic acid and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of function, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

[0270]Antisense nucleic acids have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense nucleic acid drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that nucleic acids can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans, for example to regulate expression of OST-PTP and gamma-carboxylase.

[0271]Nucleic acids in the context of this invention includes "oligonucleotides," which refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0272]While antisense nucleic acids are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e., from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense nucleic acids comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) nucleic acids (oligozymes), and other short catalytic RNAs or catalytic nucleic acids which hybridize to the target nucleic acid and modulate its expression.

[0273]The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare nucleic acids such as the phosphorothioates and alkylated derivatives.

[0274]The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, and prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder such as metabolic syndrome, glucose intolerance, diabetes, atherosclerosis, and/or obesity, which can be treated by modulating the expression of gamma-carboxylase or OST-PTP, is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. The antisense compounds and methods of the invention are useful prophylactically, e.g., to prevent or delay the appearance of metabolic syndrome, glucose intolerance, diabetes, atherosclerosis or obesity. The antisense compounds and methods of the invention are also useful to retard the progression of metabolic syndrome, glucose intolerance, diabetes, atherosclerosis or obesity.

[0275]The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention that are administered to return the level of serum insulin in diabetic patients (for example) to normal.

[0276]US Patent Application 2004/0023390 (the entire contents of which are hereby incorporated by reference as if fully set forth herein) teaches that double-stranded RNA (dsRNA) can induce sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). However, in mammalian cells, dsRNA that is 30 base pairs or longer can induce sequence-nonspecific responses that trigger a shut-down of protein synthesis and even cell death through apoptosis. Recent work shows that RNA fragments are the sequence-specific mediators of RNAi (Elbashir et al., 2001). Interference of gene expression by these small interfering RNA (siRNA) is now recognized as a naturally occurring strategy for silencing genes in C. elegans, Drosophila, plants, and in mouse embryonic stem cells, oocytes and early embryos (Cogoni et al., 1994; Baulcombe, 1996; Kennerdell, 1998; Timmons, 1998; Waterhouse et al., 1998; Wianny and Zernicka-Goetz, 2000; Yang et al., 2001; Svoboda et al., 2000).

[0277]In mammalian cell culture, a siRNA-mediated reduction in gene expression has been accomplished by transfecting cells with synthetic RNA nucleic acids (Caplan et al., 2001; Elbashir et al., 2001). The 2004/0023390 application, the entire contents of which are hereby incorporated by reference as if fully set forth herein, provides exemplary methods using a viral vector containing an expression cassette containing a pol II promoter operably-linked to a nucleic acid sequence encoding a small interfering RNA molecule (siRNA) targeted against a gene of interest.

[0278]As used herein RNAi is the process of RNA interference. A typical mRNA produces approximately 5,000 copies of a protein. RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA, preferably encoding OST-PTP or gamma-carboxylase. For example, a double-stranded short interfering RNA (siRNA) molecule is engineered to complement and match the protein-encoding nucleotide sequence of the target mRNA to be interfered with. Following intracellular delivery, the siRNA molecule associates with an RNA-induced silencing complex (RISC). The siRNA-associated RISC binds the target mRNA (such as mRNA encoding gamma-carboxylase and OST-PTP) through a base-pairing interaction and degrades it. The RISC remains capable of degrading additional copies of the targeted mRNA. Other forms of RNA can be used such as short hairpin RNA and longer RNA molecules. Longer molecules cause cell death, for example by instigating apoptosis and inducing an interferon response. Cell death was the major hurdle to achieving RNAi in mammals because dsRNAs longer than 30 nucleotides activated defense mechanisms that resulted in non-specific degradation of RNA transcripts and a general shutdown of the host cell. Using from about 20 to about 29 nucleotide siRNAs to mediate gene-specific suppression in mammalian cells has apparently overcome this obstacle. These siRNAs are long enough to cause gene suppression but not of a length that induces an interferon response. In a specific embodiment of the invention, the targets of gene suppression are the OST-PTP gene and the gene for gamma-carboxylase. siRNA molecules useful in the present invention include those sequences that bind under stringent conditions to the human gamma-carboxylase gene of SEQ ID:10 or the OST-PTP gene of SEQ ID NO:18. siRNA molecules useful in the present invention also include those sequences that bind under stringent conditions to nucleic acids that are 80%, 85%, 90%, or 95% homologous to SEQ ID NO:18.

Co-Administration of the Therapeutic Agents of the Present Invention and Other Drugs

[0279]The undercarboxylated/uncarboxylated osteocalcin and inhibitors of OST-PTP and gamma-carboxylase described herein may be co-administered to a patient with other drugs such as anti-coagulants, vasodilators, drugs used to treat atherosclerosis, drugs used to treat glucose intolerance, drugs used to treat diabetes, vitamin K inhibitors, statins, beta blockers, and other drugs used to treat diseases associated with disorders related to the OST-PTP signaling pathway, including, but not limited to metabolic syndrome, glucose intolerance, type 1 or type 2 diabetes, atherosclerosis, and obesity in amounts effective to provide therapeutic benefit of the drug in the combination therapy. The combination may provide increased, additive, or synergistic effect. The co-administration of the undercarboxylated/uncarboxylated osteocalcin, inhibitors of OST-PTP, inhibitors of gamma-carboxylase and the other drugs may be done by administration of separate pharmaceutical compositions or the the undercarboxylated/uncarboxylated osteocalcin, inhibitors of OST-PTP, inhibitors of gamma-carboxylase and the other drugs may be present in a single pharmaceutical composition.

[0280]Anticoagulants useful in the invention are exemplified by vitamin K antagonists, heparin and derivatives of heparin, and direct thrombin inhibitors. Vitamin K antagonists are exemplified by warfarin (also known under the brand names COUMADIN®, JANTOVEN®, MAREVAN®, and WARAN®), warfarin derivatives, acenocoumarol, phenprocoumon as well as phenindione. Heparin and derivatives of heparin are exemplified by low molecular weight heparin and fondaparinux. Direct thrombin inhibitors are exemplified by argatroban, lepirudin, bivalirudin and ximelagatran.

[0281]Vasodilators are useful in the present invention. Vasodilators are exemplified by adenosine, amyl nitrite and other nitrites, L-arginine, atrial natriuretic peptide (ANP), bradykinin, ethanol, endothelium-derived hyperpolarizing factor (EDHF), histamine, complement proteins C3a, C4a and C5a, niacin (nicotinic acid), nitric oxide, glyceryl trinitrate (commonly known as nitroglycerin), isosorbide mononitrate & isosorbide dinitrate, pentaerythritol tetranitrate (PETN), sodium nitroprusside, PDE5 inhibitors, sildenafil, tadalafil, vardenafil, platelet activating factor (PAF), prostacyclin (PGI₂) as well as other prostaglandins, tetrahydrocannabinol (THC), theobromine, and papaverine.

[0282]Drugs used to treat atherosclerosis are useful in the present invention. Drugs used to treat atherosclerosis are exemplified by statins, scilostazol, benzothiazepines, phenylalkylamines, dihydropyridines, epoprostenol, vitamin B3, and aspirin. Statins are further exemplified by atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Benzothiazepines are exemplified by diltiazem. Phenylalkylamines are exemplified by verapamil. Dihydropyridines are exemplified by amlodipine, felodipine, isradipine, lacidipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, and nitrendipine.

[0283]Drugs useful in the treatment of diabetes include, but are not limited to, sulfonylureas, meglitinides, D-Phenylalanine Derivatives (nateglinides), biguanides, thiazolidinediones, alpha-glucose inhibitors, Dipeptidyl peptidase 4 (DPP4) inhibitors, insulins (preferably human recombinant insulin) and incretins.

[0284]Sulfonylureas are exemplified by glimepiride, glyburide, chlorpropamide, acetohexamide, glipizide, tolbutamide, and tolazamide. Meglitinides are exemplified by Repaglinide. D-Phenylalanine Derivatives are exemplified by nateglinide. Biguanides are exemplified by metformin and metformin hydrochloride. Thiazolidinediones are exemplified by pioglitazone and rosiglitazone. Alpha-glucose inhibitors are exemplified by miglitol and acarbose. Dipeptidyl peptidase 4 (DPP4) inhibitors are exemplified by vildagliptin, sitagliptin and saxagliptin.

[0285]Generally, there are six categories of insulins: rapid-acting, short-acting, intermediate acting, long acting, very long acting, and premixed. Incretins are a type of gastrointestinal hormone that cause an increase in the amount of insulin released from the beta-cells of the islets of Langerhans after eating, even before blood glucose levels become elevated. Incretins are exemplified by glucagon-like peptide-1 (GLP-1) and Gastric inhibitory peptide (aka glucose-dependent Insulinotropic peptide or GIP).

[0286]Beta blockers are used to treat high blood pressure (hypertension), congestive heart failure (CHF), abnormal heart rhythms (arrhythmias), and chest pain (angina). Beta blockers are sometimes used in heart attack patients to prevent future heart attacks. Beta blockers work by blocking the effects of the hormone epinephrine, also known as adrenaline. As a result, the heart beats more slowly and with less force, thereby reducing blood pressure. Beta blockers also help blood vessels relax and open up to improve blood flow. Beta blockers also block the impulses that can cause an arrhythmia. There are 2 main beta receptors: beta 1 and beta 2. Some beta blockers are selective, which means that they block beta 1 receptors more than they block beta 2 receptors. Beta 1 receptors are responsible for heart rate and the strength of the heartbeat. Nonselective beta blockers block both beta 1 and beta 2 receptors. Beta 2 receptors are responsible for the function of smooth muscles; they are also the only beta receptors expressed by osteoblasts.

[0287]Brand Names and generic claims of beta blockers commonly used in the United States are: Betapace (sotalol), Blocadren (timolol), Brevibloc (esmolol), Cartrol (carteolol), Coreg (carvedilol), Corgard (nadolol), Inderal (propranolol), Inderal-LA (propranolol), Kerlone (betaxolol), Levatol (penbutolol), Lopressor (metoprolol), Normodyne (labetalol), Sectral (acebutolol), Tenormin (atenolol), Toprol-XL (metoprolol), Trandate (labetalol), Visken (pindolol), Zebeta (bisoprolol). Commonly Used Brand Names in Canada are: Apo-Atenolol (atenolol), Apo-Metoprolol (metoprolol), Apo-Propranolol (propranolol), Apo-Timol (timolol), Betaloc (metoprolol), Blocadren (timolol), Corgard (nadolol), Inderal (propranolol), Lopressor (metoprolol), Monitan (acebutolol), Novo-Atenol (atenolol), Novometoprol (metoprolol), Novo-Pindol (pindolol), Novo-Timol (timolol), Sectral (acebutolol), Sotacor (sotalol), Tenormin (atenolol), Trandate (labetalol), Trasicor (oxprenolol), Visken (pindolol).

Pharmaceutical Compositions and Administration

[0288]The present invention encompasses the use of the polypeptides, nucleic acids, antibodies, small molecules and other therapeutic agents described herein formulated in pharmaceutical compositions to administer to a subject. The therapeutic agents (also referred to as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. Such compositions typically comprise the polypeptides, nucleic acids, antibodies, small molecules and a pharmaceutically acceptable carrier. Preferably, such compositions are non-pyrogenic when administered to humans.

[0289]The pharmaceutical compositions of the invention are adminstered in an amount sufficient to modulate the the OST-PTP signaling pathway involving gamma-carboxylase, osteocalcin, insulin and adiponectin.

[0290]As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, binders, diluents, disintegrants, lubricants, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. As long as any conventional media or agent is compatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds or therapeutic agents can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, intranasal, subcutaneous, oral, inhalation, transdermal (topical), transmucosal, and rectal administration.

[0291]The term "administer" is used in its broadest sense and includes any method of introducing the compositions of the present invention into a subject. This includes producing polypeptides or polynucleotides in vivo as by transcription or translation of polynucleotides that have been exogenously introduced into a subject. Thus, polypeptides or nucleic acids produced in the subject from the exogenous compositions are encompassed in the term "administer."

[0292]Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diamine tetra acetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0293]Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where the therapeutic agents are water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0294]Sterile injectable solutions can be prepared by incorporating the active compound (e.g., undercarboxylated/uncarboxylated osteocalcin protein or anti-OST-PTP antibody) in the required amount in an appropriate solvent with one or a combination of the ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0295]Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. Depending on the specific conditions being treated, pharmaceutical compositions of the present invention for treatment of atherosclerosis or the other elements of metabolic syndrome can be formulated and administered systemically or locally. Techniques for formulation and administration can be found in "Remington: The Science and Practice of Pharmacy" (20^th edition, Gennaro (ed.) and Gennaro, Lippincott, Williams & Wilkins, 2000). For oral administration, the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the GI tract by known methods. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL®, or corn starch; a lubricant such as magnesium stearate or STEROTES®; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0296]For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0297]Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0298]If appropriate, the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0299]In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to particular cells with, e.g., monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0300]It is especially advantageous to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. "Unit dosage form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0301]As previously noted, the agent may be administered continuously by pump or frequently during the day for extended periods of time. In certain embodiments, the agent may be administered at a rate of from about 0.3-100 ng/hour, preferably about 1-75 ng/hour, more preferably about 5-50 ng/hour, and even more preferably about 10-30 ng/hour. The agent may be administered at a rate of from about 0.1-100 pg/hr, preferably about 1-75 μg/hr, more preferably about 5-50 μg/hr, and even more preferably about 10-30 μg/hr It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from monitoring the level of undercarboxylated/uncarboxylated osteocalcin and/or adiponectin and/or insulin and/or monitoring glycemia control in a biological sample, preferably blood or serum.

[0302]In an embodiment of the invention, the agent can be delivered by subcutaneous, long-term, automated drug delivery using an osmotic pump to infuse a desired dose of the agent for a desired time. Insulin pumps are widely available and are used by diabetics to automatically deliver insulin over extended periods of time. Such insulin pumps can be adapted to deliver the agent. The delivery rate of the agent to control glucose intolerance, diabetes types 1 or 2 can be readily adjusted through a large range to accommodate changing insulin requirements of an individual (e.g., basal rates and bolus doses). New pumps permit a periodic dosing manner, i.e., liquid is delivered in periodic discrete doses of a small fixed volume rather than in a continuous flow manner. The overall liquid delivery rate for the device is controlled and adjusted by controlling and adjusting the dosing period. The pump can be coupled with a continuous blood glucose monitoring device and remote unit, such as a system described in U.S. Pat. No. 6,560,471, entitled "Analyte Monitoring Device and Methods of Use." In such an arrangement, the hand-held remote unit that controls the continuous blood glucose monitoring device could wirelessly communicate with and control both the blood glucose monitoring unit and the fluid delivery device delivering therapeutic agents of the present invention.

[0303]A "therapeutically effective amount" of a protein or polypeptide, small moleclue or nucleic acid is an amount that achieves the desired therapeutic result. For example, if a therapeutic agent is administered to treat or prevent atherosclerosis, a therapeutically effective amount is an amount that ameliorates one or more symptoms of the disease, or produces at least one effect selected from the group consisting of a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, a decrease in hypertension, and the like. If a therapeutic agent is used for treating or preventing metabolic syndrome in an animal (including mammals, including humans and laboratory animals) a therapeutically effective amount is an amount that produces at least one effect selected from the group consisting of increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, weight loss, decreasing fat mass, increasing serum adiponectin, and an a decrease in or improved control of atherosclerosis.

[0304]A therapeutically effective amount of protein or polypeptide, small molecule or nucleic acid for use in the present invention typically varies and can be an amount sufficient to achieve serum therapeutic agent levels typically of between about 1 nanogram per milliliter and about 10 micrograms per milliliter in the subject, or an amount sufficient to achieve serum therapeutic agent levels of between about 1 nanogram per milliliter and about 7 micrograms per milliliter in the subject. Other preferred serum therapeutic agent levels include about 0.1 nanogram per milliliter to about 3 micrograms per milliliter, about 0.5 nanograms per milliliter to about 1 microgram per milliliter, about 1 nanogram per milliliter to about 750 nanograms per milliliter, about 5 nanograms per milliliter to about 500 nanograms per milliliter, and about 5 nanograms per milliliter to about 100 nanograms per milliliter.

[0305]Expressed as a daily dose, this amount can be between about 0.1 nanograms per kilogram body weight per day and about 20 milligrams per kilogram body weight per day, and between about 1 nanogram per kilogram body weight per day and about 10 milligrams per kilogram body weight per day. Other preferred daily dosages include about 1 nanogram per kilogram body weight per day to about 20 milligrams per kilogram body weight per day, about 5 nanograms per kilogram body weight per day to about 5 milligrams per kilogram body weight per day, about 20 nanograms per kilogram body weight per day to about 500 micrograms per kilogram body weight per day, and about 500 nanograms per kilogram body weight per day to about 100 micrograms per kilogram body weight per day. However, the skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the condition, previous treatments, the general health and/or age of the subject, and other disorders or diseases present.

[0306]In certain embodiments, the pharmaceutical compositions of the present invention comprise about 0.1 mg to 5 g, about 0.5 mg to about 1 g, about 1 mg to about 750 mg, about 5 mg to about 500 mg, or about 10 mg to about 100 mg of therapeutic agent.

[0307]Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, nucleotide or antibody can include a single treatment or, preferably, can include a series of treatments.

[0308]In certain embodiments, treatment of a subject with undercarboxylated/uncarboxylated osteocalcin leads to undercarboxylated/uncarboxylated osteocalcin being about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the total osteocalcin in the blood of the patient.

[0309]It is understood that the appropriate dose of a small molecule agent depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, and the effect which the practitioner desires the small molecule to have. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of OST-PTP or gamma-carboxylase, a relatively low dose may be prescribed at first, with the dose subsequently increased until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0310]For atherosclerosis prevention or treatment, a suitable subject can be an individual who is suspected of having, has been diagnosed as having, or is at risk of developing atherosclerosis, coronary artery disease, stroke, restenosis, vascular fibromuscular dysplasia, polyarteritis nodosa, Takayasu's arteritis, and like conditions as can be determined by one knowledgeable in the art. Another example of a suitable subject is an individual who is to undergo vascular surgery, including but not limited to vascular bypass surgery, atherectomy, endatherectomy, laser ablation, angioplasty, balloon angioplasty, cardiac allograft (cardiac transplant), insertion of a prosthesis, insertion of a graft, insertion of a stent, catheterization, or arterial blockage evaluation. Suitable routes of administration can include oral, intestinal, parenteral, transmucosal, transdermal, intramuscular, subcutaneous, transdermal, rectal, intramedullary, intrathecal, intravenous, intraventricular, intraatrial, intraaortal, intraarterial, or intraperitoneal administration. The pharmaceutical compositions of the present invention can be administered to the subject by a medical device, such as, but not limited to, catheters, balloons, implantable devices, biodegradable implants, prostheses, grafts, sutures, patches, shunts, or stents. For atherosclerosis, pharmaceutical compositions of the present invention can contain a therapeutic agent in an amount sufficient to prevent/slow down the development of atherosclerotic lesions. In one preferred embodiment, the therapeutic agent (e.g., undercarboxylated/uncarboxylated osteocalcin) can be coated on a stent for localized administration to the target area. In this situation a slow release preparation of undercarboxylated/uncarboxylated osteocalcin, for example, is preferred.

[0311]The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0312]In yet another aspect of the invention, undercarboxylated/uncarboxylated osteocalcin is administered as a pharmaceutical composition with a pharmaceutically acceptable excipient. Exemplary pharmaceutical compositions for undercarboxylated/uncarboxylated osteocalcin include injections as solutions or injections as injectable self-setting or self-gelling mineral polymer hybrids. Undercarboxylated/uncarboxylated osteocalcin may be administered using a porous crystalline biomimetic bioactive composition of calcium phosphate. See U.S. Pat. Nos. 5,830,682; 6,514,514; 6,511,958 and U.S. Pat. Pub. No.: 2006/0063699; 2006/0052327; 2003/199615; 2003/0158302; 2004/0157864; 2006/0292670; 2007/0099831 and 2006/0257492, all of which are incorporated herein in their entirety by reference.

Methods of Treatment

[0313]The present invention provides methods for modulating energy metabolism through the OST-PTP signaling pathway for treating or preventing a variety of different disorders relating to the OST-PTP signaling pathway involving gamma-carboxylase, osteocalcin, insulin and adiponectin. In particular, the methods are used to inhibit OST-PTP phosphorylase activity, reduce gamma-carboxylase activity, and/or increase undercarboxylated/uncarboxylated osteocalcin. According to the invention, the methods provide an amount of an agent effective to treat or prevent a disorder associated with the OST-PTP signaling pathway. The agent may be selected from the group consisting of small molecules, antibodies and nucleic acids. Such disorders include, but are not limited to, metabolic syndrome, glucose intolerance, diabetes type 1, diabetes type 2, atherosclerosis, and/or obesity.

[0314]In certain embodiments, the methods comprise identifying a patient in need of treatment or prevention of metabolic syndrome, glucose intolerance, diabetes type 1, diabetes type 2, vascular disorders (such as atherosclerosis), and/or obesity and then applying the methods disclosed herein to the patient.

[0315]Vascular disorders include atherosclerosis, arteriosclerosis and arteriolosclerosis. Arteriosclerosis, which means hardening (sclerosis) of the arteries (arterio-), is a general term for several diseases in which the wall of an artery becomes thicker and less elastic. There are three types: atherosclerosis, arteriolosclerosis, and Monckeberg's arteriosclerosis. Atherosclerosis, the most common type, means hardening related to atheromas, which are deposits of fatty materials. It affects medium-sized and large arteries of the brain, heart, kidneys, other vital organs, and legs. It is the most important and most common type of arteriosclerosis, a general term for several diseases in which the wall of an artery becomes thicker and less elastic.

[0316]Type 1 diabetes is usually diagnosed in children and young adults, and was previously known as juvenile diabetes. In type 1 diabetes, the body does not produce insulin. Insulin is a hormone that is needed to convert sugar (glucose), starches and other food into energy needed for daily life. Conditions associated with type 1 diabetes include hyperglycemia, hypoglycemia, ketoacidosis and celiac disease.

[0317]Type 2 diabetes is the most common form of diabetes. In type 2 diabetes, either the body does not produce enough insulin or the cells ignore the insulin. Conditions associated with type 2 diabetes include hyperglycemia and hypoglycemia.

[0318]Disorders associated with energy metabolism include diabetes, glucose intolerance, decreased insulin sensitivity, decreased pancreatic beta-cell proliferation, decreased insulin secretion, weight gain, increased fat mass and decreased serum adiponectin

[0319]The methods of the invention increase serum osteocalcin levels (preferably undercarboxylated/uncarboxylated osteocalcin), serum adiponectin levels and/or serum insulin levels. The pharmaceutical compositions may also increase glucose tolerance, increase insulin sensitivity, and/or increase pancreatic beta-cell proliferation.

[0320]As used herein, the terms "animal," "patient," or "subject" include mammals, e.g., humans, dogs, cows, horses, kangaroos, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. The preferred animal, patient, or subject is a human.

[0321]In certain embodiments, the methods comprise administering undercarboxylated/uncarboxylated osteocalcin to a patient, preferably a human. In a specific embodiment of the invention, the daily dose of undercarboxylated/uncarboxylated osteocalcin is between about 0.1 nanograms per kilogram body weight per day and about 20 milligrams per kilogram body weight per day, or between about 1 nanogram per kilogram body weight per day and about 10 milligrams per kilogram body weight per day. Other preferred daily dosages include about 1 nanogram per kilogram body weight per day to about 20 milligrams per kilogram body weight per day, about 5 nanograms per kilogram body weight per day to about 5 milligrams per kilogram body weight per day, about 20 nanograms per kilogram body weight per day to about 500 micrograms per kilogram body weight per day, and about 500 nanograms per kilogram body weight per day to about 100 micrograms per kilogram body weight per day.

[0322]In some embodiments, the daily dosage is about 0.1 milligrams per kilogram body weight per day, about 0.25 milligrams per kilogram body weight per day, about 0.5 milligrams per kilogram body weight per day, about 0.75 milligrams per kilogram body weight per day, about 1 milligram per kilogram body weight per day, about 2 milligrams per kilogram body weight per day, about 5 milligrams per kilogram body weight per day, about 10 milligrams per kilogram body weight per day, or about 20 milligrams per kilogram body weight per day.

[0323]In one embodiment of the invention, the method of treatment comprises administering to a patient in need thereof a therapeutically effective amount of undercarboxylated/uncarboxylated osteocalcin sufficient to raise the patient's blood level of undercarboxylated/uncarboxylated osteocalcin compared to the pretreatment patient level. Preferably, the patient is a human. In another embodiment, the method of treatment comprises administering to a patient in need thereof a therapeutically effective amount of undercarboxylated/uncarboxylated osteocalcin sufficient to raise the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in the patient's blood compared to the pretreatment patient ratio.

[0324]In another aspect of the invention, a method is provided for treating or preventing metabolic syndrome in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that produces at least one effect selected from the group consisting of increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, decreasing fat mass, weight loss and increasing serum adiponectin compared to pretreatment levels. Alternatively, the undercarboxylated/uncarboxylated osteocalcin is administered in an amount that produces at least one effect selected from the group consisting of a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, and a decrease in hypertension as compared to pretreatment levels. Preferably, the patient is a human.

[0325]Insulin sensitivity can be measured by the insulin tolerance test or euglycemic hyperinsulinemic clamp. Glucose tolerance can be measured by glucose tolerance tests. Insulin secretion can be measured by the glucose stimulated insulin secretion test. The most common test for glycemic control in diabetic patients is a blood glucose test done typically by using test strips and one drop of blood. To better monitor the level of glycemic control over time, one can measure hemoglobin A1c (glycosylated hemoglobin).

[0326]In a specific embodiment of the invention, a method is provided for treating or preventing type 1 or type 2 diabetes or glucose intolerance in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that produces at least one effect selected from the group comprising increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, decreasing fat mass, weight loss, and increasing serum adiponectin compared to pretreatment levels. Preferably, the patient is a human. In an embodiment of the invention, said method further comprises co-administration of anti-diabetic drugs such as insulin (preferably recombinant human insulin), incretins, sulfonylureas, meglitinides, D-Phenylalanine Derivatives (nateglinides), biguanides, thiazolidinediones, alpha-glucose inhibitors,GLP-1, GLP-1 analogues such as liraglutide, exendin-4 LY5448806 and CJC-1131, as well as dipeptidyl peptidase IV inhibitors.

[0327]Sulfonylureas are exemplified by glimepiride, glyburide, chlorpropamide, acetohexamide, glipizide, tolbutamide, and tolazamide. Meglitinides are exemplified by Repaglinide. Biguanides are exemplified by metformin and metformin hydrochloride. Thiazolidinediones are exemplified by pioglitazone and rosiglitazone. Alpha-glucose inhibitors are exemplified by miglitol and acarbose. Dipeptidyl peptidase 4 (DPP4) inhibitors are exemplified by vildagliptin, sitagliptin and saxagliptin.

[0328]Generally, there are six categories of insulins: rapid-acting, short-acting, intermediate acting, long acting, very long acting, and premixed. Incretins are a type of gastrointestinal hormone that cause an increase in the amount of insulin released from the beta-cells of the islets of Langerhans after eating, even before blood glucose levels become elevated. Incretins are exemplified by glucagon-like peptide-1 (GLP-1) and Gastric inhibitory peptide (aka glucose-dependent Insulinotropic peptide or GIP).

[0329]The present invention further provides (i) methods for treating or preventing obesity in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that decreases weight gain, decreases fat mass or results in loss of weight; (ii) methods for increasing insulin sensitivity in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that increases insulin sensitivity; (iii) methods for increasing glucose tolerance in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that increases glucose tolerance; (iv) methods for increasing insulin secretion in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that increases insulin secretion; and (v) methods for increasing beta-cell proliferation in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that increases beta-cell proliferation. Preferably, the animal is a human.

[0330]In another aspect of the invention, a method is provided for treating or preventing atherosclerosis in an animal comprising administering to an animal in need thereof undercarboxylated/uncarboxylated osteocalcin in a therapeutically effective amount that produces at least one effect selected from the group consisting of a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, and a decrease in hypertension. Preferably, the animal is a human. In an embodiment of the invention, said method further comprises co-administration of compounds used to treat atherosclerosis such as low density lipoprotein peroxidation inhibitors, antihyperlipidemic agents, anticoagulants, vasodilators, and other drugs useful in the treatment of atherosclerosis.

[0331]Anticoagulants are exemplified by vitamin K antagonists, heparin and derivatives of heparin, and direct thrombin inhibitors. Vitamin K antagonists are exemplified by warfarin (also known under the brand names COUMADIN®, JANTOVEN®, MAREVAN®, and WARAN®); acenocoumarol, phenprocoumon as well as phenindione. Heparin and derivatives of heparin are exemplified by low molecular weight heparin and fondaparinux. Direct thrombin inhibitors are exemplified by argatroban, lepirudin, bivalirudin and ximelagatran.

[0332]Vasodilators are exemplified by adenosine, amyl nitrite and other nitrites, 1-arginine, atrial natriuretic peptide (ANP), bradykinin, ethanol, endothelium-derived hyperpolarizing factor (EDHF), histamine, complement proteins C3a, C4a and C5a, niacin (nicotinic acid), nitric oxide, glyceryl trinitrate (commonly known as nitroglycerin), isosorbide mononitrate & isosorbide dinitrate, pentaerythritol tetranitrate (PETN), sodium_nitroprusside, PDE5 inhibitors, sildenafil, tadalafil, vardenafil, platelet activating factor (PAF), prostacyclin (PGI₂) as well as other prostaglandins, tetrahydrocannabinol (THC), theobromine, and papaverine.

[0333]Other drugs useful in the treatment of atherosclerosis are exemplified by statins, scilostazol, benzothiazepines, phenylalkylamines, dihydropyridines, epoprostenol, vitamin B3, and aspirin. Statins are further exemplified by atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Benzothiazepines are exemplified by diltiazem. Phenylalkylamines are exemplified by verapamil. Dihydropyridines are exemplified by amlodipine, felodipine, isradipine, lacidipine, lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine, and nitrendipine.

[0334]In yet another embodiment of the invention, when administering undercarboxylated/uncarboxylated osteocalcin, it may be desirable to co-administered drugs normally administered to treat osteoporosis. Such drugs include, for example, raloxifene, calcitonin and alendronate.

[0335]In methods wherein adiponectin is co-administered with undercarboxylated/uncarboxylated osteocalcin, the adiponectin and undercarboxylated/uncarboxylated osteocalcin may be administered in a single pharmaceutical composition. Alternatively, the undercarboxylated/uncarboxylated osteocalcin and adiponectin may be administered in separate pharmaceutical compositions. In another embodiment of the invention, adiponectin and undercarboxylated/uncarboxylated osteocalcin are administered on the same day. In yet another embodiment, the adiponectin and undercarboxylated/uncarboxylated osteocalcin are administered on different days.

[0336]In an embodiment of the invention, a method is provided for treating or preventing a disease associated with low levels of serum osteocalcin in a patient comprising administering to a patient in need thereof an amount of a beta blocker or a vitamin K blocker or a combination thereof that causes an increase in the level of serum osteocalcin compared to pretreatment levels. Preferably, the patient is a human and the serum level of undercarboxylated/uncarboxylated osteocalcin is increased.

[0337]The present invention further provides a method of treating or preventing a disease including, but not limited to, metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity in a patient comprising administering to a patient in need thereof a therapeutically effective amount of an agent that reduces OST-PTP expression or activity in osteoblasts sufficient to raise the patient's level of undercarboxylated/uncarboxylated osteocalcin. Preferably, the patient is a human.

[0338]The invention further provides a method of treating or preventing a disease selected from the group consisting of metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity comprising administering to a patient in need thereof a therapeutically effective amount of an agent that reduces gamma-carboxylase expression or activity in osteoblasts sufficient to raise the patient level of undercarboxylated/uncarboxylated osteocalcin. Preferably, the patient is a human. Preferably, the patient is a human.

[0339]In an embodiment of the invention, a method is provided for treating or preventing metabolic syndrome in a patient comprising administering to a patient in need thereof a therapeutically effective amount of an agent that reduces OST-PTP expression or activity in osteoblasts sufficient to produce at least one effect selected from the group consisting of increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, decreasing fat mass, weight loss, and increasing serum adiponectin, or which decreases or controls atherosclerosis compared to pretreatment levels. Preferably, the patient is a human.

[0340]In another aspect of the invention, a method is provided for treating or preventing type 1 or type 2 diabetes or glucose ,intolerance, in an animal comprising administering to an animal in need thereof adiponectin in a therapeutically effective amount so as to reduce OST-PTP expression or activity in osteoblasts sufficient to produce at least one effect selected from the group consisting of increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, and increasing serum adiponectin compared to pretreatment levels. Preferably, the animal is a human.

[0341]A method is provided for treating or preventing atherosclerosis in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces OST-PTP expression or activity in osteoblasts sufficient to produce at least one effect selected from the group consisting of a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, and a decrease in hypertension compared to pretreatment levels. Preferably, the animal is a human.

[0342]In different embodiments of the invention, methods are provided (i) for treating or preventing obesity in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces OST-PTP expression or activity in osteoblasts sufficient to decrease weight gain, decrease fat mass or result in weight loss compared to pretreatment levels; (ii) for treating or preventing glucose intolerance in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces OST-PTP expression or activity in osteoblasts sufficient to increase glucose tolerance compared to pretreatment levels; (iii) for increasing insulin sensitivity in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces OST-PTP expression or activity in osteoblasts sufficient to increase insulin sensitivity compared to pretreatment levels. Preferably, the animal is a human.

[0343]In another aspect of the invention, a method is provided for treating or preventing metabolic syndrome in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces gamma-carboxylase expression or activity in osteoblasts sufficient to (1) produce at least one effect selected from the group consisting of increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, decreasing fat mass, weight loss and increasing serum adiponectin compared to pretreatment levels, or (2) to produce at least one effect selected from the group consisting of a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, and a decrease in hypertension compared to pretreatment levels. Preferably, the animal is a human.

[0344]In yet another embodiment of the invention, method for is provided for treating or preventing type 1 or type 2 diabetes or glucose intolerance in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces gamma-carboxylase expression or activity in osteoblasts sufficient to produce at least one effect selected from the group consisting of increasing pancreatic beta-cell proliferation, increasing insulin secretion, increasing insulin sensitivity, increasing glucose tolerance, decreasing weight gain, decreasing fat mass, weight loss and increasing serum adiponectin compared to pretreatment levels. Preferably, the animal is a human.

[0345]A method is also provided for treating or preventing atherosclerosis in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces gamma-carboxylase expression or activity in osteoblasts sufficient to produce at least one effect selected from the group consisting of a reduction of oxidized phospholipids, a regression of atherosclerotic plaques, a decrease in inflammatory protein biosynthesis, a reduction in plasma cholesterol, a reduction in vascular smooth muscle cell (VSMC) proliferation and number, a decrease in the thickness of arterial plaque, a reduction in clinical events such as heart attack, angina, or stroke, and a decrease in hypertension compared to pretreatment levels. Preferably, the animal is a human.

[0346]The present invention is directed to methods (i) for treating or preventing obesity in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces gamma-carboxylase expression or activity in osteoblasts sufficient to cause a decrease in weight gain, a decrease in fat mass, or weight loss compared to pretreatment levels (ii) for treating or preventing glucose intolerance in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces gamma-carboxylase expression or activity in osteoblasts sufficient to increase glucose tolerance compared to pretreatment levels or (iii) for increasing insulin sensitivity in an animal comprising administering to an animal in need thereof in a therapeutically effective amount an agent that reduces gamma-carboxylase expression or activity in osteoblasts sufficient to increase insulin sensitivity compared to pretreatment levels. Preferably, the animal is a human. In an embodiment of the invention, the agent is an isolated nucleic acid that is selected from the group consisting of cDNA, antisense DNA, antisense RNA, and small interfering RNA, which nucleic acid is sufficiently complementary to the gene or mRNA encoding gamma-carboxylase to permit specific hybridization to the gene or mRNA, and wherein the hybridization prevents or reduces expression of gamma-carboxylase in osteoblasts. In another embodiment of the invention, the nucleic acid is conjugated to a phosphate group or other targeting ligand to facilitate uptake by osteoblasts.

[0347]In the methods described herein, it will be understood that "treating" a disease encompasses not only improving the disease or its symptoms but also retarding the progression of the disease or ameliorating the disease.

[0348]The present invention also encompasses the use of gene therapy for treatment of metabolic syndrome, including obesity, type 2 diabetes, glucose intolerance atherosclerosis, and type 1 diabetes. This can be accomplished by introducing a gene encoding osteocalcin or a biologically active fragment or variant thereof into a vector, and transfecting or infecting cells from a patient afflicted with the disease or at a high risk of developing the disease with the vector, according to various methods known in the art. The cells may be transfected or infected by ex vivo or by in vivo methods.

[0349]Adeno-associated virus (AAV) is one of the most promising vectors for gene therapy and may be used in the methods of the present invention. Conventional methods of gene transfer and gene therapy are described in, e.g., Gene Therapy: Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy, ed. C. P. Hodgson, Springer Verlag, 1996. AAV is an attractive vector system for human gene therapy because it is non-pathogenic for humans, it has a high frequency of integration, and it can infect non-dividing cells, thus making it useful for delivery of genes into mammalian cells both in tissue culture and in whole animals. Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-129, 1992. Recent studies have demonstrated AAV to be a potentially useful vector for gene delivery. LaFace et al., Viology, 162:483-486, 1998; Zhou et al., Exp. Hematol. (NY), 21:928-933, 1993; Flotte et al., PNAS 90:10613-10617, 1993; and Walsh et al., Blood 84:1492-1500, 1994. Recombinant AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt et al., Nature Genetics, 8:148-154, 1994; Lebkowski et al., Mol. Cell. Biol. 8:3988-3996, 1988; Samulski et al., J. Virol., 63:3822-3828, 1989; Shelling, A. N., and Smith, M. G., Gene Therapy, 1:165-169, 1994; Yoder et al., Blood, 82:suppl. 1:347A, 1994; Zhou et al., J. Exp. Med., 179:1867-1875, 1994; Hermonat, P. L. and Muzyczka, N., Proc. Natl. Acad. Sci. USA., 81:6466-6470, 1984; Tratschin et al., Mol. Cell. Biol., 4:2072-2081, 1984; McLaughlin et al., J. Virol., 62:1963-1973, 1988) as well as genes involved in human diseases (Flotte et al., Am. J. Respir. Cell Mol. Biol., 7:349-356, 1992; Luo et al., Blood, 82:suppl. 1,303A, 1994; Ohi et al., Gene, 89L:27914 282, 1990; Walsh et al., PNAS 89:7257-7261, 1992; Wei et al., Gene Therapy, 1:261-268, 1994).

[0350]In certain other embodiments, the gene of interest (e.g., osteocalcin) can be transferred into a target cell using a retroviral vector. Retroviruses refer to viruses that belong to the Retroviridae family, and include oncoviruses, foamy viruses (Russell, D. W. and Miller, A. D., J. Virol. 1996, 70:217-222; Wu, M. et al., J. Virol. 1999, 73:4498-4501), and lentiviruses (for example, HIV-1 (Naldini, L. et al., Science 1996, 272:263-267; Poeschla, E. et al., Proc. Natl. Acad. Sci. USA 1996, 93:11395-11399; Srinivasakumar, N. et al., J. Virol. 1997, 71:5841-5848; Zufferey, R., et al. Nat. Biotechnol. 1997, 15:871-875; Kim, V. N., et al., J. Virol. 1998,72:811-816) and feline immunodeficiency virus (Johnston, J. C. et al., J. Virol. 1999, 73:4991-5000; Johnston, J. and Power, C., J. Virol. 1999, 73:2491-2498; Poeschla, E. M. et al., Nat. Med. 1998, 4:354-357)). Numerous gene therapy methods that take advantage of retroviral vectors for treating a wide variety of diseases are well-known in the art (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764; Friedmann, 1989, Science, 244:1275-1281; Mulligan, 1993, Science, 260:926-932, R. Crystal, 1995, Science 270:404-410, and U.S. Pat. No. 6,899,871, Kasahara, et al. each of which are incorporated herein by reference in their entirety). An increasing number of these methods are currently being applied in human clinical trials (Morgan, R., 1993, BioPharm, 6(1):32-35; see also The Development of Human Gene Therapy, Theodore Friedmann, Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. ISBN 0-87969-528-5, which is incorporated herein by reference in its entirety).

