IDENTIFICATION, CLONING, EXPRESSION, AND A PURIFICATION OF THREE NOVEL HUMAN CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 FAMILY MEMBERS POSSESSING TRIACYLGLYCEROL LIPASE AND ACYLGLYCEROL TRANSACYLASE ACTIVITIES

Abstract

ed and characterized phospholipase A₂, referred to herein as calcium-independent phospholipase A₂zeta (iPLA₂zeta) having SEQ. ID. NO: 2 (See FIG. 1), and nucleic acid sequence (SEQ. ID. NO: 4), and calcium-independent phospholipase A₂eta (iPLA₂eta) having SEQ. ID. NO: 3 (See FIG. 1), and nucleic acid sequences (SEQ. ID. NO: 5). For the first time herein, these novel enzymes have been isolated and characterized and are involved in the catalysis, synthesis and hydrolysis of lipids in a living mammalian cell. Moreover, these enzymes iPLA₂zeta and iPLA₂eta through the process of transesterification can catalyze the net anabolic synthesis of triglycerides through a variety of metabolic precursors (e.g. monoacylglycerol, diacylglycerol and acyl CoA).

Description

[0001]This application is a divisional of U.S. application Ser. No. 11/179,363 filed Jul. 11, 2005, herein incorporated in its entirety, which claims the benefit of U.S. Ser. No. 60/586,913 filed Jul. 9, 2004, herein incorporated in its entirety. This application is also a continuation-in-part of U.S. Ser. No. 11/010,558, filed Dec. 13, 2004, herein incorporated in its entirety, which claims the benefit of U.S. Ser. No. 60/528,951 filed Dec. 11, 2003, herein incorporated in its entirety.

FIELD OF THE INVENTION

[0003]This invention relates to lipases and more particularly to human calcium independent lipases A₂, nucleic acids capably expressing such lipases, to use of such lipases as pharmacological targets in screening to identify potentially useful antiobesity drugs and drugs for other sequelae of the metabolic syndrome including at least one of atherosclerosis, diabetes and hypertension in humans and to a functional animal model useful for such screening.

BACKGROUND OF THE INVENTION

[0004]In many industrialized countries, the incidence of human obesity is ever increasing. Moreover, human obesity is a common and costly nutritional problem in the United States, Obesity is characterized clinically by the accumulation of fat tissue (at times this is referred to as body fat content).

[0005]In humans, obesity is usually defined as a body fat content greater than about 25% of the total weight for males, or greater than 30% of the total weight for females. Regardless of the cause of obesity, obesity is an ever present problem for Americans. But a fat content >18% for males and >22% for females can have untold consequences secondary to several mechanisms and disorders of metabolic function. For example, obesity can have a significant adverse impact on health care costs and provoke a higher risk of numerous illnesses, including heart attacks, strokes and diabetes.

[0006]Without being bound by theory, it is believed that obesity in humans results from an abnormal increase in white adipose tissue mass that occurs due to an increased number of adipocytes (hyperplasia) or from increased lipid mass (stored as triglycerides) accumulating in existing adipocytes. Obesity and the associated type 2 metabolic syndrome along with its clinical sequelae are among the major and the most rapidly increasing medical problems in America. However, to date, a lack of suitable adipocyte specific protein targets has unfortunately hampered progress in the development of effective therapeutic agents to combat the clinical sequelae of obesity.

[0007]Despite existing knowledge of the critical role of phospholipases and triglyceride lipases in adipocyte signaling, enhanced clinical methodology and research tools and research methods are highly needed for identifying useful drugs to treat obesity and over-weightness. It is highly desired to have technology based on the specific types of phospholipases and triglyceride lipases present in the adipocyte or their mechanisms of regulation and determine their natural substrates and roles in anabolic lipid metabolism, catabolic lipid metabolism or both (e.g. triglyceride cycling).

[0008]Additionally, a screening method and research tool is needed to identify useful drugs which can be used to reduce the fat level of a living mammal and/or to maintain the fat level at a predetermined level.

BRIEF DESCRIPTION OF THE INVENTION

[0009]This invention relates to phospholipases and more particularly to human calcium independent phospholipase A₂, hereinafter referred to as and denoted iPLA₂, to nucleic acids expressing iPLA₂, to use of iPLA₂ as a pharmacological target in screening to identify potentially useful anti-obesity drugs and drugs for other sequelae of the metabolic syndrome including at least one of atherosclerosis, diabetes and hypertension and to a functional animal model useful for such screening. This discovery has utility.

[0010]In an aspect, the invention provides for the first time isolated, novel, purified, functional, characterized and useful phospholipases A₂ referred to herein as calcium-independent lipase A₂zeta (iPLA2zeta) having SEQ. ID. NO: 2 (See FIG. 1) and nucleic acid sequence SEQ. ID. NO: 4 (See FIG. 7), and calcium-independent lipase A₂eta (iPLA₂eta) having SEQ. ID. NO: 3 (See FIG. 1) and nucleic acid sequence SEQ. ID. NO: 5 (See FIG. 8). For the first time herein, these novel enzymes have been isolated and characterized and have been discovered to be involved in the catalysis, synthesis and hydrolysis of lipids in a living mammalian cell. Moreover, these enzymes iPLA₂zeta and iPLA₂eta through the process of transesterification can each independently catalyze the net anabolic synthesis of triglycerides through a variety of metabolic precursors (e.g. monoacylglycerol, diacylglycerol and acyl CoA).

[0011]In one embodiment, the invention is directed to an isolated and characterized nucleic acid molecule comprising a set of iPLA₂zeta polynucleotides. In an aspect of this embodiment, the iPLA₂zeta polynucleotides (SEQ. ID. NO: 4) encode (and express) an iPLA₂zeta polypeptide (SEQ. ID. NO. 2).

[0012]In one embodiment, the invention is directed to an isolated characterized nucleic acid molecule comprising a set of iPLA₂eta polynucleotides. In an aspect of this embodiment, the iPLA₂eta polynucleotides (SEQ. ID. NO: 5) encode (and express) an iPLA₂eta polypeptide (SEQ. ID. NO: 3).

[0013]In one aspect, an isolated and characterized human gene (iPLA₂zeta) comprises a characterized polynucleotide having a sequence shown in SEQ. ID. NO: 4 (See FIG. 7).

[0014]In one aspect, an isolated and characterized human gene (iPLA₂eta) comprises a polynucleotide having a sequence shown in SEQ. ID. NO: 5 (See FIG. 8).

[0015]In an aspect, an isolated and characterized human protein (iPLA₂zeta) comprises a polypeptide having a sequence shown in SEQ. ID. NO: 2 (See FIG. 1).

[0016]In an aspect, an isolated and characterized human protein (iPLA₂zeta) comprises a polypeptide having a sequence shown in SEQ. ID. NO: 2 (See FIG. 1) with a histidine tag (4-12 histidine residues).

[0017]In an aspect, an isolated and characterized human protein (iPLA₂eta) comprises a polypeptide having a sequence shown in SEQ. ID. NO: 3 (See FIG. 1).

[0018]In an aspect, an isolated and characterized human protein (iPLA₂eta) comprises a polypeptide having a sequence shown in SEQ. ID. NO: 3 (See FIG. 1) with a histidine tag (4-12 histidine residues).

[0019]In an aspect, the invention comprises a set of enzymes whose activities can be effectively modulated, alone or in concert, to have salutary effects on the sequelae of lipotoxicity by altering the amount and molecular species composition of triglycerides and/or phospholipids. These beneficial effects can be realized in multiple living mammalian cell types, including but not limited to myocardium, pancreatic beta cells, and macrophages during atheromatous plaque formation.

[0020]In an aspect, a method to improve the insulin sensitivity of the organism by effectively modulating the amounts of fatty acids, fatty acyl-CoAs, and other lipid species in pancreatic beta cells, muscle, or liver which contribute to insulin resistance in Type 2 diabetes.