[0351]Efficacy of the methods of treatment described herein can be monitored by determining whether the methods ameliorate any of the symptoms of the disease being treated. Alternatively, one can monitor the level of serum undercarboxylated/uncarboxylated osteocalcin (either in absolute terms or as a ratio of undercarboxylated/uncarboxylated osteocalcin/total osteocalcin), and/or serum adiponectin, and/or serum insulin, which levels should increase in response to therapy. Alternatively efficacy can be measured by monitoring glycemia in the subject being treated.

Diagnostics

[0352]The present invention provides methods and compositions for diagnosing disorders such as those associated with decreased levels of undercarboxylated/uncarboxylated osteocalcin. Such disorders include, but are not limited to, metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity.

[0353]In a specific embodiment of the invention, a method is provided for diagnosing a patient at risk of developing glucose intolerance or diabetes comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient and a control level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from a subject that does not have glucose intolerance or diabetes, (ii) comparing the patient and control levels, and (iii) concluding that the patient is at risk of developing glucose intolerance or diabetes if the patient level is lower than the test level. In an embodiment of the invention, the diabetes is Type 1 or Type 2.

[0354]"Biological samples" include solid and body fluid samples. The biological samples of the present invention may include tissue, organs, cells, protein or membrane extracts of cells, blood or biological fluids such as blood, serum, ascites fluid or brain fluid (e.g., cerebrospinal fluid).

[0355]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing glucose intolerance or diabetes comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient; and (ii) comparing the patient level to a standard level; where, if the patient level is lower than the standard level, the patient is at risk of developing diabetes. In instances where the method is practiced on humans, the standard level can be a level of undercarboxylated/uncarboxylated osteocalcin that has been previously determined to be the normal range for people who are not at risk of developing diabetes. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample, or a tissue sample. In an embodiment of the invention, the diabetes is Type 1 or Type 2.

[0356]A "standard level" of undercarboxylated/uncarboxylated osteocalcin in humans can include values of 0.1 ng/ml to 10 ng/ml, preferably 0.2 ng/ml to 7.5 ng/ml, more preferably 0.5 ng/ml to 5 ng/ml, and even more preferably 1 ng/ml to 5 ng/ml. A standard level of undercarboxylated/uncarboxylated osteocalcin in humans can also include about 0.1 ng/ml, about 0.5 ng/ml, about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, or about 10 ng/ml.

[0357]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing glucose intolerance or diabetes comprising: (i) determining the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in a biological sample taken from the patient; and (ii) comparing the ratio to a standard ratio; where, if the patient ratio is lower than the standard ratio, the patient is at risk of developing glucose intolerance or diabetes. In certain embodiments, the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35%. In certain embodiments, the standard ratio is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. Preferably, the patient is a human. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample, or a tissue sample. In an embodiment of the invention, the diabetes is Type 1 or Type 2.

[0358]The present invention further provides a method for diagnosing a patient at risk of developing atherosclerosis comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient and a control level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from a subject that does not have atherosclerosis, (ii) comparing the patient and control levels, and (iii) concluding that the patient is at risk of developing atherosclerosis if the patient level is lower than the test level.

[0359]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing atherosclerosis comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient; and (ii) comparing the patient level to a standard level; where, if the patient level is lower than the standard level, the patient is at risk of developing atherosclerosis. In instances where the method is practiced on humans, the standard level can be a level of undercarboxylated/uncarboxylated osteocalcin that has been previously determined to be the normal range for people who are not at risk of developing atherosclerosis. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample, or a tissue sample. Preferably, the patient is a human.

[0360]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing atherosclerosis comprising: (i) determining the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in a biological sample taken from the patient; and (ii) comparing the ratio to a standard ratio; where, if the patient ratio is lower than the standard ratio, the patient is at risk of developing atherosclerosis. In certain embodiments, the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35%. In certain embodiments, the standard ratio is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. Preferably, the patient is a human. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or a tissue sample.

[0361]In yet another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing metabolic syndrome comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient and a control level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from a subject that does not have metabolic syndrome, (ii) comparing the patient and control levels, and (iii) concluding that the patient is at risk of developing metabolic syndrome if the patient level is lower than the test level.

[0362]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing metabolic syndrome comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient; and (ii) comparing the patient level to a standard level; where, if the patient level is lower than the standard level, the patient is at risk of developing metabolic syndrome. In instances where the method is practiced on humans, the standard level can be a level of undercarboxylated/uncarboxylated osteocalcin that has been previously determined to be the normal range for people who are not at risk of developing metabolic syndrome. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or a tissue sample. Preferably, the patient is a human.

[0363]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing metabolic syndrome comprising: (i) determining the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in a biological sample taken from the patient; and (ii) comparing the ratio to a standard ratio; where, if the patient ratio is lower than the standard ratio, the patient is at risk of developing metabolic syndrome. In certain embodiments, the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35%. In certain embodiments, the standard ratio is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. Preferably, the patient is a human. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or a tissue sample.

[0364]In another aspect of the invention a method for diagnosing a patient at risk of developing obesity is provided comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient and a control level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from a subject that does not have obesity, (ii) comparing the patient and control levels, and (iii) concluding that the patient is at risk of developing obesity if the patient level is lower than the test level.

[0365]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing obesity comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient; and (ii) comparing the patient level to a standard level; where, if the patient level is lower than the standard level, the patient is at risk of developing obesity. In instances where the method is practiced on humans, the standard level can be a level of undercarboxylated/uncarboxylated osteocalcin that has been previously determined to be the normal range for people who are not at risk of developing obesity. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or a tissue sample. Preferably, the patient is a human.

[0366]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing obesity comprising: (i) determining the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in a biological sample taken from the patient; and (ii) comparing the ratio to a standard ratio; where, if the patient ratio is lower than the standard ratio, the patient is at risk of developing obesity. In certain embodiments, the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35%. In certain embodiments, the standard ratio is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. Preferably, the patient is a human. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or a tissue sample.

[0367]In yet another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing a disease selected from the group consisting of glucose intolerance, impaired pancreatic beta-cell proliferation, impaired insulin secretion, and impaired insulin sensitivity comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient and a control level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from a subject that does not have the disease, (ii) comparing the patient and control levels, and (iii) concluding that the patient is at risk of developing the disease if the patient level is lower than the test level.

[0368]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing a disease selected from the group consisting of glucose intolerance, impaired pancreatic beta-cell proliferation, impaired insulin secretion, and impaired insulin sensitivity comprising: (i) determining a patient level of undercarboxylated/uncarboxylated osteocalcin in a biological sample taken from the patient; and (ii) comparing the patient level to a standard level; where, if the patient level is lower than the standard level, the patient is at risk of developing a disease selected from the group consisting of glucose intolerance, impaired pancreatic beta-cell proliferation, impaired insulin secretion, and impaired insulin sensitivity. In instances where the method is practiced on humans, the standard level can be a level of undercarboxylated/uncarboxylated osteocalcin that has been previously determined to be the normal range for people who are not at risk of developing a disease selected from the group consisting of glucose intolerance, impaired pancreatic beta-cell proliferation, impaired insulin secretion, and impaired insulin sensitivity. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or a tissue sample. Preferably, the patient is a human.

[0369]In another embodiment of the invention, a method is provided for diagnosing a patient at risk of developing a disease selected from the group consisting of glucose intolerance, impaired pancreatic beta-cell proliferation, impaired insulin secretion, and impaired insulin sensitivity comprising: (i) determining the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in a biological sample taken from the patient; and (ii) comparing the ratio to a standard ratio; where, if the patient ratio is lower than the standard ratio, the patient is at risk of developing a disease selected from the group consisting of glucose intolerance, impaired pancreatic beta-cell proliferation, impaired insulin secretion, and impaired insulin sensitivity. In certain embodiments, the standard ratio is 5%-10%, 10%-15%, 15%-20%, 20%-25%, 25%-30%, or 30%-35%. In certain embodiments, the standard ratio is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35%. Preferably, the patient is a human. In preferred embodiments, the biological sample is blood, serum, plasma, cerebrospinal fluid, urine, a cell sample or a tissue sample.

[0370]In addition to assaying for levels of undercarboxylated/uncarboxylated osteocalcin, the present invention also provides methods and compositions for diagnosing disorders associated with decreased levels of adiponectin. Such disorders include, but are not limited to, metabolic syndrome, glucose intolerance, diabetes types 1 and 2, atherosclerosis and obesity. In a specific embodiment of the invention, a method is provided for diagnosing a patient at risk of developing diabetes comprising: (i) determining a patient level of adiponectin in a biological sample taken from the patient and a control level of adiponectin in a biological sample taken from a subject that does not have diabetes, (ii) comparing the patient and control levels, and (iii) concluding that the patient is at risk of developing diabetes if the patient level is lower than the test level.

[0371]In certain embodiments, the serum level of adiponectin and insulin are both measured and, if the serum levels of adiponectin and insulin in the patient are both lower than the levels in a subject without the disease, then the patient is at risk of developing the disease. In another embodiment, serum adiponectin and the glycemic index for the patient are measured and, if the serum level of adiponectin in the patient is lower than the level in a subject without the disease and the patient also has high glycemia, then the patient is at risk of developing the disease. Alternatively, serum adiponectin and uncarboxylated osteocalcin can be measured, or serum adiponectin, uncarboxylated osteocalcin and insulin can be measured and compared to controls for diagnosis of metabolic syndrome, its components, or type 1 diabetes.

[0372]In practicing the diagnostic methods of the invention, as set forth above, the biological sample is selected from the group consisting of blood, serum, plasma, cerebral spinal fluid, a cell sample or a tissue sample. In another embodiment, the sample is derived from a human.

[0373]Assays for detecting the levels of protein expression are well known to those of skill in the art. Such assays include, for example, antibody-based immunoassays. Methods for using antibodies as disclosed herein are particularly applicable to the cells, tissues and disorders that differentially express osteocalcin, OST-PTP, or gamma-carboxylase or that are involved in conditions as otherwise discussed herein. The methods use antibodies that selectively bind to the protein of interest and its fragments or variants. For therapeutic applications, antibodies that recognize OST-PTP and reduce its ability to bind to or dephosphorylate gamma-carboxylase are preferred. For diagnostic use, antibodies against undercarboxylated/uncarboxylated osteocalcin, gamma-carboxylase, adiponectin and vitamin K are preferred. An antibody is considered to selectively bind even if it also binds to other proteins that are not substantially homologous with the protein of interest. These other proteins share homology with a fragment or domain of the protein of interest. This conservation in specific regions gives rise to antibodies that bind to both proteins by virtue of the homologous sequence. In this case, it would be understood that antibody binding to the protein of interest is still selective. In certain embodiments, however, the antibodies do not substantially bind to proteins other than the protein of interest.

[0374]The amount of an antigen (e.g., osteocalcin or other protein of interest) in a biological sample may be determined by an assay such as a radioimmunoassay, an immunoradiometric assay, and/or an enzyme immunoassay. A "radioimmunoassay" is a technique for detecting and measuring the concentration of an antigen using a labeled (e.g., radioactively labeled) form of the antigen. Examples of radioactive labels for antigens include ³H, ¹4C, and ¹²⁵I. The concentration of antigen (e.g., osteocalcin) in a sample (e.g., biological sample) is measured by having the antigen in the sample compete with a labeled (e.g., radioactively) antigen for binding to an antibody to the antigen. To ensure competitive binding between the labeled antigen and the unlabeled antigen, the labeled antigen is present in a concentration sufficient to saturate the binding sites of the antibody. The higher the concentration of antigen in the sample, the lower the concentration of labeled antigen that will bind to the antibody.

[0375]In a radioimmunoassay, to determine the concentration of labeled antigen bound to antibody, the antigen-antibody complex must be separated from the free antigen. One method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum. Another method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with formalin-killed S. aureus. Yet another method for separating the antigen-antibody complex from the free antigen is by performing a "solid-phase radioimmunoassay" where the antibbdy is linked (e.g., covalently) to Sepharose beads, polystyrene wells, polyvinylchloride wells, or microtiter wells. By comparing the concentration of labeled antigen bound to antibody to a standard curve based on samples having a known concentration of antigen, the concentration of antigen in the biological sample can be determined.

[0376]An "Immunoradiometric Assay" (IRMA) is an immunoassay in which the antibody reagent is radioactively labeled. An IRMA requires the production of a multivalent antigen conjugate, by techniques such as conjugation to a protein, e.g., rabbit serum albumin (RSA). The multivalent antigen conjugate must have at least 2 antigen residues per molecule and the antigen residues must be of sufficient distance apart to allow binding by at least two antibodies to the antigen. For example, in an IRMA the multivalent antigen conjugate can be attached to a solid surface such as a plastic sphere. Unlabeled "sample" antigen and antibody to antigen which is radioactively labeled are added to a test tube containing the multivalent antigen conjugate coated sphere. The antigen in the sample competes with the multivalent antigen conjugate for antigen antibody binding sites. After an appropriate incubation period, the unbound reactants are removed by washing and the amount of radioactivity on the solid phase is determined. The amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample.

[0377]The most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent Assay (ELISA)." The "Enzyme-Linked Immunosorbent Assay (ELISA)" is a technique for detecting and measuring the concentration of an antigen using a labeled (e.g., enzyme linked) form of the antibody. In a "sandwich ELISA", an antibody (e.g., to osteocalcin) is linked to a solid phase (e.g., a microtiter plate) and exposed to a biological sample containing antigen (e.g., osteocalcin). The solid phase is then washed to remove unbound antigen. A labeled (e.g., enzyme linked) antibody is then bound to the bound-antigen (if present) forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to the antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease, and β-galactosidase. The enzyme linked antibody reacts with a substrate to generate a colored reaction product that can be assayed.

[0378]In a "competitive ELISA," antibody is incubated with a sample containing antigen (e.g., osteocalcin). The antigen-antibody mixture is then contacted with an antigen-coated solid phase (e.g., a microtiter plate). The more antigen present in the sample, the less free antibody that will be available to bind to the solid phase. A labeled (e.g., enzyme linked) secondary antibody is then added to the solid phase to determine the amount of primary antibody bound to the solid phase.

[0379]In an "immunohistochemistry assay," a section of tissue is tested for specific proteins by exposing the tissue to antibodies that are specific for the protein that is being assayed. The antibodies are then visualized by any of a number of methods to determine the presence and amount of the protein present. Examples of methods used to visualize antibodies are, for example, through enzymes linked to the antibodies (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or β-galactosidase), or chemical methods (e.g., DAB/Substrate chromagen).

[0380]In addition to detecting levels of protein expression, the diagnostic assays of the invention may employ methods designed to detect the level of RNA expression. Levels of RNA expression may be determined using methods well known to those of skill in the art, including, for example, the use of northern blots, RT-PCR or in situ hybridizations.

[0381]Carboxylation of osteocalcin confers a greater affinity for hydroxyapatite. Typically, total osteocalcin is measured by immunoassay followed by incubation with hydroxyapatite and centrifugation. The supernatant, which contains osteocalcin that has not adsorbed to hyudroxyapatite is then measured using the same immunoassay. The results of this procedure can be expressed either as absolute concentrations or as a ratio of undercarbocylated to carboxylated osteocalcin.

[0382]Another procedure uses monoclonal antibodies that distinguish the carboxylation state of all or some of the Glu/Gla residues of osteocalcin. For example, GluOC4-5 (TaKaRa catalog no. M171) reacts with human osteocalcin with glutamic acid residues (decarboxylated) at positions 21 and 24, and does not react with react with Gla-type osteocalcin.

[0383]For a review of osteocalcin measurement methods, see Lee et al., 2000, Ann.

[0384]Clin. Biochem. 37, 432-446.

Drug Screening and Assays

[0385]Cell-based and non-cell based methods of drug screening are provided to identify candidate agents that reduce OST-PTP or gamma-carboxylase activity or expression, or increase the level of undercarboxylated/uncarboxylated osteocalcin activity or expression. Such agents find use in treating or preventing a disorder related to energy metabolism and the OST-PTP signaling pathway. Such disorders include, metabolic syndrome, glucose intolerance, type 1 or type 2 diabetes, atherosclerosis, or obesity. Such agent may be used to treat disorders characterized by decreased insulin production, deceased insulin sensitivity, and decreased glucose tolerance or increased fat mass. Such assays may also be used to assay for the effectiveness of an agent in treating or preventing a disorder related to the OST-PTP pathway.

[0386]A non-cell based screening method is provided to identify compounds that bind to OST-PTP, gamma-carboxylase or osteocalcin and thereby modulate the activity of said proteins.

[0387]A screening method is provided to identify, or assay for, an agent that binds to OST-PTP, the method comprising the steps of: (i) providing a mixture comprising OST-PTP or a fragment or variant thereof, (ii) contacting the mixture with an agent, (iii) determining whether the agent binds to the OST-PTP, and (iv) identifying the agent if it binds to the OST-PTP or a fragment or variant thereof. The method may further comprise the step of determining whether the agent reduces the ability of OST-PTP to dephosphorylate gamma-carboxylase.

[0388]A screening method is provided to identify, or assay for, an agent that binds to the phosphatase 1 domain of OST-PTP, the method comprising the steps of (i) providing a mixture comprising the phosphatase 1 domain of OST-PTP or a fragment or variant thereof, (ii) contacting the mixture with an agent, (iii) determining whether the agent binds to the phosphatase 1 domain of OST-PTP, and (iv) identifying the agent if it binds to the phosphatase 1 domain of OST-PTP or a fragment or variant thereof. The method may further comprise the step of determining whether the agent reduces the ability of OST-PTP to dephosphorylate gamma-carboxylase.

[0389]A screening method is provided to identify, or assay for, an agent that binds to gamma-carboxylase, the method comprising the steps of: (i) providing a mixture comprising the gamma-carboxylase or a fragment or variant thereof, (ii) contacting the mixture with an agent, (iii) determining whether the agent binds to the gamma-carboxylase, and (iv) identifying the agent if it binds to the gamma-carboxylase or a fragment or variant thereof The method may further comprise the step of determining whether the agent reduces gamma-carboxylase activity.

[0390]A screening method is provided to identify, or assay for, an agent that binds to osteocalcin, the method comprising, the steps of: (i) providing a mixture comprising osteocalcin or a fragment or variant thereof, (ii) contacting the mixture with an agent, (iii) determining whether the agent binds to the osteocalcin, and (iv) identifying the agent if it binds to the osteocalcin or a fragment or variant thereof. The method may further comprise the step of determining whether the agent increases osteocalcin activity.

[0391]The binding of the agent may be determined through the use of competitive binding assays. The competitor is a binding moiety known to bind to the target molecule (i.e. one of the various proteins), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the agent and the binding moiety, with the binding moiety displacing the agent.

[0392]The agent may be labeled. Either the agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 degrees Centigrade. and 40 degrees Centigrade. Incubation periods are selected for optimum activity, but may also optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

[0393]Using such assays, the competitor may be added first, followed by the agent. Displacement of the competitor is an indication that the agent is binding to one of the various proteins and thus is capable of binding to, and potentially modulating, its activity. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the agent is labeled, the presence of the label on the support indicates displacement.

[0394]In another example, the agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the agent is bound to one of the various proteins with a higher affinity. Thus, if the agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the agent is capable of binding to one of the various proteins.

[0395]The method may comprise differential screening to identify agents that are capable of modulating the activity of one of the various proteins. In such an instance, the methods comprise combining a protein and a competitor in a first sample. A second sample comprises an agent, a protein and a competitor. Addition of the agent is performed under conditions which allow the modulation of one of the various proteins. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to one of the various proteins and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to one of the various proteins.

[0396]Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.

[0397]A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.

[0398]Non-cell based screening for agents that modulate the activity of one of the various proteins may also be done. Methods for screening for an agent capable of modulating the activity of one of the various proteins comprise the steps of adding an agent to a sample of one of the various proteins, as above, and determining an alteration in the biological activity of one of the various proteins. "Modulating the activity of one of the various proteins" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, the agent should both bind to the protein (although this may not be necessary), and alter its biological or biochemical activity as defined herein.

[0399]Thus, in one example, the methods comprise combining a protein sample and an agent, and evaluating the effect on OST-PTP, gamma-carboxylase or osteocalcin. By enzyme activity, specifically OST-PTP or gamma-carboxylase activity or grammatical equivalents herein is meant one or more of the biological activities associated with the enzyme. For OST-PTP this activity is preferably the dephosphorylation of gamma-carboxylase or of the insulin receptor; for gamma-carboxylase it is the carboxylation of osteocalcin. The screening assay are designed to find agents that reduce OST-PTP or gamma-carboxylase activity, or increase levels of undercarboxylated/uncarboxylated osteocalcin and adiponectin in a biological sample taken from the transformed animal or cell.

[0400]Specifically, a screening method is provided to identify, or assay for, an agent that reduces OST-PTP activity, the method comprising the steps of: (a) providing a control and a test mixture comprising OST-PTP or a fragment or variant thereof, (b) contacting the mixture with an agent, (c) determining the level of activity of OST-PTP in the test mixture and in the control, and(d) selecting the bioactive agent if the level of OST-PTP activity in the test mixture is lower than the level in the control.

[0401]A screening method is provided to identify, or assay for, an agent that reduces gamma-carboxylase activity, the method comprising the steps of: (a) providing a control and a test mixture comprising or a fragment or variant thereof, (b) adding to the test mixture a bioactive agent under conditions that permit the bioactive agent to bind to the gamma-carboxylase or a fragment or variant thereof, (c) determining the level of activity of gamma-carboxylase in the test mixture and in the control, and (d) selecting the bioactive agent if the level of gamma-carboxylase activity in the test mixture is lower than the level in the control.

[0402]Cell-based screening methods are provided for identifying, or assaying for, agents which decrease the level of expression of the Esp gene encoding OST-PTP or the gene encoding gamma-carboxylase. Alternatively, the drug screening assays may be used to identify, or assay for, agents which increase the level of osteocalcin gene expression.

[0403]The present invention also provides a screening method to identify an agent that decarboxylates osteocalcin, the method comprising the steps of: (a) providing a control and a test mixture comprising carboxylated osteocalcin, (b) adding to the test mixture an agent, (c) determining the level of carboxylated osteocalcin in the test mixture and in the control, and (d) selecting an agent if the level of carboxylated osteocalcin in the test mixture is lower than the activity in the control.

[0404]A cell-based method is provided for identifying, or assaying for, an agent that reduces OST-PTP gene expression, the method comprising steps: (a) determining a first expression level of OST-PTP in a cell, (b) determining a second expression level of OST-PTP after contact with a test agent; and (c) comparing the first expression level with the second expression level, wherein an agent that is capable of reducing OST-PTP expression is identified if the level of expression in the first is higher than the second expression level. The level of OST-PTP gene expression may be determined by measuring the amount of the OST-PTP mRNA made or the amount of the OST-PTP protein made.

[0405]A cell-based method is provided for identifying, or assaying for, an agent that reduces gamma-carboxylase gene expression, the method comprising steps: (a) determining a first expression level of gamma-carboxylase in a cell, (b) determining a second expression level of gamma-carboxylase after contact with a test agent; and (c) comparing the first expression level with the second expression level, wherein an agent that is capable of reducing gamma-carboxylase expression is identified if the level of expression in the first is higher than the second expression level. The level of gamma-carboxylase gene expression may be determined by measuring the amount of the gamma-carboxylase mRNA made or the amount of gamma-carboxylase protein made.

[0406]A cell-based method is provided for identifying, or assaying for, an agent that increases osteocalcin gene expression, the method comprising steps: (a) determining a first expression level of osteocalcin in a cell, (b) determining a second expression level of osteocalcin expression after contact with a test agent; and (c) comparing the first expression level with the second expression level, wherein an agent that is capable of increasing osteocalcin expression is identified if the level of expression in the first is lower than the second expression level. The level osteocalcin gene expression may be determined by measuring the amount of the osteocalcin mRNA made or the amount of osteocalcin protein made.

[0407]A reporter gene may be utilized to screen for agent capable of modulating gene expression. In such assays, cells are generated that contain a gene construct wherein expression of a reporter gene is placed under the control of native gene expression regulatory elements of the native gene of interest, i.e., the OST-PTP, gamma-carboxylase or osteocalcin gene. Reporter genes include, but are not limited to CAT, LacZ, lucierase or GFP.

[0408]A cell-based method is provided for screening, or assaying, for an agent that reduces OST-PTP gene expression, the method comprising steps: (a) determining a first expression level of a reporter gene in a cell wherein expression of the reporter gene is under the control of native OST-PTP gene expression regulatory elements, (b) determining a second expression level of reporter gene exprssion after contact with a test agent; and (c) comparing the first expression level with the second expression level, wherein an agent that is capable of reducing reporter gene expression is identified if the level of expression in the first is higher than the second expression level.

[0409]A cell-based method is provided for screening, or assaying, for an agent that reduces gamma-carboxylase gene expression, the method comprising steps: (a) determining a first expression level of a reporter gene in a cell wherein expression of the reporter gene is under the control of native gamma-carboxylase gene expression regulatory elements, (b) determining a second expression level of reporter gene expression after contact with a test agent; and (c) comparing the first expression level with the second expression level, wherein an agent that is capable of reducing gamma-carboxylase gene expression is identified if the level of expression in the first is higher than the second expression level.

[0410]A cell-based method is provided for screening, or assaying, for an agent that increases osteocalcin gene expression, the method comprising steps: (a) determining a first expression level of a reporter gene in a cell wherein expression of the reporter gene is under the control of native osteocalcin gene expression regulatory elements, (b) determining a second expression level of reporter gene expression after contact with a test agent; and (c) comparing the first expression level with the second expression level, wherein an agent that is capable of increasing osteocalcin gene expression is identified if the level of expression in the first is lower than the second expression level.

[0411]Cell-based screening assays are provided for identifying agents that reduce OST-PTP or gamma-carboxylase activity.

[0412]Specifically, a cell-based method for screening for an agent that reduces OST-PTP activity is provided, the method comprising steps: (a) determining a first activity level in a first cell that expresses the phosphatase 1 domain of OST-PTP, (b) contacting a second cell that expresses the phosphatase 1 domain of OST-PTP with an agent, (c) determining a second activity level in the second cell that expresses the phosphatase 1 domain of OST-PTP; and (d) comparing the first activity level with the second activity level, wherein the agent reduces OST-PTP activity if the first activity level is higher than the second activity level. The level of OST-PTP activity may be determined by measuring the level of gamma-carboxylase activity. The level of OST-PTP activity may be determined by measuring the level of osteocalcin carboxylation.

[0413]A cell-based method for screening, or assaying, for an agent that reduces gamma-carboxylase activity is provided, the method comprising the steps: (a) determining a first activity level in a first cell that expresses gamma-carboxylase, (b) contacting a second cell that expresses gamma-carboxylase with an agent (c) determining a second activity level in the second cell that expresses gamma-carboxylase; and (d) comparing the first activity level with the second activity level, wherein the agent reduces gamma-carboxylase activity if the first activity level is higher than the second activity level. Assays for measuring gamma carboxylase activity are know to those of skill in the art (See, for example, Hubbard et al., (1989) Proc. Natl. Acad. Sci. USA 86:6893-6897; Rehemtulla et al., (1993) Proc. Natl. acad. Sci USA 90:4611-4615).

[0414]Gamma carboxylase catalyzes the posttranslational modification of specific glutamic acid residues within osteocalcin to form γ-carboxyglutamic acid residues. In an embodiment of the invention, the level of gamma carboxylase activity or decarboxylase activity is determined by measuring the level of osteocalcin carboxylation.

[0415]A cell based method is provided for screening for an agent that decarboxylates osteocalcin, the method comprising the steps of: (a) determining a first level of carboxylated osteocalcin in a first cell that expresses osteocalcin, (b) contacting a second cell that expresses carboxylated osteocalcin with an agent, (c) determining a second level of carboxylated, osteocalcin, and (d) comparing the first level of carboxylated osteocalcin with the second level of carboxylated osteocalcin, wherein the agent decarboxylates osteocalcin if the first level of carboxylated osteocalcin is higher than the second level.

[0416]Cells to be used in the screening, or assaying, methods include cells that naturally express OST-PTP, gamma-carboxylase, or osteocalcin, cells that have been genetically engineered to express (or overexpress) OST-PTP, the phosphatase 1 domain of OST-PTP, gamma-carboxylase, or osteocalcin, as well as cells derived from the transgenic animals of the present invention. Such cells include transformed osteoblast that overexpresses OST-PTP or gamma-carboxylase.

[0417]A method is provided for testing an agent's effectiveness in increasing adiponectin expression in adipocytes, comprising: (a) co-culturing osteoblasts and adipocytes, (b) contacting the osteoblasts with a candidate agent,(c) determining whether the candidate agent increases the level of expression or secretion of adiponectin or a fragment or variant thereof above a control level measured in a control co-culture in which osteoblasts are not contacted with the candidate agent, and (d) if the candidate agent increases the level of adiponectin expression or secretion above the control level, then selecting the candidate agent as an agent that increases adiponectin expression or secretion in adipocytes.

[0418]A method is provided for testing an agent's effectiveness in increasing insulin expression or secretion in pancreatic beta cells, comprising: (a) co-culturing the osteoblasts and pancreatic beta cells, (b) contacting the osteoblasts with a candidate agent, (c) determining whether the candidate agent increases the level of insulin expression or secretion above a control level of insulin expression measured in a control co-culture in which osteoblasts are not contacted with the candidate agent, and (d) if the candidate agent increases the level of insulin expression or secretion above the control level, then selecting the candidate agent as an agent that increases insulin expression or secretion in pancreatic beta cells.

[0419]A method is provided for determining the ability of a candidate agent to treat or prevent in an animal metabolic syndrome or a phenotype associated with metabolic syndrome is provided that is selected from the group comprising predisposition to type 1 or 2 diabetes, glucose intolerance, decreased insulin production, decreased insulin sensitivity, decreased glucose tolerance, atherosclerosis and increased fat mass, comprising: (a) providing a test animal and a control animal, (b) administering the candidate agent to the test animal, (c) comparing the level of undercarboxylated/uncarboxylated osteocalcin in the test animal to the level of undercarboxylated/uncarboxylated osteocalcin in the control animal, and (d) selecting the candidate agent if the level of undercarboxylated/uncarboxylated osteocalcin is higher in the test animal than in the control animal. In a specific embodiment of the invention the level of undercarboxylated/uncarboxylated osteocalcin is measured in osteoblasts.

[0420]In one example, the level of undercarboxylated/uncarboxylated osteocalcin is measured in osteoblasts. The candidate agent may be bound to a phosphate group that facilitates its uptake by osteoblasts.

[0421]A method is provided for screening a candidate agent for the ability to treat or prevent metabolic syndrome in an animal or a phenotype associated with metabolic syndrome including predisposition to type 2 diabetes, glucose intolerance, decreased insulin production, decreased insulin sensitivity, decreased glucose tolerance, atherosclerosis and increased fat mass, comprising: (a) providing a first and a second animal, (b) administering to said first animal a candidate agent, and (c) comparing the level of OST-PTP expression or activity in the first animal of step (b) that was given the candidate agent to the level of OST-PTP in the second animal of step (a) that was not administered said candidate agent; wherein a candidate agent that reduces the level of OST-PTP expression or activity is selected as an agent that has effectiveness in treating metabolic syndrome or a phenotype associated therewith.

[0422]The level of OST-PTP expression or activity may be measured in osteoblasts. Further, the candidate agent may be bound to a phosphate group that facilitates its uptake by osteoblasts.

[0423]A method is provided for screening a candidate agent for the ability to treat or prevent metabolic syndrome in an animal, or a phenotype associated with metabolic syndrome including predisposition to type 1 and 2 diabetes, glucose intolerance, decreased insulin production, decreased insulin sensitivity, atherosclerosis, decreased glucose tolerance and increased fat mass, comprising: (a) providing a first and a second animal, (b) administering to said first animal a candidate agent, and (c) comparing the level of expression or activity or secretion of osteocalcin in the first animal of step (b) that was given the candidate agent to the level of expression or activity of osteocalcin in the second animal of step (a) that was not administered said candidate agent; wherein a candidate agent that in increases expression or activity or secretion of osteocalcin or a fragment or variant thereof is selected as an agent that has effectiveness in treating metabolic syndrome or a phenotype associated therewith.

[0424]A method is provided for screening a candidate agent for the ability to treat or prevent metabolic syndrome in an animal or a phenotype associated with metabolic syndrome including predisposition to type 1 or 2 diabetes, glucose intolerance, decreased insulin production, decreased insulin sensitivity, atherosclerosis, decreased glucose tolerance and increased fat mass, comprising: (a) providing a first and a second animal, (b) administering to said first animal a candidate agent, and (c) comparing the level of expression or secretion of adiponectin or a fragment or variant thereof in the first animal of step (b) that was given the candidate agent to the level of adiponectin expression or secretion in the second animal of step (a) that was not administered said candidate agent; wherein a candidate agent that increases the level of expression or secretion of adiponectin or a fragment or variant thereof is selected as an agent that has effectiveness in treating metabolic syndrome or a phenotype associated therewith. In such a method, the level of adiponectin expression or secretion is measured in adipocytes or in serum.

[0425]A method is provided for screening a candidate agent for the ability to treat or prevent metabolic syndrome in a osteocalcin-deficient mouse, wherein the osteocalcin-deficient mouse exhibits a phenotype relative to a wild type mice, which phenotype is selected from the group comprising reduced osteocalcin expression, type 1 or 2 diabetes predisposition, decreased insulin secretion, atherosclerosis, decreased insulin sensitivity, decreased expression or secretion of adiponectin or a fragment or variant thereof, decreased glucose tolerance, and increased fat mass,comprising: (a) providing a first and a second osteocalcin-deficient mouse that are both from the same strain as the osteocalcin-deficient mouse; (b) administering to said first osteocalcin-deficient mouse a candidate agent, and (c) comparing the phenotype of the first osteocalcin-deficient mouse of step (b) that was given the candidate agent to the phenotype of said second osteocalcin-deficient mouse of step (a) that was not administered said candidate agent; wherein a candidate agent that reduces or ameliorates the phenotype is selected as an agent that has effectiveness in treating metabolic syndrome.

[0426]A method is also provided for screening a candidate agent for the ability to treat metabolic syndrome in an adiponectin-deficient mouse, wherein the adiponectin-deficient mouse exhibits a phenotype selected from the group comprising type 1 or 2 diabetes predisposition; decreased insulin secretion; decreased insulin sensitivity; atherosclerosis, decreased glucose tolerance, and increased fat mass, comprising: (a) providing a first and a second adiponectin-deficient mouse that are both from the same strain, (b) administering to said first adiponectin-deficient mouse a candidate agent, and c) comparing the phenotype of the first adiponectin-deficient mouse of step (b) that was given the candidate agent to the phenotype of said second adiponectin-deficient mouse of step (a) not administered said candidate agent; wherein a candidate agent that reduces or ameliorates the phenotype is selected as an agent that has effectiveness in treating metabolic syndrome.

[0427]A method is provided for screening for an agent suspected to reduce OST-PTP activity or expression in osteoblasts for use as a therapeutic agent for treating or preventing a disease that is a member of the group comprising metabolic syndrome, type I or II diabetes, decreased insulin secretion, decreased insulin sensitivity, decreased glucose tolerance, increased fat mass and atherosclerosis, comprising: a) obtaining a control transgenic mouse overexpressing OST-PTP selectively in osteoblasts, and a second transgenic mouse from the same strain as the control, b) subjecting the first mouse to a placebo and the second mouse to a therapeutic agent, c) assaying for the level of OST-PTP activity in a sample of osteoblasts from the first and second mice, d) comparing the level of OST-PTP activity assayed in the first mouse to that in the second mouse,and e) concluding that the agent is useful as a therapeutic compound to treat or prevent the disease if the level in the first mouse is higher than the level in the second mouse.

[0428]A method is provided for screening a agent suspected to reduce gamma-carboxylase activity or expression in osteoblasts for use as a therapeutic compound for treating or preventing a disease that is a member of the group comprising metabolic syndrome, type I or II diabetes, decreased insulin secretion, decreased insulin sensitivity, decreased glucose tolerance, increased fat mass and atherosclerosis,comprising: a) obtaining a control transgenic mouse overexpressing gamma-carboxylase selectively in osteoblasts, and a second transgenic mouse from the same strain as the control, b) subjecting the first mouse to a placebo and the second mouse to the therapeutic compound under the same conditions that allow the therapeutic compound to have an effect, c) assaying for the level of gamma-carboxylase activity in a sample of osteoblasts from the first and second mice, d) comparing the level of gamma-carboxylase activity assayed in the first mouse to that in the second mouse, e) concluding that bioactive agent is useful as a therapeutic compound for use in reducing gamma-carboxylase activity or expression in osteoblasts if the level in the first mouse is higher than the level in the second mouse. The bioactive agent may be an enzyme inhibitor.

[0429]A method is provided for screening a agent suspected of having a therapeutic use to treat or prevent a disease that is a member of the group comprising metabolic syndrome, type I or II diabetes, decreased insulin secretion, decreased insulin sensitivity, decreased glucose tolerance, increased fat mass and atherosclerosis, comprising: (a) providing an animal that has the disease, (b) determining the amount of undercarboxylated/uncarboxylated osteocalcin in a pretreatment biological sample taken from the animal, (c) administering the bioactive agent to the test animal under conditions that permit the agent to have an effect, (d) determining the amount of undercarboxylated/uncarboxylated osteocalcin in a post-treatment biological sample taken from the animal, and (e) if the bioactive agent increases the amount of undercarboxylated/uncarboxylated osteocalcin in the post-treatment biological sample compared to the pre-treatment sample, concluding that the agent has the therapeutic use.

[0430]A method is provided for screening a bioactive agent suspected of having a therapeutic use to treat or prevent a disease that is a member of the group comprising metabolic syndrome, type I or II diabetes, decreased insulin secretion, decreased insulin sensitivity, decreased glucose tolerance, increased fat mass and atherosclerosis, comprising: (a) providing an animal that has the disease, (b) determining the amount of adiponectin in a pretreatment biological sample taken from the animal, (c) administering the bioactive agent to the test animal under conditions that permit the agent to have an effect, (d) determining the amount of adiponectin in a post-treatment biological sample taken from the animal, and (e) if the bioactive agent increases the amount of adiponectin in the post-treatment biological sample compared to the pre-treatment sample, concluding that the agent has the therapeutic use.

[0431]The term "agent" or "exogeneous compound" as used herein includes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, lipid, etc., or mixtures thereof, with the capability of directly or indirectly altering the bioactivity of one of the various proteins (OST-PTP, gamma-carboxylase, osteocalcin,). Some of the agents can be used therapeutically. Generally a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

[0432]Agents for use in screening encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons, preferably less than about 500 daltons. Agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The agent often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.

[0433]Libraries of high-purity small organic ligands and peptide agonists that have well-documented pharmacological activities are available from numerous sources. One example is an NCI diversity set which contains 1,866 drug-like compounds (small, intermediate hydrophobicity). Another is an Institute of Chemistry and Cell Biology (ICCB; maintained by Harvard Medical School) set of known bioactives (467 compounds) which includes many extended, flexible compounds. Some other examples of the ICCB libraries are: Chem Bridge DiverSet E (16,320 compounds); Bionet 1 (4,800 compounds); CEREP (4,800 compounds); Maybridge 1 (8,800 compounds); Maybridge 2 (704 compounds); Maybridge HitFinder (14,379 compounds); Peakdale 1 (2,816 compounds); Peakdale 2 (352 compounds); ChemDiv Combilab and International (28,864 compounds); Mixed Commercial Plate 1 (352 compounds); Mixed Commercial Plate 2 (320 compounds); Mixed Commercial Plate 3 (251 compounds); Mixed Commercial Plate 4 (331 compounds); ChemBridge Microformat (50,000 compounds); Commercial Diversity Set1 (5,056 compounds). Other NCI Collections are: Structural Diversity Set, version 2 (1,900 compounds); Mechanistic Diversity Set (879 compounds); Open Collection 1 (90,000 compounds); Open Collection 2 (10,240 compounds); Known Bioactives Collections: NINDS Custom Collection (1,040 compounds); ICCB Bioactives 1 (489 compounds); SpecPlus Collection (960 compounds); ICCB Discretes Collections. The following ICCB compounds were collected individually from chemists at the ICCB, Harvard, and other collaborating institutions: ICCB1 (190 compounds); ICCB2 (352 compounds); ICCB3 (352 compounds); ICCB4 (352 compounds). Natural Product Extracts: NCI Marine Extracts (352 wells); Organic fractions--NCI Plant and Fungal Extracts (1,408 wells); Philippines Plant Extracts 1 (200 wells); ICCB-ICG Diversity Oriented Synthesis (DOS) Collections; DDS1 (DOS Diversity Set) (9600 wells). Compound libraries are also available from a commercial suppliers, such as ActiMol, Albany Molecular, Bachem, Sigma-Aldrich, TimTec, and others.

[0434]Known and novel pharmacological agents identified in screens may be further subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

[0435]When screeing, designing or modifying compounds, other factors to consider include the Lipinski rule-of-five (not more than 5 hydrogen bond donors (OH and NH groups); not more than 10 hydrogen bond acceptors (notably N and O); molecular weight under 500 g/mol; partition coefficient log P less than 5), and Veber criteria, which are recognized in the pharmaceutical art and relate to properties and structural features that make molecules more or less drug-like.

[0436]The agent may be a protein. By "protein" in this context is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bounds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acids" also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.

[0437]The agent may be a naturally occurring protein or fragment or variant of a naturally occurring protein. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way, libraries of prokaryotic and eukaryotic proteins may be made for screening against one of the various proteins. Libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred may be used.

[0438]Agents may be peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized agent bioactive proteinaceous agents.

[0439]The library may be fully randomized, with no sequence preferences or constants at any position. The library may be biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

[0440]The agent may be an isolated nucleic acid, preferably antisense, siRNA, or cDNA that binds to either the gene encoding the protein of interest, or its mRNA to block gene expression or mRNA translation, respectively. By "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together. Such nucleic acids will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49)10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), pohsphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference).

[0441]Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleoside 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in antisense Research", Ed. Y. S. Sanghui and P. Can Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxathine, isocytosine, isoguanine, etc.

[0442]As described above generally for proteins, nucleic acid agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes may be used as is outlined above for proteins.

[0443]The agents may be obtained from combinatorial chemical libraries, a wide variety of which are available in the literature. By "combinatorial chemical library" herein is meant a collection of diverse chemical compounds generated in a defined or random manner, generally by chemical synthesis. Millions of chemical compounds can be synthesized through combinatorial mixing.

[0444]The determination of the binding of the agent to one of the various proteins may be done in a number of ways. In a preferred embodiment, the agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of one of the various proteins to a solid support, adding a labeled agent (for example an agent comprising a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as is known in the art.

[0445]By "labeled" herein is meant that the agent is either directly or indirectly labeled with a label which provides a detectable signal, e.g. a radioisotope (such as ³H, ¹4C, ³²P, ³3P, ³⁵S, or ¹²⁵I), a fluorescent or chemiluminescent compound (such as fluorescein isothiocyanate, rhodamine, or luciferin), an enzyme (such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase), antibodies, particles such as magnetic particles, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal. Only one of the components may be labeled. Alternatively, more than one component may be labeled with different labels.

Sequence Listings

[0446]Full nucleic acid and amino acid sequence listings relevant to this application are listed below. Transgenic mice and isolated cells from them (especially osteoblasts and adipocytes) that over or under express any of the listed nucleic acids (cDNA for Esp, osteocalcin, adiponectin, gamma-carboxylase, apolipoprotein E) can be made using routine methods known in the art and described herein, including knock in and knock out mice. In certain instances, nucleic acids are inserted into the genome of the host organism operably connected to and under the control of a promoter and regulatory elements (endogenous or heterogeneous) that will cause the organism to over express the nucleic acid gene or mRNA. One example of an exogenous/heterogeneous promoter included in the transfecting vector carrying the gene to be amplified is alpha 1(I) collagen. Many such promoters are known in the art. Human osteoblasts can be transfected with vectors carrying the cDNA for human Esp or human osteocalcin (or fragments or variants thereof) operably linked to known promoters and regulatory elements that cause the transfected human osteoblast to overexpress osteocalcin (or fragments or variants thereof). Disclosed herein are transgenic mice and mouse cells, and transfected human cells over expressing osteocalcin (or fragments or variants thereof), OST-PTP or gamma-carboxylase. Also disclosed herein are double mutant mice that have deletions of one or both alleles for osteocalcin, Esp, gamma-carboxylase and adiponectin, and various combinations of double mutants. Also disclosed herein are vectors carrying the cDNA or mRNA encoding the proteins for insertion into the genome of a target animal or cell. Such vectors can optionally include promoters and regulatory elements operably linked to the cDNA or mRNA. By "operably linked" is meant that promoters and regulatory elements are connected to the cDNA or mRNA in such a way as to permit expression of the cDNA or mRNA under the control of the promoters and regulatory elements.

[0447]Antisense and small interfering RNAs for use in reducing expression of OST-PTP and gamma-carboxylase thereby treating or preventing metabolic syndrome or a component thereof in an animal or type 1 diabetes, can be made that specifically hybridize to the gene and mRNA encoding OST-PTP or gamma-carboxylase, respectively. The sequence for mouse (OST-PTP, Ptprv) cDNA is set forth in SEQ ID NO:18. The amino acid sequence for OST-PTP, Ptprv) protein is set forth in SEQ ID NO:19. This cDNA will hybridize with mRNA for OST-PTP and thereby interfere with its translation. Reducing OST-PTP expression will increase undercarboxylate/uncarboxylated osteocalcin. The cDNA for mouse gamma-carboxylase is identified by SEQ ID NO:12, and its amino acid sequence is SEQ ID NO:13. This cDNA will hybridize with mRNA for gamma-carboxylase and thereby interfere with its translation and is a preferred embodiment. The cDNA for human gamma-carboxylase is identified by SEQ ID NO:10, and the amino acid sequence is SEQ ID NO:11. Human gamma-carboxylase cDNA can be used therapeutically to reduce gamma-carboxylase expression to treat or prevent metabolic syndrome and its components and type 1 or type 2 diabetes.

Examples

[0448]The invention is illustrated herein by the experiments described above and by the following examples, which should not be construed as limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. Those skilled in the art will understand that this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will fully convey the invention to those skilled in the art. Many modifications and other embodiments of the invention will come to mind in one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing description. Although specific terms are employed, they are used as in the art unless otherwise indicated.

Materials and Methods

[0449]Esp-nLacZ mice refers to an Esp-deficient mouse model in which one (+/-) or both alleles (-/-) for OST-PTP have been inactivated in all of the cells in the animal. The nLacZ (or LacZ) mouse is made by homologous recombination of a targeted OST-PTP allele with a transgene having a sequence encoding a nuclear-localized LacZ cassette, which is homologously recombined into exon 6 of the OST-PTP allele, such that the transgene is in frame with the OST-PTP gene, and expression of the transgene is operably linked to the native gene expression regulatory sequences of the OST-PTP allele. Esp KI (Knock In)=Esp nLacZ (-/-) mice.

[0450]Esp-nLacZ mice were generated using a targeting vector designed to insert a nuclear-localized LacZ (nLacZ) cassette into exon 6 such that LacZ was in frame with OST-PTP sequence (Dacquin et al., 2004; Ducy et al., 1996). Genomic clones spanning the entire mouse Ptprv gene were isolated from a mouse genomic library (129ola strain) by using fragments of the mouse cDNA (Lee et al., [1996]). A targeting vector was constructed that contains an HPRT hypoxanthine guanine phosphoribosyl transferase minigene selection cassette, an internal ribosomal entry site (Mountford et al., 1994), and a reporter containing SV40 nuclear localization sequences fused to the LacZ gene (nLacZ). Into this were cloned 4.4 kb of homology from the 5' end of the gene and 1.9 kb of homology from the 3' end of the gene. Gene targeting was conducted by using standard techniques (Joyner, 1999) in E14Tg2A feeder-independent embryonic stem (ES) cells (Hooper et al., 1987). Targeted ES cells were selected in HAT (10 microM hypoxanthine, 9 microM aminopterin, 20 microM thymidine) selection medium as previously described (Thompson et al., 1989). Tissue culture medium was GMEM (Glasgow Modified Eagles Media; Gibco) supplemented with 10% fetal calf serum (FCS), 0.1 mM 2 mercaptoethanol, 1 mM sodium pyruvate, and approximately 10³ U/ml of leukemia inhibitory factor. A total of 5×10⁶ cells were electroporated in 800 microliters of phosphate buffered saline (PBS) with 20 micrograms of NotI linearized vector DNA at 800 V and 3 microFD by using a Gene Pulser (Bio-Rad) and plated onto gelatin coated 10-cm tissue culture plates. After 48 hr, the cells were transferred to HAT selection medium. Targeted ES cell clones were identified by Southern hybridization using radiolabeled cDNA fragments complementary to regions outside the homology of the targeting vector both 5' and 3' of the integration site and by using a LacZ probe to check for single copy integration. Targeted ES cells were injected in C57BL/6 blastocysts, which were subsequently transferred into foster mothers. Chimeric males were mated with MF1 strain females, and Southern blot analysis or polymerase chain reaction (PCR) of tail tip DNA from grey offspring was used to identify heterozygous animals. The mutation was crossed to the MF1 strain for five generations to provide heterozygous mice for subsequent analysis. This mutation resulted in deletion of most of OST-PTP extracellular domain, its transmembrane and intracellular domains (1). This type of mutant allele is referred to as the Esp nLacZ mutant allele or as the Esp KI (Knock In) mutant allele. In the Esp nLacZ mutant mice, one (+/-) or both alleles (-/-) for OST-PTP have been inactivated in all of the cells in the animal thereby interfering with OST-PTP expression.

[0451]"Esp osb mutant mice" are Esp-deficient mouse models in which one (+/-) or both alleles (-/-) for OST-PTP have been deleted or knocked out from osteoblasts only in the animal, thereby blocking synthesis of OST-PTP selectively in osteoblasts. This is not to be confused with the ob mutant which is lacking one or both alleles of leptin. An Esp osb mouse carries a disruption in one or both endogenous OST-PTP alleles in which exons 24 to 35 encoding the phosphatase domain of the OST-PTP allele have been deleted and replaced by a neomycin resistance gene floxed by loxP sites in one (+/-) or both alleles (-/-) for OST-PTP.

[0452]A targeting vector harboring LoxP sites within introns 23 and 35 as well as a foxed neomycin resistance cassette was electroporated into ES cells. Targeted ES cells were injected in 129Sv/EV blastocysts to generate chimeric mice harboring the floxed allele (Esp_flox). Esp_flox/+ mice were crossed with α1(I)collagen-Cre mice to generate Esp_ob-/+ mice and their progeny was intercrossed to obtain Esp_ob-/- mice. The mice harboring floxed Esp alleles can be crossed with transgenic mice expressing the recombinase under the control of any promoter of interest to specifically inactivate the Esp gene in the cells where this promoter is active. In the Esp_ob, one (+/-) or both alleles (-/-) for OST-PTP have been inactivated in osteoblasts only thereby interfering with OST-PTP expression only in these cells. Molecular analysis showed that recombination occurred at high frequency at the Esp locus in osteoblasts but not in any other tissues or cell types including testis, adipocytes or pancreas beta-cells (FIGS. 1C and 1D). Northern blot analysis verified that it was a null allele while Souther blot hybridization was used to demonstrate the efficiency of Esp excision in osteoblasts (FIG. 1C). Quantitative RT-PCR and Western analysis failed to detect Esp mRNA or OST-PTP protein, respectively, in bone of Esp_ob-/- mice while both Esp mRNA and OST-PTP protein were present in the testis of Esp_osb-/- mice (FIG. 1D). These data indicate that an osteoblast-specific inactivation of Esp was achieved.

[0453]As used herein, "Esp-deficient mice" means either of two strains of transgenic mice in which both alleles for osteotesticular protein tyrosine phosphatase OST-PTP (encoded by the Esp gene) have been deleted (knocked out) as in the Esp osb -/- mouse, or have been disrupted or (knocked in) as in the Esp-nLacz -/- mouse.

[0454]FIG. 22 shows certain details of the method for generating Esp_osb-/- mice and normal bone formation in Esp-/- animals. FIG. 23 compares 1 month-old WT and Esp-/- mice with respect to various metabolic and physiologic parameters: serum levels of C-peptide (A), serum Glucagon level (left) and glucagons content in pancreas (right) (B) and serum levels of IGF-1 (C), PYY (D), and amylin (E) in Esp-/- mice; (F) Ratio of muscle mass over body mass calculated by proton magnetic resonance spectroscopy (¹H-MRS) in 10 week-old WT and Esp-/- mice; (G) Representative images of proton ¹H-MRS of 10 week-old WT and Esp-/- mice; (H) Food intake per day in 1 month- and 3 month-old Esp-/- and WT mice; (I and J) Comparison of expression level by real time PCR (I) and of serum levels (J) of TNF-α (left) and IL-6 (right) in 1 month-old Esp-/- and WT mice; and (K) Serum leptin (left) and resistin (right) levels in 1 month-old Esp-/- and WT mice. In all panels data represent the means ±SD of experiments. *, P<0.01 (t-test).

[0455]FIG. 24 shows the anatomy of the destruction of VMH nuclei by GTG. FIG. 25 shows that there is no cell transdifferentiation during the co-culture assays. FIG. 26 shows that Osteocalcin expression is bone specific. In situ hybridization analysis of osteocalcin and Esp expression in pancreas of 18.5 dpc embryos show that neither gene is expressed in pancreas. Insulin expression was used as a positive control. Hematoxylin-eosin staining of adjacent sections was used to assess tissues integrity. Real time PCR analysis of osteocalcin expression in osteoblasts, adipocytes, and pancreatic islets collected from 1 month-old WT mice showed that osteocalcin is not expressed in adipocytes or islets.

[0456]Generation of Collagen alpha 1(I)-PTP and Collagen alpha 1(I)-PTP_ED transgenic mice. Transgenic mice over expressing either the full length Esp cDNA (alpha1(I) collagen--OST-PTP) or a truncated version of this cDNA encoding only the OST-PTP extracellular (also herein referred to as the soluble domain) domain (alpha 1(I) collagen--OST-PTP_EC mice) were generated. The extracellular domain is also herein referred to as the soluble domain (SD). These cDNA genes were under the control of the osteoblast-specific regulatory elements of the alpha 1(I) collagen to make mice that over express ESP (OST-PTP) or the OST-PTP extracellular domain in osteoblast in vivo.

[0457]At 1 month of age, the alpha 1(I) collagen--Esp transgenic mice displayed an increase in serum glucose both after fasting and after feeding, a decrease in insulin serum level after feeding, and a decrease in energy expenditure. Accordingly, glucose tolerance tests (GTT) showed that alpha1(I) collagen--Esp mice were glucose-intolerant while insulin tolerance tests (ITT) established that they were insulin-resistant (FIG. 4). Altogether the phenotype of the transgenic mice is the mirror image (the opposite) of the one observed in Esp-deficient mice. Moreover, this Esp cDNA full transcript transgene corrected all the metabolic abnormalities in Esp-deficient, diabetes-resistant mice. Transgenic mice over-expressing either the full-length Esp cDNA (alpha1(I) collagen--Esp) or a truncated version of this cDNA encoding only the OST-PTP extracellular domain are herein referred to as the soluble domain) domain (alpha 1(I) collagen--Esp_EC mice.

[0458]Generation of ApoE-PTP, ApoE-PTP_SD (also named ApoE-PTP_ED) transgenic mice. The full-length mouse Esp cDNA or fragment of the Esp mouse cDNA encoding amino acids 1 to 1111 of the extracellular domain (ED) was cloned into a vector directing liver-specific expression using the promoter of the ApoE gene. In contrast to expression of the full cDNA transcript for OST-PTP, Apolipoprotein E--OST-PTP_EC transgenic mice that express a truncated version of this cDNA encoding only OST-PTP extracellular domain were indistinguishable from wild type mice. These experiments further prove that OST-PTP regulates energy metabolism through its intracellular phosphatase domain.

[0459]Generation of Osteocalcin-deficient (also named Ocn-/- or Bgp-/-) mice. "Osteocalcin-deficient mice" means a strain of mice in which both osteocalcin alleles were deleted. In the osteocalcin deficient transgenic mice described herein, Exon 4 of osteocalcin gene 1 (OG1) coding for the mature protein, and the entire osteocalcin gene 2 (OG2) sequence were deleted, while osteocalcin-related gene (ORG) was left in place. Correct targeting resulted in the replacement of the entire mature osteocalcin protein-coding sequences by the pGKNeo selection cassette.

[0460]Generation of Osteocalcin-/- mice was previously reported (Ducy et al., 1996). Exon 4 of osteocalcin gene 1 (OG1) coding for the mature protein, and the entire osteocalcin gene 2 (OG2) sequence were deleted, while osteocalcin-related gene (ORG) was left in place. Correct targeting resulted in the replacement of the entire mature osteocalcin protein-coding sequences by the pGKNeo selection cassette. Analysis of these mice is reported FIGS. 5-7 and Table 1.

[0461]Generation of Adiponectin-deficient Mice and Ocn+/-; Adiponectin +/- mice. Adiponectin-deficient mice were generated according to a previously described strategy (Maeda et al., 2002) where Exons 2 and 3 of either one (+/-) or both (-/-) alleles of the adiponectin gene were deleted. Adiponectin +/- or -/- were then crossed with Ocn-/- or +/- mice to generate Adiponectin+/-;Ocn+/- mice. Analysis of these mice is reported FIG. 6.

[0462]Generation of SAP-Adiponectin transgenic Mice. Transgenic mice may be generated which overexpress adiponectin. Such a transgenic mouse's genome carries heterogeneous cDNA encoding adiponectin under the control of the regulatory elements of the mouse serum amyloid protein (SAP) gene, that produces an effect, relative to a wild-type effect, that is selected from the group consisting of an increase in adiponectin production, secretion and activity. In some cases, the cDNA is defined by SEQ ID NO:8. Constructs for use in generating such a mouse include one comprising the cDNA for adiponectin under the control of the serum amyloid protein promoter, which construct is designated pSAP-Adipo. Cells, including adipocytes, may be isolated from such transgenic animals.

[0463]To generate mice that over express adiponectin, the mouse cDNA for adiponectin was subcloned upstream of a cassette containing the human SAP promoter and rabbit β-globin non-coding exon/intron (FIG. 28). Fat pad weight was measured in WT and adiponectin transgenic pups and mice of each sex at 3 months of age (FIG. 28D). Food intake and energy expenditure were assessed in WT and Sap-Adiponectin transgenic mice to ascertain if the increase in energy expenditure observed in Esp-deficient mice is due solely to their increase in adiponectin serum levels. It was also verified that increasing serum adiponectin level will not affect appetite. To that end, metabolic cages and equipment were used. Serum glucose level was measured at birth, 2, 4, 8, and 16 weeks of age in WT and Sap-Adiponectin transgenic mice. In adult mice, this was done both after fasting and after feeding. In the same samples, serum insulin and adiponectin levels were measured (FIGS. 28C and 28E). Serum leptin levels were measured in serum of adult mice. Insulin sensitivity was assessed by insulin tolerance test (FIG. 28F): mice were fasted for six hours, injected IP with insulin (0.2 U/kg BW) and glucose levels were measured at indicated times as described (Mauvais-Jarvis et al., 2002). ITT data are presented as percentage of initial blood glucose concentration. Insulin secretion was assayed both by a glucose tolerance test performed following glucose intraperitoneal injection and by a glucose stimulated insulin secretion. Blood samples were obtained at 0, 2, 5, 15, and 30 minutes for GSIS or at 0, 15, 30, 60, and 120 minutes for GTT after intraperitoneal injection of 2 g/kg dextrose. Whole blood glucose values were determined using an automatic glucose monitor. Histological analysis. We have observed that in Esp-deficient mice there are fewer adipocytes than in WT mice yet they are larger, suggesting that they cannot release fat. The same analysis may be performed in 1 and 2 month old WT and Sap-Adiponectin transgenic mice. To ascertain that the large size of the adipocytes betrays their inability to release fat, WT, Esp-deficient and Sap-Adiponectin 1 month-old mice may be fasted for 16 or 24 hours and measured for free fatty acid (FFA) serum levels. It is expected that FFA serum levels will not increase in Esp-deficient and Sap-Adiponectin as it will in WT mice.

[0464]Generation of Sap-Insulin transgenic mice. A transgenic mouse is disclosed herein whose genome carries cDNA encoding full length mouse insulin under the control of the promoter and regulatory elements of the mouse serum amyloid protein (SAP) gene, that produces an effect, relative to a wild-type, comprising increased insulin expression and secretion.

[0465]To generate mice that over express insulin, the mouse cDNA for insulin was subcloned upstream of a cassette containing the human SAP promoter and rabbit β-globin non-coding exon/intron. These transgenic mice were analyzed using the same batteries of metabolic/molecular tests, including than the ones used for studying the Sap-Adiponectin transgenic mice. These studies are presented in FIG. 29.

[0466]Substrate Trapping. Plasmids for substrate trapping experiments were made as follows: Rat OST-PTP sequences encoding the first phosphatase domain (a.a 1116-a.a 1412) were cloned into the BamHI site of pGEX 4T3 (Amersham) encoding GST (Glutathione S-Transferase). This construct (GST-PTP) was used to generate Asp1316Ala GST-PTP DA, which is a catalytic mutant form which leads to the stabilization of the enzyme-substrate interaction, by site directed mutagenesis. The mutation was made in the phosphatase 1 domain that is known to mediate the dephosphorylation function of this class of phosphatases. The GST-PTP.sup.D1316A mutant has reduced phosphatase activity but increased substrate binding ability compared to wild type OST-PTP. It can thus retain, i.e. "Trap," the substrate better than the wild type protein. Cells expressing the mutant OST-PTP.sup.D1316A will trap any substrate that is the usual target of OST-PTP, but the mutant enzyme cannot dephosphorylate the substrate. It therefore holds onto the substrate without releasing it. Protein complexes for each experimental condition were then pulled down by centrifugation, washed 4 times and analyzed by western blot.

[0467]For the substrate-trapping experiments, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton, 0.1% CHAPS, 5 mM iodoacetic acid, 10 mM sodium phosphate, 10 mM NaF). Cell lysates were incubated with either GST, GST-PTP^WT (a fusion of GST with the phosphatase domain I of OST-PTP), or with GST-PTP.sup.D1316A (a trapping mutant of the Asp of the phosphatase domain I). Recombinant proteins bound to Sepharose beads for 1 hour at 4° C. (insulin receptor trapping) or for 2 h at 4° C. (gamma-carboxylase substrate trapping). Precipitates were collected, washed four times with lysis buffer and resolved on SDS-PAGE, followed by western blotting. Insulin receptor (InsR) was detected using rabbit anti-insulin receptor antibodies (Santa-Cruz, C-19) and GST was detected by mouse anti-GST antibodies (Santa-Cruz). Gamma-carboxylase was detected using rabbit anti-gamma-carboxylase antibody.

[0468]OST-PTP substrates are insulin receptor and gamma-carboxylase. To determine if OST-PTP acts through gamma-carboxylase, we conducted substrate-trapping experiments in primary osteoblasts. Differentiated primary osteoblasts (d10) were cultured for 10 days in alpha MEM/10% fetal bovine serum (FBS) supplemented with ascorbic acid (100 micrograms/ml) and Beta-glycerophosphate (5 mM). They were then starved for 24 hoursμ in the same medium supplemented with 1% FBS only and treated with pervanadate (100 μM), an irreversible protein-tyrosine phosphatase inhibitor, and 20% FBS for 30 minutes. Cell lysates were incubated for 2 h at 4° C. with either GST, GST-PTP^WT or GST-PTP.sup.D1316A. Different amounts of the total cell extract were also loaded as control.

[0469]Transformed cells that over express full-length or truncated OST-PTP. Eukaryotic expression vectors that express flag-tagged full-length OST-PTP or flag-tagged truncated OST-PTP containing only its extracellular domain (OST-PTP_EC) were used to perform DNA permanent transfection experiments in ROS (rat osteoblast cells) 17/2.8 osteoblastic cells transfected with these flag-tagged vectors, and as a negative control in COS 7 cells. Following selection, and isolation of clones of cells that have incorporated each of the two genes in chromosomes (flag-tagged full-length OST-PTP or flag-tagged truncated OST-PTP containing only its extracellular domain) it was verified that the genes were transcribed and that the proteins were made using RT-PCR and Western blot analysis of cell lysates respectively. The cells were then cultured in a serum-free medium overnight. The supernatant of cells transfected with the empty vector, or vector encoding the full length or the truncated Esp cDNA was isolated and a Western analysis was performed using a commercially available anti-Flag antibody.

[0470]Bacterial expression vectors for osteocalcin production. We have generated prokaryotic expression vectors for GST-tagged mouse osteocalcin, GST-tagged human osteocalcin, GST-tagged mutants of mouse and human osteocalcin and GST-tagged truncation mutants of mouse and human osteocalcin.

[0471]Diet and GTG induced obesity and type 2 diabetes. For diet-induced obesity, male and female six-week old WT and osteocalcin-deficient mice (n=10 per group) were fed for 4, 6, 8, or 12 weeks with either a normal or a "western" diet" of 45% fat, 35% carbohydrate, and 20% protein. For GTG-induced obesity, male and female 4 week-old WT and osteocalcin-deficient mice (n=10 per group) were injected with 0.5 mg/kg of GTG and sacrificed at 12 weeks of age. In both type of experiments, WT and mutant mice were analyzed as follows. Physical inspection: Whole body weight of each mouse at the start of the experiment and every week thereafter until sacrifice was measured. Food intake: This parameter was assessed to ascertain in particular that GTG lesions induce an increase in food intake. To that end, metabolic cages and equipment were used. Metabolic studies: Serum glucose and insulin levels were measured after fasting overnight and after feeding. Serum adiponectin and leptin levels were also measured in each mouse. Insulin secretion was assayed both by a glucose tolerance test (GTT) performed following glucose intraperitoneal injection and or glucose stimulated insulin secretion test (GSIS). Blood samples were obtained at 0, 2, 5, 15 and 30 minutes or at 0, 15, 30, 60 and 120 minutes following intraperitoneal injection of 2 g/kg dextrose for GTT. Whole blood glucose values were determined using an automatic glucose monitor. Molecular analysis: Expression of multiple markers of insulin sensitivity in hepatocytes, adipocytes and myoblasts at the end of each experiment was measured.

[0472]Co-culture of osteoblasts and adipocytes to study regulation of adiponectin expression/secretion by osteocalcin. A co-culture assay was developed between osteoblasts and adipocytes to analyze modifications in adiponectin expression. We used in this assay osteoblast from WT, Esp-deficient or osteocalcin-deficient mice along with primary adipocytes taken from any of these same mice. As a negative control, we co-cultured mouse embryonic fibroblasts of each genotype with adipocytes. Osteoblasts and fibroblasts were prepared according standard protocols that have been routinely used in the laboratory for the last twelve years (Ducy and Karsenty 1995), incorporated by reference as if set forth fully herein. Osteoblasts or fibroblasts were plated at 70% confluence in alpha MEM, 10% fetal bovine serum (FBS) 36 h prior to the beginning of the experiment. Prior to adding adipocytes, culture medium was changed to decrease the FBS concentration to 1%. Adipocytes were added for 0, 2, 4, 8, or 12 hours the following morning. At the end of the experiment, adipocytes that were present as non adherent cells were collected by centrifuging the culture medium. Adipocytes were used to extract RNA and to measure by real time the PCR expression of adiponectin and possibly other adipocyte-derived hormones including leptin. Culture medium was used to measure osteocalcin, adiponectin, leptin and other adipokine levels.

[0473]Co-culture of osteoblasts and beta-cells to study regulation of insulin expression/secretion by osteocalcin. A co-culture assay between osteoblasts and pancreatic beta-cells was developed to analyze modifications in insulin expression. Osteoblasts from WT, Esp-deficient or osteocalcin-deficient mice were used along with pancreatic beta-cells taken from any of these same mice. As a negative control, mouse embryonic fibroblasts of each genotype were co-cultured with adipocytes. Osteoblasts and fibroblasts were prepared according standard protocols that have been routinely used in the laboratory for the last twelve years. (Duey and Karsenty 1995, incorporated by reference as if set forth fully herein). Osteoblasts or fibroblasts were plated at 70% confluence in alpha MEM 10% fetal bovine serum (FBS) 36 h prior to the beginning of the experiment. Prior to adding beta-cells, culture medium was changed to decrease the FBS concentration to 1%. Beta-cells were added for 0, 2, 4, 8, or 12 hours the following morning. At the end of the experiment, beta-cells that were present as non adherent cells were collected by centrifuging the culture medium. Beta-cells were used to extract RNA and to measure by real time the PCR expression of insulin and other beta-cell-derived hormones as well as the expression of molecules known to regulate insulin expression and cell proliferation. Culture medium was used to measure osteocalcin, adiponectin, insulin and other cytokine levels.

[0474]Metabolic studies. For glucose tolerance test (GTT), glucose (2 g/kg body weight (BW)) was injected intraperitoneally (IP) after an overnight fast and blood glucose was monitored using blood glucose strips and the Accu-Check glucometer (Roche) at indicated times. For glucose stimulated insulin secretion test (GSIS), glucose (3 g/kg BW) was injected IP after an overnight fast; sera were collected from tails and insulin measured as described (Mauvais-Jarvis et al., 2000). For insulin tolerance test (ITT), mice were fasted for six hours, injected IP with insulin (0.2 U/kg BW) and blood glucose levels were measured at indicated times as described (Mauvais-Jarvis et al., 2002). ITT data are presented as percentage of initial blood glucose concentration. Gold thioglucose (600 mg/kg BW, USP) was injected IP after an overnight fast, mice were sacrificed 3 months later for analysis. Streptozotocin (150 mg/ml single injection, Sigma) was injected IP and blood glucose measured as described above every 2 days thereafter. After 8 days, pancreases were isolated to measure insulin content as previously described (Mauvais-Jarvis et al., 2000). Food intake was measured using metabolic cages as the daily change of food weight. Energy expenditure was measured using metabolic cages connected to a calorimeter (Columbus Instrument). Heat values (Kcal/Hr) were recorded over 2 days and reported to each mouse BW.

[0475]Laboratory measurements. Blood was collected by heart puncture of isoflurane anesthetized mice in the fed and fasted states. Colorimetric assays were used to measure serum levels of free fatty acids (Wako Chemicals) and of triglycerides (Sigma). Serum levels of insulin (Crystal Chem Inc. kit), adiponectin (Linco kit), leptin (Crystal Chem Inc. kit) and resistin (Linco kit) were quantified by ELISA, osteocalcin levels by IRMA (Immunotopics kit). There is no IRA, IRMA, or ELISA designed to differentiate carboxylated from undercarboxylated osteocalcin in mice. The existing kits measure total osteocalcin, but cannot specifically recognize undercarboxylated osteocalcin. Therefore, hydroxyapatite (HA) resin was used to separate the two forms. The carboxylated form is the only one that binds to the HA.

[0476]Mouse islets and adipocytes isolation. Islets were isolated using a Histopaque gradient (1077, Sigma). In brief, after clamping the common bile duct at its entrance to the duodenum, lmg/ml collagenase P (Sigma) in M199 medium (GIBCO) was injected into the duct. The swollen pancreas was surgically removed and incubated at 37° C. for 17 min. Digested pancreata were dispersed by pipetting and rinsed twice with the same medium. After filtering the tissue suspension through a Spectra-mesh (400 μm), the digested tissue was resuspended in Histopaque and overlaid with M199 medium. The sample was then centrifuged at 1,700 g for 20 min, and the islets were collected from the interface. The recovered material was washed twice with cold M199 medium, resuspended in M199/1% NCS or aMEM/1% FBS (GIBCO) medium and cultured at 37° C. in 5% CO₂.

[0477]Primary adipocytes were isolated from epididymal fat pads by collagenase digestion. Briefly, minced adipose tissue was digested by lmg/ml collagenase P in KRP Buffer (20 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 1 mM CaCl₂, 0.6 mM Na₂HPO₄, 0.4 mM NaH₂PO₄, 2.5 mM D-glucose, 2% BSA, pH 7.4) for 1 h at 37° C. The isolated cells were washed twice with KRP Buffer before being cultured in αMEM/1% FBS at 37° C. in 5% CO₂.

[0478]Cell culture experiments. Primary osteoblasts were prepared from calvaria of 5 day-old pups as previously described (Ducy et al., 2000a) and were cultured in αMEM/10% FBS in the, presence of 100 μg/ml ascorbic acid and 5 mM β-glycerophosphate for 5 days. Skin fibroblasts were isolated by collagenase digestion (0.5 mg/ml) and were cultured in quadratureMEM/10% FBS. Twenty-four hours before addition of primary islets (or adipocytes), osteoblasts (or fibroblasts) were placed in αMEM/1% FBS. For warfarin treatment, ROS17/2.8 osteoblastic cells were maintained in DMEM/F12/10% FBS until being supplemented with 50 μM warfarin or vehicle in DMEM/F12/1% FBS for 48 h prior to co-culture with adipocytes. After 4 h of co-culture, either in the presence or absence of (1 μm) culture inserts (Falcon) islets (or adipocytes) were collected for RNA isolation using TRIZOL (Invitrogen).

[0479]Gene expression analyses. All gene expression analyses were performed using real time PCR. DNAse I-treated total RNA was converted to cDNA with the SuperScript III kit (Invitrogen). Real-time PCR were performed using the Taq SYBR Green Supermix with ROX (Biorad) on an MX3000 instrument (Stratagene); beta-actin amplification was used as an internal reference for each sample. All primers were from SuperArray.

[0480]Osteocalcin/hydroxyapatite (HA) binding assay. Sera from 1 month-old mice, obese patients or supernatant from warfarin-treated osteoblast cultures were added to HA slurry to achieve a final concentration of 25 mg slurry/ml. After 15 min (mouse sera, supernatant) or 30 min (human sera), HA beads were pelleted by centrifugation and HA-bound osteocalcin was eluted with 0.5M sodium phosphate buffer, pH 8.0. Osteocalcin present in eluates and initial samples was measured by IRMA. Values represent percentage of HA-bound osteocalcin over initial osteocalcin content. Hauschka, P. V., et al., Physiol Review 69, 990-1047 (1989).

[0481]Statistical analyses. Results are given as means±standard deviations except in FIGS. 2B and 5F where means±standard errors of the mean are shown. Statistical analyses were performed using unpaired, two-tailed Student's t or ANOVA tests followed by post hoc tests. A p value <0.05 was considered significant and is indicated by a star in all figures unless otherwise indicated.

[0482]Recombinant Osteocalcin. Recombinant osteocalcin was bacterially produced and purified on glutathione beads according to standard procedures. Osteocalcin was then cleaved from the GST subunit using thrombin digestion. Thrombin contamination was removed using an affinity column. The purity of the product was qualitatively assessed by SDS-PAGE. Bacteria do not have a gamma-carboxylase gene. Therefore, recombinant osteocalcin produced in bacteria is always completely undercarboxylated at all three sites. Osteocalcin can be made in many ways known in the art, including being chemically synthesized, since it can be made without gamma-carboxylation when chemically synthesized.

[0483]Human studies. This study enrolled a group of obese and non-obese Caucasian women participating in a Clinical investigation performed at the Center of Research on Human Nutrition, Hotel-Dieu Hospital, Paris, France (PHRC protocol N° A0R076). This study was approved by the Ethics Committees of Hotel-Dieu (Paris). All subjects gave their informed consent. Subjects were weight stable for at least 3 months before the investigation day. Clinical and biochemical parameters were assessed in the morning (8:00 am) at the fasting state.

[0484]Histology. Frozen sections of livers were cryoembedded, sectioned at 5 μm and stained with Oil red O. Fat and pancreatic tissues were fixed overnight in 10% neutral formalin, embedded in paraffin, and sectioned at 5 μm. Histology sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry was performed using rabbit anti-insulin (SantaCruz, 1:100) and mouse anti-Ki67 (Vector, 1:100) antibodies and ABC Elite kits (Vector). Hypothalamic histology was performed as described (Takeda et al., 2002). To evaluate cell sizes or numbers, 5 to 10 sections (each 50 micrometers apart) were analyzed using a 40× objective on a Leica microscope outfitted with a CCD camera (SONY). Images were processed using the Osteomeasure software. Beta-cell area represents the surface positive for insulin immunostaining divided by the total pancreatic surface. Beta-cell mass was calculated as beta-cell area multiplied by pancreatic weight. At least 3 mice were analyzed per condition. Tibia anterior muscles were fixed in 4% PFA/2% glutaraldehyde/0.1 M sodium cacodylate ph 7.3, post-fixed in 1% osmium tetraoxide and embedded in epoxy resin (Epon). Ultrathin sections were stained in 4% aqueous Uranyl Acetate and 2 min in Reynolds' Lead Citrate and examined with a JEOL 2000FX. Ten electron micrographs per mouse were digitized and the area of each clearly distinguishable mitochondrion was analyzed using ImageJ software. Fifteen to 25 individual mitochondria were measured in 4 mice of each genotype.

[0485]Results

[0486]Generation and perinatal lethality of Esp-/- mouse models. To study OST-PTP, Esp was disrupted in a classical way (Esp-nLacZ) (Dacquin et al., 2004) and in an osteoblast-specific manner (Esp_ob-/-) by deleting exons 24 to 35 that encode the phosphatase domain using the LoxP/Cre recombinase technology (FIG. 22A). Mice harboring Esp floxed alleles were crossed with α1(1) collagen-Cre mice (Dacquin et al., 2002) to generate osteoblast-specific Esp-deficient mice (Esp_ob-/-) (FIG. 22B). Southern blot analysis showed that recombination occurred at high frequency at the Esp locus in osteoblasts (FIG. 1C). Accordingly, Esp expression was reduced nearly 90% in Esp_ob-/- osteoblasts and was unaffected in testis, the other site of Esp expression (FIG. 1D). Esp expression could not be detected in adipocytes or pancreatic beta-cells (data not shown). These data established that an osteoblast-specific inactivation of Esp was achieved. For the sake of clarity, reference to Esp-/- mice will be made when both Esp-nLacZ and Esp_ob-/- mice were studied.

[0487]When analyzed at weaning, intercrosses of Esp-/- mice in either a 129Sv/EV or a C57BL/6 genetic background yielded only about 25% of Esp-/- mice (FIG. 1F). To determine if this early post-natal lethality was due to a delay in skeletal development, skeletal preparations of newborn wild-type (WT) and Esp-/- pups were stained. No abnormality of bone formation was detected that could explain this lethality (FIGS. 22D-22F). Experiments were conducted to determine whether Esp-/- pup lethality could be due to a maternal effect, possibly a humoral abnormality. If it were the case, mutant pups born from homozygous mutant mothers should die at a higher frequency than those born from heterozygous mothers. That is precisely what was observed. While lethality of Esp-/- pups born from Esp+/- mothers never reached 15%, up to 35% of Esp-/- pups born from Esp-/- mothers died before weaning (FIG. 1F). These data indicate that the lethality of Esp-/- pups was caused in part by a maternal effect.

[0488]Increased beta-cell proliferation and insulin secretion in Esp-/- mice. To determine if the maternal effect responsible of Esp-/- mice perinatal lethality was caused by a humoral abnormality, metabolic parameters were measured in newborn pups prior to milk ingestion. Esp-/- pups, regardless of genetic background, sex, and type of deletion performed, showed only one abnormality: a 3-fold reduction of blood glucose levels (FIG. 1G). In some mutant pups blood glucose levels were even too low to be detected. Albeit less severe, a significant decrease in blood glucose level was also observed in 1 and 3 month-old Esp-/- mice after feeding (FIG. 1G). This hypoglycemia was explained by a significant hyperinsulinemia in newborn, 1 and 3 month-old fed Esp-/- mice (FIG. 1H). On the other hand, expression of Glucagon, a hormone secreted by pancreatic beta-cells, was normal (FIG. 23B), thus indicating that Esp mutation affects beta-cells specifically.

[0489]To establish more firmly that there was an increase in insulin secretion in the Esp-/- mice, intraperitoneal (IP) glucose stimulated insulin secretion tests (GSIS) were performed at 1 and 3 months of age. These assays showed that insulin secretion was enhanced by the absence of OST-PTP (FIGS. 1H and 1L). To assess how this increase in insulin secretion affects the ability to dispose of a glucose load, glucose tolerance tests were performed following IP injection of glucose (2 g/kg of body weight) after an overnight fast (GTT). These tests revealed that 1 and 3 month-old Esp-/- mice had a significantly higher tolerance to glucose than WT mice (FIG. 1J).

[0490]Histological and immunochemical analyses showed an increase in pancreas insulin content, the number of islets, islet size and overall of beta-cell mass in the Esp-/- pancreas (FIGS. 1K and 1L). A TUNEL assay failed to detect any abnormal apoptosis, and Ki67 immunostaining performed in 5 day-old pups (P5) and 1 month-old mice showed that beta-cell proliferation was increased 60 to 300% in Esp-/- mice (FIG. 1M). These data demonstrate that OST-PTP expressed in osteoblasts influences a pathway regulating beta-cell proliferation.