[0021]In an aspect, a method to attenuate the development and progression of atherosclerosis and vascular disease by altering the lipid composition of plasma and modifying the lipid metabolism of critical cells that promote atherogenesis (e.g. macrophages, smooth muscle cells, and platelets),

[0022]In an aspect, a method of protecting against or modifying the deleterious sequelae of heart attacks or strokes by altering the lipid composition of heart or brain cells to withstand episodes of ischemia, attack by free radicals, or effects due to dysfunctional lipid metabolism.

[0023]In an aspect, this discovery comprises a method to measure the types and amounts (assay) of different lipase activities in fat cells and their inhibition by BEL or other suitable pharmacologic agents.

[0024]In an aspect, a method of treating a living mammal to reduce obesity, which comprises administering an effective amount of iPLA₂zeta and/or iPLA₂eta inhibitor thereto or by administering an agent which changes the lipase to transacylase activity ratio.

[0025]In an aspect, a genetically engineered expression vector comprises a gene or part of the sequence of a human gene (iPLA₂zeta) comprising an isolated and characterized polynucleotide having a sequence shown in SEQ ID NO: 4.

[0026]In an aspect, the gene encodes a protein, iPLA₂zeta, comprising a polypeptide having a sequence shown in SEQ ID NO: 2 (FIG. 1). In an aspect, the gene is operatively linked to a capable viable promoter element.

[0027]In an aspect, a genetically engineered expression vector comprises a gene or part of the sequence of a human gene (iPLA₂eta) comprising a polynucleotide having a sequence shown in SEQ ID NO: 5. In an aspect, the gene encodes a protein (iPLA₂eta) comprising a polypeptide having a sequence shown in SEQ ID NO: 3 (FIG. 1). In an aspect, the gene is operatively linked to a capable viable promoter element.

[0028]In another aspect, a method of medically treating a mammal comprises administering an anti-obesity (drug or pharmaceutical) to the mammal in therapeutically effective amounts as an inhibitor.

[0029]In another aspect, a method of medically treating a living mammal comprises administering a therapeutically effective amount of a moiety such as a compound (drug or pharmaceutical) which inhibits iPLA₂zeta and/or iPLA₂eta expression to the mammal which results in a different isoform expression or different enzymatic activity or post-translational modification.

[0030]In another aspect, a method of treating obesity, which comprises administering an agent selected from the group consisting of iPLA₂epsilon (adiponutrin), iPLA₂zeta (TTS-2.2), and iPLA₂eta (GS2) which changes the transacylase to lipase ratio of any or a combination of these three enzymes in a metabolic setting. In an aspect, the metabolic setting is a living animal or animal model.

[0031]In an aspect, a pharmaceutical composition is provided comprising a compound which effectively inhibits or counteracts iPLA₂epsilon (adiponutrin), iPLA₂zeta (TTS-2.2), and iPLA₂eta (GS2) expression, activity, phospholipase A₂ activity, hydrolysis or transesterification activity or transesterification in a living mammal.

[0032]In an aspect, a pharmaceutical kit comprises a container housing a compound which inhibits at least one of iPLA₂epsilon (adiponutrin), iPLA₂zeta (TTS-2.2), and iPLA₂eta (GS2) expression hydrolytic activity, phospholipase A₂ activity, or transesterification activity and optionally a carrier.

[0033]In another embodiment, the present invention is directed to a method of modulating fatty acid utilization in a patient. In an aspect, the patient is a living human patient. In this aspect, the method comprises increasing or decreasing iPLA₂zeta and/or iPLA₂eta activity in the patient. Patients in need of such treatment include those patients suffering from one of diabetes and/or obesity. Preferably, this method comprises administering to the patient a substance (compound) in an effective amount which blocks or inhibits expression of iPLA₂zeta and/or iPLA₂eta mass or activity.

[0034]In an aspect a method of identifying an agent which changes the ratio of transacylase to lipase activity in a living mammal by administering a compound to a mammal and determining if the transacylase to lipase ratio was changed by lipid analysis and if the ratio was changed then determining that the drug is an anti-obesity drug.

[0035]In an aspect, the invention comprises a method for ameliorating at least one symptom of a symptomatology comprising obesity and clinical manifestation of the type 2 metabolic syndrome in a living human which comprises treating a living human cell expressing iPLA₂zeta and/or iPLA₂eta in a pharmacologically effective manner with a pharmacologically effective amount of a drug which alters (increases or decreases) iPLA₂zeta and/or iPLA₂eta expression or activates or inhibits iPLA₂zeta and/or iPLA₂eta enzymatic activity.

[0036]A method of treating at least one of an overweight and obese disorders in a living animal or animal model, the method comprises administering to a subject (in need of such treatment) a therapeutically effective amount of a composition comprising an inhibitor of human iPLA₂zeta and/or iPLA₂eta.

[0037]A method of treating at least one of an overweight and obese disorders in a living animal or animal model, the method comprises administering to a subject (in need of such treatment) a therapeutically effective amount of a composition comprising an activator of human iPLA₂zeta and/or iPLA₂eta.

[0038]A screening and/or research tool useful to identify drugs useful to treat obesity. In a further aspect, a method (and/or screening or research tool) of identifying and/or for an anti-obesity drug comprises administering a drug to an animal and determining if there has been any change in iPLA₂zeta and/or iPLA₂eta expression, hydrolysis activity, phospholipase A₂ activity, or transesterification activity, or metabolic futile cycling and if so determining that the drug is an anti-obesity drug.

[0039]A method of practicing medicine which comprises administering a therapeutic amount of a drug to a patient at risk for obesity or being obese, the drug being an inhibitor of human iPLA₂zeta and/or iPLA₂eta.

[0040]A method of practicing medicine which comprises administering a therapeutic amount of a drug to a patient at risk for obesity or being obese, the drug being an activator of human iPLA₂zeta and/or iPLA₂eta.

[0041]A method of providing therapy to a patient in need thereof which comprises administering a drug to a patient at risk for obesity, the drug being an inhibitor of the expressing of human iPLA₂zeta and/or iPLA₂eta.

[0042]A method of providing therapy to a patient in need thereof which comprises administering a drug to a patient at risk for obesity, the drug being an activator of the expressing of human iPLA₂zeta and/or iPLA₂eta.

[0043]A method for treating a diabetic patient which comprises administering a drug in an effective amount to modulate iPLA₂zeta and/or iPLA₂eta expression whereby the insulin requirement of the patient is decreased

[0044]A method of treating diabetes which comprises administering a drug in an effective amount to modulate iPLA₂zeta and/or iPLA₂eta expression whereby the insulin requirement of the patient being treated for diabetes is decreased.

[0045]In an aspect, the present discovery encompasses genetically engineered cells capable of identifying substances which modulate iPLA₂zeta or iPLA₂eta expression in a living cell. In an aspect, such cells comprise a promoter operably linked to the iPLA₂zeta gene or the iPLA₂eta gene and a reporter gene. This reporter gene preferably encodes an enzyme capable of being detected by at least one of a suitable radiometric, fluorometric or luminometric assay such as, for example, a reporter sequence encoding a luciferase. In an aspect, the promoter sequence is a baculovirus promoter sequence and the cells are Sf9 cells.

[0046]In an aspect, the invention comprises a method for prioritizing the therapeutic capability of drugs of putative efficacy against obesity, comprising administering drugs to a living animal system which is actively expressing iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta, measuring any modulation of the iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression by a TAG or free fatty acid/glycerol analysis of an effect and determining if the modulation was an increase or a decrease or no change in iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression level. If the modulation is determined to be a decrease then determining that the drug was effective in inhibiting iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression, a value is assigned to that modulation and is thereafter compared to the modulation of other drugs. In an aspect, a prioritization can be set up by comprising the magnitudes of the various respective modulations and a hierarchy of drugs can be established. From this, it is possible to establish a priority of work on the drugs.