[0491]Increased insulin sensitivity in Esp-/- mice. To determine whether the enhanced ability of Esp-/- mice to dispose of a glucose load was secondary to an increase in insulin sensitivity, insulin tolerance tests (ITT) were performed. Insulin sensitivity, defined by the drop in blood glucose level following IP insulin injection, was significantly increased in 1 and 3 month-old Esp-/- compared to WT mice (FIG. 2A). Accordingly, expression of molecular markers of insulin sensitivity in fat (PPARα, PPARγ), liver (Foxa2, PPARα) and skeletal muscle (Pgc-1α, Nrf-1, Mcad) were also markedly increased in Esp-/- compared to WT mice. Pepck expression was decreased in Esp-/- liver indicating that gluconeogenesis was inhibited in this organ (FIG. 2E). It was speculated that as a result of these molecular events energy expenditure was increased in Esp-/- mice (FIG. 2G). In all analyses, heterozygous Esp+/- mice behaved as their WT littermates.

[0492]The experimental data show that Esp (OST-PTP) inactivation causes hypoglycemia, potentially lethal in newborn pups, that is associated with an increase in insulin secretion and sensitivity. That these abnormalities were observed to the same extent in both Esp-nLacZ-/- and in Esp_ob-/- mice established that it is the Esp gene expressed in osteoblasts, and not in any other cells or tissues, that is responsible for the metabolic phenotype.

[0493]One and 3 month-old Esp-/- mice displayed another phenotypic abnormality; their fat pads were significantly lighter than those of their WT littermates (FIG. 2F). Serum triglyceride levels were also lower in Esp-/- than in WT mice (FIG. 2H). Since Esp is not expressed in fat and food intake is normal in Esp-/- mice (FIG. 23H), this decrease in fat mass is secondary to the increase in insulin sensitivity. Although there were fewer adipocytes in Esp-/- than in WT mice (WT, 93.2±10.7×10³ adipocytes/fat pad (n=5); Esp-/-, 37±5.1×10³ adipocytes/fat pad (n=3)) they were larger (FIG. 21). To understand this phenotype the expression of multiple molecular markers was studied. C/EBPα, Srebp1c, Fatty acid synthase (FAS) and Lipoprotein lipase (LPL) were similarly expressed in Esp-/- and WT adipocytes, showing that adipogenesis, lipogenesis and fat uptake were not overtly affected by the mutation (FIG. 2J). In contrast, expression of molecular markers of insulin sensitivity (PPARγ and the regulator of fat oxidation PPARα) was increased, thus explaining enhanced insulin sensitivity without fat accumulation. Furthermore, expression of Perilipin and Triglyceride lipase (TGL), two anti-lipolytic proteins, was markedly decreased in Esp-/- compared to WT adipocytes (FIG. 2J) indicating that lipolysis is inhibited in Esp-/- mice. Accordingly, the serum level of free fatty acid did not increase following an overnight fast in Esp-/- mice as it did in WT littermates (FIG. 2K). The combination of increased insulin sensitivity and fat oxidation with inhibition of fat release from adipocytes synergized to produce the observed phenotype of low adiposity with large adipocytes in Esp-/- mice. These results are consistent with the increase in insulin secretion in Esp-deficient mice because insulin is a potent inhibitor of lipolysis.

[0494]Increased adiponectin expression in Esp-/- mice. Experiments were conducted to determine whether there was a humoral basis for the increase in insulin sensitivity observed in Esp-/- mice. Expression and serum levels of Resistin, an adipokine mediating insulin resistance, were virtually unaffected by Esp deletion. The same was true for leptin, an insulin-sensitizing hormone (Friedman and Halaas, 1998; Steppan et al., 2001) (FIGS. 2L and 23K). This latter observation is in agreement with the fact that food intake was normal in Esp-/- mice (FIG. 23H). By contrast, expression and serum levels of adiponectin, an adipokine able to enhance sensitivity to insulin (Yamauchi et al., 2001), were respectively increased three and two-fold in Esp-/- mice at birth, 1 and 3 months of age regardless of their sex and genetic background (FIGS. 2L and 23M). Accordingly, it was observed that expression of adiponectin target genes such as Acyl-CoA Oxidase, PPARα and Ucp2 was increased in Esp-/- mice (FIG. 2N) (Kadowaki and Yamauchi, 2005). This increase in adiponectin expression and serum levels provides one mechanism to explain the increase in insulin sensitivity observed in Esp-/- mice.

[0495]In summary, Esp inactivation caused hypoglycemia as a result of increased pancreatic beta-cell proliferation, enhanced insulin secretion and improved insulin sensitivity in peripheral tissues with decreased adiposity. That these abnormalities were observed in both Esp-nLacZ-/- and Esp_ob-/- mice demonstrated that the skeleton via osteoblasts is involved in regulating glucose homeostasis.

[0496]Esp-/- mice are protected from obesity and glucose intolerance. The increase in insulin secretion and sensitivity characterizing Esp-/- mice raised the prospect that these mutant mice could be protected from obesity and diabetes. Esp-nLacZ-/- and Esp_ob-/- showed identical metabolic and molecular abnormalities. In some experiments only one or the other model were tested so for the sake of clarity we will refer to Esp-/- in this case.

[0497]First, gold thioglucose (GTG) was injected in 1 month-old mice to induce specific lesions in the ventromedial hypothalamus (Brecher et al., 1965). As expected, GTG induced ventromedial hypothalamic lesions (FIG. 24) and hyperphagia (FIG. 3A) in both WT and Esp-/- mice. When analyzed 3 months after injection, GTG-treated WT mice were obese and their fat pad mass and serum triglyceride levels were significantly increased. GTT and ITT analyses showed that glucose intolerance and insulin resistance also increased (FIGS. 3E-3F). By contrast, GTG-treated Esp-/- mice were not obese, had fat pad mass and serum triglyceride levels similar to those of PBS-treated WT mice, and they displayed no evidence of glucose intolerance or of insulin insensitivity (FIGS. 3E-3F).

[0498]Next, 1 month-old WT and Esp-deficient mice were fed with a high fat diet (HFD) (58% fat kcal) for six weeks. It was discovered that body weights were significantly lower in Esp-nLacZ-/- mice than in WT mice at the end of this six week period (FIGS. 3G-3I). Glucose tolerance testing (GTT) demonstrated that after being fed a HFD for six weeks Esp-nLacZ-/- mice kept a normal tolerance to glucose, and insulin sensitivity determined by ITT remained normal. By contrast these parameters were altered in WT mice fed a high fat diet (HFD).

[0499]Whether the increase in insulin sensitivity could protect Esp-/- mice from pancreatic beta-cell failure was determined. To that end, mice were injected with streptozotocin (STZ) to provoke oxidative stress in beta-cells and cell death as are seen in type 2 diabetes (Le May et al., 2006). STZ treatment markedly decreased pancreas insulin content and insulin serum level in both genotypes (FIGS. 3J and 3K). Eight days after STZ injection, 3 of the 7 STZ-treated WT mice had died and all the surviving ones had serum glucose levels above 500 mg/dl (FIGS. 3L and 3M). On the other hand, only one STZ-treated Esp-/- mouse died during this period and the blood glucose level of the surviving ones did not exceed 250 mg/dl. Unlike STZ-treated WT mice, glucose could not be detected in urine of STZ-injected Esp-/- mice (FIG. 3N). Since both STZ-treated WT and Esp-/- mice had a major decrease in islet insulin content, the absence of an overt diabetic phenotype in STZ-treated Esp-/- mice showed that their increase in insulin sensitivity occurred independently of their increase in insulin secretion. These results establish that Esp function (OST-PTP) is required for the development of obesity and glucose intolerance in mice.

[0500]Esp influences the biological activity of an osteoblast-secreted molecule. The next question was how Esp, through its expression in osteoblasts could regulate insulin secretion and sensitivity. Cell-based assays failed to provide evidence that the OST-PTP extracellular domain could be either cleaved and secreted or expressed independently of the phosphatase domain. Therefore, COS cells that do not normally express Esp, were transfected with vectors expressing either a full-length flag tagged OST-PTP or its flag-tagged extracellular domain only. The cells were transfected using the standard calcium phosphate method well known in the art. At the end of the experiment, supernatant was collected, cells were lysed and both supernatant and cell lysate assayed for the presence of OST-PTP. Western blot analysis using either cell lysates or cell supernatants was then performed. Recombinant full-length or truncated proteins were detected in cell lysates but never in the supernatants, showing that OST-PTP extracellular domain is not normally secreted by cells. An antibody was made against the OST-PTP extracellular domain to be able to perform these experiments; certain embodiments of this invention are directed to this antibody and to other antibodies that bind to the OST-PTP extracellular domain. The OST-PTP extracellular domain is accessible to antibodies because it is not sequestered inside the cell membrane. An antibody against the transmembrane domain of OST also exists. Both of these antibodies are polyclonal and could be administered to an animal to inhibit OST-PTP, thereby increasing osteocalcin activity, which in turn increases adiponectin production and secretion from adipocytes, which in turn increases insulin production and sensitivity. Of course, monoclonal antibodies can be used as well.

[0501]To further study OST-PTP function, transgenic mice expressing either full-length OST-PTP or its extracellular domain only in osteoblasts were generated and analysed. Transgenic mice overexpressing full-length Esp cDNA selectively in osteoblasts (alpha1(I)-OST-PTP mice) were made that displayed decreased beta-cell proliferation, lower beta-cell mass, hypoinsulinemia in the fed state and impaired insulin secretion in response to glucose (FIGS. 4A-C). They also showed lower adiponectin serum concentrations (FIG. 4B). As a result, alpha1(I)-OST-PTP mice developed hyperglycemia on regular chow, glucose intolerance and insulin resistance (FIGS. 4B, 4D and 4E). The fact that this phenotype, which is the minor image of the one observed in Esp-/- mice, is only observed in transgenic mice over expressing full-length OST-PTP shows that the phosphatase activity of OST-PTP is required to affect glucose homeostasis. Furthermore, the fact that these mice over expressed Esp in osteoblasts further supports the conclusion that OST-PTP regulates the bioactivity of an osteoblast-derived secreted molecule that in turn regulates glucose homeostasis. By contrast, alpha₁(I) collagen-Esp_sd mice, which express only OST-PTP extracellular domain, had no energy metabolism abnormalities of any kind. These results taken with the well-described fact that OST-PTP phosphatase domain is an active one, show that it is through its phosphatase domain and not through its extracellular domain that OST-PTP regulates insulin secretion and adiponectin expression and further confirm that OST-PTP act on the regulation of energy metabolism via its expression in osteoblasts.

[0502]Apolipoprotein E--OST-PTP_EC transgenic mice were also generated that express the OST-PTP extracellular domain and release it into the general circulation. The apolipoprotein E promoter was used to direct Esp expression in liver cells thereby causing release of the OST-PTP extracellular domain into the general circulation. These transgenic mice were indistinguishable from wild type mice, further proving that OST-PTP regulates energy metabolism through its intracellular phosphatase domain and its expression in osteoblasts.

[0503]To further prove that osteoblasts secrete a factor that acts on pancreatic beta-cells and adipocytes, osteoblasts, which are adherent cells, were co-cultured with either pancreatic islets or adipocytes, which are non-adherent cells. Co-culture of differentiated WT osteoblasts with islets isolated from WT mice increased insulin expression in islets 40% (FIG. 4F). In full agreement with the increase in insulin secretion observed in Esp-/- mice, Esp-/- osteoblasts further enhanced insulin expression (FIG. 4F). Osteoblasts or fibroblasts were also co-cultured with adipocytes. WT osteoblasts, but not fibroblasts, increased expression of adiponectin and Esp-/- osteoblasts were twice as potent as WT osteoblasts in enhancing adiponectin expression (FIG. 4G). In this assay, adiponectin was the only adipokine whose expression was affected (FIG. 4G). Control experiments using WT osteoblasts co-cultured with Esp-/- islets or adipocytes showed the same increase in insulin and adiponectin expression as seen when using WT islets or adipocytes (FIG. 4H).

[0504]To establish that osteoblasts influence insulin and adiponectin expression via the release of secreted molecule(s), additional experiments were performed. First, osteoblasts were co-cultured with either islets or adipocytes using a filter preventing cell-cell contact. Second, islets and adipocytes were co-cultured in the presence of supernatant of primary osteoblast cultures. In both cases, a significant increase in insulin and adiponectin expression was observed (FIGS. 41 and 4J). Taken together, these data indicate that Esp expressed in osteoblasts regulates the expression or activity of a secreted molecule that affects insulin and adiponectin expression in beta-cells and adipocytes.

[0505]Osteocalcin is the osteoblast-derived secreted molecule that increases proliferation, insulin secretion and insulin sensitivity. To identify the molecule(s) secreted by osteoblasts that regulate glucose homeostasis, energy metabolism parameters were analysed in mutant mouse strains lacking osteocalcin, an osteoblast-specific secreted molecule present in serum. In earlier studies, it was observed that, upon their generation, oc-/- mice were abnormally fat. Ducy et al Nature 1996, herein incorporated by reference. At the time there was no explanation for why these animals were so fat and therefore the obesity aspect of these mice was observed but not published. Both homozygous (Oc-/-) and heterozygous strains (Oc+/-) were made.

[0506]Osteocalcin is one of the major non-collagenous proteins made by osteoblasts and is also an osteoblast-specific molecule. Like many secreted proteins, including peptide hormones, osteocalcin is generated as pre-pro-osteocalcin and undergoes cleavage and post-translational modifications in the cytoplasm before being secreted. In addition, osteocalcin belongs to the family of gla proteins in which some glutamic acid residues are carboxylated by a gamma-carboxylase to form gla residues. Hence the other name of osteocalcin: bone gla protein (BGP). Gla residues confer on gla proteins a high affinity for mineral ions.

[0507]Osteocalcin-/- mice had higher blood glucose and lower insulin serum levels than WT mice (FIGS. 5A and 5B). Insulin secretion and sensitivity as well as glucose tolerance analyzed by GSIS, GTT and ITT were all decreased in Osteocalcin-/- mice, as was energy expenditure (FIGS. 5C-5E and 5G). Accordingly, the expression of genes involved in insulin action was decreased in skeletal muscle and liver while Pepck expression was increased (FIG. 5H). Islet size and number, beta-cell mass, pancreas insulin content and insulin immunoreactivity were all markedly decreased in Osteocalcin-/- mice (FIG. 5I). Beta-cell proliferation measured by Ki67 immunostaining was decreased two fold in Osteocalcin-/- pancreas in P5 pups and at 3 months of age (FIG. 5I). Accompanying this marked decrease in beta-cell proliferation, insulin secretion and sensitivity, was an increase in fat pad mass, adipocyte number (WT, 93.2±10.7×10³ adipocytes/fat pad (n=5); Osteocalcin-/-, 125.6±10.6×10³ adipocytes/fat pad (n=3)) and serum triglyceride levels (FIGS. 5J and 5K). Adiponectin expression and serum levels were significantly lower in Osteocalcin-/- than in WT mice, especially considering their increased fat pad mass, while expression of other adipokines was not affected (FIGS. 5L and 5M). Expression of molecular targets of adiponectin action was decreased in Osteocalcin-/- mice (FIG. 5N). However, Osteocalcin+/- mice were undistinguishable from WT littermates (data not shown). The cDNA sequence for mouse adiponectin is SEQ ID NO:8; and it identified also by amino acid SEQ ID NO:9. The cDNA sequence for human adiponectin is SEQ ID NO:6; and it identified also by amino acid SEQ ID NO:7.

[0508]To demonstrate that osteocalcin is the molecule secreted by osteoblasts that affects insulin and adiponectin expression, further co-culture experiments were performed. Unlike WT osteoblasts, Osteocalcin-/- osteoblasts failed to enhance expression of insulin and adiponectin in islets and adipocytes, respectively (FIGS. 5O and 5P). In a converse experiment, forced expression of osteocalcin in COS cells allowed these cells to increase insulin expression in islets and adiponectin expression in adipocytes (FIG. 5Q). WT immature osteoblasts, that do not express osteocalcin (Ducy et al., 2000b) were co-cultured with either islets or adipocytes. These cells failed to induce either insulin or adiponectin expression (FIG. 5R). Taken together, these data provide genetic and cellular evidence indicating that osteocalcin is the molecule secreted by differentiated osteoblasts that regulates insulin and adiponectin expression.

[0509]Osteocalcin regulates insulin sensitivity through adiponectin. To determine whether insulin and adiponectin both contribute, independently of each other, to the metabolic phenotype of the Osteocalcin-/- mice, two related questions were asked. First, does osteocalcin regulate adiponectin expression independently of its action on insulin secretion, and if so, does the decrease in adiponectin expression noted in the Osteocalcin-/- mice explain the decrease in insulin sensitivity? If both hypotheses are correct, then compound heterozygote Osteocalcin+/-; Adiponectin+/- mice should have lower expression of adiponectin than WT littermates and should show a decrease in insulin sensitivity similar to the one observed in the Osteocalcin-/- or in the Adiponectin-/- mice (Maeda et al., 2002). Certain embodiments are directed to these heterozygous transgenic strains.

[0510]As shown in FIGS. 6A-D, insulin sensitivity was markedly decreased in Osteocalcin+/-; Adiponectin+/- mice while blood glucose levels, insulin serum levels and insulin secretion as determined by GSIS test remained within the normal range. Adiponectin serum levels were also significantly decreased in Osteocalcin+/-; Adiponectin+/- compared to WT or single heterozygote mice (FIG. 6E). These observations are consistent with the notion that osteocalcin regulates insulin sensitivity at least in part through its regulation of adiponectin expression and secretion.

[0511]To show that the increase in insulin sensitivity and decrease in fat weight observed in the Esp-deficient mice was secondary to the increase in adiponectin expression, Sap-Adiponectin transgenic mice harboring a two-fold increase in serum adiponectin level similar to the one observed in Esp-deficient mice were generated. The Sap-Adiponectin transgenic mice also showed the phenotype of low fat pad weight, high energy expenditure and metabolic and molecular evidence of increased insulin sensitivity similar to those observed in Esp-deficient mice (FIG. 22). This result shows that the increase in adiponectin expression was the main identifiable cause of the increase in insulin production and sensitivity in Esp-deficient mice. Certain embodiments of the invention are thus directed to human cells transfected with the gene for adiponectin under the control of a promoter that causes the cell to over express adiponectin.

[0512]OST-PTP regulates osteocalcin bioactivity by influencing indirectly its carboxylation. The metabolic phenotype of Osteocalcin-/- mice is the mirror image of the one observed in Esp-/- mice suggesting that in the latter there is a gain of osteocalcin activity. To further prove that Esp-deficient mice (OST-PTP-/-) are a model of a gain of activity of osteocalcin, double mutants were made by introducing additional mutations into Esp-deficient transgenic mice, specifically by making them osteocalcin+/-.

[0513]It was hypothesized that the metabolic abnormalities of Esp-/- mice would be reversible by reducing osteocalcin expression. This is exactly what was observed: Esp-/- mice lacking one allele of osteocalcin showed a remarkable reversal of all their metabolic abnormalities such as blood glucose, insulin and adiponectin serum levels, glucose tolerance, insulin secretion and sensitivity (FIGS. 7A-F). Ki67 staining showed that beta-cell proliferation was also reduced in these mutant mice (FIG. 7G).

[0514]Indeed, Esp-/-; Ocn+/- mice display a decrease in insulin synthesis and sensitivity compared to Esp-/- without any osteocalcin deletion, showing a fully corrected/normalization of all metabolic abnormalities of the Esp-/- mice compared to wt mice. This experiment established genetically that OST-PTP and osteocalcin are in the same signaling cascade, and that the Esp_ob-/- mouse phenotype is a model of a gain of activity of osteocalcin. In other words, the metabolic phenotype seen in Esp_ob-/- mice is due to an increase in osteocalcin activity.

[0515]Because osteocalcin expression and serum levels were normal in Esp-/- mice, OST-PTP regulation of osteocalcin expression was ruled out (FIG. 20) In contrast, Esp-/- mice showed a decreased ratio of serum carboxylated osteocalcin to total osteocalcin (FIG. 7H). Carboxylated osteocalcin has a higher affinity for hydroxyapatite (HA) than undercarboxylated osteocalcin (Hauschka et al., 1989; Price, 1989). An assay was used where carboxylated osteocalcin is measured as the % of total osteocalcin able to bind to hydroxyapatite (HA). This assay showed that this value is decreased by 20% in Esp-/- mice compared to wt mice. In the presence of an equal amount of total osteocalcin, this means that undercarboxylated osteocalcin is increased 20% in Esp-/- mice compared to WT.

[0516]This experiment suggested that OST-PTP influences osteocalcin function by regulating its degree of gamma-carboxylation and that it was the undercarboxylated form of osteocalcin that regulated glucose homeostasis. To determine whether the was the case, two additional experiments were performed. WT primary osteoblasts were treated with warfarin, an inhibitor of gamma-carboxylation (Bergner, 2005) prior to and during co-culture assays. This treatment resulted in a marked decrease in the percentage of osteocalcin bound to HA, indicating that, as expected, these osteoblasts secrete less carboxylated osteocalcin (FIG. 7I). Nevertheless, and despite secreting less osteocalcin than WT osteoblasts (+vehicle, 10 ng/ml; +warfarin, 2 ng/ml) (Hauschka et al., 1989), warfarin-treated osteoblasts induced adiponectin expression to a significantly higher extent than vehicle-treated osteoblasts (FIG. 7J). Second, carboxylated osteocalcin and bacterially produced mouse osteocalcin, which is not carboxylated, were used in cell-based assays. While carboxylated osteocalcin failed to induce adiponectin expression, bacterially produced osteocalcin did (FIG. 7K). Likewise, undercarboxylated osteocalcin induced insulin expression as well as expression of Cyclin D1, a molecular marker of beta-cell proliferation (Kushner et al., 2005) (FIG. 7L). Lastly, we studied human obese patients that are hyperinsulinemic but not diabetic (FIG. 7M). The amount of uncarboxylated osteocalcin was significantly increased in these patients while osteocalcin serum levels were not affected (FIGS. 7M-O). Taken together these data indicate that OST-PTP influences osteocalcin bioactivity by enhancing its degree of carboxylation.

[0517]OST-PTP affects enzymes involved in the carboxylation process. A mandatory event in every function of any cell type is the ability of intracellular proteins to be phosphorylated by protein kinases and/or dephosphorylated by protein phosphatase. In particular phosphorylation of tyrosine residues accounts for 0.1% of the total cellular phosphoamino acid content; as a result protein tyrosine phosphatases (PTPs) are critically important intracellular proteins (23).

[0518]Protein tyrosine phosphatases can be schematically grouped into four classes: the classical receptor-like PTPs that have an extracellular domain that sometimes is cleaved (RPTPs); OST-PTP is a receptor-like PTP. Other classes include the classical non-receptor PTPs, the dual specificity PTPs and the low molecular weight PTPs (24). There are approximately 20 RPTPs in the human genome. RPTPs that are predominantly localized in the plasma membrane can be involved in cell to cell functions, cell-cell adhesion and in hormone signaling. However, two questions remain often unanswered regarding their biology. One is to determine the identity of substrate(s) for their phosphatase activity and the second one is to identify their ligands.

[0519]Results suggested that OST-PTP could dephosphorylate a specific substrate present in osteoblasts, thereby increasing the substrate's expression and/or activity. This substrate would then be released by osteoblasts and signal to pancreatic beta-cells and adipocytes, thereby affecting insulin secretion and sensitivity. While osteocalcin was a logical target candidate for OST-PTP physiologically speaking, osteocalcin is not phosphorylated. It was thus eliminated as a direct target.

[0520]To decipher how OST-PTP might influence osteocalcin activity we asked whether it was regulating gamma-carboxylation of osteocalcin, which is the main post-translational modification known for this molecule (Hauschka et al., 1989). This post-translational modification occurs both in rodent and in human; Poser et al analyzed the primary structure of human osteocalcin, and reported that human osteocalcin is a mixture of Glu⁷ osteocalcin (herein "Oc-glu") with the 17-position being glutamic acid, and Gla⁷ osteocalcin with the 17-position being gamma-carboxyglutamic acid (herein "BGP", also bone Gla-protein) [Poser, J. W. et al., Proc. Natl. Acad. Sci. U.S., 255, 8685-8691 (1980)]. Gla residues usually confer on proteins a high affinity for mineral ions. However, loss and gain of function experiments failed to identify a function for osteocalcin in extracellular matrix mineralization in vivo (Ducy et al., 1996; Murshed et al., 2004).

[0521]OST-PTP Substrates Include Insulin Receptor and Gamma-Carboxylase. A computer search revealed that gamma-carboxylase, also known as vitamin K-dependent gamma-glutamyl carboxylase, has PTP consensus sites. This enzyme catalyzes the conversion of glutamic acid to gamma-carboxyglutamic acid in substrate proteins like osteocalcin. To determine if OST-PTP acts on gamma-carboxylase, substrate-trapping experiments were conducted in COS cells, Ros17/2.8 osteoblast cells and differentiated primary osteoblasts. d10 bone-derived cells were cultured for 10 days in alpha MEM/10% fetal bovine serum (FBS) supplemented with ascorbic acid (100 micrograms/ml) and beta-glycerophosphate (5 mM)). These cells were then starved for 24 hours in alpha MEM culture medium supplemented with 1% FBS only and treated with pervanadate (100 mM), an irreversible protein-tyrosine phosphatase inhibitor, and 20% FBS for 30 minutes. Cell lysates were incubated for 2 h at 4° C. with either GST, GST-PTP^WT or GST-PTP.sup.D1316A. Different amounts of the total cell extract were also loaded as control.

[0522]The results in FIG. 9 showed that the mutant enzyme GST-PTP.sup.D1316A trapped gamma-carboxylase, thereby demonstrating that gamma-carboxylase is a substrate of OST-PTP. This, however, does not mean that gamma-carboxylase is the only substrate of OST-PTP. There was no binding in the GST lane because there is no PTP transfected. It is a control to show that if there is trapping, it is not due to the GST part of any GST fusion protein. There was also no trapping with GST-PTP^WT because this form dephosphorylated the substrate gamma-carboxylase, which is then released. A band is clearly seen in the lane having the mutant GST-PTP.sup.D1316A because the mutation engineered a defect in OST-PTP phosphatase activity that allowed the substrate to irreversibly bind to and be retained by the enzyme.

[0523]These results show that gamma-carboxylase is a substrate for OST-PTP in osteoblasts. This enabled elucidation of part of the biochemical pathway through which OST-PTP regulates osteocalcin bioactivity: OST-PTP dephosphorylates gamma-carboxylase, thereby activating it. Activated gamma-carboxylase in turn causes an increase in carboxylated osteocalcin. There is less dephosphorylated active gamma-carboxylase in OST-PTP-deficient mice, which leads to secretion of more undercarboxylated osteocalcin. This explains why the OST-PTP-deficient mice have elevated levels of undercarboxylated osteocalcin, which itself causes resistance to metabolic syndrome and diabetes.

[0524]Using the same substrate trapping assay, it was also discovered that the insulin receptor which is expressed in osteoblasts is a substrate for OST-PTP (FIG. 8). The results of the substrate-trapping experiments show that mutated OST-PTP (GST-PTP^DA) interacts with the insulin receptor (InsR) expressed in COS cells (left upper panel) and ROS17/2.8 osteoblasts cells (right upper panel) (third lane). By contrast, WT OST-PTP (GST-PTP^WT) did not interact with the insulin receptor (second lane). The same amount of GST fusion proteins were used for substrate trapping.

[0525]Human Patient Data. FIG. 7O shows that human obese patients that are hyperinsulinemic but not diabetic, have significantly elevated levels of undercarboxylated osteocalcin (about 30% higher) compared to normal patients, even though osteocalcin serum levels (7M) are about the same. This shows that in mice and in humans the level of carboxylation of osteocalcin influences its bioactivity. FIG. 7O further shows that obese non-diabetic patients have an increase in undercarboxylated osteocalcin compared to patients who are obese and diabetic. The ratio of carboxylated osteocalcin compared to total osteocalcin was measured in serum from non-medicated normal, obese non-diabetic and obese-diabetic patients.

[0526]An in vivo experiment was conducted in which the effect of undercarboxylated osteocalcin on glycemia was monitored. Wild type mice were infused with 3 different amounts of mouse recombinant undercarboxylated osteocalcin or placebo (PBS) subcutaneously for 28 days (0.3, 1 and 3 ng/hour). Compared to the control animal infused with placebo, all three doses of undercarboxylated osteocalcin decreased glycemia in vivo over the 28 day period (FIG. 10).

[0527]In another in vivo experiment, the effect of uncarboxylated osteocalcin on glucose tolerance was investigated. Wild type mice were infused subcutaneously with either 0.3 or 3 ng/hour doses of recombinant uncarboxylated osteocalcin or PBS for 14 days before receiving a single injection of glucose. Blood glucose was measured thereafter at the indicated times. The results show that both doses of uncarboxylated osteocalcin increased glucose tolerance above control levels over the 120 minute time period following the glucose injection (FIG. 11).

[0528]The effect of uncarboxylated osteocalcin on insulin sensitivity was also examined. Wild type mice were infused subcutaneously with 0.3 or 3 ng/hour doses of recombinant osteocalcin or PBS for 18 days before receiving a single injection of insulin. Blood glucose was measured thereafter at the indicated times from 0-120 minutes after injection. The results show that insulin sensitivity was increased by both doses of uncarboxylated osteocalcin (FIG. 12).

[0529]In another in vivo experiment, the effect of uncarboxylated osteocalcin on body weight and fat pad mass was monitored (FIG. 13). Wild type mice were infused subcutaneously for 28 days with PBS or uncarboxylated osteocalcin at 0.3, 1 or 3 ng/hour. The results show that body weight was slightly reduced by uncarboxylated osteocalcin with the highest dose being the most effective. (FIG. 13) Gonadal fat pad mass, measured after 28 days, decreased by about %18 with 3 ng/hour uncarboxylated osteocalcin treatment. The other doses did not significantly decrease fat pad mass in that period.

[0530]The effect of uncarboxylated osteocalcin on GTG-induced obesity was investigated (FIG. 14). Wild type mice were injected with gold thioglucose (GTG) to induce hyperphagia and obesity or vehicle. Two weeks later they were implanted with subcutaneous osmotic pumps infusing lng/hr of recombinant uncarboxylated osteocalcin or PBS for 28 days. Body weight gain was significantly reduced with both doses of uncarboxylated osteocalcin by the first time point checked, 7 days, and remained lower than controls for the entire 28 day period. At 28 days, body weight was reduced by about 15% with uncarboxylated osteocalcin treatment.

[0531]A fragment of uncarboxylated osteocalcin is biologically active. Experiments were performed to test whether truncated osteocalcin was as effective as full-length uncarboxylated osteocalcin in stimulating adiponectin secretion from mouse adipocytes in vitro. Wild type adipocytes were treated for 4 hours with recombinant full-length osteocalcin (1-46) or a truncated form (1-36) (having a deletion of the first ten amino acids from the C-terminal end) or vehicle. Adiponectin expression was then quantified by real time PCR. The results show that full-length uncarboxylated osteocalcin produced about a 1.5 fold increase, and the 1-36 fragment of uncarboxylated osteocalcin produced about a 1.8 fold increase (FIG. 15). Thus, the full length molecule is not needed for biological activity; at least up to 10 amino acids can be deleted from the C-terminal end of the mouse osteocalcin molecule to achieve the same biological effects on adipocytes and beta-cells. Certain embodiments of the invention are directed to osteocalcin from which the first ten amino acids from the C-terminal end have been deleted, preferably human osteocalcin, preferably undercarboxylated osteocalcin.

[0532]The primary sequence of osteocalcin is highly conserved among species and it is one of the ten most abundant proteins in the human body (FIG. 21), suggesting that its function is preserved throughout evolution. Conserved features importantly include 3 Gla residues at positions 17, 21, and 24, a disulfide bridge between Cys23 and Cys29, and most species contain a hydroxyproline at position 9. The N-terminus of osteocalcin shows highest sequence variation in comparison to other parts of the molecule. The high degree of conservation of human and mouse osteocalcin underscores the relevance of the mouse as an animal model for the human, in both healthy and diseased states, and validates our claims to the therapeutic and diagnostic use of osteocalcin to treat or prevent metabolic syndrome or any of its components and type 1 diabetes.

[0533]Vitamin K and Statins Increase Osteocalcin. Vitamin K is required for gamma-carboxylation. Warfarin and other COUMADIN® derivatives block vitamin K-dependent gamma-carboxylation, thus increasing the level of active, undercarboxylated osteocalcin. This is in agreement with data showing that warfarin-treated osteoblasts produce elevated levels of undercarboxylated osteocalcin compared to vehicle-treated osteoblasts (FIG. 7I). Others have shown that four weeks of treatment of osteoporotic patients with Vitamin K caused a dramatic percentage mean decrease in undercarboxylated osteocalcin of 85% compared to controls without Vitamin K treatment. Vitamin D had no significant effect alone or when administered together with Vitamin K. Takahashi, et al, Clinical Endocrinology (2001) 54, 291-224. See also Sugiyama, T., J Bone Miner Metabolism (2001) 19,146-159. This observation suggests that warfarin or another COUMADIN® derivative could be used to block vitamin K-dependent gamma-carboxylation and increase the level of undercarboxylated osteocalcin in patients with the goal to prevent/treat metabolic disorders.

[0534]Warfarin, sold as the brand name COUMADIN®, is used as an oral anticoagulant that inhibits the synthesis of clotting factors, thus preventing blood clot formation. However, COUMADIN® can cause bleeding and necrosis (gangrene) of the skin. Many drugs, both prescription and nonprescription (OTC), can affect the anticoagulant action of COUMADIN®. Some medications can enhance the action of COUMADIN® and cause excessive blood thinning and life-threatening bleeding. A few examples of such medications include Aspirin, TYLENOL®, alcohol, ibuprofen (MOTRIN®), cimetidine (TAGAMET®), oxandrolone (OXANDRIN®), certain vitamins, and antibiotics.

[0535]Others have shown that the statin ZOCOR® (simvastatin at 20 mg/day in humans) significantly increased serum levels of osteocalcin (p value less than 0.05) after four weeks of treatment, although undercarboxylated osteocalcin could not be distinguished from intact osteocalcin. Chan, M. H., et al., J Clin Endocrinology and Metabolism (2001) Vol 86(9), 4556-59. Even though there is no experimental proof that the level of undercarboxylated osteocalcin was increased by statins, a significant increase in the overall expression of osteocalcin, could cause a saturation of the gamma-carboxylase activity and an inability to carboxylate all of the osteocalcin produced. As a result statins could indirectly increase the amount of undercarboxylated osteocalcin released in the blood. Moreover, administering statins together with drugs that block gamma-carboxylation, such as warfarin, which blocks vitamin K, or inhibitors of OST-PTP and gamma-carboxylase, could work together to elevate serum undercarboxylated osteocalcin and have therapeutic use. Statins and vitamin K inhibitors could be administered in a single preparation or in separate preparations.

[0536]Therefore certain aspects of the present invention are directed to the use of vitamin K inhibitors and statins to increase undercarboxylated osteocalcin levels in serum, and to their therapeutic use in treating metabolic syndrome and its various components.

[0537]The sympathetic nervous system positively regulates OST-PTP expression. It was discovered that sympathetic nervous system (SNS) activity positively regulates Esp expression in osteoblasts. Indeed, FIG. 16 shows that stimulation of SNS signaling with isoproterenol, a beta adrenergic receptor agonist, increased Esp expression by about 80% by 4 hours, and that this increase remains steady even at 8 hours. However, increased SNS activity did not increase expression of gamma-carboxylase (ggcx), vkor (an enzyme involved in recycling of vitamin K that is necessary for ggcx activity) or osteocalcin. This experiment shows that SNS signaling positively regulates Esp expression in osteoblasts. Thus, decreasing sympathetic activity should lead to a decrease in Esp expression and thereby to an increase in the undercarboxylated, active form of osteocalcin.

[0538]In vivo experiments using ob/ob mice, which have low sympathetic activity, confirmed this hypothesis, and showed that there is a genetic link between leptin (the product of the ob gene) and osteocalcin. It has been shown that leptin signals to osteoblasts via the SNS. Thus, ob/ob mice are a model of decreased SNS signaling on osteoblasts. It has been shown that ob/ob mice display an increase in insulin before they develop any other metabolic abnormalities. This increase could be due to decreased SNS activity on osteoblasts, which as a result would express less OST-PTP and secrete more bioactive undercarboxylated osteocalcin, leading to increased insulin expression. The level of serum insulin was measured in one week old mice having various genotypes: WT mice, ob -/+ mice (hemizygous for obesity), ob/ob mice, Bgp -/+ (hemizygous for osteocalcin), BGP-/- mice, and ob/ob mice that are also Bgp-/- (Ocn deficient). One week old mice were chosen because ob/ob mice at one week are not yet obese and they are relatively normal metabolically, except for having high serum insulin levels. The results show that ob/ob mice indeed have increased serum insulin levels but if both alleles of the Bgp gene (encoding osteocalcin) are deleted in ob/ob mice their serum insulin levels returns to normal. FIG. 17. This experiment demonstrates that the increase in insulin observed in ob/ob mice is dependent on osteocalcin.

[0539]Taken together the results presented above mean that administering beta blockers, which decrease SNS activity, will likewise decrease Esp expression and thereby increase the level of undercarboxylated osteocalcin. As such they could be used to prevent/treat metabolic disorders via an increase in osteocalcin activity. Beta blockers have been used clinically for a long time, therefore the amounts that are safe for human use are established. Routine experimentation will determine the optimum amount of a particular beta blocker to administer to achieve increased levels of serum undercarboxylated osteocalcin. New beta-blockers, targeting more preferentially the cells of the skeleton could also be developed to more specifically increase osteocalcin activity and reduce the risk of side effects.

TABLE-US-00004 TABLE 2 Wild-type Esp-/- Ocn-/- (n = 4) (n = 3) (n = 4) Basal hepatic glucose 13.2 ± 1.9 16.6 ± 1.3 15.5 ± 1.8 production (mg/kg/min) Clamp hepatic glucose 10.3 ± 1.9 -5.5 ± 1.8* 16.1 ± 1.9* production (mg/kg/min) Glucose turnover 41.8 ± 1.3 50.6 ± 2.6* 38.2 ± 3.7 (mg/kg/min) Glycogen synthesis 19.2 ± 2.7 19.4 ± 3.2 11.5 ± 1.3* (mg/kg/min) Muscle glucose uptake 229 ± 29 358 ± 65* 188 ± 15 (nmol/g/min) White adipose 16.7 ± 2.7 31.7 ± 8.2* 7.5 ± 0.9* glucose uptake (nmol/g/min) Brown adipose 2022 ± 205 3330 ± 263* ND glucose uptake (nmol/g/min) *p ≦ 0.05, Student t-test, ND, not done

Analysis of 3 month-old Esp-/- and Ocn-/- mice by hyperinsulinemic-euglycemic clamps.