[0047]In an aspect, the invention comprises a method for prioritizing the therapeutic capability of drugs of putative efficacy against obesity, comprising administering drugs to a living animal system which is actively expressing iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta, measuring any modulation of the iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression by a TAG or free fatty acid/glycerol analysis of an effect and determining if the modulation was an increase or a decrease or no change in iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression level. If the modulation is determined to be a decrease then determining that the drug was effective in promoting iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression, a value is assigned to that modulation and is thereafter compared to the modulation of other drugs. In an aspect, a prioritization can be set up by comprising the magnitudes of the various respective modulations and a hierarchy of drugs can be established. From this, it is possible to establish a priority of work on the drugs.

[0048]The present discovery includes a method and research tool for identifying substances which modulate iPLA₂zeta expression. In an aspect, the screening method and research tool comprises a screening method contacting a candidate substance with cells capably expressing iPLA₂zeta or a fragment thereof, and measuring the expression of iPLA₂zeta or a fragment thereof by the cells by an analysis of an effluent for the TAG content.

[0049]The present discovery includes a method and research tool for identifying substances which modulate iPLA₂eta expression. In an aspect, the screening method and research tool comprises a screening method contacting a candidate substance with cells capably expressing iPLA₂eta or a fragment thereof, and measuring the expression of iPLA₂eta or a fragment thereof by the cells by an analysis of an effluent for the TAG content.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050]FIG. 1 depicts Amino Acid Sequence Alignment of Human iPLA₂epsilon (Adiponutrin; SEQ ID NO: 1), iPLA₂zeta (TTS-2.2; SEQ ID NO: 2), and iPLA₂eta (GS2; SEQ ID NO: 3). H_x (SEQ ID NO: 30) denotes the position of the histidine tag where x=0 (no histidine tag) or an integer ranging from 4 to 12. For human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), x=6.

[0051]FIG. 2 depicts Western Analysis of the Expression and Subcellular Localization of Recombinant Human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) in Sf9 Cells.

[0052]FIG. 3 depicts Triolein Lipase Activity of Sf9 Subcellular Fractions Containing Recombinant Human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29).

[0053]FIG. 4 depicts Affinity purified Human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) Catalyze Transacylation of Mono-olein to Form Diolein and Triolein.

[0054]FIG. 5 depicts Inhibition of Recombinant Human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) Triolein Lipase Activity by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL).

[0055]FIG. 6 depicts Quantitative PCR of iPLA₂epsilon and iPLA₂zeta Message in Mouse 3T3-L1 Preadipocytes and iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta Message in Human SW872 Lipsarcoma Cells.

[0056]FIG. 7 depicts the Nucleotide Sequence (SEQ ID NO: 4) (and translated polypeptide sequence; SEQ ID NO: 6) of Human iPLA₂zeta (ITS-2.2).

[0057]FIG. 8 depicts the Nucleotide Sequence (SEQ ID NO: 5) (and translated polypeptide sequence; SEQ ID NO: 7) of Human iPLA₂eta (GS2).

[0058]FIG. 9 shows Schematic Diagram of Adipocyte Acyl-CoA-Independent Triglyceride Cycling in a living human. The inventors have discovered that acyl-equivalents are stored in the adipocyte primarily in the form of triglycerides which can be synthesized by iPLA₂epsilon (adiponutrin), iPLA₂zeta and iPLA₂eta through an acyl-CoA independent transacylation mechanism which transfers fatty acyl moieties from monoacylglycerol (MAG) or diacylglycerol (DAG) acyl-donors to MAG and DAG acyl-acceptor intermediates to form triacylglycerols (TAG) and that, alternatively, hydrolysis of a single TAG fatty acyl moiety is catalyzed by iPLA₂epsilon, iPLA₂zeta, iPLA₂eta to form DAG which can then be further degraded to MAG and glycerol by either iPLA₂epsilon, iPLA₂zeta, iPLA₂eta or other intracellular lipases (e.g. hormone sensitive lipase (HSL)). Thus these enzymes contribute substantially to triglyceride homeostasis in the adipocyte.

[0059]FIG. 10 and FIG. 11 provide Nucleotide and Deduced Amino Acid Sequences of Our Newly Discovery Human Adiponutrin (iPLA₂epsilon) and variants thereof A. Glu434 Variant (refSNP ID=2294918(g); Sequence ID+AK025665 (nucleotide)) B. Lys434 Variant (refSNP ID=2294918(a); Sequence ID=AL138578.2 (nucleotide): NP_--079501 (protein)). The depicted nucleotide coding sequences (lower case letters) of human adiponutrin (1446 bp) encode for polypeptides of 481 amino acids (upper case letters). The amino acid encoded for each adiponutrin (iPLA₂epsilon) allelic variant is boxed. The conserved nucleotide binding (GCGFLG; SEQ ID NO: 12) and lipase (GASAG; SEQ ID NO: 13) consensus sequences are indicated with dashed and solid lines, respectively. The catalytic serine (Ser-47) is depicted to illustrate the native (where R═H) or acylated enzyme (where R=any fatty acyl moiety).

[0060]FIG. 10 and FIG. 11 show our novel nucleic acids and our novel enzymes herein as iPLA₂epsilon SEQ ID#'s are at the top of FIGS. 10 and 11. SEQ ID NO. 9 is FIG. 10 (listed protein sequence); SEQ ID NO. 11 is FIG. 11 (listed protein sequence); SEQ ID NO. 8 is FIG. 10 (listed nucleotide sequence); and SEQ ID NO. 10 is FIG. 11 (listed nucleotide sequence) such variants are included herein as iPLA₂epsilon.

DETAILED DESCRIPTION OF THE INVENTION

[0061]This invention relates generally to the identification of phospholipases A₂/nonhormone sensitive lipases (HSL) in triacylglycerol lipase activities and transacylase activities.

[0062]Sequence database searches for proteins containing calcium-independent phospholipase A₂ (iPLA₂) nucleotide (G/AxGxxG) and lipase (GxSxG) consensus motifs identified a novel subfamily of three putative iPLA₂ family members designated iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta (adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein, we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA₂ subfamily in Sf9 cells and demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta also possesses acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H tetrahydro-pyran-2-one (BEL), a mechanism-based suicide substrate inhibitor of all known iPLA₂s, inhibits the triglyceride lipase activity of each of the three isofoms similarly (IC₅₀=0.1-0.5 microM). Quantitative PCR revealed dramatically increased expression of iPLA₂zeta and iPLA₂eta transcripts in differentiating 3T3-L1 adipocytes and identified the presence of all three iPLA₂ isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.

[0063]In an aspect, the invention provides for the first time isolated novel and purified and characterized phospholipases A₂, referred to herein as calcium-independent lipases A₂zeta (iPLA₂zeta) having SEQ ID NO: 2 (See FIG. 1) and nucleic acid sequence SEQ ID NO: 4 (See FIG. 7), and calcium-independent lipases A₂eta (iPLA₂eta) having SEQ ID NO: 3 (See FIG. 1) and nucleic acid sequence SEQ ID NO: 5 (See FIG. 8). For the first time herein, these novel enzymes has been isolated and characterized and is involved in the catalysis (hydrolysis) and synthesis (transesterification) of lipids in a living mammalian cell. Moreover, these enzymes, iPLA₂zeta, and iPLA₂eta, through the process of transesterification can catalyze the net anabolic synthesis of triglycerides through a variety of metabolic precursor's (e.g. monoacylglycerol, diacylglycerol and acyl CoA).

[0064]Further, the inventors have discovered a medical treatment for combating obesity and over-weightness in humans which comprises effectively administering an inhibiting amount of a compound which promotes (increases) or blocks (inhibits) human iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression or enzymatic activity in a living cell.

[0065]The inventors have discovered a screening method and research tool for identifying drugs which are useful to successfully hold weight in a living mammal or if desired to reduce weight gain.

[0066]As used herein the term "putative" means deemed to be, supposed, reputed to be an inhibitor (repressor) of the expression of iPLA₂zeta in iPLA₂zeta expressible tissue such as in adipose tissue of a transgenic mouse or a sample tissue thereof or a sample adequately representative thereof.

[0067]As used herein the term "putative" means deemed to be, supposed, reputed to be an inhibitor (repressor) of the expression of iPLA₂eta in iPLA₂eta expressible tissue such as in adipose tissue or sample tissue thereof or a sample adequately representative thereof.

[0068]As used herein, the term "compound" includes cell(s), compounds, irons/anions, cations and salts.

[0069]As used herein, the term "tissue" includes tissue, cells and collections of a multiplicity of homogenous or nearly homogenous cell lines or a sample thereof or a representative sample thereof. In an aspect the tissue is a living mammalian tissue such as in a tissue culture or living mammal or in a living transgenic mouse.

[0070]As used herein, the term "peptide" is any of a group of compounds comprising two or more amino acids linked by chemical bonding between their respective carboxyl and amino groups. The term "peptide" includes peptides and proteins that are of sufficient length and composition to affect a biological response, e.g. antibody production or cytokine activity whether or not the peptide is a hapten. The term "peptide" includes modified amino acids, such modifications including, but not limited to, phosphorylation, glycosylation, acylation, prenylation, lipidation and methylation.

[0071]As used herein, the term "polypeptide" is any of a group of natural or synthetic polymers made up of amino acids chemically linked together such as peptides linked together. The term "polypeptide" includes peptide, translated nucleic acid and fragments thereof.

[0072]As used herein, the term "polynucleotide" includes nucleotide sequences and partial sequences, DNA, cDNA, RNA variant isoforms, splice variants, allelic variants and fragments thereof.

[0073]As used herein, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein when referring to a translated nucleic acid (e.g. a gene product). The term "polypeptide" includes proteins. The term "protein" includes the native (or wild-type) protein as well as a histidine-tagged protein. The term "protein" includes histidine-tagged proteins in which the number of histidine residues ranges from 4 to 12.

[0074]As used herein, the term "isolated polypeptide" includes a polypeptide essentially and substantially free from contaminating cellular components.

[0075]As used herein, the term "isolated protein" includes a protein that is essentially free from contamination cellular components normally associated with the protein in nature.

[0076]As used herein, the term "nucleic acid" refers to oligonucleotides or polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) as well as analogs of either RNA or DNA, for example made from nucleotide analogs any of which are in single or double stranded form.

[0077]As used herein, the term "patient" and subject" are synonymous and are used interchangeably herein.

[0078]As used herein, the term "expression" includes the biosynthesis of a product as an expression product from a gene such as the transcription of a structural gene into mRNA and the translation of mRNA into at least one peptide or at least one polypeptide.

[0079]As used herein, the term "mammal" includes living animals including humans and non-human animals such as murine, porcine, canine and feline.

[0080]As used herein, the term "sample" means a viable sample of biological tissue or fluid and is not limited to adipose tissue. Biological samples may include representative sections of tissues.

[0081]As used herein, the term "target protein" includes an amino acid sequence expressed in a target cell such as in an adipocyte. In an aspect, the target protein is a protein having a sequence shown in SEQ. ID. NO: 1, SEQ. ID. NO: 2 or SEQ. ID. NO: 3.

[0082]As used herein, the term "antisense" means a strand of RNA whose sequence of bases is complementary to messenger RNA.

[0083]As used herein, the term "siRNA" means short interfering RNA.

[0084]The phrase "a sequence encoding a gene product" refers to a nucleic acid that contains sequence information, e.g., for a structural RNA such as rRNA, a tRNA, the primary amino acid sequence of a specific protein or peptide, a binding site for a transacting regulatory agent, an antisense RNA or a ribozyme. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to conform with codon preference in a specific host cell.

[0085]By "host cell" is meant a cell which contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, (e.g. Xenopus), or mammalian cells such as HEK293, CHO, HeLa and the like.

[0086]As used herein a "therapeutic amount" is an amount of a moiety such as a drug or compound which produces a desired or detectable therapeutic effect on or in a mammal administered with the moiety.

[0087]The term "recombinant" when used with reference to a cell, or protein, nucleic acid, or vector, includes reference to a cell, protein, or nucleic acid, or vector, that has been modified by the introduction of a heterologous nucleic acid, the alteration of a native nucleic acid to a form not native to that cell, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes and proteins that are not found within the native (non-recombinant) forms of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0088]An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, with a series of specific nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.

[0089]Genetic knockout of hormone sensitive lipase (HSL) in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Sequence database searches for proteins containing calcium-independent phospholipase A₂ (iPLA₂) nucleotide (G/AxGxxG) and lipase (GxSxG) consensus motifs identified a novel subfamily of three putative iPLA₂ family members designated iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta (adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein, we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA₂ subfamily in Sf9 cells and demonstrate that each possesses abundant TAG lipase activity.

[0090]Moreover, iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta also possesses acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2Htetrahydropyran-2-one (BEL), a mechanism-based suicide substrate inhibitor of all known iPLA₂s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC₅₀=0.1-0.5 microM). Quantitative PCR revealed dramatically increased expression of iPLA₂epsilon and iPLA₂zeta transcripts in differentiating 3T3-L1 adipocytes and identified the presence of all three iPLA₂ isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.

[0091]Obesity and its associated clinical sequelae (e.g. type 2 diabetes, atherosclerosis, and hypertension) represent the major and most rapidly expanding health epidemic in industrialized nations (1-4). Obesity results from an abnormal increase in white adipose tissue mass, primarily in the form of triglycerides, and, in humans, is thought to be caused by a complex array of genetic, environmental and hormonal factors (1,4). Under conditions of obesity, serum non-esterified fatty acids are elevated, contributing to the accumulation of triglycerides in non-adipose tissues (e.g. hepatic, myocardial, and pancreatic) and to the development of the type 2 metabolic syndrome (5). The combined effects of excess cellular triglycerides, fatty acyl-CoAs, and free fatty acids are believed to be primary mediators of the lipotoxic effects of obesity which include decreased insulin sensitivity, increased oxidative stress, reduced metabolic capacity, and increased rates of apoptosis in multiple organ systems (5-8).

[0092]Triacylglycerol/fatty acid recycling is an important mechanism by which adipocytes modulate fatty acyl flux in response to changing metabolic conditions (9, 10). The TAG metabolic cycle encompasses both de novo triacylglycerol synthesis, which is thought to be mediated primarily through the concerted activities of glycolytic/glyceroneogenic enzymes, acyl-CoA dependent acyltransferases, and phosphatidic acid phosphatases (10-12), and TAG hydrolysis catalyzed by triacylglycerol lipases. Hormone sensitive lipase (HSL) was the first intracellular lipase to be purified and cloned (13), having since been extensively characterized in terms of its substrate selectivity and mechanisms of regulation (14,15). Results from these studies have emphasized the role of this enzyme in meeting increased systemic demand for free fatty acids through its activation by phosphorylation by protein kinase A (16-18), the exbracellular signal-regulated kinase pathway (19) and/or by interactions with various proteins partners (14,15). Genetic knockout of HSL in mice has revealed that HSL catalyzes the rate determining step in the hydrolysis of adipose tissue diacylglycerol (DAG) since DAG, but not TAG, accumulates in these animals (20). Furthermore, TAG is hydrolyzed less efficiently than DAG by HSL in in vitro assays (16) and measurement of TAG lipase activity in adipose tissue of HSL knockout mice demonstrates the existence of other as yet unknown TAG lipase(s) (20-23).