TABLE-US-00005 TABLE 3 SEQ ID NO: Amino GenBank cDNA Acid Accession No: Human Osteocalcin cDNA 1 2 NM_199173 Mouse osteocalcin gene 1 3 5 NM_007541 Mouse osteocalcin gene 2 4 5 NM_001032298 Human Adiponectin 6 7 NM_004797 Mouse Adiponectin 8 9 NM_009605 Human Gamma-glutamyl 10 11 NM_000821 carboxylase Mouse Gamma-glutamyl 12 13 NM_019802 carboxylase Human ApoE 14 15 NM_000041 Mouse ApoE 16 17 NM_009696 Mouse Esp (OST-PTP, Ptprv) 18 19 NM_007955 E. coli β-galactosidase 20 21 Rat (OST-PTP, Ptprv) 24 25 NM_033099

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Sequence CWU 1

251498DNAHomo sapiensCDS(19)..(318) 1cgcagccacc gagacacc atg aga gcc ctc aca ctc ctc gcc cta ttg gcc 51 Met Arg Ala Leu Thr Leu Leu Ala Leu Leu Ala 1 5 10ctg gcc gca ctt tgc atc gct ggc cag gca ggt gcg aag ccc agc ggt 99Leu Ala Ala Leu Cys Ile Ala Gly Gln Ala Gly Ala Lys Pro Ser Gly 15 20 25gca gag tcc agc aaa ggt gca gcc ttt gtg tcc aag cag gag ggc agc 147Ala Glu Ser Ser Lys Gly Ala Ala Phe Val Ser Lys Gln Glu Gly Ser 30 35 40gag gta gtg aag aga ccc agg cgc tac ctg tat caa tgg ctg gga gcc 195Glu Val Val Lys Arg Pro Arg Arg Tyr Leu Tyr Gln Trp Leu Gly Ala 45 50 55cca gtc ccc tac ccg gat ccc ctg gag ccc agg agg gag gtg tgt gag 243Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Arg Glu Val Cys Glu60 65 70 75ctc aat ccg gac tgt gac gag ttg gct gac cac atc ggc ttt cag gag 291Leu Asn Pro Asp Cys Asp Glu Leu Ala Asp His Ile Gly Phe Gln Glu 80 85 90gcc tat cgg cgc ttc tac ggc ccg gtc tagggtgtcg ctctgctggc 338Ala Tyr Arg Arg Phe Tyr Gly Pro Val 95 100ctggccggca accccagttc tgctcctctc caggcaccct tctttcctct tccccttgcc 398cttgccctga cctcccagcc ctatggatgt ggggtcccca tcatcccagc tgctcccaaa 458taaactccag aagaggaatc tgaaaaaaaa aaaaaaaaaa 4982100PRTHomo sapiens 2Met Arg Ala Leu Thr Leu Leu Ala Leu Leu Ala Leu Ala Ala Leu Cys1 5 10 15Ile Ala Gly Gln Ala Gly Ala Lys Pro Ser Gly Ala Glu Ser Ser Lys 20 25 30Gly Ala Ala Phe Val Ser Lys Gln Glu Gly Ser Glu Val Val Lys Arg 35 40 45Pro Arg Arg Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro 50 55 60Asp Pro Leu Glu Pro Arg Arg Glu Val Cys Glu Leu Asn Pro Asp Cys65 70 75 80Asp Glu Leu Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe 85 90 95Tyr Gly Pro Val 1003494DNAmus musculus 3agaacagaca agtcccacac agcagcttgg cccagaccta gcagacacca tgaggaccat 60ctttctgctc actctgctga ccctggctgc gctctgtctc tctgacctca cagatgccaa 120gcccagcggc cctgagtctg acaaagcctt catgtccaag caggagggca ataaggtagt 180gaacagactc cggcgctacc ttggagcctc agtccccagc ccagatcccc tggagcccac 240ccgggagcag tgtgagctta accctgcttg tgacgagcta tcagaccagt atggcttgaa 300gaccgcctac aaacgcatct atggtatcac tatttaggac ctgtgctgcc ctaaagccaa 360actctggcag ctcggctttg gctgctctcc gggacttgat cctccctgtc ctctctctct 420gccctgcaag tatggatgtc acagcagctc caaaataaag ttcagatgag gaagtgcaaa 480aaaaaaaaaa aaaa 4944470DNAmus musculusCDS(49)..(333) 4gaacagacaa gtcccacaca gcagcttggt gcacacctag cagacacc atg agg acc 57 Met Arg Thr 1ctc tct ctg ctc act ctg ctg gcc ctg gct gcg ctc tgt ctc tct gac 105Leu Ser Leu Leu Thr Leu Leu Ala Leu Ala Ala Leu Cys Leu Ser Asp 5 10 15ctc aca gat ccc aag ccc agc ggc cct gag tct gac aaa gcc ttc atg 153Leu Thr Asp Pro Lys Pro Ser Gly Pro Glu Ser Asp Lys Ala Phe Met20 25 30 35tcc aag cag gag ggc aat aag gta gtg aac aga ctc cgg cgc tac ctt 201Ser Lys Gln Glu Gly Asn Lys Val Val Asn Arg Leu Arg Arg Tyr Leu 40 45 50gga gcc tca gtc ccc agc cca gat ccc ctg gag ccc acc cgg gag cag 249Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr Arg Glu Gln 55 60 65tgt gag ctt aac cct gct tgt gac gag cta tca gac cag tat ggc ttg 297Cys Glu Leu Asn Pro Ala Cys Asp Glu Leu Ser Asp Gln Tyr Gly Leu 70 75 80aag acc gcc tac aaa cgc atc tac ggt atc act att taggacctgt 343Lys Thr Ala Tyr Lys Arg Ile Tyr Gly Ile Thr Ile 85 90 95gctgccctaa agccaaactc tggcagctcg gctttggctg ctctccggga cttgatcctc 403cctgtcctct ctctctgccc tgcaagtatg gatgtcacag cagctccaaa ataaagttca 463gatgagg 470595PRTmus musculus 5Met Arg Thr Leu Ser Leu Leu Thr Leu Leu Ala Leu Ala Ala Leu Cys1 5 10 15Leu Ser Asp Leu Thr Asp Pro Lys Pro Ser Gly Pro Glu Ser Asp Lys 20 25 30Ala Phe Met Ser Lys Gln Glu Gly Asn Lys Val Val Asn Arg Leu Arg 35 40 45Arg Tyr Leu Gly Ala Ser Val Pro Ser Pro Asp Pro Leu Glu Pro Thr 50 55 60Arg Glu Gln Cys Glu Leu Asn Pro Ala Cys Asp Glu Leu Ser Asp Gln65 70 75 80Tyr Gly Leu Lys Thr Ala Tyr Lys Arg Ile Tyr Gly Ile Thr Ile 85 90 9564592DNAhomo sapiensCDS(85)..(816) 6aggctgttga ggctgggcca tctcctcctc acttccattc tgactgcagt ctgtggttct 60gattccatac cagaggggct cagg atg ctg ttg ctg gga gct gtt cta ctg 111 Met Leu Leu Leu Gly Ala Val Leu Leu 1 5cta tta gct ctg ccc ggt cat gac cag gaa acc acg act caa ggg ccc 159Leu Leu Ala Leu Pro Gly His Asp Gln Glu Thr Thr Thr Gln Gly Pro10 15 20 25gga gtc ctg ctt ccc ctg ccc aag ggg gcc tgc aca ggt tgg atg gcg 207Gly Val Leu Leu Pro Leu Pro Lys Gly Ala Cys Thr Gly Trp Met Ala 30 35 40ggc atc cca ggg cat ccg ggc cat aat ggg gcc cca ggc cgt gat ggc 255Gly Ile Pro Gly His Pro Gly His Asn Gly Ala Pro Gly Arg Asp Gly 45 50 55aga gat ggc acc cct ggt gag aag ggt gag aaa gga gat cca ggt ctt 303Arg Asp Gly Thr Pro Gly Glu Lys Gly Glu Lys Gly Asp Pro Gly Leu 60 65 70att ggt cct aag gga gac atc ggt gaa acc gga gta ccc ggg gct gaa 351Ile Gly Pro Lys Gly Asp Ile Gly Glu Thr Gly Val Pro Gly Ala Glu 75 80 85ggt ccc cga ggc ttt ccg gga atc caa ggc agg aaa gga gaa cct gga 399Gly Pro Arg Gly Phe Pro Gly Ile Gln Gly Arg Lys Gly Glu Pro Gly90 95 100 105gaa ggt gcc tat gta tac cgc tca gca ttc agt gtg gga ttg gag act 447Glu Gly Ala Tyr Val Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr 110 115 120tac gtt act atc ccc aac atg ccc att cgc ttt acc aag atc ttc tac 495Tyr Val Thr Ile Pro Asn Met Pro Ile Arg Phe Thr Lys Ile Phe Tyr 125 130 135aat cag caa aac cac tat gat ggc tcc act ggt aaa ttc cac tgc aac 543Asn Gln Gln Asn His Tyr Asp Gly Ser Thr Gly Lys Phe His Cys Asn 140 145 150att cct ggg ctg tac tac ttt gcc tac cac atc aca gtc tat atg aag 591Ile Pro Gly Leu Tyr Tyr Phe Ala Tyr His Ile Thr Val Tyr Met Lys 155 160 165gat gtg aag gtc agc ctc ttc aag aag gac aag gct atg ctc ttc acc 639Asp Val Lys Val Ser Leu Phe Lys Lys Asp Lys Ala Met Leu Phe Thr170 175 180 185tat gat cag tac cag gaa aat aat gtg gac cag gcc tcc ggc tct gtg 687Tyr Asp Gln Tyr Gln Glu Asn Asn Val Asp Gln Ala Ser Gly Ser Val 190 195 200ctc ctg cat ctg gag gtg ggc gac caa gtc tgg ctc cag gtg tat ggg 735Leu Leu His Leu Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly 205 210 215gaa gga gag cgt aat gga ctc tat gct gat aat gac aat gac tcc acc 783Glu Gly Glu Arg Asn Gly Leu Tyr Ala Asp Asn Asp Asn Asp Ser Thr 220 225 230ttc aca ggc ttt ctt ctc tac cat gac acc aac tgatcaccac taactcagag 836Phe Thr Gly Phe Leu Leu Tyr His Asp Thr Asn 235 240cctcctccag gccaaacagc cccaaagtca attaaaggct ttcagtacgg ttaggaagtt 896gattattatt tagttggagg cctttagata ttattcattc atttactcat tcatttattc 956attcattcat caagtaactt taaaaaaatc atatgctatg ttcccagtcc tggggagctt 1016cacaaacatg accagataac tgactagaaa gaagtagttg acagtgctat tttgtgccca 1076ctgtctctcc tgatgctcat atcaatccta taaggcacag ggaacaagca ttctcctgtt 1136tttacagatt gtatcctgag gctgagagag ttaagtgaat gtctaaggtc acacagtatt 1196aagtgacagt gctagaaatc aaacccagag ctgtggactt tgttcactag actgtgccct 1256tttatagagg tacatgttct ctttggagtg ttggtaggtg tctgtttccc acctcacctg 1316agagccattg aatttgcctt cctcatgaat taaaacctcc cccaagcaga gcttcctcag 1376agaaagtggt tctatgatga agtcctgtct tggaaggact actactcaat ggcccctgca 1436ctactctact tcctcttacc tatgtccctt ctcatgcctt tccctccaac ggggaaagcc 1496aactccatct ctaagtgctg aactcatccc tgttcctcaa ggccacctgg ccaggagctt 1556ctctgatgtg atatccactt tttttttttt tgagatggag tctcactctg tcacccaggc 1616tggagtacag tgacacgacc tcggctcact gcagcctcct tctcctgggt ccaagcaatt 1676attgtgcctc agcctcccga gtagctgaga cttcaggtgc attccaccac acatggctaa 1736tttttgtatt tttagtagaa atggggtttc gtcatgttgg ccaggctggt ctcgaactcc 1796tggcctaggt gatccacccg cctcgacctc ccaaagtgct gggattacag gcatgagcca 1856ccatgcccag tcgatatctc actttttatt ttgccatgga tgagagtcct gggtgtgagg 1916aacacctccc accaggctag aggcaactgc ccaggaagga ctgtgcttcc gtcacctcta 1976aatcccttgc agatccttga taaatgcctc atgaagacca atctcttgaa tcccatatct 2036acccagaatt aactccattc cagtctctgc atgtaatcag ttttatccac agaaacattt 2096tcattttagg aaatccctgg ttttaagtat caatccttgt tcagctggac aatatgaatc 2156ttttccactg aagttaggga tgactgtgat tttcagaaca cgtccagaat ttttcatcaa 2216gaaggtagct tgagcctgaa atgcaaaacc catggaggaa ttctgaagcc attgtctcct 2276tgagtaccaa cagggtcagg gaagactggg cctcctgaat ttattattgt tctttaagaa 2336ttacaggttg aggtagttga tggtggtaaa cattctctca ggagacaata actccagtga 2396tgttcttcaa agattttagc aaaaacagag taaatagcat tctctatcaa tatataaatt 2456taaaaaacta tctttttgct tacagtttta aattctgaac aattctctct tatatgtgta 2516ttgctaatca ttaaggtatt attttttcca catataaagc tttgtctttt tgttgttgtt 2576gttgttttta agatggagtt tccctctgtt gccaggctag agtgcagtgg catgatctcg 2636gcttactgca acctttgcct cccaggttca agcgattctt ctgcctcagc ctcccgagta 2696gctgggacca caggtgccta ccaccatgcc aggctaattt ttgtattttt agtaaagaca 2756gggtttcacc atattggcca ggctggtctc gaactcctga ccttgtgatc tgcccgcctc 2816catttttgtt gttatttttt gagaaagata gatatgaggt ttagagaggg atgaagaggt 2876gagagtaagc cttgtgttag tcagaactct gtgttgtgaa tgtcattcac aacagaaaac 2936ccaaaatatt atgcaaacta ctgtaagcaa gaaaaataaa ggaaaaatgg aaacatttat 2996tcctttgcat aatagaaatt accagagttg ttctgtcttt agataaggtt tgaaccaaag 3056ctcaaaacaa tcaagaccct tttctgtatg tccttctgtt ctgccttccg cagtgtaggc 3116tttaccctca ggtgctacac agtatagttc tagggtttcc ctcccgatat caaaaagact 3176gtggcctgcc cagctctcgt atccccaagc cacaccatct ggctaaatgg acatcatgtt 3236ttctggtgat gcccaaagag gagagaggaa gctctctttc ccagatgccc cagcaagtgt 3296aaccttgcat ctcattgctc tggctgagtt gtgtgcctgt ttctgaccaa tcactgagtc 3356aggaggatga aatattcata ttgacttaat tgcagcttaa gttaggggta tgtagaggta 3416ttttccctaa agcaaaattg ggacactgtt atcagaaata ggagagtgga tgatagatgc 3476aaaataatac ctgtccacaa caaactctta atgctgtgtt tgagctttca tgagtttccc 3536agagagacat agctggaaaa ttcctattga ttttctctaa aatttcaaca agtagctaaa 3596gtctggctat gctcacagtc tcacatctgg ttggggtggg ctccttacag aacacgcttt 3656cacagttacc ctaaactctc tggggcaggg ttattccttt gtggaaccag aggcacagag 3716agagtcaact gaggccaaaa gaggcctgag agaaactgag gtcaagattt caggattaat 3776ggtcctgtga tgctttgaag tacaattgtg gatttgtcca attctcttta gttctgtcag 3836cttttgcttc atatatttta gcgctctatt attagatata tacatgttta gtattatgtc 3896ttattggtgc atttactctc ttatcattat gtaatgtcct tctttatctg tgataatttt 3956ctgtgttctg aagtctactt tgtctaaaaa taacatacgc actcaacttc cttttctttc 4016ttccttcctt tctttcttcc ttcctttctt tctctctctc tctctttcct tccttccttc 4076ctccttttct ttctctctct ctctctctct ctttttttga cagactctcg ttctgtggcc 4136ctggctggag ttcagtggtg tgatcttggc tcactgctac ctctaccatg agcaattctc 4196ctgcctcagc ctcccaagta gctggaacta caggctcatg ccactgcgcc cagctaattt 4256ttgtattttt cgtagagacg gggtttcacc acattcgtca ggttggtttc aaactcctga 4316ctttgtgatc cacccgcctc ggcctcccaa agtgctggga ttacaggcat gagccatcac 4376acctggtcaa ctttcttttg attagtgttt ttgtggtata tctttttcca tcatgttact 4436ttaaatatat ctatattatt gtatttaaaa tgtgtttctt acagactgca tgtagttggg 4496tataattttt atccagtcta aaaatatctg tcttttaatt ggtgtttaga caatttatat 4556ttaataaaat tgttgaattt aaaaaaaaaa aaaaaa 45927244PRThomo sapiens 7Met Leu Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His1 5 10 15Asp Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 20 25 30Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly 35 40 45His Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 50 55 60Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile65 70 75 80Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 90 95Ile Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg 100 105 110Ser Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met 115 120 125Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp 130 135 140Gly Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe145 150 155 160Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe 165 170 175Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn 180 185 190Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly 195 200 205Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu 210 215 220Tyr Ala Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr225 230 235 240His Asp Thr Asn81233DNAmus musculusCDS(115)..(855) 8taagctgggg tctgcctgtc cccatgagta ccagactaat gagacctggc cactttctcc 60tcatttctgt ctgtacgatt gtcagtggat ctgacgacac caaaagggct cagg atg 117 Met 1cta ctg ttg caa gct ctc ctg ttc ctc tta atc ctg ccc agt cat gcc 165Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His Ala 5 10 15gaa gat gac gtt act aca act gaa gag cta gct cct gct ttg gtc cct 213Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val Pro 20 25 30cca ccc aag gga act tgt gca ggt tgg atg gca ggc atc cca gga cat 261Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly His 35 40 45cct ggc cac aat ggc aca cca ggc cgt gat ggc aga gat ggc act cct 309Pro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro50 55 60 65gga gag aag gga gag aaa gga gat gca ggt ctt ctt ggt cct aag ggt 357Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys Gly 70 75 80gag aca gga gat gtt gga atg aca gga gct gaa ggg cca cgg ggc ttc 405Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly Phe 85 90 95ccc gga acc cct ggc agg aaa gga gag cct gga gaa gcc gct tat gtg 453Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr Val 100 105 110tat cgc tca gcg ttc agt gtg ggg ctg gag acc cgc gtc act gtt ccc 501Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val Pro 115 120 125aat gta ccc att cgc ttt act aag atc ttc tac aac caa cag aat cat 549Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His130 135 140 145tat gac ggc agc act ggc aag ttc tac tgc aac att ccg gga ctc tac 597Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu Tyr 150 155 160tac ttc tct tac cac atc acg gtg tac atg aaa gat gtg aag gtg agc 645Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser 165 170 175ctc ttc aag aag gac aag gcc gtt ctc ttc acc tac gac cag tat cag 693Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr Gln 180 185 190gaa aag aat gtg gac cag gcc tct ggc tct gtg ctc ctc cat ctg gag 741Glu Lys Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu 195 200 205gtg gga gac caa gtc tgg ctc cag gtg tat ggg gat ggg gac cac aat 789Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His Asn210 215 220 225gga ctc tat gca gat aac gtc aac gac tct aca ttt act ggc ttt ctt 837Gly Leu Tyr Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe Leu 230 235 240ctc tac cat gat acc aac tgactgcaac tacccatagc ccatacacca 885Leu Tyr His Asp Thr Asn 245ggagaatcat ggaacagtcg acacactttc agcttagttt gagagattga ttttattgct 945tagtttgaga gtcctgagta ttatccacac gtgtactcac ttgttcatta aacgacttta 1005taaaaaataa tttgtgttcc tagtccagaa aaaaaggcac tccctggtct ccacgactct 1065tacatggtag caataacaga

atgaaaatca catttggtat gggggcttca caatattcgc 1125atgactgtct ggaagtagac catgctattt ttctgctcac tgtacacaaa tattgttcac 1185ataaacccta taatgtaaat atgaaataca gtgattactc ttctcact 12339247PRTmus musculus 9Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His1 5 10 15Ala Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 20 25 30Pro Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 40 45His Pro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 50 55 60Pro Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys65 70 75 80Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 90 95Phe Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr 100 105 110Val Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val 115 120 125Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn 130 135 140His Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu145 150 155 160Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val 165 170 175Ser Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr 180 185 190Gln Glu Lys Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu 195 200 205Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His 210 215 220Asn Gly Leu Tyr Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe225 230 235 240Leu Leu Tyr His Asp Thr Asn 245103236DNAhomo sapiensCDS(29)..(2302) 10gtgacccacc tgcctcctcc gcagagca atg gcg gtg tct gcc ggg tcc gcg 52 Met Ala Val Ser Ala Gly Ser Ala 1 5cgg acc tcg ccc agc tca gat aaa gta cag aaa gac aag gct gaa ctg 100Arg Thr Ser Pro Ser Ser Asp Lys Val Gln Lys Asp Lys Ala Glu Leu 10 15 20atc tca ggg ccc agg cag gac agc cga ata ggg aaa ctc ttg ggt ttt 148Ile Ser Gly Pro Arg Gln Asp Ser Arg Ile Gly Lys Leu Leu Gly Phe25 30 35 40gag tgg aca gat ttg tcc agt tgg cgg agg ctg gtg acc ctg ctg aat 196Glu Trp Thr Asp Leu Ser Ser Trp Arg Arg Leu Val Thr Leu Leu Asn 45 50 55cga cca acg gac cct gca agc tta gct gtc ttt cgt ttt ctt ttt ggg 244Arg Pro Thr Asp Pro Ala Ser Leu Ala Val Phe Arg Phe Leu Phe Gly 60 65 70ttc ttg atg gtg cta gac att ccc cag gag cgg ggg ctc agc tct ctg 292Phe Leu Met Val Leu Asp Ile Pro Gln Glu Arg Gly Leu Ser Ser Leu 75 80 85gac cgg aaa tac ctt gat ggg ctg gat gtg tgc cgc ttc ccc ttg ctg 340Asp Arg Lys Tyr Leu Asp Gly Leu Asp Val Cys Arg Phe Pro Leu Leu 90 95 100gat gcc cta cgc cca ctg cca ctt gac tgg atg tat ctt gtc tac acc 388Asp Ala Leu Arg Pro Leu Pro Leu Asp Trp Met Tyr Leu Val Tyr Thr105 110 115 120atc atg ttt ctg ggg gca ctg ggc atg atg ctg ggc ctg tgc tac cgg 436Ile Met Phe Leu Gly Ala Leu Gly Met Met Leu Gly Leu Cys Tyr Arg 125 130 135ata agc tgt gtg tta ttc ctg ctg cca tac tgg tat gtg ttt ctc ctg 484Ile Ser Cys Val Leu Phe Leu Leu Pro Tyr Trp Tyr Val Phe Leu Leu 140 145 150gac aag aca tca tgg aac aac cac tcc tat ctg tat ggg ttg ttg gcc 532Asp Lys Thr Ser Trp Asn Asn His Ser Tyr Leu Tyr Gly Leu Leu Ala 155 160 165ttt cag cta aca ttc atg gat gca aac cac tac tgg tct gtg gac ggt 580Phe Gln Leu Thr Phe Met Asp Ala Asn His Tyr Trp Ser Val Asp Gly 170 175 180ctg ctg aat gcc cat agg agg aat gcc cac gtg ccc ctt tgg aac tat 628Leu Leu Asn Ala His Arg Arg Asn Ala His Val Pro Leu Trp Asn Tyr185 190 195 200gca gtg ctc cgt ggc cag atc ttc att gtg tac ttc att gcg ggt gtg 676Ala Val Leu Arg Gly Gln Ile Phe Ile Val Tyr Phe Ile Ala Gly Val 205 210 215aaa aag ctg gat gca gac tgg gtt gaa ggc tat tcc atg gaa tat ttg 724Lys Lys Leu Asp Ala Asp Trp Val Glu Gly Tyr Ser Met Glu Tyr Leu 220 225 230tcc cgg cac tgg ctc ttc agt ccc ttc aaa ctg ctg ttg tct gag gag 772Ser Arg His Trp Leu Phe Ser Pro Phe Lys Leu Leu Leu Ser Glu Glu 235 240 245ctg act agc ctg ctg gtc gtg cac tgg ggt ggg ctg ctg ctt gac ctc 820Leu Thr Ser Leu Leu Val Val His Trp Gly Gly Leu Leu Leu Asp Leu 250 255 260tca gct ggt ttc ctg ctc ttt ttt gat gtc tca aga tcc att ggc ctg 868Ser Ala Gly Phe Leu Leu Phe Phe Asp Val Ser Arg Ser Ile Gly Leu265 270 275 280ttc ttt gtg tcc tac ttc cac tgc atg aat tcc cag ctt ttc agc att 916Phe Phe Val Ser Tyr Phe His Cys Met Asn Ser Gln Leu Phe Ser Ile 285 290 295ggt atg ttc tcc tac gtc atg ctg gcc agc agc cct ctc ttc tgc tcc 964Gly Met Phe Ser Tyr Val Met Leu Ala Ser Ser Pro Leu Phe Cys Ser 300 305 310cct gag tgg cct cgg aag ctg gtg tcc tac tgc ccc cga agg ttg caa 1012Pro Glu Trp Pro Arg Lys Leu Val Ser Tyr Cys Pro Arg Arg Leu Gln 315 320 325caa ctg ttg ccc ctc aag gca gcc cct cag ccc agt gtt tcc tgt gtg 1060Gln Leu Leu Pro Leu Lys Ala Ala Pro Gln Pro Ser Val Ser Cys Val 330 335 340tat aag agg agc cgg ggc aaa agt ggc cag aag cca ggg ctg cgc cat 1108Tyr Lys Arg Ser Arg Gly Lys Ser Gly Gln Lys Pro Gly Leu Arg His345 350 355 360cag ctg gga gct gcc ttc acc ctg ctc tac ctc ctg gag cag cta ttc 1156Gln Leu Gly Ala Ala Phe Thr Leu Leu Tyr Leu Leu Glu Gln Leu Phe 365 370 375ctg ccc tat tct cat ttt ctc acc cag ggc tat aac aac tgg aca aat 1204Leu Pro Tyr Ser His Phe Leu Thr Gln Gly Tyr Asn Asn Trp Thr Asn 380 385 390ggg ctg tat ggc tat tcc tgg gac atg atg gtg cac tcc cgc tcc cac 1252Gly Leu Tyr Gly Tyr Ser Trp Asp Met Met Val His Ser Arg Ser His 395 400 405cag cac gtg aag atc acc tac cgt gat ggc cgc act ggc gaa ctg ggc 1300Gln His Val Lys Ile Thr Tyr Arg Asp Gly Arg Thr Gly Glu Leu Gly 410 415 420tac ctt aac cct ggg gta ttt aca cag agt cgg cga tgg aag gat cat 1348Tyr Leu Asn Pro Gly Val Phe Thr Gln Ser Arg Arg Trp Lys Asp His425 430 435 440gca gac atg ctg aag caa tat gcc act tgc ctg agc cgc ctg ctt ccc 1396Ala Asp Met Leu Lys Gln Tyr Ala Thr Cys Leu Ser Arg Leu Leu Pro 445 450 455aag tat aat gtc act gag ccc cag atc tac ttt gat att tgg gtc tcc 1444Lys Tyr Asn Val Thr Glu Pro Gln Ile Tyr Phe Asp Ile Trp Val Ser 460 465 470atc aat gac cgc ttc cag cag agg att ttt gac cct cgt gtg gac atc 1492Ile Asn Asp Arg Phe Gln Gln Arg Ile Phe Asp Pro Arg Val Asp Ile 475 480 485gtg cag gcc gct tgg tca ccc ttt cag cgc aca tcc tgg gtg caa cca 1540Val Gln Ala Ala Trp Ser Pro Phe Gln Arg Thr Ser Trp Val Gln Pro 490 495 500ctc ttg atg gac ctg tct ccc tgg agg gcc aag tta cag gaa atc aag 1588Leu Leu Met Asp Leu Ser Pro Trp Arg Ala Lys Leu Gln Glu Ile Lys505 510 515 520agc agc cta gac aac cac act gag gtg gtc ttc att gca gat ttc cct 1636Ser Ser Leu Asp Asn His Thr Glu Val Val Phe Ile Ala Asp Phe Pro 525 530 535gga ctg cac ttg gag aat ttt gtg agt gaa gac ctg ggc aac act agc 1684Gly Leu His Leu Glu Asn Phe Val Ser Glu Asp Leu Gly Asn Thr Ser 540 545 550atc cag ctg ctg cag ggg gaa gtg act gtg gag ctt gtg gca gaa cag 1732Ile Gln Leu Leu Gln Gly Glu Val Thr Val Glu Leu Val Ala Glu Gln 555 560 565aag aac cag act ctt cga gag gga gaa aaa atg cag ttg cct gct ggt 1780Lys Asn Gln Thr Leu Arg Glu Gly Glu Lys Met Gln Leu Pro Ala Gly 570 575 580gag tac cat aag gtg tat acg aca tca cct agc cct tct tgc tac atg 1828Glu Tyr His Lys Val Tyr Thr Thr Ser Pro Ser Pro Ser Cys Tyr Met585 590 595 600tac gtc tat gtc aac act aca gag ctt gca ctg gag caa gac ctg gca 1876Tyr Val Tyr Val Asn Thr Thr Glu Leu Ala Leu Glu Gln Asp Leu Ala 605 610 615tat ctg caa gaa tta aag gaa aag gtg gag aat gga agt gaa aca ggg 1924Tyr Leu Gln Glu Leu Lys Glu Lys Val Glu Asn Gly Ser Glu Thr Gly 620 625 630cct cta ccc cca gag ctg cag cct ctg ttg gaa ggg gaa gta aaa ggg 1972Pro Leu Pro Pro Glu Leu Gln Pro Leu Leu Glu Gly Glu Val Lys Gly 635 640 645ggc cct gag cca aca cct ctg gtt cag acc ttt ctt aga cgc caa caa 2020Gly Pro Glu Pro Thr Pro Leu Val Gln Thr Phe Leu Arg Arg Gln Gln 650 655 660agg ctc cag gag att gaa cgc cgg cga aat act cct ttc cat gag cga 2068Arg Leu Gln Glu Ile Glu Arg Arg Arg Asn Thr Pro Phe His Glu Arg665 670 675 680ttc ttc cgc ttc ttg ttg cga aag ctc tat gtc ttt cgc cgc agc ttc 2116Phe Phe Arg Phe Leu Leu Arg Lys Leu Tyr Val Phe Arg Arg Ser Phe 685 690 695ctg atg act tgt atc tca ctt cga aat ctg ata tta ggc cgt cct tcc 2164Leu Met Thr Cys Ile Ser Leu Arg Asn Leu Ile Leu Gly Arg Pro Ser 700 705 710ctg gag cag ctg gcc cag gag gtg act tat gca aac ttg aga ccc ttt 2212Leu Glu Gln Leu Ala Gln Glu Val Thr Tyr Ala Asn Leu Arg Pro Phe 715 720 725gag gca gtt gga gaa ctg aat ccc tca aac acg gat tct tca cat tct 2260Glu Ala Val Gly Glu Leu Asn Pro Ser Asn Thr Asp Ser Ser His Ser 730 735 740aat cct cct gag tca aat cct gat cct gtc cac tca gag ttc 2302Asn Pro Pro Glu Ser Asn Pro Asp Pro Val His Ser Glu Phe745 750 755tgaagggggc cagatgttgg gtgcagatgt agaagcagcc agtcacagac ccattctatg 2362caatggacat ttatttgaaa aaaattctca aaagtttttt tttttttttt gggggggcgg 2422ggttctaaag ctgtttttaa ctccgagatt acaacttaga ggaaccaagg aaataaagca 2482aataagattt aacaacccaa gattaagagg ccaggaagag gttagacgca atgtgaaact 2542gtcctcctag gataaggttt aaagtggctt tttgggggct gggtgccgtg gctcacgcct 2602gtaatcccag cattttggga ggctgaggtg ggcagatcac ttgaggccag gagttcgaga 2662ccagcctggc caacatggca aaaccccttc tctactaaaa atacaaaaat tagccagacg 2722tggtggtggg tgcctgtaat ccaactaccc aggaggctga ggcatgagaa tcgcttgggc 2782ccaggaggtg gaggttgcag tgagccgaga tcgagccact gcactcctgg gcaacagagc 2842aagacttcgt ctcaaaataa ataaataaag tggctcttgg ggaaaagcaa tttaatgtac 2902cacgatgaat agctaactgt tcccaagtgt ttgctatgtg caacacaccg cgtgagcagt 2962gttacctgca ttattacatt aggctgagag gtaaaataat ttgcccgaag acatacagct 3022agtgacgaat ggactgatgg tttgaactta acgtctattt gacttaaggt cctgcaccct 3082gccacttgta attttcagaa tcactgataa tctgaaataa tgcagcttaa aacatgtttt 3142cttaattaaa agtataaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3202aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 323611758PRThomo sapiens 11Met Ala Val Ser Ala Gly Ser Ala Arg Thr Ser Pro Ser Ser Asp Lys1 5 10 15Val Gln Lys Asp Lys Ala Glu Leu Ile Ser Gly Pro Arg Gln Asp Ser 20 25 30Arg Ile Gly Lys Leu Leu Gly Phe Glu Trp Thr Asp Leu Ser Ser Trp 35 40 45Arg Arg Leu Val Thr Leu Leu Asn Arg Pro Thr Asp Pro Ala Ser Leu 50 55 60Ala Val Phe Arg Phe Leu Phe Gly Phe Leu Met Val Leu Asp Ile Pro65 70 75 80Gln Glu Arg Gly Leu Ser Ser Leu Asp Arg Lys Tyr Leu Asp Gly Leu 85 90 95Asp Val Cys Arg Phe Pro Leu Leu Asp Ala Leu Arg Pro Leu Pro Leu 100 105 110Asp Trp Met Tyr Leu Val Tyr Thr Ile Met Phe Leu Gly Ala Leu Gly 115 120 125Met Met Leu Gly Leu Cys Tyr Arg Ile Ser Cys Val Leu Phe Leu Leu 130 135 140Pro Tyr Trp Tyr Val Phe Leu Leu Asp Lys Thr Ser Trp Asn Asn His145 150 155 160Ser Tyr Leu Tyr Gly Leu Leu Ala Phe Gln Leu Thr Phe Met Asp Ala 165 170 175Asn His Tyr Trp Ser Val Asp Gly Leu Leu Asn Ala His Arg Arg Asn 180 185 190Ala His Val Pro Leu Trp Asn Tyr Ala Val Leu Arg Gly Gln Ile Phe 195 200 205Ile Val Tyr Phe Ile Ala Gly Val Lys Lys Leu Asp Ala Asp Trp Val 210 215 220Glu Gly Tyr Ser Met Glu Tyr Leu Ser Arg His Trp Leu Phe Ser Pro225 230 235 240Phe Lys Leu Leu Leu Ser Glu Glu Leu Thr Ser Leu Leu Val Val His 245 250 255Trp Gly Gly Leu Leu Leu Asp Leu Ser Ala Gly Phe Leu Leu Phe Phe 260 265 270Asp Val Ser Arg Ser Ile Gly Leu Phe Phe Val Ser Tyr Phe His Cys 275 280 285Met Asn Ser Gln Leu Phe Ser Ile Gly Met Phe Ser Tyr Val Met Leu 290 295 300Ala Ser Ser Pro Leu Phe Cys Ser Pro Glu Trp Pro Arg Lys Leu Val305 310 315 320Ser Tyr Cys Pro Arg Arg Leu Gln Gln Leu Leu Pro Leu Lys Ala Ala 325 330 335Pro Gln Pro Ser Val Ser Cys Val Tyr Lys Arg Ser Arg Gly Lys Ser 340 345 350Gly Gln Lys Pro Gly Leu Arg His Gln Leu Gly Ala Ala Phe Thr Leu 355 360 365Leu Tyr Leu Leu Glu Gln Leu Phe Leu Pro Tyr Ser His Phe Leu Thr 370 375 380Gln Gly Tyr Asn Asn Trp Thr Asn Gly Leu Tyr Gly Tyr Ser Trp Asp385 390 395 400Met Met Val His Ser Arg Ser His Gln His Val Lys Ile Thr Tyr Arg 405 410 415Asp Gly Arg Thr Gly Glu Leu Gly Tyr Leu Asn Pro Gly Val Phe Thr 420 425 430Gln Ser Arg Arg Trp Lys Asp His Ala Asp Met Leu Lys Gln Tyr Ala 435 440 445Thr Cys Leu Ser Arg Leu Leu Pro Lys Tyr Asn Val Thr Glu Pro Gln 450 455 460Ile Tyr Phe Asp Ile Trp Val Ser Ile Asn Asp Arg Phe Gln Gln Arg465 470 475 480Ile Phe Asp Pro Arg Val Asp Ile Val Gln Ala Ala Trp Ser Pro Phe 485 490 495Gln Arg Thr Ser Trp Val Gln Pro Leu Leu Met Asp Leu Ser Pro Trp 500 505 510Arg Ala Lys Leu Gln Glu Ile Lys Ser Ser Leu Asp Asn His Thr Glu 515 520 525Val Val Phe Ile Ala Asp Phe Pro Gly Leu His Leu Glu Asn Phe Val 530 535 540Ser Glu Asp Leu Gly Asn Thr Ser Ile Gln Leu Leu Gln Gly Glu Val545 550 555 560Thr Val Glu Leu Val Ala Glu Gln Lys Asn Gln Thr Leu Arg Glu Gly 565 570 575Glu Lys Met Gln Leu Pro Ala Gly Glu Tyr His Lys Val Tyr Thr Thr 580 585 590Ser Pro Ser Pro Ser Cys Tyr Met Tyr Val Tyr Val Asn Thr Thr Glu 595 600 605Leu Ala Leu Glu Gln Asp Leu Ala Tyr Leu Gln Glu Leu Lys Glu Lys 610 615 620Val Glu Asn Gly Ser Glu Thr Gly Pro Leu Pro Pro Glu Leu Gln Pro625 630 635 640Leu Leu Glu Gly Glu Val Lys Gly Gly Pro Glu Pro Thr Pro Leu Val 645 650 655Gln Thr Phe Leu Arg Arg Gln Gln Arg Leu Gln Glu Ile Glu Arg Arg 660 665 670Arg Asn Thr Pro Phe His Glu Arg Phe Phe Arg Phe Leu Leu Arg Lys 675 680 685Leu Tyr Val Phe Arg Arg Ser Phe Leu Met Thr Cys Ile Ser Leu Arg 690 695 700Asn Leu Ile Leu Gly Arg Pro Ser Leu Glu Gln Leu Ala Gln Glu Val705 710 715 720Thr Tyr Ala Asn Leu Arg Pro Phe Glu Ala Val Gly Glu Leu Asn Pro 725 730 735Ser Asn Thr Asp Ser Ser His Ser Asn Pro Pro Glu Ser Asn Pro Asp 740 745 750Pro Val His Ser Glu Phe 755122905DNAmus musculusCDS(75)..(2345) 12agacagcaag tctaagtctg gaggttccac tgggtccgac ctggctgcag agaggctcac 60ctgtccctgc agtc atg gct gtg cac cgc ggc tcc gca ctg gtt gct ccc 110 Met Ala Val His Arg Gly Ser Ala Leu Val Ala Pro 1 5 10gcc tca gat aaa gta cag aaa aac aag tct gca cag aca