[0093]Although the biosynthesis of triglycerides is believed to be mediated primarily by an array of acyl-CoA-dependent enzymes in pathways utilizing either glycerol phosphate, dihydroxyacetone phosphate, or monoacylglycerol as initial acyl acceptors, the relative contribution of acyl-CoA independent transacylases utilizing mono- and diacylglycerols as acyl donors/acceptors in the synthesis of cellular TAG is largely unknown. Intestinal enterocytes contain an sn-1,2(2,3)-diacylglycerol transacylase which has been suggested to be important for the acyl-CoA independent transacylation of monoacylglycerol and diacylglycerol leading to the production of triacylglycerol for incorporation into chylomicrons (24,25). However, despite the apparent importance of acylglycerol transacylation in intestinal lipid transport and non-HSL TAG lipases in adipocyte lipid homeostasis, the molecular identities of the polypeptides catalyzing these reactions is currently unknown.

[0094]In the process of searching for novel calcium-independent phospholipases A₂ by protein sequence homology searches for candidate enzymes containing the iPLA₂ dual signature nucleotide (G/AxGxxG) and active site lipase (GxSxG) motifs, we identified a subfamily of putative iPLA₂ enzymes (previously named adiponutrin, TTS-2.2, and GS2) of previously unknown catalytic function which each contain an N-terminal patatin (iPLA₂alpha) homology domain as determined by protein family analysis (FIG. 1). One of these proteins, adiponutrin, has received much attention as an adipocyte specific protein which is down-regulated by either fasting (26) or treatment with thiazolodinediones (27) and is acutely upregulated by re-feeding a high carbohydrate (26,28) or high protein diet (29). Moreover, mouse adiponutrin mRNA is dramatically up-regulated during 3T3-L1 adipocyte differentiation (26) and TTS-2.2 has been shown to be associated with lipid droplets in CHO K2 cells (30).

[0095]In this application, we describe the cloning, heterolgous expression, and affinity purification of human iPLA₂epsilon (adiponutrin), iPLA₂zeta (TTS-2.2), and iPLA₂eta (GS2) in SfP cells. Furthermore, we demonstrate that the expressed recombinant enzymes hydrolyze triolein and are able to transfer the donor acyl moiety of mono-olein to mono-olein or diolein acceptors to form diolein or triolein, respectively. Expression of iPLA₂zeta message is markedly upregulated during 3T3-L1 differentiation and parallels the dramatic induction of iPLA₂epsilon expression in this cell line. In addition, all three iPLA₂ mRNAs are present in human liposarcoma cells. Collectively, these results identify a novel class of triglyceride lipaseltransacylase enzymes which likely participate in adipocyte triglyceride fatty acyl liberation, recycling, and lipid homeostasis.

[0096]Exemplary embodiments are described in the following examples. It is intended that the specification, together with the examples, be considered exemplary only.

EXAMPLES

[0097]Materials--Grace's insect medium and Bac-to-Bac baculoviral system reagents were obtained from Invitrogen. Restriction enzymes were purchased from Roche. Nucleotide sequencing was performed by the Nucleic Acid Chemistry Laboratory at Washington University. 1-palmitoyl-2-[1-¹⁴C]-oleoyl-sn-glycerol-3-phosphocholine, and [1-¹⁴C]-oleoyl-glycerol were purchased from American Radiolabeled Chemicals. [9,10-³H(N)]-triolein was obtained from Perkin Elmer and was re-purified before use utilizing a Vydac C18 Pharmaceutical HPLC column equilibrated with acetonitrile/dichloromethane (55:45) as the mobile phase. BEL was obtained from Cayman Chemical. Most other reagents were purchased from Fisher Scientific or Sigma.

[0098]Cloning of Human iPLA₂epsilon (Adiponutrin), iPLA₂zeta (TTS-2.21, and iPLA₂eta (GS2)--Human adipocyte Marathon-Ready cDNA (Clontech) was used as a template for PCR to obtain full-length cDNA for iPLA₂epsilon. PCR primers were designed to introduce a 5' Kozak sequence, a C-terminal 6×His tag (SEQ ID NO: 29) at the 3'-end of the iPLA₂ coding sequence and to incorporate EcoRI and SalI restriction sites for subcloning into the baculoviral expression vector pFASTBacl. Full-length human iPLA₂zeta (TTS-2.2) and iPLA₂eta (GS2) were amplified by PCR (with primers to introduce 5' Kozak sequences) from ATCC IMAGE clones 4875483 and 4717901, respectively and subcloned into pcDNAV5HisB. The insert including the in-frame 3' (H is)₆ (SEQ ID NO: 29) coding sequence was then excised from this vector utilizing BarnH1 and Pmel restriction sites for ligation into the baculoviral expression vector pFASTBacl. After sequencing the insert and flanking sequences on both strands of the iPLA²-pFASTBacl constructs to ensure the sequence integrity of the construct, a bacrnid construct was prepared using the Bac-to-Bac Baculovirus Expression System protocol (Invitrogen) for subsequent Cellfectin-mediated transfection of Sf9 cells in 35 mm plates to produce infectious recombinant baculovirus. Amplified recombinant baculovirus was then used to infect a spinner culture of Sf9 cells for 72 h and the supernatant was collected as a high titer viral stock.

[0099]Expression of Human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) in Sf9 cells and Subcellular Fractionation--Sf9 cells (100 ml culture volume) at a density of approximately 1×10⁶ cells/ml were infected with either control baculovirus or recombinant baculovirus encoding human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) at an multiplicity of infection of approximately 1. Forty-eight hours post-infection, cells were harvested by centrifugation (900 rpm×10 min), washed once in Grace's insect medium without serum, re-pelleted, and resuspended in 10 ml lysis buffer (25 mM sodium phosphate, pH 7.8 containing 20% glycerol and 2 mM 2-mercaptoethanol). Cells were lysed by sonication (30×1 s bursts at 40% power) and centrifuged at 100,000×g for 1 h to separate cytosolic and membrane fractions. Cellular membranes were resuspended in a volume of lysis buffer equivalent to the volume of the cytosol fraction.

[0100]Co²+-Affinity Column Chromatography of iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29). The cytosolic fraction (30 ml obtained from 300 ml of cultured Sf9 cells) containing recombinant human iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), or iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) was mixed by inversion with 3 ml of TALON-Co²+ resin for 1 h at 4° C. The resin-cytosol suspension was then poured into an empty Pharmacia column (1.5×10 cm) and washed with 10 column volumes of lysis buffer containing 500 mM NaCl (Buffer A). Recombinant iPLA₂ His-tagged proteins were eluted utilizing a gradient of imidazole (200 mM final concentration) in 50 ml of Buffer A. Fractions were collected and assayed for phospholipase A₂ and triolein lipase activities as described below.

[0101]Assay for Calcium-Independent Phospholipase A₂ Activity--Sample fractions were incubated in 100 mM Tris-HCl, pH 7.2 containing 4 mM EGTA (200 microliters final volume) for 5 min at 37° C. in the presence of 1-palmitoyl-2-[1-¹⁴C]-oleoyl-sn-glycero-3-phosphocholine introduced by ethanolic injection. Reactions were terminated by addition of 100 microliters of butanol and extraction of the radiolabeled product and remaining substrate into the butanol layer by vigorous vortexing. Samples were spotted on LK6 Silica Gel 60 Å TLC plates, overlaid with oleic acid standard, dried, and developed in petroleum etherlethyl etherlacetic acid (70:30:1). The region of the plate corresponding to the oleic acid standard (visualized by iodine staining) was scraped into scintillation vials and quantified by liquid scintillation spectrometry.