tca gga ctg 158Ala Ser Asp Lys Val Gln Lys Asn Lys Ser Ala Gln Thr Ser Gly Leu 15 20 25aaa cag ggc agc cga atg gag aaa att tta ggg ttt gaa tgg aca gat 206Lys Gln Gly Ser Arg Met Glu Lys Ile Leu Gly Phe Glu Trp Thr Asp 30 35 40tta tct agc tgg cag agt gtc gtg acc ctg ctt aac aaa cca acg gac 254Leu Ser Ser Trp Gln Ser Val Val Thr Leu Leu Asn Lys Pro Thr Asp45 50 55 60cct gca aac ctg gct gtc ttt cgt ttt ctc ttt gct ttc ttg atg ctg 302Pro Ala Asn Leu Ala Val Phe Arg Phe Leu Phe Ala Phe Leu Met Leu 65 70 75ctg gac att ccc cag gaa cgc ggc ctt agc tcc ctg gac cga aaa tac 350Leu Asp Ile Pro Gln Glu Arg Gly Leu Ser Ser Leu Asp Arg Lys Tyr 80 85 90ttg gat ggg ctg gat gtg tgc cgt ttc ccc ttg ctg gat gcc ttg cgc 398Leu Asp Gly Leu Asp Val Cys Arg Phe Pro Leu Leu Asp Ala Leu Arg 95 100 105cca ctg cca ctg gac tgg atg tat ctt gtc tac acc atc atg ttt ctg 446Pro Leu Pro Leu Asp Trp Met Tyr Leu Val Tyr Thr Ile Met Phe Leu 110 115 120ggg gca ctg ggc atg atg ctg ggg cta tgc tac cgg cta agc tgt gtg 494Gly Ala Leu Gly Met Met Leu Gly Leu Cys Tyr Arg Leu Ser Cys Val125 130 135 140tta ttc ctg cta ccg tac tgg tac gtg ttt ctc ctg gac aag act tcg 542Leu Phe Leu Leu Pro Tyr Trp Tyr Val Phe Leu Leu Asp Lys Thr Ser 145 150 155tgg aac aat cac tcc tat ctg tat ggt ttg ttg gcc ttt cag ttg aca 590Trp Asn Asn His Ser Tyr Leu Tyr Gly Leu Leu Ala Phe Gln Leu Thr 160 165 170ttc atg gat gca aac cac tac tgg tct gtg gat ggc ttg ctg aat gcc 638Phe Met Asp Ala Asn His Tyr Trp Ser Val Asp Gly Leu Leu Asn Ala 175 180 185cga aag aag aat gct cac gtg ccc ctt tgg aac tac aca gtt ctg cgt 686Arg Lys Lys Asn Ala His Val Pro Leu Trp Asn Tyr Thr Val Leu Arg 190 195 200ggc cag atc ttc atc gtg tac ttc atc gcg ggt gtg aag aag ctc gat 734Gly Gln Ile Phe Ile Val Tyr Phe Ile Ala Gly Val Lys Lys Leu Asp205 210 215 220gct gac tgg gtt ggg ggc tac tcc atg gag cac ctg tcc cgg cac tgg 782Ala Asp Trp Val Gly Gly Tyr Ser Met Glu His Leu Ser Arg His Trp 225 230 235ctc ttc agt ccc ttc aag ctg gtg ttg tcg gag gag ctg aca agc ctg 830Leu Phe Ser Pro Phe Lys Leu Val Leu Ser Glu Glu Leu Thr Ser Leu 240 245 250ctg gta gta cac tgg tgt ggg ctt ctc ctt gac ctc tcg gct ggc ttc 878Leu Val Val His Trp Cys Gly Leu Leu Leu Asp Leu Ser Ala Gly Phe 255 260 265ctg ctc ttc ttt gat gcc tcc aga ccc gtc ggc ctg ttc ttc gtg tcc 926Leu Leu Phe Phe Asp Ala Ser Arg Pro Val Gly Leu Phe Phe Val Ser 270 275 280tac ttt cac tgc atg aac tcg cag ctc ttc agc atc ggg atg ttt ccc 974Tyr Phe His Cys Met Asn Ser Gln Leu Phe Ser Ile Gly Met Phe Pro285 290 295 300tat gtc atg ctg gcc agc agc cct ctc ttc tgc tca gct gaa tgg cct 1022Tyr Val Met Leu Ala Ser Ser Pro Leu Phe Cys Ser Ala Glu Trp Pro 305 310 315cgg aag ttg gta gcc cga tgc ccg aaa agg ctg caa gag ctg ctg ccc 1070Arg Lys Leu Val Ala Arg Cys Pro Lys Arg Leu Gln Glu Leu Leu Pro 320 325 330acc aaa gcc gct cct cgg cct agt gct tcc tgt gtg tat aag agg tcc 1118Thr Lys Ala Ala Pro Arg Pro Ser Ala Ser Cys Val Tyr Lys Arg Ser 335 340 345cgg ggc aaa gct ggc ccg aag ccc ggg ctg cgc cac cag ctg gga gcc 1166Arg Gly Lys Ala Gly Pro Lys Pro Gly Leu Arg His Gln Leu Gly Ala 350 355 360atc ttc acc ctg ctc tac ctc cta gag cag ctc ttc ctg ccc tat tcc 1214Ile Phe Thr Leu Leu Tyr Leu Leu Glu Gln Leu Phe Leu Pro Tyr Ser365 370 375 380cac ttc ctg acc cag ggt tac aat aac tgg aca aat ggg ctg tat ggc 1262His Phe Leu Thr Gln Gly Tyr Asn Asn Trp Thr Asn Gly Leu Tyr Gly 385 390 395tat tcc tgg gac atg atg gtg cac tcc cgc tcc cac cag cac gta aag 1310Tyr Ser Trp Asp Met Met Val His Ser Arg Ser His Gln His Val Lys 400 405 410atc acc tac cgc gac ggc ctc acg ggc gag cta ggc tac ctt aac cct 1358Ile Thr Tyr Arg Asp Gly Leu Thr Gly Glu Leu Gly Tyr Leu Asn Pro 415 420 425ggg gta ttc aca cag agc cgg cga tgg aag gat cat gca gac atg ctg 1406Gly Val Phe Thr Gln Ser Arg Arg Trp Lys Asp His Ala Asp Met Leu 430 435 440aag caa tat gcc act tgc ctg agc ctc ctg ctt ccc aag tac aat gtc 1454Lys Gln Tyr Ala Thr Cys Leu Ser Leu Leu Leu Pro Lys Tyr Asn Val445 450 455 460act gag ccc cag atc tac ttt gat att tgg gtc tcc atc aat gac cgc 1502Thr Glu Pro Gln Ile Tyr Phe Asp Ile Trp Val Ser Ile Asn Asp Arg 465 470 475ttc cag cag agg ctt ttt gac cct cgt gtg gac atc gtg cag gct gtc 1550Phe Gln Gln Arg Leu Phe Asp Pro Arg Val Asp Ile Val Gln Ala Val 480 485 490tgg tcc ccc ttc cag cgc aca cct tgg gtg cag cca ctc ttg atg gat 1598Trp Ser Pro Phe Gln Arg Thr Pro Trp Val Gln Pro Leu Leu Met Asp 495 500 505tta tct ccc tgg agg acc aag tta cag gat att aag agc agt ctg gac 1646Leu Ser Pro Trp Arg Thr Lys Leu Gln Asp Ile Lys Ser Ser Leu Asp 510 515 520aac cac acc gag gtg gtc ttc att gca gat ttc cct ggg ctt cac ttg 1694Asn His Thr Glu Val Val Phe Ile Ala Asp Phe Pro Gly Leu His Leu525 530 535 540gag aat ttt gtg agt gaa gac ctg ggc aac act agc atc cag ctg ctg 1742Glu Asn Phe Val Ser Glu Asp Leu Gly Asn Thr Ser Ile Gln Leu Leu 545 550 555cag gga gaa gtc acc gtg gaa ttg gtg gca gaa cag aaa aat cag act 1790Gln Gly Glu Val Thr Val Glu Leu Val Ala Glu Gln Lys Asn Gln Thr 560 565 570ctt caa gaa gga gag aaa atg cag ttg cct gct gga gag tac cat aaa 1838Leu Gln Glu Gly Glu Lys Met Gln Leu Pro Ala Gly Glu Tyr His Lys 575 580 585gtc tat act gta tca tct agt cct tcc tgc tac atg tac gtc tat gtc 1886Val Tyr Thr Val Ser Ser Ser Pro Ser Cys Tyr Met Tyr Val Tyr Val 590 595 600aac act aca gag gtc gca ctg gag caa gac ctg gca tat ctg caa gaa 1934Asn Thr Thr Glu Val Ala Leu Glu Gln Asp Leu Ala Tyr Leu Gln Glu605 610 615 620tta aag gag aag gtg gag aac gga agt gaa aca ggg ccc ctg cct cca 1982Leu Lys Glu Lys Val Glu Asn Gly Ser Glu Thr Gly Pro Leu Pro Pro 625 630 635gaa ctt cag cct ctt ttg gaa ggg gaa gta aaa ggg ggc cct gag cca 2030Glu Leu Gln Pro Leu Leu Glu Gly Glu Val Lys Gly Gly Pro Glu Pro 640 645 650aca cct ctg gtc caa act ttt ctc aga cga cag agg aag ctc caa gaa 2078Thr Pro Leu Val Gln Thr Phe Leu Arg Arg Gln Arg Lys Leu Gln Glu 655 660 665att gaa cgc agg cga aat agc cct ttc cat gag cga ttt ctc cgc ttc 2126Ile Glu Arg Arg Arg Asn Ser Pro Phe His Glu Arg Phe Leu Arg Phe 670 675 680gtg ctg cga aag ctc tac gtc ttt cga cgc agc ttc ctg atg act cga 2174Val Leu Arg Lys Leu Tyr Val Phe Arg Arg Ser Phe Leu Met Thr Arg685 690 695 700att tca ctc cga aac ctg cta tta ggc cgc cct tcc cta gag caa cta 2222Ile Ser Leu Arg Asn Leu Leu Leu Gly Arg Pro Ser Leu Glu Gln Leu 705 710 715gcc caa gag gtg aca tat gca aac ttg cga cca ttt gaa cca gtt gat 2270Ala Gln Glu Val Thr Tyr Ala Asn Leu Arg Pro Phe Glu Pro Val Asp 720 725 730gag tca agt gct tca aac aca gat tct tca aat cac ccg tca gag cca 2318Glu Ser Ser Ala Ser Asn Thr Asp Ser Ser Asn His Pro Ser Glu Pro 735 740 745gat tct gag cat gtt cac tct gag ttc tgagggatgt acagatgctc 2365Asp Ser Glu His Val His Ser Glu Phe 750 755tgtgcagatg tgggggcagc ctgttatagg cttattgtct acgcaaagaa catatttttg 2425gagaaaaatg atatgggaca ggctttcaca gtacagccca ggctggcctc aaactcatgg 2485ttggtccctc tgcttcagcc tgttttgtaa ttacatagta tcaccaaacc tagttgcttt 2545tccctttaca ttttttcccc ttataagttc tttaaaatta tagcttacat tttttctttt 2605ttcttttttt tttttttgta ttttttcttt gtcaagacag gtctctctct gtgtagcact 2665ggctgtcctg gaactcactc tgtagtccag gctggcctcc aactcagaaa ttctcctgcc 2725tctgcctccc aagtgctggg attaaaggtg tgtgccacca cgccccactg ggcttttagt 2785ttttatagac aagatttctc catgtagacc agaccagctc tcctgagtgc tgaaattaaa 2845ggcacgggac atcactacct ggctttctta ttaaacttgt tttagtggtc tcaacaaaaa 290513757PRTmus musculus 13Met Ala Val His Arg Gly Ser Ala Leu Val Ala Pro Ala Ser Asp Lys1 5 10 15Val Gln Lys Asn Lys Ser Ala Gln Thr Ser Gly Leu Lys Gln Gly Ser 20 25 30Arg Met Glu Lys Ile Leu Gly Phe Glu Trp Thr Asp Leu Ser Ser Trp 35 40 45Gln Ser Val Val Thr Leu Leu Asn Lys Pro Thr Asp Pro Ala Asn Leu 50 55 60Ala Val Phe Arg Phe Leu Phe Ala Phe Leu Met Leu Leu Asp Ile Pro65 70 75 80Gln Glu Arg Gly Leu Ser Ser Leu Asp Arg Lys Tyr Leu Asp Gly Leu 85 90 95Asp Val Cys Arg Phe Pro Leu Leu Asp Ala Leu Arg Pro Leu Pro Leu 100 105 110Asp Trp Met Tyr Leu Val Tyr Thr Ile Met Phe Leu Gly Ala Leu Gly 115 120 125Met Met Leu Gly Leu Cys Tyr Arg Leu Ser Cys Val Leu Phe Leu Leu 130 135 140Pro Tyr Trp Tyr Val Phe Leu Leu Asp Lys Thr Ser Trp Asn Asn His145 150 155 160Ser Tyr Leu Tyr Gly Leu Leu Ala Phe Gln Leu Thr Phe Met Asp Ala 165 170 175Asn His Tyr Trp Ser Val Asp Gly Leu Leu Asn Ala Arg Lys Lys Asn 180 185 190Ala His Val Pro Leu Trp Asn Tyr Thr Val Leu Arg Gly Gln Ile Phe 195 200 205Ile Val Tyr Phe Ile Ala Gly Val Lys Lys Leu Asp Ala Asp Trp Val 210 215 220Gly Gly Tyr Ser Met Glu His Leu Ser Arg His Trp Leu Phe Ser Pro225 230 235 240Phe Lys Leu Val Leu Ser Glu Glu Leu Thr Ser Leu Leu Val Val His 245 250 255Trp Cys Gly Leu Leu Leu Asp Leu Ser Ala Gly Phe Leu Leu Phe Phe 260 265 270Asp Ala Ser Arg Pro Val Gly Leu Phe Phe Val Ser Tyr Phe His Cys 275 280 285Met Asn Ser Gln Leu Phe Ser Ile Gly Met Phe Pro Tyr Val Met Leu 290 295 300Ala Ser Ser Pro Leu Phe Cys Ser Ala Glu Trp Pro Arg Lys Leu Val305 310 315 320Ala Arg Cys Pro Lys Arg Leu Gln Glu Leu Leu Pro Thr Lys Ala Ala 325 330 335Pro Arg Pro Ser Ala Ser Cys Val Tyr Lys Arg Ser Arg Gly Lys Ala 340 345 350Gly Pro Lys Pro Gly Leu Arg His Gln Leu Gly Ala Ile Phe Thr Leu 355 360 365Leu Tyr Leu Leu Glu Gln Leu Phe Leu Pro Tyr Ser His Phe Leu Thr 370 375 380Gln Gly Tyr Asn Asn Trp Thr Asn Gly Leu Tyr Gly Tyr Ser Trp Asp385 390 395 400Met Met Val His Ser Arg Ser His Gln His Val Lys Ile Thr Tyr Arg 405 410 415Asp Gly Leu Thr Gly Glu Leu Gly Tyr Leu Asn Pro Gly Val Phe Thr 420 425 430Gln Ser Arg Arg Trp Lys Asp His Ala Asp Met Leu Lys Gln Tyr Ala 435 440 445Thr Cys Leu Ser Leu Leu Leu Pro Lys Tyr Asn Val Thr Glu Pro Gln 450 455 460Ile Tyr Phe Asp Ile Trp Val Ser Ile Asn Asp Arg Phe Gln Gln Arg465 470 475 480Leu Phe Asp Pro Arg Val Asp Ile Val Gln Ala Val Trp Ser Pro Phe 485 490 495Gln Arg Thr Pro Trp Val Gln Pro Leu Leu Met Asp Leu Ser Pro Trp 500 505 510Arg Thr Lys Leu Gln Asp Ile Lys Ser Ser Leu Asp Asn His Thr Glu 515 520 525Val Val Phe Ile Ala Asp Phe Pro Gly Leu His Leu Glu Asn Phe Val 530 535 540Ser Glu Asp Leu Gly Asn Thr Ser Ile Gln Leu Leu Gln Gly Glu Val545 550 555 560Thr Val Glu Leu Val Ala Glu Gln Lys Asn Gln Thr Leu Gln Glu Gly 565 570 575Glu Lys Met Gln Leu Pro Ala Gly Glu Tyr His Lys Val Tyr Thr Val 580 585 590Ser Ser Ser Pro Ser Cys Tyr Met Tyr Val Tyr Val Asn Thr Thr Glu 595 600 605Val Ala Leu Glu Gln Asp Leu Ala Tyr Leu Gln Glu Leu Lys Glu Lys 610 615 620Val Glu Asn Gly Ser Glu Thr Gly Pro Leu Pro Pro Glu Leu Gln Pro625 630 635 640Leu Leu Glu Gly Glu Val Lys Gly Gly Pro Glu Pro Thr Pro Leu Val 645 650 655Gln Thr Phe Leu Arg Arg Gln Arg Lys Leu Gln Glu Ile Glu Arg Arg 660 665 670Arg Asn Ser Pro Phe His Glu Arg Phe Leu Arg Phe Val Leu Arg Lys 675 680 685Leu Tyr Val Phe Arg Arg Ser Phe Leu Met Thr Arg Ile Ser Leu Arg 690 695 700Asn Leu Leu Leu Gly Arg Pro Ser Leu Glu Gln Leu Ala Gln Glu Val705 710 715 720Thr Tyr Ala Asn Leu Arg Pro Phe Glu Pro Val Asp Glu Ser Ser Ala 725 730 735Ser Asn Thr Asp Ser Ser Asn His Pro Ser Glu Pro Asp Ser Glu His 740 745 750Val His Ser Glu Phe 755141223DNAhomo sapiensCDS(84)..(1034) 14gggatccttg agtcctactc agccccagcg gaggtgaagg acgtccttcc ccaggagccg 60actggccaat cacaggcagg aag atg aag gtt ctg tgg gct gcg ttg ctg gtc 113 Met Lys Val Leu Trp Ala Ala Leu Leu Val 1 5 10aca ttc ctg gca gga tgc cag gcc aag gtg gag caa gcg gtg gag aca 161Thr Phe Leu Ala Gly Cys Gln Ala Lys Val Glu Gln Ala Val Glu Thr 15 20 25gag ccg gag ccc gag ctg cgc cag cag acc gag tgg cag agc ggc cag 209Glu Pro Glu Pro Glu Leu Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln 30 35 40cgc tgg gaa ctg gca ctg ggt cgc ttt tgg gat tac ctg cgc tgg gtg 257Arg Trp Glu Leu Ala Leu Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val 45 50 55cag aca ctg tct gag cag gtg cag gag gag ctg ctc agc tcc cag gtc 305Gln Thr Leu Ser Glu Gln Val Gln Glu Glu Leu Leu Ser Ser Gln Val 60 65 70acc cag gaa ctg agg gcg ctg atg gac gag acc atg aag gag ttg aag 353Thr Gln Glu Leu Arg Ala Leu Met Asp Glu Thr Met Lys Glu Leu Lys75 80 85 90gcc tac aaa tcg gaa ctg gag gaa caa ctg acc ccg gtg gcg gag gag 401Ala Tyr Lys Ser Glu Leu Glu Glu Gln Leu Thr Pro Val Ala Glu Glu 95 100 105acg cgg gca cgg ctg tcc aag gag ctg cag gcg gcg cag gcc cgg ctg 449Thr Arg Ala Arg Leu Ser Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu 110 115 120ggc gcg gac atg gag gac gtg tgc ggc cgc ctg gtg cag tac cgc ggc 497Gly Ala Asp Met Glu Asp Val Cys Gly Arg Leu Val Gln Tyr Arg Gly 125 130 135gag gtg cag gcc atg ctc ggc cag agc acc gag gag ctg cgg gtg cgc 545Glu Val Gln Ala Met Leu Gly Gln Ser Thr Glu Glu Leu Arg Val Arg 140 145 150ctc gcc tcc cac ctg cgc aag ctg cgt aag cgg ctc ctc cgc gat gcc 593Leu Ala Ser His Leu Arg Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala155 160 165 170gat gac ctg cag aag cgc ctg gca gtg tac cag gcc ggg gcc cgc gag 641Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu 175 180 185ggc gcc gag cgc ggc ctc agc gcc atc cgc gag cgc ctg ggg ccc ctg 689Gly Ala Glu Arg Gly Leu Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu 190 195 200gtg gaa cag ggc cgc gtg cgg gcc gcc act gtg ggc tcc ctg gcc ggc 737Val Glu Gln Gly Arg Val Arg Ala Ala Thr Val Gly Ser Leu Ala Gly 205 210 215cag ccg cta cag gag cgg gcc cag gcc tgg ggc gag cgg ctg cgc gcg 785Gln Pro Leu Gln Glu Arg Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala 220 225 230cgg atg gag gag atg ggc agc cgg acc cgc gac cgc ctg gac gag gtg 833Arg Met Glu Glu Met Gly Ser Arg Thr Arg Asp Arg Leu Asp Glu Val235 240 245 250aag gag cag gtg gcg gag gtg cgc gcc aag

ctg gag gag cag gcc cag 881Lys Glu Gln Val Ala Glu Val Arg Ala Lys Leu Glu Glu Gln Ala Gln 255 260 265cag ata cgc ctg cag gcc gag gcc ttc cag gcc cgc ctc aag agc tgg 929Gln Ile Arg Leu Gln Ala Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp 270 275 280ttc gag ccc ctg gtg gaa gac atg cag cgc cag tgg gcc ggg ctg gtg 977Phe Glu Pro Leu Val Glu Asp Met Gln Arg Gln Trp Ala Gly Leu Val 285 290 295gag aag gtg cag gct gcc gtg ggc acc agc gcc gcc cct gtg ccc agc 1025Glu Lys Val Gln Ala Ala Val Gly Thr Ser Ala Ala Pro Val Pro Ser 300 305 310gac aat cac tgaacgccga agcctgcagc catgcgaccc cacgccaccc 1074Asp Asn His315cgtgcctcct gcctccgcgc agcctgcagc gggagaccct gtccccgccc cagccgtcct 1134cctggggtgg accctagttt aataaagatt caccaagttt cacgcaaaaa aaaaaaaaaa 1194aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 122315317PRThomo sapiens 15Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys1 5 10 15Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25 30Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu 35 40 45Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln 50 55 60Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala65 70 75 80Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu 85 90 95Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120 125Val Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg145 150 155 160Lys Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg 165 170 175Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu 180 185 190Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val 195 200 205Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 210 215 220Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly225 230 235 240Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295 300Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His305 310 315161104DNAmus musculusCDS(46)..(978) 16ggacttgttt cggaaggagc tgactggcca atcacaattg cgaag atg aag gct ctg 57 Met Lys Ala Leu 1tgg gcc gtg ctg ttg gtc aca ttg ctg aca gga tgc cta gcc gag gga 105Trp Ala Val Leu Leu Val Thr Leu Leu Thr Gly Cys Leu Ala Glu Gly5 10 15 20gag ccg gag gtg aca gat cag ctc gag tgg caa agc aac caa ccc tgg 153Glu Pro Glu Val Thr Asp Gln Leu Glu Trp Gln Ser Asn Gln Pro Trp 25 30 35gag cag gcc ctg aac cgc ttc tgg gat tac ctg cgc tgg gtg cag acg 201Glu Gln Ala Leu Asn Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr 40 45 50ctg tct gac cag gtc cag gaa gag ctg cag agc tcc caa gtc aca caa 249Leu Ser Asp Gln Val Gln Glu Glu Leu Gln Ser Ser Gln Val Thr Gln 55 60 65gaa ctg acg gca ctg atg gag gac act atg acg gaa gta aag gct tac 297Glu Leu Thr Ala Leu Met Glu Asp Thr Met Thr Glu Val Lys Ala Tyr 70 75 80aaa aag gag ctg gag gaa cag ctg ggt cca gtg gcg gag gag aca cgg 345Lys Lys Glu Leu Glu Glu Gln Leu Gly Pro Val Ala Glu Glu Thr Arg85 90 95 100gcc agg ctg ggc aaa gag gtg cag gcg gca cag gcc cga ctc gga gcc 393Ala Arg Leu Gly Lys Glu Val Gln Ala Ala Gln Ala Arg Leu Gly Ala 105 110 115gac atg gag gat cta cgc aac cga ctc ggg cag tac cgc aac gag gtg 441Asp Met Glu Asp Leu Arg Asn Arg Leu Gly Gln Tyr Arg Asn Glu Val 120 125 130cac acc atg ctg ggc cag agc aca gag gag ata cgg gcg cgg ctc tcc 489His Thr Met Leu Gly Gln Ser Thr Glu Glu Ile Arg Ala Arg Leu Ser 135 140 145aca cac ctg cgc aag atg cgc aag cgc ttg atg cgg gat gcc gat gat 537Thr His Leu Arg Lys Met Arg Lys Arg Leu Met Arg Asp Ala Asp Asp 150 155 160ctg cag aag cgc cta gct gtg tac aag gca ggg gca cgc gag ggc gcc 585Leu Gln Lys Arg Leu Ala Val Tyr Lys Ala Gly Ala Arg Glu Gly Ala165 170 175 180gag cgc ggt gtg agt gcc atc cgt gag cgc ctg ggg cct ctg gtg gag 633Glu Arg Gly Val Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu 185 190 195caa ggt cgc cag cgc act gcc aac cta ggc gct ggg gcc gcc cag cct 681Gln Gly Arg Gln Arg Thr Ala Asn Leu Gly Ala Gly Ala Ala Gln Pro 200 205 210ctg cgc gat cgc gcc cag gct ttt ggt gac cgc atc cga ggg cgg ctg 729Leu Arg Asp Arg Ala Gln Ala Phe Gly Asp Arg Ile Arg Gly Arg Leu 215 220 225gag gaa gtg ggc aac cag gcc cgt gac cgc cta gag gag gtg cgt gag 777Glu Glu Val Gly Asn Gln Ala Arg Asp Arg Leu Glu Glu Val Arg Glu 230 235 240cac atg gag gag gtg cgc tcc aag atg gag gaa cag acc cag caa ata 825His Met Glu Glu Val Arg Ser Lys Met Glu Glu Gln Thr Gln Gln Ile245 250 255 260cgc ctg cag gcg gag atc ttc cag gcc cgc ctc aag ggc tgg ttc gag 873Arg Leu Gln Ala Glu Ile Phe Gln Ala Arg Leu Lys Gly Trp Phe Glu 265 270 275cca ata gtg gaa gac atg cat cgc cag tgg gca aac ctg atg gag aag 921Pro Ile Val Glu Asp Met His Arg Gln Trp Ala Asn Leu Met Glu Lys 280 285 290ata cag gcc tct gtg gct acc aac ccc atc atc acc cca gtg gcc cag 969Ile Gln Ala Ser Val Ala Thr Asn Pro Ile Ile Thr Pro Val Ala Gln 295 300 305gag aat caa tgagtatcct tctcctgtcc tgcaacaaca tccatatcca 1018Glu Asn Gln 310gccaggtggc cctgtctcaa gcacctctct ggccctctgg tggcccttgc ttaataaaga 1078ttctccgagc aaaaaaaaaa aaaaaa 110417311PRTmus musculus 17Met Lys Ala Leu Trp Ala Val Leu Leu Val Thr Leu Leu Thr Gly Cys1 5 10 15Leu Ala Glu Gly Glu Pro Glu Val Thr Asp Gln Leu Glu Trp Gln Ser 20 25 30Asn Gln Pro Trp Glu Gln Ala Leu Asn Arg Phe Trp Asp Tyr Leu Arg 35 40 45Trp Val Gln Thr Leu Ser Asp Gln Val Gln Glu Glu Leu Gln Ser Ser 50 55 60Gln Val Thr Gln Glu Leu Thr Ala Leu Met Glu Asp Thr Met Thr Glu65 70 75 80Val Lys Ala Tyr Lys Lys Glu Leu Glu Glu Gln Leu Gly Pro Val Ala 85 90 95Glu Glu Thr Arg Ala Arg Leu Gly Lys Glu Val Gln Ala Ala Gln Ala 100 105 110Arg Leu Gly Ala Asp Met Glu Asp Leu Arg Asn Arg Leu Gly Gln Tyr 115 120 125Arg Asn Glu Val His Thr Met Leu Gly Gln Ser Thr Glu Glu Ile Arg 130 135 140Ala Arg Leu Ser Thr His Leu Arg Lys Met Arg Lys Arg Leu Met Arg145 150 155 160Asp Ala Asp Asp Leu Gln Lys Arg Leu Ala Val Tyr Lys Ala Gly Ala 165 170 175Arg Glu Gly Ala Glu Arg Gly Val Ser Ala Ile Arg Glu Arg Leu Gly 180 185 190Pro Leu Val Glu Gln Gly Arg Gln Arg Thr Ala Asn Leu Gly Ala Gly 195 200 205Ala Ala Gln Pro Leu Arg Asp Arg Ala Gln Ala Phe Gly Asp Arg Ile 210 215 220Arg Gly Arg Leu Glu Glu Val Gly Asn Gln Ala Arg Asp Arg Leu Glu225 230 235 240Glu Val Arg Glu His Met Glu Glu Val Arg Ser Lys Met Glu Glu Gln 245 250 255Thr Gln Gln Ile Arg Leu Gln Ala Glu Ile Phe Gln Ala Arg Leu Lys 260 265 270Gly Trp Phe Glu Pro Ile Val Glu Asp Met His Arg Gln Trp Ala Asn 275 280 285Leu Met Glu Lys Ile Gln Ala Ser Val Ala Thr Asn Pro Ile Ile Thr 290 295 300Pro Val Ala Gln Glu Asn Gln305 310185555DNAmus musculusCDS(316)..(5430) 18ggctgtggga gagcagaaga ggagctggaa gagcagccta caacagctgt cgggagggac 60cagggctagt tcacacttgg aagctgggat gccaggaccg gccctcctgc ctctctcggt 120ctccatcggc ctcctggtca gctcactcca cactgagacg attctgaagt aagatgctcc 180tggctcctca cagactctgc tacaagagac agagtgaagt gtccccaggg ctcagagcct 240ttgactctgc tccttccctt cccacggctg agttggcaca ggagcacctg ggtgagctgc 300accagactta agaag atg agg ccc ctg att ctg tta gct gcc ctc ctc tgg 351 Met Arg Pro Leu Ile Leu Leu Ala Ala Leu Leu Trp 1 5 10ctc cag gac tct ttg gcc cag gaa gat gta tgc tca tcc ttg gat ggg 399Leu Gln Asp Ser Leu Ala Gln Glu Asp Val Cys Ser Ser Leu Asp Gly 15 20 25agc cca gac agg cag ggt gga ggt cca cct ctg agt gtg aac gtc agc 447Ser Pro Asp Arg Gln Gly Gly Gly Pro Pro Leu Ser Val Asn Val Ser 30 35 40agc cgc gga aag cct acc agc ctg ttt ctg agc tgg gta gct gca gag 495Ser Arg Gly Lys Pro Thr Ser Leu Phe Leu Ser Trp Val Ala Ala Glu45 50 55 60cca ggt gga ttt gac tat gcc ctc tgc ctc agg gct atg aac ttg tcg 543Pro Gly Gly Phe Asp Tyr Ala Leu Cys Leu Arg Ala Met Asn Leu Ser 65 70 75ggt ttt cca gaa ggg caa cag ctc caa gct cat acc aac gag tcc agc 591Gly Phe Pro Glu Gly Gln Gln Leu Gln Ala His Thr Asn Glu Ser Ser 80 85 90ttt gag ttc cat ggc ctg gtg cca ggg agt cgc tac cag ctg gaa ctg 639Phe Glu Phe His Gly Leu Val Pro Gly Ser Arg Tyr Gln Leu Glu Leu 95 100 105act gtc cta aga ccc tgt tgg cag aat gtc aca att acc ctc act gct 687Thr Val Leu Arg Pro Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala 110 115 120cga act gcc cct aca gtg gtc cgt gga ctg caa ctg cat agc act ggg 735Arg Thr Ala Pro Thr Val Val Arg Gly Leu Gln Leu His Ser Thr Gly125 130 135 140agc cca gcc agc ctg gaa gcc tca tgg agc gat gcc tct ggg gat caa 783Ser Pro Ala Ser Leu Glu Ala Ser Trp Ser Asp Ala Ser Gly Asp Gln 145 150 155gac agc tat caa ctt ctc ctc tac cac ccg gaa tcc cac act ctg gca 831Asp Ser Tyr Gln Leu Leu Leu Tyr His Pro Glu Ser His Thr Leu Ala 160 165 170tgt aat gtc tct gtg tcc cct gac acc ctg tct tac aat ttt ggt gac 879Cys Asn Val Ser Val Ser Pro Asp Thr Leu Ser Tyr Asn Phe Gly Asp 175 180 185ctc ttg cca ggt agt cag tat gtc ttg gag gtt atc acc tgg gct ggc 927Leu Leu Pro Gly Ser Gln Tyr Val Leu Glu Val Ile Thr Trp Ala Gly 190 195 200agt ctc cat gcg aag act agc atc ctc caa tgg aca gag cct gtc cct 975Ser Leu His Ala Lys Thr Ser Ile Leu Gln Trp Thr Glu Pro Val Pro205 210 215 220cct gat cac cta aca ctg cgt gcc ttg ggt acc agt agc ctg caa gcc 1023Pro Asp His Leu Thr Leu Arg Ala Leu Gly Thr Ser Ser Leu Gln Ala 225 230 235ttc tgg aac agc tct gaa ggg gcc acc tgg ttt cac ctg ata ctt aca 1071Phe Trp Asn Ser Ser Glu Gly Ala Thr Trp Phe His Leu Ile Leu Thr 240 245 250gac ctc cta gag ggt acc aac ctg acc aaa gtg gtc aga caa ggc atc 1119Asp Leu Leu Glu Gly Thr Asn Leu Thr Lys Val Val Arg Gln Gly Ile 255 260 265tca acc cac acc ttc ctt cgc ctg tct ccg ggt aca cct tac cag ctg 1167Ser Thr His Thr Phe Leu Arg Leu Ser Pro Gly Thr Pro Tyr Gln Leu 270 275 280aag atc tgt gct gct gct ggg ccc cac cag att tgg gga ccc aat gcc 1215Lys Ile Cys Ala Ala Ala Gly Pro His Gln Ile Trp Gly Pro Asn Ala285 290 295 300act gag tgg acc tat ccc tct tac cca tct gac ctg gtg ctg acc ccc 1263Thr Glu Trp Thr Tyr Pro Ser Tyr Pro Ser Asp Leu Val Leu Thr Pro 305 310 315tta tgg aat gag ctc tgg gca agc tgg aag gca ggg cag gga gcc cgg 1311Leu Trp Asn Glu Leu Trp Ala Ser Trp Lys Ala Gly Gln Gly Ala Arg 320 325 330gat ggc tat gta ctg aag tta agt ggg cca gtg gag aat aca act act 1359Asp Gly Tyr Val Leu Lys Leu Ser Gly Pro Val Glu Asn Thr Thr Thr 335 340 345ctg ggt cct gag gag tgc aac gct gtc ttc cca ggg ccc ctg cct cca 1407Leu Gly Pro Glu Glu Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro 350 355 360gga cac tac act ttg ggg ctg agg gtt cta gct gga cct tat gat gcc 1455Gly His Tyr Thr Leu Gly Leu Arg Val Leu Ala Gly Pro Tyr Asp Ala365 370 375 380tgg gta gag ggc agt atc tgg ctg gct gaa tct gct gct cgt ccc atg 1503Trp Val Glu Gly Ser Ile Trp Leu Ala Glu Ser Ala Ala Arg Pro Met 385 390 395gag gtc cct ggt gcc aga ctg tgg cta gaa gga ctg gaa gct act aag 1551Glu Val Pro Gly Ala Arg Leu Trp Leu Glu Gly Leu Glu Ala Thr Lys 400 405 410caa cct ggg aga cgg gcg ctg ctc tat tct gtt gat gcc cca ggc ctc 1599Gln Pro Gly Arg Arg Ala Leu Leu Tyr Ser Val Asp Ala Pro Gly Leu 415 420 425cta ggg aac atc tct gtg tct tct ggt gcc act cat gtc acc ttc tgt 1647Leu Gly Asn Ile Ser Val Ser Ser Gly Ala Thr His Val Thr Phe Cys 430 435 440ggc ttg gta ccc gga gcg cac tac agg gtg gac att gcc tca tcc atg 1695Gly Leu Val Pro Gly Ala His Tyr Arg Val Asp Ile Ala Ser Ser Met445 450 455 460gga gac atc act cag agc ctc aca ggc tac aca agt ccc ctg cca cca 1743Gly Asp Ile Thr Gln Ser Leu Thr Gly Tyr Thr Ser Pro Leu Pro Pro 465 470 475cag tct ctg gag atc atc agc cgg aac agc cca tct gac ctg act atc 1791Gln Ser Leu Glu Ile Ile Ser Arg Asn Ser Pro Ser Asp Leu Thr Ile 480 485 490ggt tgg gct cca gca cca ggg cag atg gaa ggt tat aag gtc acc tgg 1839Gly Trp Ala Pro Ala Pro Gly Gln Met Glu Gly Tyr Lys Val Thr Trp 495 500 505cat cag gat ggc agc cag agg tca cct ggc gac ctt gtt gac ttg ggc 1887His Gln Asp Gly Ser Gln Arg Ser Pro Gly Asp Leu Val Asp Leu Gly 510 515 520cct gac att tcg agc ctg act ctg aaa tct ctg gta cct ggt tcc tgc 1935Pro Asp Ile Ser Ser Leu Thr Leu Lys Ser Leu Val Pro Gly Ser Cys525 530 535 540tac acc gtg tca gca tgg gcc tgg tct ggg aac ctc agc tct gac tct 1983Tyr Thr Val Ser Ala Trp Ala Trp Ser Gly Asn Leu Ser Ser Asp Ser 545 550 555cag aag att cac agt tgc acc cgt ccc gct cct ccc acc aac ctg agc 2031Gln Lys Ile His Ser Cys Thr Arg Pro Ala Pro Pro Thr Asn Leu Ser 560 565 570ctg ggc ttt gcc cac cag cct gca aca ctg agg gct tcc tgg tgt cac 2079Leu Gly Phe Ala His Gln Pro Ala Thr Leu Arg Ala Ser Trp Cys His 575 580 585cca ccg ggt ggc agg gat gcc ttt cag tta cgg ctt tac agg ctg agg 2127Pro Pro Gly Gly Arg Asp Ala Phe Gln Leu Arg Leu Tyr Arg Leu Arg 590 595 600ccc ctg aca ctg gaa agt gag aag atc cta tcc cag gag gcc cag aac 2175Pro Leu Thr Leu Glu Ser Glu Lys Ile Leu Ser Gln Glu Ala Gln Asn605 610 615 620ttc tcc tgg gcc cag ctg cct gca ggc tat gaa ttc cag gta cag ctg 2223Phe Ser Trp Ala Gln Leu Pro Ala Gly Tyr Glu Phe Gln Val Gln Leu 625 630 635tct acc ttg tgg ggg tcg gag gag agc ggc agt gcc aac acc aca ggc 2271Ser Thr Leu Trp Gly Ser Glu Glu Ser Gly Ser Ala Asn Thr Thr Gly 640 645 650tgg aca ccc ccc tca gct cct aca ttg gta aat gtg acc agt gaa gcc 2319Trp Thr Pro Pro Ser Ala Pro Thr Leu Val Asn Val Thr Ser Glu Ala 655 660 665ccc acc cag ctc cac gta tcc tgg gtc cac gct gct ggg gac cgg agc 2367Pro Thr Gln Leu His Val Ser Trp Val His Ala Ala Gly Asp Arg Ser 670 675

680agc tac caa gtg acc cta tac cag gag agc act cgg aca gcc acc agc 2415Ser Tyr Gln Val Thr Leu Tyr Gln Glu Ser Thr Arg Thr Ala Thr Ser685 690 695 700att gtg ggg ccc aag gca gac agc aca agc ttt tgg ggt ttg act cct 2463Ile Val Gly Pro Lys Ala Asp Ser Thr Ser Phe Trp Gly Leu Thr Pro 705 710 715ggc act aag tac aag gtg gaa gcc atc tcc tgg gct ggg ccc ctt tac 2511Gly Thr Lys Tyr Lys Val Glu Ala Ile Ser Trp Ala Gly Pro Leu Tyr 720 725 730act gca gca gcc aac gtt tct gct tgg acc tac cca ctc aca ccc aat 2559Thr Ala Ala Ala Asn Val Ser Ala Trp Thr Tyr Pro Leu Thr Pro Asn 735 740 745gag ctg ctc gcc tct atg cag gca ggc agt gct gtg gtt aac ctg gcc 2607Glu Leu Leu Ala Ser Met Gln Ala Gly Ser Ala Val Val Asn Leu Ala 750 755 760tgg ccc agt ggt ccc ttg ggg caa ggg aca tgc cat gcc caa ctc tca 2655Trp Pro Ser Gly Pro Leu Gly Gln Gly Thr Cys His Ala Gln Leu Ser765 770 775 780gat gct gga cac ctt tca tgg gag caa ccg ctg tcg cta ggc caa gac 2703Asp Ala Gly His Leu Ser Trp Glu Gln Pro Leu Ser Leu Gly Gln Asp 785 790 795ctc ctc atg cta agg aat ctt ata cca gga cat acg gtt tca ttg tct 2751Leu Leu Met Leu Arg Asn Leu Ile Pro Gly His Thr Val Ser Leu Ser 800 805 810gtg aag tgt cgg gca gga cca ctc cag gcc tcc act cac ccc ctg gtg 2799Val Lys Cys Arg Ala Gly Pro Leu Gln Ala Ser Thr His Pro Leu Val 815 820 825ctg tct gta gag cct ggc cct gtg gaa gat gtg ttc tgt caa cct gag 2847Leu Ser Val Glu Pro Gly Pro Val Glu Asp Val Phe Cys Gln Pro Glu 830 835 840gcc acc tac ctg tcc ctg aac tgg acg atg cct act gga gat gtg gct 2895Ala Thr Tyr Leu Ser Leu Asn Trp Thr Met Pro Thr Gly Asp Val Ala845 850 855 860gtc tgt ctg gtg gag gta gag cag ctg gtg cca gga ggg agc gct cat 2943Val Cys Leu Val Glu Val Glu Gln Leu Val Pro Gly Gly Ser Ala His 865 870 875ttt gtc ttc cag gtc aac acc tcg gag gat gca ctt ctg ctg ccc aac 2991Phe Val Phe Gln Val Asn Thr Ser Glu Asp Ala Leu Leu Leu Pro Asn 880 885 890ttg acg ccc acc act tct tac cgc ctt agc ctc act gtg ctg ggt ggg 3039Leu Thr Pro Thr Thr Ser Tyr Arg Leu Ser Leu Thr Val Leu Gly Gly 895 900 905aat cgc cag tgg agc cgg gcg gtt acc ctg gtg tgc act act tct gct 3087Asn Arg Gln Trp Ser Arg Ala Val Thr Leu Val Cys Thr Thr Ser Ala 910 915 920gag gtt tgg cac ccc cca gag cta gct gag gcc ccc cag gtg gag ctg 3135Glu Val Trp His Pro Pro Glu Leu Ala Glu Ala Pro Gln Val Glu Leu925 930 935 940ggg aca ggg atg ggt gtg aca gtc aca cgt ggc atg ttt ggt aaa gat 3183Gly Thr Gly Met Gly Val Thr Val Thr Arg Gly Met Phe Gly Lys Asp 945 950 955gac ggg cag atc cag tgg tat ggc ata att gcc acc atc aac atg aca 3231Asp Gly Gln Ile Gln Trp Tyr Gly Ile Ile Ala Thr Ile Asn Met Thr 960 965 970ctg gcc cag cct tcc cag gaa gcc atc aac cac aca tgg tat gac cac 3279Leu Ala Gln Pro Ser Gln Glu Ala Ile Asn His Thr Trp Tyr Asp His 975 980 985tac tat aga gga cat gac tcc tac ctg gct ctc ctg ttc cca aac ccc 3327Tyr Tyr Arg Gly His Asp Ser Tyr Leu Ala Leu Leu Phe Pro Asn Pro 990 995 1000ttc tac cca gag cct tgg gct gtg cca aga tcc tgg aca gta cct 3372Phe Tyr Pro Glu Pro Trp Ala Val Pro Arg Ser Trp Thr Val Pro1005 1010 1015gtg ggt aca gag gac tgt gac aac acc cag gag ata tgc aat ggg 3417Val Gly Thr Glu Asp Cys Asp Asn Thr Gln Glu Ile Cys Asn Gly1020 1025 1030cat ctc aag cca ggc ttc cag tat agg ttc agc att gca gcc ttt 3462His Leu Lys Pro Gly Phe Gln Tyr Arg Phe Ser Ile Ala Ala Phe1035 1040 1045agt agg ctc agc tct cca gag acc atc ctg gcc ttc tcc gcc ttc 3507Ser Arg Leu Ser Ser Pro Glu Thr Ile Leu Ala Phe Ser Ala Phe1050 1055 1060tca gag cct cag gct agc atc tct ctg gtg gcc atg ccc ctg aca 3552Ser Glu Pro Gln Ala Ser Ile Ser Leu Val Ala Met Pro Leu Thr1065 1070 1075gtt atg atg ggg act gtg gtg ggc tgc atc atc att gtg tgt gca 3597Val Met Met Gly Thr Val Val Gly Cys Ile Ile Ile Val Cys Ala1080 1085 1090gtg cta tgc ttg ttg tgc cgg cgg cgc ctg aag gga cca agg tca 3642Val Leu Cys Leu Leu Cys Arg Arg Arg Leu Lys Gly Pro Arg Ser1095 1100 1105gag aag aat ggc ttt tcc cag gag ttg atg cct tac aac ctg tgg 3687Glu Lys Asn Gly Phe Ser Gln Glu Leu Met Pro Tyr Asn Leu Trp1110 1115 1120cgg acc cat cgg ccc atc ccc agc cat agc ttc cgg cag agc tat 3732Arg Thr His Arg Pro Ile Pro Ser His Ser Phe Arg Gln Ser Tyr1125 1130 1135gag gcc aag agt gca cgt gca cac cag gcc ttc ttc cag gaa ttt 3777Glu Ala Lys Ser Ala Arg Ala His Gln Ala Phe Phe Gln Glu Phe1140 1145 1150gag gag ctg aag gag gtg ggc aag gac cag ccc aga cta gag gct 3822Glu Glu Leu Lys Glu Val Gly Lys Asp Gln Pro Arg Leu Glu Ala1155 1160 1165gag cat cct gcc aac atc acc aag aac cgg tac cca cac gtg cta 3867Glu His Pro Ala Asn Ile Thr Lys Asn Arg Tyr Pro His Val Leu1170 1175 1180cct tat gac cac tcc agg gtc agg ctg acc cag cta tca gga gag 3912Pro Tyr Asp His Ser Arg Val Arg Leu Thr Gln Leu Ser Gly Glu1185 1190 1195cct cat tct gac tac atc aat gcc aac ttc atc cca ggc tat agc 3957Pro His Ser Asp Tyr Ile Asn Ala Asn Phe Ile Pro Gly Tyr Ser1200 1205 1210cac cca cag gag atc att gcc acc cag ggg cct ctc aaa aag acg 4002His Pro Gln Glu Ile Ile Ala Thr Gln Gly Pro Leu Lys Lys Thr1215 1220 1225gtc gag gac ttc tgg cgg ttg gtg tgg gag cag caa gtc cac gtg 4047Val Glu Asp Phe Trp Arg Leu Val Trp Glu Gln Gln Val His Val1230 1235 1240atc atc atg cta act gtg ggc atg gag aat ggg cgg gta ctg tgt 4092Ile Ile Met Leu Thr Val Gly Met Glu Asn Gly Arg Val Leu Cys1245 1250 1255gag cac tac tgg cca gtc aac tcc acg cct gtc acc cac ggt cac 4137Glu His Tyr Trp Pro Val Asn Ser Thr Pro Val Thr His Gly His1260 1265 1270atc acc acc cac ctc ctg gca gag gaa tct gag gac gag tgg acc 4182Ile Thr Thr His Leu Leu Ala Glu Glu Ser Glu Asp Glu Trp Thr1275 1280 1285agg agg gaa ttc cag ctg cag cac ggt gca gag caa aaa cag agg 4227Arg Arg Glu Phe Gln Leu Gln His Gly Ala Glu Gln Lys Gln Arg1290 1295 1300cgc gtg aag cag ctg cag ttc acg acc tgg cca gac cac agt gtc 4272Arg Val Lys Gln Leu Gln Phe Thr Thr Trp Pro Asp His Ser Val1305 1310 1315ccc gag gct ccc agc tct ctg ctc gct ttt gtg gaa ctg gtg cag 4317Pro Glu Ala Pro Ser Ser Leu Leu Ala Phe Val Glu Leu Val Gln1320 1325 1330gag gag gtg aag gca act cag ggc aag ggg ccc atc ctg gtg cat 4362Glu Glu Val Lys Ala Thr Gln Gly Lys Gly Pro Ile Leu Val His1335 1340 1345tgc agt gcg ggt gtg ggc agg aca ggc acc ttt gtg gct ctc tta 4407Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Phe Val Ala Leu Leu1350 1355 1360ccg gct gtt cga caa cta gag gaa gaa cag gtg gtc gat gtg ttc 4452Pro Ala Val Arg Gln Leu Glu Glu Glu Gln Val Val Asp Val Phe1365 1370 1375aac act gtg tac ata ctc cgg ctg cac cgg ccc ctc atg atc cag 4497Asn Thr Val Tyr Ile Leu Arg Leu His Arg Pro Leu Met Ile Gln1380 1385 1390acc ttg agt caa tac atc ttc ctg cac agc tgc ctg ctg aac aag 4542Thr Leu Ser Gln Tyr Ile Phe Leu His Ser Cys Leu Leu Asn Lys1395 1400 1405att ctg gaa ggg ccc tct gac gcc tca gac tcc ggc ccc atc cct 4587Ile Leu Glu Gly Pro Ser Asp Ala Ser Asp Ser Gly Pro Ile Pro1410 1415 1420gtg atg aat ttt gca caa gct tgt gcc aag agg gca gcc aat gcc 4632Val Met Asn Phe Ala Gln Ala Cys Ala Lys Arg Ala Ala Asn Ala1425 1430 1435aat gcc ggt ttc ttg aag gag tac agg ctc ctg aag cag gcc atc 4677Asn Ala Gly Phe Leu Lys Glu Tyr Arg Leu Leu Lys Gln Ala Ile1440 1445 1450aag gat gag act ggc tct ctg ctg ccc tct cct gac tat aat cag 4722Lys Asp Glu Thr Gly Ser Leu Leu Pro Ser Pro Asp Tyr Asn Gln1455 1460 1465aac agc atc gcc tcc tgt cat cat tct cag gag cag ttg gcc ctg 4767Asn Ser Ile Ala Ser Cys His His Ser Gln Glu Gln Leu Ala Leu1470 1475 1480gtg gag gag agc cct gct gat aac atg ctg gca gcc tcg ctc ttc 4812Val Glu Glu Ser Pro Ala Asp Asn Met Leu Ala Ala Ser Leu Phe1485 1490 1495cct ggt ggg ccg tct ggt cgc gac cat gtg gtg ctg act ggc tcg 4857Pro Gly Gly Pro Ser Gly Arg Asp His Val Val Leu Thr Gly Ser1500 1505 1510gcc gga cca aag gaa ctc tgg gaa atg gtg tgg gaa cat ggc gcc 4902Ala Gly Pro Lys Glu Leu Trp Glu Met Val Trp Glu His Gly Ala1515 1520 1525tat gtg ctt gtc tcc ctg ggt ctg cct gat acc aag gag aag cca 4947Tyr Val Leu Val Ser Leu Gly Leu Pro Asp Thr Lys Glu Lys Pro1530 1535 1540caa gac atc tgg cca atg gag atg cag cct att gtc aca gac atg 4992Gln Asp Ile Trp Pro Met Glu Met Gln Pro Ile Val Thr Asp Met1545 1550 1555gtg aca gtg cac aga gtg gct gag agc aac aca gct ggc tgg ccc 5037Val Thr Val His Arg Val Ala Glu Ser Asn Thr Ala Gly Trp Pro1560 1565 1570agt acc ctc atc aga gtt ata cat ggg gac agt ggg acg gaa agg 5082Ser Thr Leu Ile Arg Val Ile His Gly Asp Ser Gly Thr Glu Arg1575 1580 1585cag gtt caa tgc ctg cag ttt cca cac tgc gag act ggg agt gag 5127Gln Val Gln Cys Leu Gln Phe Pro His Cys Glu Thr Gly Ser Glu1590 1595 1600ctc cca gct aac acc cta ctg acc ttc ctt gat gct gtg ggc cag 5172Leu Pro Ala Asn Thr Leu Leu Thr Phe Leu Asp Ala Val Gly Gln1605 1610 1615tgc tgc tcc cgg ggc aat agc aag aag cca ggg acc ctg ctc agt 5217Cys Cys Ser Arg Gly Asn Ser Lys Lys Pro Gly Thr Leu Leu Ser1620 1625 1630cac tcc agc aag gtc aca aac cag ctg agc acc ttc ttg gct atg 5262His Ser Ser Lys Val Thr Asn Gln Leu Ser Thr Phe Leu Ala Met1635 1640 1645gaa cag ctg cta cag caa gca ggg acc gag cgc aca gtg gat gtc 5307Glu Gln Leu Leu Gln Gln Ala Gly Thr Glu Arg Thr Val Asp Val1650 1655 1660ttc agt gtg gcc ctg aag cag aca cag gcc tgt ggc ctt aag acc 5352Phe Ser Val Ala Leu Lys Gln Thr Gln Ala Cys Gly Leu Lys Thr1665 1670 1675cca acg ctg gag cag tat atc tac ctc tac aac tgt ctg aac agc 5397Pro Thr Leu Glu Gln Tyr Ile Tyr Leu Tyr Asn Cys Leu Asn Ser1680 1685 1690gca ttg agg aac agg ctg ccc cga gct agg aag tgaccttgcc 5440Ala Leu Arg Asn Arg Leu Pro Arg Ala Arg Lys1695 1700 1705ctgctaggca tcacgttcca gcaatccacc caggcctggc ttccccagga gaacagatct 5500attcggcctc acgctgtcaa agggcagagt ctgggaataa agggtaaatc tcgag 5555191705PRTmus musculus 19Met Arg Pro Leu Ile Leu Leu Ala Ala Leu Leu Trp Leu Gln Asp Ser1 5 10 15Leu Ala Gln Glu Asp Val Cys Ser Ser Leu Asp Gly Ser Pro Asp Arg 20 25 30Gln Gly Gly Gly Pro Pro Leu Ser Val Asn Val Ser Ser Arg Gly Lys 35 40 45Pro Thr Ser Leu Phe Leu Ser Trp Val Ala Ala Glu Pro Gly Gly Phe 50 55 60Asp Tyr Ala Leu Cys Leu Arg Ala Met Asn Leu Ser Gly Phe Pro Glu65 70 75 80Gly Gln Gln Leu Gln Ala His Thr Asn Glu Ser Ser Phe Glu Phe His 85 90 95Gly Leu Val Pro Gly Ser Arg Tyr Gln Leu Glu Leu Thr Val Leu Arg 100 105 110Pro Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala Arg Thr Ala Pro 115 120 125Thr Val Val Arg Gly Leu Gln Leu His Ser Thr Gly Ser Pro Ala Ser 130 135 140Leu Glu Ala Ser Trp Ser Asp Ala Ser Gly Asp Gln Asp Ser Tyr Gln145 150 155 160Leu Leu Leu Tyr His Pro Glu Ser His Thr Leu Ala Cys Asn Val Ser 165 170 175Val Ser Pro Asp Thr Leu Ser Tyr Asn Phe Gly Asp Leu Leu Pro Gly 180 185 190Ser Gln Tyr Val Leu Glu Val Ile Thr Trp Ala Gly Ser Leu His Ala 195 200 205Lys Thr Ser Ile Leu Gln Trp Thr Glu Pro Val Pro Pro Asp His Leu 210 215 220Thr Leu Arg Ala Leu Gly Thr Ser Ser Leu Gln Ala Phe Trp Asn Ser225 230 235 240Ser Glu Gly Ala Thr Trp Phe His Leu Ile Leu Thr Asp Leu Leu Glu 245 250 255Gly Thr Asn Leu Thr Lys Val Val Arg Gln Gly Ile Ser Thr His Thr 260 265 270Phe Leu Arg Leu Ser Pro Gly Thr Pro Tyr Gln Leu Lys Ile Cys Ala 275 280 285Ala Ala Gly Pro His Gln Ile Trp Gly Pro Asn Ala Thr Glu Trp Thr 290 295 300Tyr Pro Ser Tyr Pro Ser Asp Leu Val Leu Thr Pro Leu Trp Asn Glu305 310 315 320Leu Trp Ala Ser Trp Lys Ala Gly Gln Gly Ala Arg Asp Gly Tyr Val 325 330 335Leu Lys Leu Ser Gly Pro Val Glu Asn Thr Thr Thr Leu Gly Pro Glu 340 345 350Glu Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro Gly His Tyr Thr 355 360 365Leu Gly Leu Arg Val Leu Ala Gly Pro Tyr Asp Ala Trp Val Glu Gly 370 375 380Ser Ile Trp Leu Ala Glu Ser Ala Ala Arg Pro Met Glu Val Pro Gly385 390 395 400Ala Arg Leu Trp Leu Glu Gly Leu Glu Ala Thr Lys Gln Pro Gly Arg 405 410 415Arg Ala Leu Leu Tyr Ser Val Asp Ala Pro Gly Leu Leu Gly Asn Ile 420 425 430Ser Val Ser Ser Gly Ala Thr His Val Thr Phe Cys Gly Leu Val Pro 435 440 445Gly Ala His Tyr Arg Val Asp Ile Ala Ser Ser Met Gly Asp Ile Thr 450 455 460Gln Ser Leu Thr Gly Tyr Thr Ser Pro Leu Pro Pro Gln Ser Leu Glu465 470 475 480Ile Ile Ser Arg Asn Ser Pro Ser Asp Leu Thr Ile Gly Trp Ala Pro 485 490 495Ala Pro Gly Gln Met Glu Gly Tyr Lys Val Thr Trp His Gln Asp Gly 500 505 510Ser Gln Arg Ser Pro Gly Asp Leu Val Asp Leu Gly Pro Asp Ile Ser 515 520 525Ser Leu Thr Leu Lys Ser Leu Val Pro Gly Ser Cys Tyr Thr Val Ser 530 535 540Ala Trp Ala Trp Ser Gly Asn Leu Ser Ser Asp Ser Gln Lys Ile His545 550 555 560Ser Cys Thr Arg Pro Ala Pro Pro Thr Asn Leu Ser Leu Gly Phe Ala 565 570 575His Gln Pro Ala Thr Leu Arg Ala Ser Trp Cys His Pro Pro Gly Gly 580 585 590Arg Asp Ala Phe Gln Leu Arg Leu Tyr Arg Leu Arg Pro Leu Thr Leu 595 600 605Glu Ser Glu Lys Ile Leu Ser Gln Glu Ala Gln Asn Phe Ser Trp Ala 610 615 620Gln Leu Pro Ala Gly Tyr Glu Phe Gln Val Gln Leu Ser Thr Leu Trp625 630 635 640Gly Ser Glu Glu Ser Gly Ser Ala Asn Thr Thr Gly Trp Thr Pro Pro 645 650 655Ser Ala Pro Thr Leu Val Asn Val Thr Ser Glu Ala Pro Thr Gln Leu 660 665 670His Val Ser Trp Val His Ala Ala Gly Asp Arg Ser Ser Tyr Gln Val 675 680 685Thr Leu Tyr Gln Glu Ser Thr Arg Thr Ala Thr Ser Ile Val Gly Pro 690 695 700Lys Ala Asp Ser Thr Ser Phe Trp Gly Leu Thr Pro Gly Thr Lys Tyr705 710 715 720Lys Val Glu Ala Ile Ser Trp Ala Gly Pro Leu Tyr Thr Ala Ala Ala 725 730 735Asn Val Ser Ala Trp Thr Tyr Pro Leu Thr Pro Asn Glu Leu Leu Ala 740 745 750Ser Met Gln Ala Gly Ser Ala Val Val Asn Leu Ala Trp Pro Ser Gly 755 760 765Pro Leu Gly Gln Gly Thr Cys His Ala Gln Leu Ser Asp Ala Gly His 770 775

780Leu Ser Trp Glu Gln Pro Leu Ser Leu Gly Gln Asp Leu Leu Met Leu785 790 795 800Arg Asn Leu Ile Pro Gly His Thr Val Ser Leu Ser Val Lys Cys Arg 805 810 815Ala Gly Pro Leu Gln Ala Ser Thr His Pro Leu Val Leu Ser Val Glu 820 825 830Pro Gly Pro Val Glu Asp Val Phe Cys Gln Pro Glu Ala Thr Tyr Leu 835 840 845Ser Leu Asn Trp Thr Met Pro Thr Gly Asp Val Ala Val Cys Leu Val 850 855 860Glu Val Glu Gln Leu Val Pro Gly Gly Ser Ala His Phe Val Phe Gln865 870 875 880Val Asn Thr Ser Glu Asp Ala Leu Leu Leu Pro Asn Leu Thr Pro Thr 885 890 895Thr Ser Tyr Arg Leu Ser Leu Thr Val Leu Gly Gly Asn Arg Gln Trp 900 905 910Ser Arg Ala Val Thr Leu Val Cys Thr Thr Ser Ala Glu Val Trp His 915 920 925Pro Pro Glu Leu Ala Glu Ala Pro Gln Val Glu Leu Gly Thr Gly Met 930 935 940Gly Val Thr Val Thr Arg Gly Met Phe Gly Lys Asp Asp Gly Gln Ile945 950 955 960Gln Trp Tyr Gly Ile Ile Ala Thr Ile Asn Met Thr Leu Ala Gln Pro 965 970 975Ser Gln Glu Ala Ile Asn His Thr Trp Tyr Asp His Tyr Tyr Arg Gly 980 985 990His Asp Ser Tyr Leu Ala Leu Leu Phe Pro Asn Pro Phe Tyr Pro Glu 995 1000 1005Pro Trp Ala Val Pro Arg Ser Trp Thr Val Pro Val Gly Thr Glu 1010 1015 1020Asp Cys Asp Asn Thr Gln Glu Ile Cys Asn Gly His Leu Lys Pro 1025 1030 1035Gly Phe Gln Tyr Arg Phe Ser Ile Ala Ala Phe Ser Arg Leu Ser 1040 1045 1050Ser Pro Glu Thr Ile Leu Ala Phe Ser Ala Phe Ser Glu Pro Gln 1055 1060 1065Ala Ser Ile Ser Leu Val Ala Met Pro Leu Thr Val Met Met Gly 1070 1075 1080Thr Val Val Gly Cys Ile Ile Ile Val Cys Ala Val Leu Cys Leu 1085 1090 1095Leu Cys Arg Arg Arg Leu Lys Gly Pro Arg Ser Glu Lys Asn Gly 1100 1105 1110Phe Ser Gln Glu Leu Met Pro Tyr Asn Leu Trp Arg Thr His Arg 1115 1120 1125Pro Ile Pro Ser His Ser Phe Arg Gln Ser Tyr Glu Ala Lys Ser 1130 1135 1140Ala Arg Ala His Gln Ala Phe Phe Gln Glu Phe Glu Glu Leu Lys 1145 1150 1155Glu Val Gly Lys Asp Gln Pro Arg Leu Glu Ala Glu His Pro Ala 1160 1165 1170Asn Ile Thr Lys Asn Arg Tyr Pro His Val Leu Pro Tyr Asp His 1175 1180 1185Ser Arg Val Arg Leu Thr Gln Leu Ser Gly Glu Pro His Ser Asp 1190 1195 1200Tyr Ile Asn Ala Asn Phe Ile Pro Gly Tyr Ser His Pro Gln Glu 1205 1210 1215Ile Ile Ala Thr Gln Gly Pro Leu Lys Lys Thr Val Glu Asp Phe 1220 1225 1230Trp Arg Leu Val Trp Glu Gln Gln Val His Val Ile Ile Met Leu 1235 1240 1245Thr Val Gly Met Glu Asn Gly Arg Val Leu Cys Glu His Tyr Trp 1250 1255 1260Pro Val Asn Ser Thr Pro Val Thr His Gly His Ile Thr Thr His 1265 1270 1275Leu Leu Ala Glu Glu Ser Glu Asp Glu Trp Thr Arg Arg Glu Phe 1280 1285 1290Gln Leu Gln His Gly Ala Glu Gln Lys Gln Arg Arg Val Lys Gln 1295 1300 1305Leu Gln Phe Thr Thr Trp Pro Asp His Ser Val Pro Glu Ala Pro 1310 1315 1320Ser Ser Leu Leu Ala Phe Val Glu Leu Val Gln Glu Glu Val Lys 1325 1330 1335Ala Thr Gln Gly Lys Gly Pro Ile Leu Val His Cys Ser Ala Gly 1340 1345 1350Val Gly Arg Thr Gly Thr Phe Val Ala Leu Leu Pro Ala Val Arg 1355 1360 1365Gln Leu Glu Glu Glu Gln Val Val Asp Val Phe Asn Thr Val Tyr 1370 1375 1380Ile Leu Arg Leu His Arg Pro Leu Met Ile Gln Thr Leu Ser Gln 1385 1390 1395Tyr Ile Phe Leu His Ser Cys Leu Leu Asn Lys Ile Leu Glu Gly 1400 1405 1410Pro Ser Asp Ala Ser Asp Ser Gly Pro Ile Pro Val Met Asn Phe 1415 1420 1425Ala Gln Ala Cys Ala Lys Arg Ala Ala Asn Ala Asn Ala Gly Phe 1430 1435 1440Leu Lys Glu Tyr Arg Leu Leu Lys Gln Ala Ile Lys Asp Glu Thr 1445 1450 1455Gly Ser Leu Leu Pro Ser Pro Asp Tyr Asn Gln Asn Ser Ile Ala 1460 1465 1470Ser Cys His His Ser Gln Glu Gln Leu Ala Leu Val Glu Glu Ser 1475 1480 1485Pro Ala Asp Asn Met Leu Ala Ala Ser Leu Phe Pro Gly Gly Pro 1490 1495 1500Ser Gly Arg Asp His Val Val Leu Thr Gly Ser Ala Gly Pro Lys 1505 1510 1515Glu Leu Trp Glu Met Val Trp Glu His Gly Ala Tyr Val Leu Val 1520 1525 1530Ser Leu Gly Leu Pro Asp Thr Lys Glu Lys Pro Gln Asp Ile Trp 1535 1540 1545Pro Met Glu Met Gln Pro Ile Val Thr Asp Met Val Thr Val His 1550 1555 1560Arg Val Ala Glu Ser Asn Thr Ala Gly Trp Pro Ser Thr Leu Ile 1565 1570 1575Arg Val Ile His Gly Asp Ser Gly Thr Glu Arg Gln Val Gln Cys 1580 1585 1590Leu Gln Phe Pro His Cys Glu Thr Gly Ser Glu Leu Pro Ala Asn 1595 1600 1605Thr Leu Leu Thr Phe Leu Asp Ala Val Gly Gln Cys Cys Ser Arg 1610 1615 1620Gly Asn Ser Lys Lys Pro Gly Thr Leu Leu Ser His Ser Ser Lys 1625 1630 1635Val Thr Asn Gln Leu Ser Thr Phe Leu Ala Met Glu Gln Leu Leu 1640 1645 1650Gln Gln Ala Gly Thr Glu Arg Thr Val Asp Val Phe Ser Val Ala 1655 1660 1665Leu Lys Gln Thr Gln Ala Cys Gly Leu Lys Thr Pro Thr Leu Glu 1670 1675 1680Gln Tyr Ile Tyr Leu Tyr Asn Cys Leu Asn Ser Ala Leu Arg Asn 1685 1690 1695Arg Leu Pro Arg Ala Arg Lys 1700 1705203078DNAEscherichia coliCDS(1)..(3078) 20acc atg att acg gat tca ctg gcc gtc gtt tta caa cgt cgt gac tgg 48Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp Trp1 5 10 15gaa aac cct ggc gtt acc caa ctt aat cgc ctt gca gca cat ccc cct 96Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro 20 25 30ttc gcc agc tgg cgt aat agc gaa gag gcc cgc acc gat cgc cct tcc 144Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser 35 40 45caa cag ttg cgc agc ctg aat ggc gaa tgg cgc ttt gcc tgg ttt ccg 192Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro 50 55 60gca cca gaa gcg gtg ccg gaa agc tgg ctg gag tgc gat ctt cct gag 240Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu65 70 75 80gcc gat act gtc gtc gtc ccc tca aac tgg cag atg cac ggt tac gat 288Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr Asp 85 90 95gcg ccc atc tac acc aac gta acc tat ccc att acg gtc aat ccg ccg 336Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn Pro Pro 100 105 110ttt gtt ccc acg gag aat ccg acg ggt tgt tac tcg ctc aca ttt aat 384Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr Ser Leu Thr Phe Asn 115 120 125gtt gat gaa agc tgg cta cag gaa ggc cag acg cga att att ttt gat 432Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe Asp 130 135 140ggc gtt aac tcg gcg ttt cat ctg tgg tgc aac ggg cgc tgg gtc ggt 480Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn Gly Arg Trp Val Gly145 150 155 160tac ggc cag gac agt cgt ttg ccg tct gaa ttt gac ctg agc gca ttt 528Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe Asp Leu Ser Ala Phe 165 170 175tta cgc gcc gga gaa aac cgc ctc gcg gtg atg gtg ctg cgt tgg agt 576Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met Val Leu Arg Trp Ser 180 185 190gac ggc agt tat ctg gaa gat cag gat atg tgg cgg atg agc ggc att 624Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp Arg Met Ser Gly Ile 195 200 205ttc cgt gac gtc tcg ttg ctg cat aaa ccg act aca caa atc agc gat 672Phe Arg Asp Val Ser Leu Leu His Lys Pro Thr Thr Gln Ile Ser Asp 210 215 220ttc cat gtt gcc act cgc ttt aat gat gat ttc agc cgc gct gta ctg 720Phe His Val Ala Thr Arg Phe Asn Asp Asp Phe Ser Arg Ala Val Leu225 230 235 240gag gct gaa gtt cag atg tgc ggc gag ttg cgt gac tac cta cgg gta 768Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg Val 245 250 255aca gtt tct tta tgg cag ggt gaa acg cag gtc gcc agc ggc acc gcg 816Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly Thr Ala 260 265 270cct ttc ggc ggt gaa att atc gat gag cgt ggt ggt tat gcc gat cgc 864Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg 275 280 285gtc aca cta cgt ctg aac gtc gaa aac ccg aaa ctg tgg agc gcc gaa 912Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu 290 295 300atc ccg aat ctc tat cgt gcg gtg gtt gaa ctg cac acc gcc gac ggc 960Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu His Thr Ala Asp Gly305 310 315 320acg ctg att gaa gca gaa gcc tgc gat gtc ggt ttc cgc gag gtg cgg 1008Thr Leu Ile Glu Ala Glu Ala Cys Asp Val Gly Phe Arg Glu Val Arg 325 330 335att gaa aat ggt ctg ctg ctg ctg aac ggc aag ccg ttg ctg att cga 1056Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile Arg 340 345 350ggc gtt aac cgt cac gag cat cat cct ctg cat ggt cag gtc atg gat 1104Gly Val Asn Arg His Glu His His Pro Leu His Gly Gln Val Met Asp 355 360 365gag cag acg atg gtg cag gat atc ctg ctg atg aag cag aac aac ttt 1152Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn Asn Phe 370 375 380aac gcc gtg cgc tgt tcg cat tat ccg aac cat ccg ctg tgg tac acg 1200Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr385 390 395 400ctg tgc gac cgc tac ggc ctg tat gtg gtg gat gaa gcc aat att gaa 1248Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu 405 410 415acc cac ggc atg gtg cca atg aat cgt ctg acc gat gat ccg cgc tgg 1296Thr His Gly Met Val Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp 420 425 430cta ccg gcg atg agc gaa cgc gta acg cga atg gtg cag cgc gat cgt 1344Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg 435 440 445aat cac ccg agt gtg atc atc tgg tcg ctg ggg aat gaa tca ggc cac 1392Asn His Pro Ser Val Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His 450 455 460ggc gct aat cac gac gcg ctg tat cgc tgg atc aaa tct gtc gat cct 1440Gly Ala Asn His Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp Pro465 470 475 480tcc cgc ccg gtg cag tat gaa ggc ggc gga gcc gac acc acg gcc acc 1488Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala Thr 485 490 495gat att att tgc ccg atg tac gcg cgc gtg gat gaa gac cag ccc ttc 1536Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln Pro Phe 500 505 510ccg gct gtg ccg aaa tgg tcc atc aaa aaa tgg ctt tcg cta cct gga 1584Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly 515 520 525gag acg cgc ccg ctg atc ctt tgc gaa tac gcc cac gcg atg ggt aac 1632Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn 530 535 540agt ctt ggc ggt ttc gct aaa tac tgg cag gcg ttt cgt cag tat ccc 1680Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro545 550 555 560cgt tta cag ggc ggc ttc gtc tgg gac tgg gtg gat cag tcg ctg att 1728Arg Leu Gln Gly Gly Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile 565 570 575aaa tat gat gaa aac ggc aac ccg tgg tcg gct tac ggc ggt gat ttt 1776Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe 580 585 590ggc gat acg ccg aac gat cgc cag ttc tgt atg aac ggt ctg gtc ttt 1824Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu Val Phe 595 600 605gcc gac cgc acg ccg cat cca gcg ctg acg gaa gca aaa cac cag cag 1872Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His Gln Gln 610 615 620cag ttt ttc cag ttc cgt tta tcc ggg caa acc atc gaa gtg acc agc 1920Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser625 630 635 640gaa tac ctg ttc cgt cat agc gat aac gag ctc ctg cac tgg atg gtg 1968Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His Trp Met Val 645 650 655gcg ctg gat ggt aag ccg ctg gca agc ggt gaa gtg cct ctg gat gtc 2016Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val 660 665 670gct cca caa ggt aaa cag ttg att gaa ctg cct gaa cta ccg cag ccg 2064Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro 675 680 685gag agc gcc ggg caa ctc tgg ctc aca gta cgc gta gtg caa ccg aac 2112Glu Ser Ala Gly Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn 690 695 700gcg acc gca tgg tca gaa gcc ggg cac atc agc gcc tgg cag cag tgg 2160Ala Thr Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp705 710 715 720cgt ctg gcg gaa aac ctc agt gtg acg ctc ccc gcc gcg tcc cac gcc 2208Arg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala 725 730 735atc ccg cat ctg acc acc agc gaa atg gat ttt tgc atc gag ctg ggt 2256Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly 740 745 750aat aag cgt tgg caa ttt aac cgc cag tca ggc ttt ctt tca cag atg 2304Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met 755 760 765tgg att ggc gat aaa aaa caa ctg ctg acg ccg ctg cgc gat cag ttc 2352Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe 770 775 780acc cgt gca ccg ctg gat aac gac att ggc gta agt gaa gcg acc cgc 2400Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg785 790 795 800att gac cct aac gcc tgg gtc gaa cgc tgg aag gcg gcg ggc cat tac 2448Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr 805 810 815cag gcc gaa gca gcg ttg ttg cag tgc acg gca gat aca ctt gct gat 2496Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp 820 825 830gcg gtg ctg att acg acc gct cac gcg tgg cag cat cag ggg aaa acc 2544Ala Val Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly Lys Thr 835 840 845tta ttt atc agc cgg aaa acc tac cgg att gat ggt agt ggt caa atg 2592Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met 850 855 860gcg att acc gtt gat gtt gaa gtg gcg agc gat aca ccg cat ccg gcg 2640Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His Pro Ala865 870 875 880cgg att ggc ctg aac tgc cag ctg gcg cag gta gca gag cgg gta aac 2688Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn 885 890 895tgg ctc gga tta ggg ccg caa gaa aac tat ccc gac cgc ctt act gcc 2736Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala 900 905 910gcc tgt ttt gac cgc tgg gat ctg cca ttg tca gac atg tat acc ccg 2784Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro 915 920 925tac gtc ttc ccg agc gaa aac ggt ctg cgc tgc ggg acg cgc gaa ttg 2832Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu 930 935 940aat tat ggc cca cac cag tgg cgc ggc gac ttc cag ttc aac atc agc 2880Asn Tyr Gly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser945