[0102]Assay for Triolein Lipase Activity--Sample fractions were incubated in 85 mM potassium phosphate, pH 7.0 containing 2 mM EDTA and 2 mM DTT for 15 min at 37° C. in the presence of a suspension of 100 microM [9,10-³H(N)]-triolein (100 microCi/micromol) in 25 microM egg yolk lecithin, and 100 microM sodium taurocholate. In some reactions, BEL was added at the indicated concentrations and incubated with enzyme at room temperature for 3 min prior to the addition of radiolabeled substrate. After extraction of radiolabeled reaction products and remaining substrate into butanol, samples were spotted on TLC plates, overlaid with oleic acid standard, dried, and developed in chloroformlmethanol/NH₄OH (65:25:5). The region of the plate corresponding to the oleic acid standard (visualized by iodine staining) was scraped into scintillation vials and quantified by liquid scintillation spectrometry.

[0103]Assay for Acylglycerol Transacylase Activity--Highly purified CO²+-TALON affinity chromatographic fractions containing iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), or iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) (40 microliters) were incubated in 85 mM potassium phosphate, pH 7.0 (200 microliters final volume) for 15 min at 37° C. in the presence of 10 microM [1-¹⁴C]-mono-olein (acyl donor), 25 microM acyl acceptor (mono-olein or diolein), 25 microM egg yolk lecithin, and 25 microM sodium taurocholate. After extraction of radiolabeled reaction products and remaining substrate into butanol, samples were spotted on TLC plates, overlaid with trioleiddiolein standards, dried, and developed in petroleum etherlethyl etherlacetic acid (75:25:1). The regions of the plate corresponding to either the diolein, triolein, and fatty acid standards (visualized by iodine staining) were scraped into scintillation vials and quantified by liquid scintillation spectrometry.

[0104]Quantitative PCR of iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta Message in Differentiating 3T3-1 Adipocytes and SW872 Human Liposacrcoma Cells-3T3-L1 pre-adipocytes were cultured and differentiated as previously described (31). Human SW872 liposarcoma cells were cultured as previously described (32). 3T3-E1 cells at day 0 through day 6 of differentiation (2 day intervals) or SW872 cells were washed twice with ice-cold phosphate buffered saline and RNA was prepared following the RNeasy (Qiagen) protocol as described by the manufacturer. RNA (0.1-2 micrograms) was reverse transcribed using MultiScribe reverse transcriptase (TaqMan Gold RT-PCR kit, Applied Biosystems) by incubation for 10 min at 25° C. followed by 30 min at 48° C. and a final step of 5 min at 95° C. 20 ng of the resultant cDNA was used for each quantitative polymerase chain reaction. Primer/probe sets for quantitative PCR were designed using Primer Express software from PE Biosystems. Probes were 5' labeled with reporter dye FAM (6-carboxylfluorescein), and 3' labeled with quenching dye, TAMRA (6-carboxytetramethylrhodamine). Human iPLA₂epsilon forward (5'-GGCAAAATAGGCATCTCTCTTACC-3'; SEQ ID NO: 14) and reverse (5'-GGAGGGATAAGGCCACTGTAGA-3'; SEQ ID NO: 15) primers were paired with probe (5'-AACATACCAAGGCATCCACGACTTCGTC-3'; SEQ ID NO: 16). Human iPLA₂zeta forward (5'-CCACGGCGCTGGTCAC-3'; SEQ ID NO: 17) and reverse (5-GCAGGACCTTCAGCAGGAAAC-3'; SEQ ID NO: 18) primers were paired with probe (5'-TGGCACCAGCCTCACCCAGGCAGAC-3'; SEQ ID NO: 19). Human iPLA₂eta forward (5'-GCACAGAAAATGAGGATTATTAAAGG-3'; SEQ ID NO: 20) and reverse (5'-CGCTGCAAATGATAGGTTGATG-3'; SEQ ID NO: 21) primers were paired with probe (5'-TGCTTCATTCTAGCTGTAGCACTGCGAGCAAC-3'; SEQ ID NO: 22). Mouse iPLA₂epsilon forward (5'-ACTGCACGCGGTCACCTT-3'; SEQ ID NO: 23) and reverse (5'-CACGAGGTCCATGAGGATCTC-3'; SEQ ID NO: 24) primers were paired with probe (5'-TGTGCAGTCT-CCCTCTCGGCCGTATAAT-3'; SEQ ID NO: 25). Mouse iPLA₂zeta forward (5'-GCCACAGCGCTGGTCACT-3'; SEQ ID NO: 26) and reverse (5'-CCTCCTTGGACACCTCAATAATG-3'; SEQ ID NO: 27) primers were paired with probe (5'-CCTGCCTGGGTGAAGCAGGTGC-3'; SEQ ID NO: 28). Quantitative PCR was carried out using TaqMan PCR reagents (Applied Biosystems) as recommended by the manufacturer with GAPDH primers and probe as an internal standard. Each PCR amplification was performed in triplicate for 2 min at 50° C., 10 min at 95° C., followed by 40 cycles of 15 s at 95° C. and 1 min at 60° C.

[0105]Other Methods--Proteins were separated by SDS-PAGE according to the method of Laemmli (33). For Western analyses, the separated proteins in SDS-PAGE gels were transferred to polyvinylidene difluoride membranes and subsequently probed with a mouse monoclonal anti-His₆ antibody (BD Biosciences) in conjunction with an anti-mouse IgG-horseradish peroxidase conjugate. Protein concentrations were determined by the Bradford protein assay (Bio-Rad) using bovine serum albumin as standard.

Results

[0106]Searches of the protein sequence database for novel iPLA₂ family members possessing both nucleotide (G/AxGxxG) and lipase (GxSxG) consensus motifs identified a group of three human proteins (adiponutrin, TTS-2.2, and GS2) which contained patatin (iPLA₂alpha) homology domains (FIG. 1). On the basis of their similarity to iPLA₂alpha, we designated adiponutrin, TTS-2.2, and GS2 as iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta, respectively. To determine if the presence of the dual signature nucleotide and lipase motifs of human iPLA₂epsilon, iPLA₂zeta and iPLA₂eta proteins correctly identified novel lipase family members, we heterologously expressed each of the three C-terminal His₆-tagged proteins (6×His tag disclosed as SEQ ID NO: 29) individually in an Sf9 cell baculovirus expression system. Western analysis of Sf9 cells infected with baculoviruses encoding human either iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), or iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) proteins revealed the presence of 53, 58, and 28 kDa (respectively) immunoreactive bands, corresponding to their predicted molecular weights, utilizing an anti-His₆ (6×His tag disclosed as SEQ ID NO: 29) monoclonal antibody (FIG. 2). In contrast, these immunoreactive bands were not present in Sf9 cells infected with wild-type empty vector (pFB) baculovirus, Subcellular fractionation of the infected Sf9 cells demonstrated that the majority (70-90%) of the expressed iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) proteins were associated with the membrane fraction (FIG. 3). The presence of apparently soluble forms of each of the proteins in the Sf9 cell cytosolic fraction simplified purification procedures (described below) since high concentrations of detergents utilized for solubilization are known to inhibit other iPLA₂ family members. Differences in the expression levels of iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta could arise from multiple factors including altered rates of transcription, translation, and/or differences in mRNA or protein stability.

[0107]As potential members of the iPLA₂ family of enzymes, iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) expressed in Sf9 cells were initially assayed for iPLA₂ activity utilizing 1-palmitoyl-2-[1-¹⁴C]-oleoyl-sn-glycerol-3-phosphocholine as substrate. Results from these experiments indicated that the cytosol and membrane fractions containing iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29) possessed modest calcium-independent PLA₂ activity (20-50 pmolmin^-1mg protein^-1) relative to pFastBac control Sf9 subcellular fractions (data not shown). However, similar assays with iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) did not yield detectable amounts of [1-¹⁴C]-oleic acid released from 1-palmitoyl-2-[1-¹⁴C]-oleoyl-sn-glycerol-3-phosphocholine in comparison to control reactions. Inclusion of Ca²+ and/or ATP (a known stabilizer and activator of iPLA₂β activity) did not measurably increase iPLA₂ activity relative to control samples. Subsequent experiments with affinity purified iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) with 1-palmitoyl-2-[1-¹⁴C]-linoleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-[1-¹⁴C]-arachidonyl-sn-glycerol-3-phosphocholine revealed phospholipase A₂ activity from 67 to 134 pmolmin^-1mg protein^-1 (see below for details).

[0108]Since the founding member of the iPLA₂ family, patatin (iPLA₂alpha), hydrolyzes both phospho- and neutral lipid substrates (34,35) and is able to catalyze transesterification reactions (36), we believe that iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta might possess neutral lipid lipase and/or transacylase activity. Moreover, the similar positional location of the nucleotide and lipase motifs near the N-terminus (FIG. 1) and the induced expression of adiponutrin (iPLA₂epsilon) in either differentiating 3T3-L1 adipocytes (26) or rat adipose tissue following meal feeding (28,29) further suggested that triacylglycerol was a potential substrate for this polypeptide. To address this possibility, each of the cytosol and membrane fractions containing iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) were incubated in the presence of a sonicated suspension of phosphatidylcholine, sodium taurocholate, and [³H]-triolein at 37° C. for 15 min. Robust hydrolysis of [³H]-triolein, as determined by the release of [³H]-oleic acid, was observed in cytosolic and membrane fractions containing either iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta isoforms in comparison to pFB control fractions which exhibited very low triolein lipase activity (FIG. 3). The iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) cytosolic fractions possessed greater triolein lipase activity in comparison to their membrane counterparts which was surprising considering that the majority of protein mass was present in the membrane fraction as determined by Western analysis (FIG. 2). This difference in activity may reflect the presence of a membrane-associated inhibitor, substrate dilution, or more likely that a large percentage of the membrane associated iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) is unable to effectively interact with the triolein substrate in this in vitro prepared emulsion. In contrast, the triolein lipase activities of the iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) cytosolic and membrane fractions were more proportionate to the relative amount of immunoreative protein in each fraction (FIGS. 2 and 3). Assuming similar degrees of immunoreactivity of the monoclonal HiS₆ (SEQ ID NO: 29) antibody toward the His-tagged proteins, iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) possesses approximately 5-10 fold greater triolein lipase measured specific activity relative to iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) under the conditions employed (FIGS. 2 and 3). In addition, a large number of factors could contribute to different specific activities in vivo, including differential substrate presentation, subcellular localization, post-translational modifications, and protein-protein interactions.

[0109]Since patatin can catalyze ATP and acyl-CoA-independent transacylation reactions (34-36), we considered the possibility that iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta may be involved in the cycling of acyl equivalents between triacylglycerol/diacylglycerol/monoacylglycerol pools. Accordingly, we sought to determine if the three members of this iPLA₂ subfamily could catalyze the transfer of the oleoyl moiety from mono-olein (donor) to a mono-olein or diolein acceptor to form diolein or triolein, respectively. To this end, we affinity purified each His-tagged iPLA₂ isoform utilizing co²+ TALON affinity chromatography. The triolein lipase specific activities within peak fractions following elution of iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) from the CO₂+-charged TALON column were 8, 9, and 52 nmol oleic acidmin^-1mg^-1 protein, respectively, under the conditions employed, representing an approximate 50 to 100-fold purification from the crude cytosol. Incubation of each of the affinity purified iPLA₂ His-tagged family members with [1-¹⁴C]-mono-olein demonstrated the synthesis of radiolabeled diolein (FIG. 4A) which was not observed with pFB control column fractions. Consistent with the lipase activity measurements, affinity purified iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) displayed approximately 10-fold greater transacylation specific activity than iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) (FIGS. 4A and 4B). Remarkably, [¹⁴C]-triolein was observed as product in these incubations (FIG. 4B), indicating that iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta were each capable of catalyzing sequential transacylation reactions to form triolein (MOG+MOG→DOG+glycerol and MOG+DOG→TOG+glycerol). Addition of exogenous 1,2- or 1,3 diolein as acyl acceptor increased the amount of [¹⁴C]-triolein formed utilizing [1-¹⁴C]-mono-olein as acyl donor in the presence of iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta substantiating each transacylation reaction independently (FIG. 4B). Under the conditions examined, no detectable preference for either 1,2-diolein or 1,3-diolein as acyl acceptor for iPLA₂-catalyzed triolein synthesis were observed.

[0110]Although Sf9 cell cytosolic and membrane fractions containing iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) did not display measurable iPLA₂ activity (above control samples) utilizing 1-palmitoyl-2-[1-¹⁴C]-oleoyl-sn-glycerol-3-phosphocholine as substrate, we measured the PLA₂ activity of the affinity purified proteins with several different phospholipid substrates including those with polyunsaturated fatty acids at the sn-2 position. Incubation of iPLA₂epsilon(His)₆ (6×His tag disclosed as SEQ ID NO: 29), iPLA₂zeta(His)₆ (6×His tag disclosed as SEQ ID NO: 29), and iPLA₂eta(His)₆ (6×His tag disclosed as SEQ ID NO: 29) with 1-palmitoyl-2-[1-¹⁴C]-linoleoyl-sn-glycerol-3-phosphocholine or 1-palmitoyl-2-[1-¹⁴C]-arachidonyl-sn-glycerol-3-phosphocholine for 30 min resulted in hydrolysis at rates of 57±3, 57±19, and 77±23 pmol linoleic acidmin^-1mg protein^-1 or 100±4, 119±6, and 134±42 pmol arachidonic acidmin^-1mg protein^-1, respectively. We specifically point out that these results do not preclude the possibility that these iPLA₂ isoforms have greater phospholipase A₂ activity in vivo or in vitro with other phospholipid substrates, protein partners, or under different assay conditions.

[0111]The mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) has been previously demonstrated to inhibit iPLA₂beta and iPLA₂gamma activity at sub to low micromolar concentrations (37-39). To determine whether the three new iPLA₂ family members were inhibitable by BEL, each enzyme was pre-incubated with 0.1-2 microM BEL or ethanol vehicle alone prior to measuring triolein lipase activity (FIG. 5). Remarkably, BEL is a highly potent inhibitor for iPLA₂epsilon (IC₅₀≈0.1 microM), iPLA₂zeta (IC₅₀≈0.5 microM), and iPLA₂eta (IC₅₀≈0.1 microM) (FIG. 5). Thus, iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta are likely inhibited by BEL by a similar mechanism to that of iPLA₂beta and iPLA₂gamma. Considering the hydrophobic nature of the acyl-glycerol substrates of iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta identified herein, it is not surprising that BEL would have access to the active sites of these enzymes.

[0112]To further examine the potential roles of iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta in adipocyte biology, we determined the mRNA levels of each isoform in both human SW872 liposarcoma cells and differentiating 3T3-L1 adipocytes. Previous Northern analyses have demonstrated the dramatic upregulation of adiponutrin (iPLA₂epsilon) mRNA during 3T3-L 1 adipocyte differentiation (26). Consistent with prior work, quantitative PCR utilizing primers for mouse iPLA₂epsilon revealed a marked increase in message by day 6 (FIG. 6A). Similarly, quantitative PCR analysis of mouse iPLA₂zeta in differentiating 3T3-L1 adipocytes demonstrated a 10-fold increase in message by day 6 (FIG. 6B). Since the mouse genome does not contain an obvious iPLA₂eta paralog, we were not able to determine the expression levels of this isoform in 3T3-L1 cells. Human SW872 liposarcorna cells have been previously utilized in the study of lipoprotein receptor-mediated cholesterol ester homeostasis (32,40,41). Quantitative PCR utilizing primers for iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta demonstrated high levels of expression for all three iPLA₂ isofoms in this cell line (FIG. 6c).