950 955 960cgc tac agt caa cag caa ctg atg gaa acc agc cat cgc cat ctg ctg 2928Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His Leu Leu 965 970 975cac gcg gaa gaa ggc aca tgg ctg aat atc gac ggt ttc cat atg ggg 2976His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met Gly 980 985 990att ggt ggc gac gac tcc tgg agc ccg tca gta tcg gcg gaa ttc cag 3024Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Phe Gln 995 1000 1005ctg agc gcc ggt cgc tac cat tac cag ttg gtc tgg tgt caa aaa 3069Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys 1010 1015 1020taa taa taa 3078211023PRTEscherichia coli 21Thr Met Ile Thr Asp Ser Leu Ala Val Val Leu Gln Arg Arg Asp Trp1 5 10 15Glu Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro 20 25 30Phe Ala Ser Trp Arg Asn Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser 35 40 45Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp Phe Pro 50 55 60Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu Cys Asp Leu Pro Glu65 70 75 80Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly Tyr Asp 85 90 95Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn Pro Pro 100 105 110Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr Ser Leu Thr Phe Asn 115 120 125Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile Ile Phe Asp 130 135 140Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn Gly Arg Trp Val Gly145 150 155 160Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe Asp Leu Ser Ala Phe 165 170 175Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met Val Leu Arg Trp Ser 180 185 190Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp Arg Met Ser Gly Ile 195 200 205Phe Arg Asp Val Ser Leu Leu His Lys Pro Thr Thr Gln Ile Ser Asp 210 215 220Phe His Val Ala Thr Arg Phe Asn Asp Asp Phe Ser Arg Ala Val Leu225 230 235 240Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu Arg Val 245 250 255Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly Thr Ala 260 265 270Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala Asp Arg 275 280 285Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys Leu Trp Ser Ala Glu 290 295 300Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu His Thr Ala Asp Gly305 310 315 320Thr Leu Ile Glu Ala Glu Ala Cys Asp Val Gly Phe Arg Glu Val Arg 325 330 335Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu Ile Arg 340 345 350Gly Val Asn Arg His Glu His His Pro Leu His Gly Gln Val Met Asp 355 360 365Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn Asn Phe 370 375 380Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp Tyr Thr385 390 395 400Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala Asn Ile Glu 405 410 415Thr His Gly Met Val Pro Met Asn Arg Leu Thr Asp Asp Pro Arg Trp 420 425 430Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met Val Gln Arg Asp Arg 435 440 445Asn His Pro Ser Val Ile Ile Trp Ser Leu Gly Asn Glu Ser Gly His 450 455 460Gly Ala Asn His Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val Asp Pro465 470 475 480Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr Ala Thr 485 490 495Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln Pro Phe 500 505 510Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu Pro Gly 515 520 525Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met Gly Asn 530 535 540Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln Tyr Pro545 550 555 560Arg Leu Gln Gly Gly Phe Val Trp Asp Trp Val Asp Gln Ser Leu Ile 565 570 575Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly Asp Phe 580 585 590Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu Val Phe 595 600 605Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His Gln Gln 610 615 620Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val Thr Ser625 630 635 640Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His Trp Met Val 645 650 655Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly Glu Val Pro Leu Asp Val 660 665 670Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro Gln Pro 675 680 685Glu Ser Ala Gly Gln Leu Trp Leu Thr Val Arg Val Val Gln Pro Asn 690 695 700Ala Thr Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln Gln Trp705 710 715 720Arg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser His Ala 725 730 735Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu Leu Gly 740 745 750Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser Gln Met 755 760 765Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp Gln Phe 770 775 780Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly Val Ser Glu Ala Thr Arg785 790 795 800Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly His Tyr 805 810 815Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu Ala Asp 820 825 830Ala Val Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly Lys Thr 835 840 845Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly Gln Met 850 855 860Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His Pro Ala865 870 875 880Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu Arg Val Asn 885 890 895Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu Thr Ala 900 905 910Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr Thr Pro 915 920 925Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg Glu Leu 930 935 940Asn Tyr Gly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn Ile Ser945 950 955 960Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His Leu Leu 965 970 975His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His Met Gly 980 985 990Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu Phe Gln 995 1000 1005Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln Lys 1010 1015 10202249PRThomo sapiens 22Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu1 5 10 15Glu Pro Arg Arg Glu Val Cys Glu Leu Asn Pro Asp Cys Asp Glu Leu 20 25 30Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly Pro 35 40 45Val2349PRThomo sapiensVARIANT(17)..(17)Xaa can be any amino acid. 23Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu1 5 10 15Xaa Pro Arg Arg Xaa Val Cys Xaa Leu Asn Pro Asp Cys Asp Glu Leu 20 25 30Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly Pro 35 40 45Val245136DNARattus norvegicusCDS(1)..(5136) 24atg agg ccc ctg att ctg tta gct gcc ctc ctc tgg ctc cag ggc ttt 48Met Arg Pro Leu Ile Leu Leu Ala Ala Leu Leu Trp Leu Gln Gly Phe1 5 10 15ttg gcc gag gac gac gca tgc tca tcc ttg gaa ggg agc cca gac agg 96Leu Ala Glu Asp Asp Ala Cys Ser Ser Leu Glu Gly Ser Pro Asp Arg 20 25 30cag ggt gga ggt cca ctt ctg agt gtg aac gtc agt agc cat gga aag 144Gln Gly Gly Gly Pro Leu Leu Ser Val Asn Val Ser Ser His Gly Lys 35 40 45tct acc agc ctg ttt ctg agc tgg gta gct gca gag ctg ggc gga ttt 192Ser Thr Ser Leu Phe Leu Ser Trp Val Ala Ala Glu Leu Gly Gly Phe 50 55 60gac tat gcc ctc agc ctc agg agt gtg aac tcc tca ggt tct cca gaa 240Asp Tyr Ala Leu Ser Leu Arg Ser Val Asn Ser Ser Gly Ser Pro Glu65 70 75 80ggg caa cag ctc cag gct cac aca aat gag tcc ggc ttt gag ttc cat 288Gly Gln Gln Leu Gln Ala His Thr Asn Glu Ser Gly Phe Glu Phe His 85 90 95ggc ctg gtg cca ggg agt cgc tac cag cta aaa ctg act gtc cta aga 336Gly Leu Val Pro Gly Ser Arg Tyr Gln Leu Lys Leu Thr Val Leu Arg 100 105 110ccc tgt tgg cag aat gtc aca att acc ctc act gcc cga act gcc ccg 384Pro Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala Arg Thr Ala Pro 115 120 125aca gtg gtc cgt gga ctg cag ctg cat agc gct ggg agc cca gcc agg 432Thr Val Val Arg Gly Leu Gln Leu His Ser Ala Gly Ser Pro Ala Arg 130 135 140ctg gaa gcc tcg tgg agt gat gcc cct gga gat caa gac agc tac caa 480Leu Glu Ala Ser Trp Ser Asp Ala Pro Gly Asp Gln Asp Ser Tyr Gln145 150 155 160ctt ctc ctc tac cac ctg gaa tcc caa act ctg gca tgc aat gtc tct 528Leu Leu Leu Tyr His Leu Glu Ser Gln Thr Leu Ala Cys Asn Val Ser 165 170 175gtg tcc cct gac acc ctg tct tac agt ttt ggc gac ctt ttg cca ggt 576Val Ser Pro Asp Thr Leu Ser Tyr Ser Phe Gly Asp Leu Leu Pro Gly 180 185 190act cag tat gtc ttg gag gtt atc acc tgg gct ggc agt ctc cat gcg 624Thr Gln Tyr Val Leu Glu Val Ile Thr Trp Ala Gly Ser Leu His Ala 195 200 205aag act agt atc ctc cag tgg aca gag cct gtc cct cct gat cac cta 672Lys Thr Ser Ile Leu Gln Trp Thr Glu Pro Val Pro Pro Asp His Leu 210 215 220gca cta cgt gcc ttg ggt acc agt agc ctg caa gcc ttc tgg aac agc 720Ala Leu Arg Ala Leu Gly Thr Ser Ser Leu Gln Ala Phe Trp Asn Ser225 230 235 240tct gaa ggg gcc acc tcg ttt cac ctg atg ctc aca gac ctc ctc ggg 768Ser Glu Gly Ala Thr Ser Phe His Leu Met Leu Thr Asp Leu Leu Gly 245 250 255ggc acc aac acg act gcg gtg atc aga caa ggg gtc tcg acc cac acc 816Gly Thr Asn Thr Thr Ala Val Ile Arg Gln Gly Val Ser Thr His Thr 260 265 270ttt ctt cac cta tct ccg ggt aca cct cat gag ctg aag att tgt gct 864Phe Leu His Leu Ser Pro Gly Thr Pro His Glu Leu Lys Ile Cys Ala 275 280 285tct gct ggg ccc cac cag atc tgg gga ccc agt gcc acc gag tgg acc 912Ser Ala Gly Pro His Gln Ile Trp Gly Pro Ser Ala Thr Glu Trp Thr 290 295 300tat ccc tct tac cca tct gac ctg gtg ctg act ccc tta cgg aat gag 960Tyr Pro Ser Tyr Pro Ser Asp Leu Val Leu Thr Pro Leu Arg Asn Glu305 310 315 320ctc tgg gcc agc tgg aag gca ggg ctg gga gcc cgg gac ggc tat gta 1008Leu Trp Ala Ser Trp Lys Ala Gly Leu Gly Ala Arg Asp Gly Tyr Val 325 330 335ctg aag tta agt ggg cca atg gag agt acg tct acc ctg ggc ccg gaa 1056Leu Lys Leu Ser Gly Pro Met Glu Ser Thr Ser Thr Leu Gly Pro Glu 340 345 350gag tgc aat gca gtc ttc cca ggg ccc ctg cct ccg gga cac tac act 1104Glu Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro Gly His Tyr Thr 355 360 365ttg cag ctg aag gtt cta gct gga cct tat gat gcc tgg gtg gag ggc 1152Leu Gln Leu Lys Val Leu Ala Gly Pro Tyr Asp Ala Trp Val Glu Gly 370 375 380agt acc tgg ctg gct gaa tct gct gcc ctt ccc agg gag gtc cct ggt 1200Ser Thr Trp Leu Ala Glu Ser Ala Ala Leu Pro Arg Glu Val Pro Gly385 390 395 400gcc aga ctg tgg cta gat gga ctg gaa gct tcc aag cag cct ggg aga 1248Ala Arg Leu Trp Leu Asp Gly Leu Glu Ala Ser Lys Gln Pro Gly Arg 405 410 415cgg gcg cta ctc tat tct gac gat gcc cca ggc tcc cta ggg aac atc 1296Arg Ala Leu Leu Tyr Ser Asp Asp Ala Pro Gly Ser Leu Gly Asn Ile 420 425 430tct gtg ccc tct ggt gcc act cac gtc att ttc tgt ggc ctg gta cct 1344Ser Val Pro Ser Gly Ala Thr His Val Ile Phe Cys Gly Leu Val Pro 435 440 445gga gcc cac tat agg gtg gac att gcc tca tcc acg ggg gac atc tct 1392Gly Ala His Tyr Arg Val Asp Ile Ala Ser Ser Thr Gly Asp Ile Ser 450 455 460cag agc atc tca ggc tat aca agt ccc ctg cca ccg cag tca ctg gag 1440Gln Ser Ile Ser Gly Tyr Thr Ser Pro Leu Pro Pro Gln Ser Leu Glu465 470 475 480gtc atc agc agg agc agc cca tct gac ctg act att gct tgg ggt cca 1488Val Ile Ser Arg Ser Ser Pro Ser Asp Leu Thr Ile Ala Trp Gly Pro 485 490 495gca cca ggg cag ctg gaa ggt tat aag gtt acc tgg cat cag gat ggc 1536Ala Pro Gly Gln Leu Glu Gly Tyr Lys Val Thr Trp His Gln Asp Gly 500 505 510agc cag agg tct cct ggc gac ctt gtt gac ttg ggc cct gac act ttg 1584Ser Gln Arg Ser Pro Gly Asp Leu Val Asp Leu Gly Pro Asp Thr Leu 515 520 525agc ctg act ctg aaa tct ctg gta ccc ggc tcc tgc tac acc gtg tca 1632Ser Leu Thr Leu Lys Ser Leu Val Pro Gly Ser Cys Tyr Thr Val Ser 530 535 540gca tgg gcc tgg gcc ggg aac ctc gac tct gac tct cag aag att cac 1680Ala Trp Ala Trp Ala Gly Asn Leu Asp Ser Asp Ser Gln Lys Ile His545 550 555 560agc tgc acc cgc ccc gct cct ccc acc aac ctg agt ctg ggc ttt gcc 1728Ser Cys Thr Arg Pro Ala Pro Pro Thr Asn Leu Ser Leu Gly Phe Ala 565 570 575cac cag cct gcg gca ctg aag gct tcc tgg tat cac cca ccg ggt ggc 1776His Gln Pro Ala Ala Leu Lys Ala Ser Trp Tyr His Pro Pro Gly Gly 580 585 590agg gat gcc ttt cac tta cgg ctt tac agg ctg agg cct ctg aca ctg 1824Arg Asp Ala Phe His Leu Arg Leu Tyr Arg Leu Arg Pro Leu Thr Leu 595 600 605gaa agt gag aag gtc cta cct cgg gag gcc cag aac ttc tcc tgg gcc 1872Glu Ser Glu Lys Val Leu Pro Arg Glu Ala Gln Asn Phe Ser Trp Ala 610 615 620cag ctg act gca ggc tgt gag ttc cag gta cag ctg tct acc ttg tgg 1920Gln Leu Thr Ala Gly Cys Glu Phe Gln Val Gln Leu Ser Thr Leu Trp625 630 635 640ggg tct gag aga agc agc agt gcc aac gcc aca ggc tgg aca ccc cct 1968Gly Ser Glu Arg Ser Ser Ser Ala Asn Ala Thr Gly Trp Thr Pro Pro 645 650 655tca gct cct aca ctg gta aac gtg acc agc gat gct cct acc cag ctc 2016Ser Ala Pro Thr Leu Val Asn Val Thr Ser Asp Ala Pro Thr Gln Leu 660 665 670caa gta tcc tgg gcc cac gtt cct ggg ggc cgg agc cgc tac caa gtg 2064Gln Val Ser Trp Ala His Val Pro Gly Gly Arg Ser Arg Tyr Gln Val 675 680 685acc cta tac cag gag agt acc cgg aca gcc acc agc atc atg ggg ccc 2112Thr Leu Tyr Gln Glu Ser Thr Arg Thr Ala Thr Ser Ile Met Gly Pro 690 695 700aag gaa gat ggc acg agc ttt ttg ggt ttg act cct ggc act aag tac 2160Lys Glu Asp Gly Thr Ser Phe Leu Gly Leu Thr Pro Gly Thr Lys Tyr705 710 715 720aag gtg gaa gtc atc tcc tgg gct ggg ccc ctc tac act gca gca gcc 2208Lys Val Glu Val Ile Ser Trp Ala Gly Pro Leu Tyr Thr Ala Ala Ala 725 730 735aac gtt tct gcc tgg acc tac cca ctc ata ccc aat gag ctg ctc gtg 2256Asn Val Ser Ala Trp Thr Tyr Pro Leu Ile Pro Asn Glu Leu Leu Val 740 745 750tca atg cag gca ggc agt

gct gtg gtt aac ctg gcc tgg ccc agt ggt 2304Ser Met Gln Ala Gly Ser Ala Val Val Asn Leu Ala Trp Pro Ser Gly 755 760 765ccc ctg ggg caa ggg gca tgc cac gcc caa ctc tca gat gct gga cac 2352Pro Leu Gly Gln Gly Ala Cys His Ala Gln Leu Ser Asp Ala Gly His 770 775 780ctc tca tgg gag caa ccc ctg aaa cta ggc caa gag ctc ttc atg cta 2400Leu Ser Trp Glu Gln Pro Leu Lys Leu Gly Gln Glu Leu Phe Met Leu785 790 795 800agg gat ctc aca cca gga cat acc atc tcg atg tca gtg agg tgt cgg 2448Arg Asp Leu Thr Pro Gly His Thr Ile Ser Met Ser Val Arg Cys Arg 805 810 815gca ggg ccg ctc cag gcc tct acg cac ctt gtg gtg ctg tct gtg gag 2496Ala Gly Pro Leu Gln Ala Ser Thr His Leu Val Val Leu Ser Val Glu 820 825 830cct ggc cct gtg gaa gat gtg ctc tgt cat cca gag gcc acc tac ctg 2544Pro Gly Pro Val Glu Asp Val Leu Cys His Pro Glu Ala Thr Tyr Leu 835 840 845gcc ctg aac tgg acg atg cct gct gga gac gtg gat gtc tgt ctg gtg 2592Ala Leu Asn Trp Thr Met Pro Ala Gly Asp Val Asp Val Cys Leu Val 850 855 860gtg gta gag cgg ctg gtg ccg gga ggg ggc act cat ttt gtc ttc cag 2640Val Val Glu Arg Leu Val Pro Gly Gly Gly Thr His Phe Val Phe Gln865 870 875 880gtc aac acc tca ggg gat gct ctt ctg ttg ccc aac ttg atg ccc acc 2688Val Asn Thr Ser Gly Asp Ala Leu Leu Leu Pro Asn Leu Met Pro Thr 885 890 895act tct tac cgc ctt agc ctc acc gtt ctg ggc agg aat agt cgg tgg 2736Thr Ser Tyr Arg Leu Ser Leu Thr Val Leu Gly Arg Asn Ser Arg Trp 900 905 910agc cgg gcg gtt tcc ctg gtg tgc agt act tct gct gag gct tgg cac 2784Ser Arg Ala Val Ser Leu Val Cys Ser Thr Ser Ala Glu Ala Trp His 915 920 925ccc cca gag cta gct gag ccc ccc cag gtg gag ctg ggg aca ggg atg 2832Pro Pro Glu Leu Ala Glu Pro Pro Gln Val Glu Leu Gly Thr Gly Met 930 935 940ggt gtg aca gtc atg cgt ggc atg ttt ggt aaa gat gac ggg cag atc 2880Gly Val Thr Val Met Arg Gly Met Phe Gly Lys Asp Asp Gly Gln Ile945 950 955 960cag tgg tat ggc ata att gcc acc atc aac atg acg ctg gcc cag cct 2928Gln Trp Tyr Gly Ile Ile Ala Thr Ile Asn Met Thr Leu Ala Gln Pro 965 970 975tcc cgg gaa gcc atc aat tac aca tgg tat gac cac tac tat aga gga 2976Ser Arg Glu Ala Ile Asn Tyr Thr Trp Tyr Asp His Tyr Tyr Arg Gly 980 985 990tgt gag tcc ttc ctg gct ctc ctg ttc cca aac ccc ttc tac cca gag 3024Cys Glu Ser Phe Leu Ala Leu Leu Phe Pro Asn Pro Phe Tyr Pro Glu 995 1000 1005cct tgg gct ggg cca aga tcc tgg aca gta cct gtg ggt act gag 3069Pro Trp Ala Gly Pro Arg Ser Trp Thr Val Pro Val Gly Thr Glu 1010 1015 1020gac tgt gac aac acc caa gag ata tgc aat ggg cgt ctc aag tca 3114Asp Cys Asp Asn Thr Gln Glu Ile Cys Asn Gly Arg Leu Lys Ser 1025 1030 1035ggc ttc cag tat agg ttc agc gtt gtg gcc ttt agt agg ctc aac 3159Gly Phe Gln Tyr Arg Phe Ser Val Val Ala Phe Ser Arg Leu Asn 1040 1045 1050act cca gag acc atc ctc gcc ttc tcg gcc ttc tca gag ccc cgg 3204Thr Pro Glu Thr Ile Leu Ala Phe Ser Ala Phe Ser Glu Pro Arg 1055 1060 1065gcc agc atc tct ctg gcg atc att ccc ctg aca gtt atg ctg ggg 3249Ala Ser Ile Ser Leu Ala Ile Ile Pro Leu Thr Val Met Leu Gly 1070 1075 1080gct gtg gtg ggc agc att gtc att gtg tgt gca gtg cta tgc ttg 3294Ala Val Val Gly Ser Ile Val Ile Val Cys Ala Val Leu Cys Leu 1085 1090 1095ctc cgc tgg cgg tgc ctg aag gga cca aga tca gag aag gat ggc 3339Leu Arg Trp Arg Cys Leu Lys Gly Pro Arg Ser Glu Lys Asp Gly 1100 1105 1110ttt tcc aag gag ctg atg cct tac aac ctg tgg cgg acc cat cgg 3384Phe Ser Lys Glu Leu Met Pro Tyr Asn Leu Trp Arg Thr His Arg 1115 1120 1125cct atc ccc atc cat agc ttc cgg cag agc tat gag gcc aag agc 3429Pro Ile Pro Ile His Ser Phe Arg Gln Ser Tyr Glu Ala Lys Ser 1130 1135 1140gca cat gca cac cag acc ttc ttc cag gaa ttt gag gag ttg aag 3474Ala His Ala His Gln Thr Phe Phe Gln Glu Phe Glu Glu Leu Lys 1145 1150 1155gag gta ggc aag gac cag ccc cga cta gag gct gag cat ccg gac 3519Glu Val Gly Lys Asp Gln Pro Arg Leu Glu Ala Glu His Pro Asp 1160 1165 1170aac atc atc aag aac cgg tac cca cac gtg ctg ccc tat gac cac 3564Asn Ile Ile Lys Asn Arg Tyr Pro His Val Leu Pro Tyr Asp His 1175 1180 1185tcc agg gtc agg ctg acc cag cta cca gga gag cct cat tct gac 3609Ser Arg Val Arg Leu Thr Gln Leu Pro Gly Glu Pro His Ser Asp 1190 1195 1200tac atc aat gcc aac ttc atc cca ggc tat agc cac aca cag gag 3654Tyr Ile Asn Ala Asn Phe Ile Pro Gly Tyr Ser His Thr Gln Glu 1205 1210 1215atc att gcc acc cag ggg cct ctc aaa aag acg cta gag gac ttc 3699Ile Ile Ala Thr Gln Gly Pro Leu Lys Lys Thr Leu Glu Asp Phe 1220 1225 1230tgg cgg ttg gta tgg gag cag caa gtc cac gtg atc atc atg ctg 3744Trp Arg Leu Val Trp Glu Gln Gln Val His Val Ile Ile Met Leu 1235 1240 1245act gtg ggc atg gag aac ggg cgg gta ctg tgt gag cac tac tgg 3789Thr Val Gly Met Glu Asn Gly Arg Val Leu Cys Glu His Tyr Trp 1250 1255 1260cca gcc aac tcc acg cct gtt act cac ggt cac atc acc atc cac 3834Pro Ala Asn Ser Thr Pro Val Thr His Gly His Ile Thr Ile His 1265 1270 1275ctc ctg gca gag gag cct gag gat gag tgg acc agg agg gaa ttc 3879Leu Leu Ala Glu Glu Pro Glu Asp Glu Trp Thr Arg Arg Glu Phe 1280 1285 1290cag ctg cag cac ggt acc gag caa aaa cag agg cga gtg aag cag 3924Gln Leu Gln His Gly Thr Glu Gln Lys Gln Arg Arg Val Lys Gln 1295 1300 1305ctg cag ttc act acc tgg cca gac cac agt gtc ccg gag gct ccc 3969Leu Gln Phe Thr Thr Trp Pro Asp His Ser Val Pro Glu Ala Pro 1310 1315 1320agc tct ctg ctc gct ttt gta gaa ctg gta cag gag cag gtg cag 4014Ser Ser Leu Leu Ala Phe Val Glu Leu Val Gln Glu Gln Val Gln 1325 1330 1335gcc act cag ggc aag gga ccc atc ctg gtg cat tgc agt gct ggc 4059Ala Thr Gln Gly Lys Gly Pro Ile Leu Val His Cys Ser Ala Gly 1340 1345 1350gtg ggg agg aca ggc acc ttt gtg gct ctc ttg cgg cta ctg cga 4104Val Gly Arg Thr Gly Thr Phe Val Ala Leu Leu Arg Leu Leu Arg 1355 1360 1365caa cta gag gaa gag aag gtg gcc gat gtg ttc aac act gtg tac 4149Gln Leu Glu Glu Glu Lys Val Ala Asp Val Phe Asn Thr Val Tyr 1370 1375 1380ata ctc cgg ttg cac cgg ccc ctc atg atc cag acc ctg agt caa 4194Ile Leu Arg Leu His Arg Pro Leu Met Ile Gln Thr Leu Ser Gln 1385 1390 1395tac atc ttc ctg cac agt tgc ctg ctg aac aag att ctg gaa ggg 4239Tyr Ile Phe Leu His Ser Cys Leu Leu Asn Lys Ile Leu Glu Gly 1400 1405 1410ccc cct gac agc tcc gac tcc ggc ccc atc tct gtg atg gat ttt 4284Pro Pro Asp Ser Ser Asp Ser Gly Pro Ile Ser Val Met Asp Phe 1415 1420 1425gca cag gct tgt gcc aag agg gca gcc aac gcc aat gct ggt ttc 4329Ala Gln Ala Cys Ala Lys Arg Ala Ala Asn Ala Asn Ala Gly Phe 1430 1435 1440ttg aag gag tac aag ctc ctg aag cag gcc atc aag gat ggg act 4374Leu Lys Glu Tyr Lys Leu Leu Lys Gln Ala Ile Lys Asp Gly Thr 1445 1450 1455ggc tct ctg ctg ccc cct cct gac tac aat cag aac agc att gtc 4419Gly Ser Leu Leu Pro Pro Pro Asp Tyr Asn Gln Asn Ser Ile Val 1460 1465 1470tcc cgt cgt cat tct cag gag cag ttc gcc ctg gtg gag gag tgc 4464Ser Arg Arg His Ser Gln Glu Gln Phe Ala Leu Val Glu Glu Cys 1475 1480 1485cct gag gat agc atg ctg gaa gcc tca ctc ttc cct ggt ggt ccg 4509Pro Glu Asp Ser Met Leu Glu Ala Ser Leu Phe Pro Gly Gly Pro 1490 1495 1500tct ggt tgt gat cat gtg gtg ctg act ggc tca gcc gga cca aag 4554Ser Gly Cys Asp His Val Val Leu Thr Gly Ser Ala Gly Pro Lys 1505 1510 1515gaa ctc tgg gaa atg gtg tgg gag cat gat gcc cat gtg ctc gtc 4599Glu Leu Trp Glu Met Val Trp Glu His Asp Ala His Val Leu Val 1520 1525 1530tcc ctg ggc ctg cct gat acc aag gag aag cca cca gac atc tgg 4644Ser Leu Gly Leu Pro Asp Thr Lys Glu Lys Pro Pro Asp Ile Trp 1535 1540 1545cca gtg gag atg cag cct att gtc aca gac atg gtg aca gtg cac 4689Pro Val Glu Met Gln Pro Ile Val Thr Asp Met Val Thr Val His 1550 1555 1560aga gtg tct gag agc aac aca aca act ggc tgg ccc agc acc ctc 4734Arg Val Ser Glu Ser Asn Thr Thr Thr Gly Trp Pro Ser Thr Leu 1565 1570 1575ttc aga gtc ata cac ggg gag agt gga aag gaa agg cag gtt caa 4779Phe Arg Val Ile His Gly Glu Ser Gly Lys Glu Arg Gln Val Gln 1580 1585 1590tgc ctg caa ttt cca tgc tct gag tct ggg tgt gag ctc cca gct 4824Cys Leu Gln Phe Pro Cys Ser Glu Ser Gly Cys Glu Leu Pro Ala 1595 1600 1605aac acc cta ctg acc ttc ctt gat gct gtg ggc cag tgc tgc ttc 4869Asn Thr Leu Leu Thr Phe Leu Asp Ala Val Gly Gln Cys Cys Phe 1610 1615 1620cgg ggc aag agc aag aag cca ggg acc ctg ctc agc cac tcc agc 4914Arg Gly Lys Ser Lys Lys Pro Gly Thr Leu Leu Ser His Ser Ser 1625 1630 1635aaa aac aca aac cag ctg ggc acc ttc ttg gct atg gaa cag ctg 4959Lys Asn Thr Asn Gln Leu Gly Thr Phe Leu Ala Met Glu Gln Leu 1640 1645 1650tta cag caa gca ggg aca gag cgc aca gtg gac gtc ttc aat gtg 5004Leu Gln Gln Ala Gly Thr Glu Arg Thr Val Asp Val Phe Asn Val 1655 1660 1665gcc ctg aag cag tca cag gcc tgc ggc ctt atg acc cca aca ctg 5049Ala Leu Lys Gln Ser Gln Ala Cys Gly Leu Met Thr Pro Thr Leu 1670 1675 1680gag cag tat atc tac ctc tac aac tgt ctg aac agc gca ctg ctg 5094Glu Gln Tyr Ile Tyr Leu Tyr Asn Cys Leu Asn Ser Ala Leu Leu 1685 1690 1695aac ggg ctg ccc aga gct ggg aag tgg cct gcg ccc tgc tag 5136Asn Gly Leu Pro Arg Ala Gly Lys Trp Pro Ala Pro Cys 1700 1705 1710251711PRTRattus norvegicus 25Met Arg Pro Leu Ile Leu Leu Ala Ala Leu Leu Trp Leu Gln Gly Phe1 5 10 15Leu Ala Glu Asp Asp Ala Cys Ser Ser Leu Glu Gly Ser Pro Asp Arg 20 25 30Gln Gly Gly Gly Pro Leu Leu Ser Val Asn Val Ser Ser His Gly Lys 35 40 45Ser Thr Ser Leu Phe Leu Ser Trp Val Ala Ala Glu Leu Gly Gly Phe 50 55 60Asp Tyr Ala Leu Ser Leu Arg Ser Val Asn Ser Ser Gly Ser Pro Glu65 70 75 80Gly Gln Gln Leu Gln Ala His Thr Asn Glu Ser Gly Phe Glu Phe His 85 90 95Gly Leu Val Pro Gly Ser Arg Tyr Gln Leu Lys Leu Thr Val Leu Arg 100 105 110Pro Cys Trp Gln Asn Val Thr Ile Thr Leu Thr Ala Arg Thr Ala Pro 115 120 125Thr Val Val Arg Gly Leu Gln Leu His Ser Ala Gly Ser Pro Ala Arg 130 135 140Leu Glu Ala Ser Trp Ser Asp Ala Pro Gly Asp Gln Asp Ser Tyr Gln145 150 155 160Leu Leu Leu Tyr His Leu Glu Ser Gln Thr Leu Ala Cys Asn Val Ser 165 170 175Val Ser Pro Asp Thr Leu Ser Tyr Ser Phe Gly Asp Leu Leu Pro Gly 180 185 190Thr Gln Tyr Val Leu Glu Val Ile Thr Trp Ala Gly Ser Leu His Ala 195 200 205Lys Thr Ser Ile Leu Gln Trp Thr Glu Pro Val Pro Pro Asp His Leu 210 215 220Ala Leu Arg Ala Leu Gly Thr Ser Ser Leu Gln Ala Phe Trp Asn Ser225 230 235 240Ser Glu Gly Ala Thr Ser Phe His Leu Met Leu Thr Asp Leu Leu Gly 245 250 255Gly Thr Asn Thr Thr Ala Val Ile Arg Gln Gly Val Ser Thr His Thr 260 265 270Phe Leu His Leu Ser Pro Gly Thr Pro His Glu Leu Lys Ile Cys Ala 275 280 285Ser Ala Gly Pro His Gln Ile Trp Gly Pro Ser Ala Thr Glu Trp Thr 290 295 300Tyr Pro Ser Tyr Pro Ser Asp Leu Val Leu Thr Pro Leu Arg Asn Glu305 310 315 320Leu Trp Ala Ser Trp Lys Ala Gly Leu Gly Ala Arg Asp Gly Tyr Val 325 330 335Leu Lys Leu Ser Gly Pro Met Glu Ser Thr Ser Thr Leu Gly Pro Glu 340 345 350Glu Cys Asn Ala Val Phe Pro Gly Pro Leu Pro Pro Gly His Tyr Thr 355 360 365Leu Gln Leu Lys Val Leu Ala Gly Pro Tyr Asp Ala Trp Val Glu Gly 370 375 380Ser Thr Trp Leu Ala Glu Ser Ala Ala Leu Pro Arg Glu Val Pro Gly385 390 395 400Ala Arg Leu Trp Leu Asp Gly Leu Glu Ala Ser Lys Gln Pro Gly Arg 405 410 415Arg Ala Leu Leu Tyr Ser Asp Asp Ala Pro Gly Ser Leu Gly Asn Ile 420 425 430Ser Val Pro Ser Gly Ala Thr His Val Ile Phe Cys Gly Leu Val Pro 435 440 445Gly Ala His Tyr Arg Val Asp Ile Ala Ser Ser Thr Gly Asp Ile Ser 450 455 460Gln Ser Ile Ser Gly Tyr Thr Ser Pro Leu Pro Pro Gln Ser Leu Glu465 470 475 480Val Ile Ser Arg Ser Ser Pro Ser Asp Leu Thr Ile Ala Trp Gly Pro 485 490 495Ala Pro Gly Gln Leu Glu Gly Tyr Lys Val Thr Trp His Gln Asp Gly 500 505 510Ser Gln Arg Ser Pro Gly Asp Leu Val Asp Leu Gly Pro Asp Thr Leu 515 520 525Ser Leu Thr Leu Lys Ser Leu Val Pro Gly Ser Cys Tyr Thr Val Ser 530 535 540Ala Trp Ala Trp Ala Gly Asn Leu Asp Ser Asp Ser Gln Lys Ile His545 550 555 560Ser Cys Thr Arg Pro Ala Pro Pro Thr Asn Leu Ser Leu Gly Phe Ala 565 570 575His Gln Pro Ala Ala Leu Lys Ala Ser Trp Tyr His Pro Pro Gly Gly 580 585 590Arg Asp Ala Phe His Leu Arg Leu Tyr Arg Leu Arg Pro Leu Thr Leu 595 600 605Glu Ser Glu Lys Val Leu Pro Arg Glu Ala Gln Asn Phe Ser Trp Ala 610 615 620Gln Leu Thr Ala Gly Cys Glu Phe Gln Val Gln Leu Ser Thr Leu Trp625 630 635 640Gly Ser Glu Arg Ser Ser Ser Ala Asn Ala Thr Gly Trp Thr Pro Pro 645 650 655Ser Ala Pro Thr Leu Val Asn Val Thr Ser Asp Ala Pro Thr Gln Leu 660 665 670Gln Val Ser Trp Ala His Val Pro Gly Gly Arg Ser Arg Tyr Gln Val 675 680 685Thr Leu Tyr Gln Glu Ser Thr Arg Thr Ala Thr Ser Ile Met Gly Pro 690 695 700Lys Glu Asp Gly Thr Ser Phe Leu Gly Leu Thr Pro Gly Thr Lys Tyr705 710 715 720Lys Val Glu Val Ile Ser Trp Ala Gly Pro Leu Tyr Thr Ala Ala Ala 725 730 735Asn Val Ser Ala Trp Thr Tyr Pro Leu Ile Pro Asn Glu Leu Leu Val 740 745 750Ser Met Gln Ala Gly Ser Ala Val Val Asn Leu Ala Trp Pro Ser Gly 755 760 765Pro Leu Gly Gln Gly Ala Cys His Ala Gln Leu Ser Asp Ala Gly His 770 775 780Leu Ser Trp Glu Gln Pro Leu Lys Leu Gly Gln Glu Leu Phe Met Leu785 790 795 800Arg Asp Leu Thr Pro Gly His Thr Ile Ser Met Ser Val Arg Cys Arg 805 810 815Ala Gly Pro Leu Gln Ala Ser Thr His Leu Val Val Leu Ser Val Glu 820 825 830Pro Gly Pro Val Glu Asp Val Leu Cys His Pro Glu Ala Thr Tyr Leu 835 840 845Ala Leu Asn Trp Thr Met Pro Ala Gly Asp Val Asp Val Cys Leu Val 850 855 860Val Val Glu Arg Leu Val Pro Gly Gly Gly Thr His Phe Val Phe Gln865 870 875 880Val Asn Thr Ser Gly Asp Ala Leu Leu Leu

Pro Asn Leu Met Pro Thr 885 890 895Thr Ser Tyr Arg Leu Ser Leu Thr Val Leu Gly Arg Asn Ser Arg Trp 900 905 910Ser Arg Ala Val Ser Leu Val Cys Ser Thr Ser Ala Glu Ala Trp His 915 920 925Pro Pro Glu Leu Ala Glu Pro Pro Gln Val Glu Leu Gly Thr Gly Met 930 935 940Gly Val Thr Val Met Arg Gly Met Phe Gly Lys Asp Asp Gly Gln Ile945 950 955 960Gln Trp Tyr Gly Ile Ile Ala Thr Ile Asn Met Thr Leu Ala Gln Pro 965 970 975Ser Arg Glu Ala Ile Asn Tyr Thr Trp Tyr Asp His Tyr Tyr Arg Gly 980 985 990Cys Glu Ser Phe Leu Ala Leu Leu Phe Pro Asn Pro Phe Tyr Pro Glu 995 1000 1005Pro Trp Ala Gly Pro Arg Ser Trp Thr Val Pro Val Gly Thr Glu 1010 1015 1020Asp Cys Asp Asn Thr Gln Glu Ile Cys Asn Gly Arg Leu Lys Ser 1025 1030 1035Gly Phe Gln Tyr Arg Phe Ser Val Val Ala Phe Ser Arg Leu Asn 1040 1045 1050Thr Pro Glu Thr Ile Leu Ala Phe Ser Ala Phe Ser Glu Pro Arg 1055 1060 1065Ala Ser Ile Ser Leu Ala Ile Ile Pro Leu Thr Val Met Leu Gly 1070 1075 1080Ala Val Val Gly Ser Ile Val Ile Val Cys Ala Val Leu Cys Leu 1085 1090 1095Leu Arg Trp Arg Cys Leu Lys Gly Pro Arg Ser Glu Lys Asp Gly 1100 1105 1110Phe Ser Lys Glu Leu Met Pro Tyr Asn Leu Trp Arg Thr His Arg 1115 1120 1125Pro Ile Pro Ile His Ser Phe Arg Gln Ser Tyr Glu Ala Lys Ser 1130 1135 1140Ala His Ala His Gln Thr Phe Phe Gln Glu Phe Glu Glu Leu Lys 1145 1150 1155Glu Val Gly Lys Asp Gln Pro Arg Leu Glu Ala Glu His Pro Asp 1160 1165 1170Asn Ile Ile Lys Asn Arg Tyr Pro His Val Leu Pro Tyr Asp His 1175 1180 1185Ser Arg Val Arg Leu Thr Gln Leu Pro Gly Glu Pro His Ser Asp 1190 1195 1200Tyr Ile Asn Ala Asn Phe Ile Pro Gly Tyr Ser His Thr Gln Glu 1205 1210 1215Ile Ile Ala Thr Gln Gly Pro Leu Lys Lys Thr Leu Glu Asp Phe 1220 1225 1230Trp Arg Leu Val Trp Glu Gln Gln Val His Val Ile Ile Met Leu 1235 1240 1245Thr Val Gly Met Glu Asn Gly Arg Val Leu Cys Glu His Tyr Trp 1250 1255 1260Pro Ala Asn Ser Thr Pro Val Thr His Gly His Ile Thr Ile His 1265 1270 1275Leu Leu Ala Glu Glu Pro Glu Asp Glu Trp Thr Arg Arg Glu Phe 1280 1285 1290Gln Leu Gln His Gly Thr Glu Gln Lys Gln Arg Arg Val Lys Gln 1295 1300 1305Leu Gln Phe Thr Thr Trp Pro Asp His Ser Val Pro Glu Ala Pro 1310 1315 1320Ser Ser Leu Leu Ala Phe Val Glu Leu Val Gln Glu Gln Val Gln 1325 1330 1335Ala Thr Gln Gly Lys Gly Pro Ile Leu Val His Cys Ser Ala Gly 1340 1345 1350Val Gly Arg Thr Gly Thr Phe Val Ala Leu Leu Arg Leu Leu Arg 1355 1360 1365Gln Leu Glu Glu Glu Lys Val Ala Asp Val Phe Asn Thr Val Tyr 1370 1375 1380Ile Leu Arg Leu His Arg Pro Leu Met Ile Gln Thr Leu Ser Gln 1385 1390 1395Tyr Ile Phe Leu His Ser Cys Leu Leu Asn Lys Ile Leu Glu Gly 1400 1405 1410Pro Pro Asp Ser Ser Asp Ser Gly Pro Ile Ser Val Met Asp Phe 1415 1420 1425Ala Gln Ala Cys Ala Lys Arg Ala Ala Asn Ala Asn Ala Gly Phe 1430 1435 1440Leu Lys Glu Tyr Lys Leu Leu Lys Gln Ala Ile Lys Asp Gly Thr 1445 1450 1455Gly Ser Leu Leu Pro Pro Pro Asp Tyr Asn Gln Asn Ser Ile Val 1460 1465 1470Ser Arg Arg His Ser Gln Glu Gln Phe Ala Leu Val Glu Glu Cys 1475 1480 1485Pro Glu Asp Ser Met Leu Glu Ala Ser Leu Phe Pro Gly Gly Pro 1490 1495 1500Ser Gly Cys Asp His Val Val Leu Thr Gly Ser Ala Gly Pro Lys 1505 1510 1515Glu Leu Trp Glu Met Val Trp Glu His Asp Ala His Val Leu Val 1520 1525 1530Ser Leu Gly Leu Pro Asp Thr Lys Glu Lys Pro Pro Asp Ile Trp 1535 1540 1545Pro Val Glu Met Gln Pro Ile Val Thr Asp Met Val Thr Val His 1550 1555 1560Arg Val Ser Glu Ser Asn Thr Thr Thr Gly Trp Pro Ser Thr Leu 1565 1570 1575Phe Arg Val Ile His Gly Glu Ser Gly Lys Glu Arg Gln Val Gln 1580 1585 1590Cys Leu Gln Phe Pro Cys Ser Glu Ser Gly Cys Glu Leu Pro Ala 1595 1600 1605Asn Thr Leu Leu Thr Phe Leu Asp Ala Val Gly Gln Cys Cys Phe 1610 1615 1620Arg Gly Lys Ser Lys Lys Pro Gly Thr Leu Leu Ser His Ser Ser 1625 1630 1635Lys Asn Thr Asn Gln Leu Gly Thr Phe Leu Ala Met Glu Gln Leu 1640 1645 1650Leu Gln Gln Ala Gly Thr Glu Arg Thr Val Asp Val Phe Asn Val 1655 1660 1665Ala Leu Lys Gln Ser Gln Ala Cys Gly Leu Met Thr Pro Thr Leu 1670 1675 1680Glu Gln Tyr Ile Tyr Leu Tyr Asn Cys Leu Asn Ser Ala Leu Leu 1685 1690 1695Asn Gly Leu Pro Arg Ala Gly Lys Trp Pro Ala Pro Cys 1700 1705 1710

Claims

1-122. (canceled)

123. A method for treating or preventing a disorder selected from the group consisting of diabetes, metabolic syndrome, glucose intolerance, and obesity in a mammal, said method comprising administering an effective amount of uncarboxylated/undercarboxylated osteocalcin or a fragment thereof.

124. The method of claim 123, wherein the disorder is type 1 diabetes.

125. The method of claim 123, wherein the disorder is type 2 diabetes.

126. The method of claim 123, wherein the disorder is obesity.

127. The method of claim 123, wherein the disorder is metabolic syndrome.

128. The method of claim 123, wherein the uncarboxylated/undercarboxylated osteocalcin is administered in an amount effective to produce an effect selected from the group consisting of an increase in glucose tolerance, an increase in insulin production, an increase insulin sensitivity, an increase in pancreatic beta-cell proliferation, and an increase in adiponectin serum level.

129. The method of claim 123, wherein the mammal is a human.

130. The method of claim 123, wherein the uncarboxylated/undercarboxylated osteocalcin is administered in combination with an agent used to prevent bone loss.

131. The method of claim 123, wherein at least one of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21, and 24 of mature human osteocalcin is not carboxylated.

132. The method of claim 123, wherein all three of the glutamic acids in the undercarboxylated/uncarboxylated osteocalcin at the positions corresponding to positions 17, 21, and 24 of mature human osteocalcin are not carboxylated.

133. The method of claim 123, wherein the undercarboxylated/uncarboxylated osteocalcin is a preparation of undercarboxylated/uncarboxylated osteocalcin in which more than about 20% of the total Glu residues at the positions corresponding to positions 17, 21, and 24 of mature human mature human osteocalcin in the preparation are not carboxylated.

134. The method of claim 123, wherein the undercarboxylated/uncarboxylated osteocalcin shares at least 80% amino acid sequence identity with mature human osteocalcin when the undercarboxylated/uncarboxylated osteocalcin and mature human osteocalcin are aligned for maximum sequence homology.

135. The method of claim 123, wherein the undercarboxylated/uncarboxylated osteocalcin comprises the mature human osteocalcin protein.

136. The method of claim 123, wherein the undercarboxylated/uncarboxylated osteocalcin is a polypeptide selected from the group consisting of:(a) a fragment comprising mature human osteocalcin missing the first 10 amino acids from the N-terminal end;(b) a fragment comprising mature human osteocalcin missing the last 10 amino acids from the C-terminal end;(c) a fragment comprising amino acids 62-90 of SEQ ID NO:2;(d) a fragment comprising amino acids 1-36 of mature human osteocalcin;(e) a fragment comprising amino acids 13-26 of mature human osteocalcin;(f) a fragment comprising amino acids 13-46 of mature human osteocalcin; and(g) variants of the above.

137. The method of claim 136, wherein the undercarboxylated/uncarboxylated osteocalcin is a fragment comprising mature human osteocalcin missing the first 10 amino acids from the N-terminal end and missing the last 10 amino acids from the C-terminal end of mature human osteocalcin.

138. The method of claim 136, wherein the undercarboxylated/uncarboxylated osteocalcin is a fragment consisting of amino acids 1-36 of mature human osteocalcin.

139. The method of claim 136, wherein the undercarboxylated/uncarboxylated osteocalcin is a fragment consisting of amino acids 13-26 of mature human osteocalcin.

140. The method of claim 136, wherein the undercarboxylated/uncarboxylated osteocalcin is a fragment consisting of amino acids 13-46 of mature human osteocalcin.

141. The method of claim 123, wherein the undercarboxylated/uncarboxylated osteocalcin has reduced binding to hydroxyapatite compared to carboxylated osteocalcin.

142. A method for treating or preventing a vascular disorder in a mammal comprising administering an effective amount of uncarboxylated/undercarboxylated osteocalcin.

143. A method of diagnosing a patient at risk of developing a disease related to energy metabolism and the OST-PTP signaling pathway comprising (i) determining the ratio of undercarboxylated/uncarboxylated osteocalcin to total osteocalcin in a biological sample from the patient; and (ii) comparing the ratio to a standard ratio; wherein, if the patient ratio is lower than the standard ratio, the patient is at risk of developing a disease related to the OST-PTP signaling pathway.

144. A method for increasing insulin sensitivity, increasing insulin production, increasing glucose tolerance, increasing pancreatic beta-cell proliferation, or increasing adiponectin serum level in a mammal comprising administering an effect amount of uncarboxylated/undercarboxylated osteocalcin.

145. A pharmaceutical composition for treating or preventing a disorder selected from the group consisting of diabetes, metabolic syndrome, glucose intolerance, and obesity in a mammal, said pharmaceutical composition comprising an effective amount of uncarboxylated/undercarboxylated osteocalcin.

146. The pharmaceutical composition of claim 145, wherein the uncarboxylated/undercarboxylated osteocalcin is in an amount effective to produce an effect selected from the group consisting of an increase in glucose tolerance, an increase in insulin production, an increase insulin sensitivity, an increase in pancreatic beta-cell proliferation, and an increase in adiponectin serum level.

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