Discussion

[0113]Progress in understanding adipocyte highlyceride homeostasis has been hindered by the difficulty in determining the diversity and chemical identities of non-HSL TAG lipases present in adipocytes (20,22,23). In this application, we describe the cloning, heterologous expression, and affinity purification of three novel human iPLA₂ family members (epsilon, zeta, and eta) and demonstrate that each possesses robust triglyceride lipase activity. Furthermore, iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta each catalyze transacylation of a mono-olein donor to a diolein acceptor to produce TAG, thus representing a previously unrecognized acyl-CoA independent pathway for triglyceride biosynthesis in adipocytes. Importantly, iPLA₂epsilon (adiponutrin) expression has been previously identified as adipocyte-specific (26) and iPLA₂zeta (TTS-2.2) protein has been demonstrated to be enriched in CHO K2 cell liposomes (30). Herein, we demonstrate that both iPLA₂epsilon (adiponutrin) and iPLA₂zeta (TTS-2.2) transcripts are induced several-fold during 3T3-L1 preadipocyte differentiation and that mRNA encoding all three (iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta) are present in SW872 human liposarcoma cells.

[0114]Immunohistochemical analysis of 3T3-L1 and CHO cells expressing recombinant mouse adiponutrin (iPLA₂epsilon) revealed that the protein was present at the periphery of the plasma membrane in punctuate granular structures and not surrounding the lipid droplets in these cells (26). However, the chemical function of the polypeptide was unknown making determination of its biologic role difficult. Furthermore, subcellular fractionation of these cells demonstrated that adiponutrin was localized predominantly to the membrane fraction and was predicted to be an integral membrane protein (26). Proteomic studies identified TTS-2.2 (iPLA₂zeta) as a component of CHO K2 lipid droplets likely involved in lipid metabolism (30). Subcellular fractionation of human iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta in Sf9 cells revealed that the majority of each of these iPLA₂ isoforms is membrane-associated which is not unexpected given the hydrophobic nature of their lipid substrates.

[0115]Mouse adiponutrin was first identified by differential hybridization as a mRNA species that was strongly induced during differentiation of 3T3-L1 cells into adipocytes (26). Furthermore, adiponutrin mRNA was demonstrated to be exclusively expressed in adipose tissue (both white and brown), was dramatically increased after feeding (relative to the fasted state where it is virtually absent), and is inappropriately upregulated in genetic models of obesity (26,28). Subsequent studies have shown that expression of adiponutrin mRNA is rapidly induced in rats fed high sucrose (28) or high protein diets (29), but not a diet high in saturated or unsaturated fatty acids (29). Although the significance of the dietinduced regulation of adiponutrin is not known with certainty at present, the acutely coordinated responses of adiponutrin mRNA to feeding and fasting make it likely that adiponutrin participates in TAG recycling in the adipocyte.

[0116]In an aspect, the invention provides for the first time an isolated novel and purified and characterized phospholipases A₂, referred to herein as calcium-independent lipase A₂zeta (iPLA₂zeta) having SEQ ID NO: 2 (See FIG. 1) and nucleic acid sequence SEQ ID NO: 4 (See FIG. 7), and calcium-independent lipase A₂eta (iPLA₂eta) having SEQ ID NO: 3 (See FIG. 1) and nucleic acid sequence SEQ ID NO: 5 (See FIG. 8). For the first time herein, these novel enzymes has been isolated and characterized and is involved in the catalysis, synthesis and hydrolysis of lipids in a living mammalian cell. Moreover, these enzymes, iPLA₂zeta, and iPLA₂eta, through the process of transesterification can catalyze the net anabolic synthesis of triglycerides through a variety of metabolic precursors (e.g. monoacylglycerol, diacylglycerol and acyl CoA).

[0117]In one embodiment, the invention is directed to an isolated nucleic acid molecule comprising a set of iPLA₂zeta polynucleotides. In an aspect of this embodiment, the iPLA₂zeta polynucleotides (SEQ. ID. NO: 4) encode and express an iPLA₂zeta polypeptide (SEQ. ID. NO. 2).

[0118]In one embodiment, the invention is directed to an isolated nucleic acid molecule comprising a set of iPLA₂eta polynucleotides. In an aspect of this embodiment, the iPLA₂eta polynucleotides (SEQ. ID. NO: 5) encode and express an iPLA₂eta polypeptide (SEQ. ID. NO: 3).

[0119]In one aspect, an isolated and characterized human gene (iPLA₂zeta) comprises a polynucleotide having a sequence shown in SEQ. ID. NO: 4 (See FIG. 7).

[0120]In one aspect, an isolated and characterized human gene (iPLA₂eta) comprises a polynucleotide having a sequence shown in SEQ. ID. NO: 5 (See FIG. 8).

[0121]In an aspect, an isolated and characterized human protein (iPLA₂zeta) comprises a polypeptide having a sequence shown in SEQ. ID. NO: 2 (See FIG. 1).

[0122]In an aspect, an isolated and characterized human protein (iPLA₂eta) comprises a polypeptide having a sequence shown in SEQ, ID, NO: 3 (See FIG. 1).

[0123]In an aspect, a method of treating a living mammal to reduce obesity, comprises administering an effective amount iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta inhibitor thereto or an agent which changes the lipase to transacylase ratio.

[0124]In an aspect, a genetically engineered expression vector comprises a gene or part of the sequence of a human gene comprising a polynucleotide (iPLA₂zeta) having a sequence shown in SEQ. ID. NO: 4 (FIG. 7). In an aspect, the gene encodes a protein comprising a polypeptide having a sequence shown in SEQ. ID. NO: 2 (FIG. 1). In an aspect, the gene is operatively linked to a capable viable promoter element.

[0125]In an aspect, a genetically engineered expression vector comprises a gene or part of the sequence of a human gene comprising a polynucleotide (iPLA₂eta) having a sequence shown in SEQ. ID. NO: 5 (FIG. 8). In an aspect, the gene encodes a protein comprising a polypeptide having a sequence shown in SEQ. ID. NO: 3 (FIG. 1). In an aspect, the gene is operatively linked to a capable viable promoter element.

[0126]In another aspect, a method of medically treating a mammal comprises administering an anti-obesity (drug or pharmaceutical) in therapeutically effective amounts as an inhibitor to the mammal.

[0127]In another aspect, a method of medically treating a living mammal comprises administering a therapeutically effective amount of a compound (drug or pharmaceutical) which inhibits iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression to the mammal or results in a different isoform expression or different enzymatic activity or post-translational modification.

[0128]In another aspect, a method of medically treating a living mammal comprises administering a therapeutically effective amount of a compound (drug or pharmaceutical) which enhances iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression to the mammal or results in a different isoform expression or different enzymatic activity or post-translational modification.

[0129]In another aspect, a method of treating obesity, comprising administering an agent which changes the transacylase to lipase activity ratio in a metabolic setting. In an aspect, the metabolic setting is an animal or animal model.

[0130]In an aspect, a pharmaceutical composition is provided comprising a compound which effectively inhibits or counteracts iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression, hydrolytic activity, phospholipase A₂ activity, or transesterification activity in a living mammal.

[0131]In an aspect, a pharmaceutical kit comprises a container housing a compound which inhibits iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression, hydrolytic activity, phospholipase A₂ activity, or transesterification activity.

[0132]In an aspect, a pharmaceutical kit comprises a container housing a compound which enhances iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression, hydrolytic activity, phospholipase A₂ activity, or transesterification activity.

[0133]In another embodiment, the present invention is directed to a method of modulating fatty acid utilization in a patient. In an aspect, the patient is a living human patient. In this aspect, the method comprises increasing or decreasing iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta activity in the patient. Patients in need of such treatment include those patients suffering from one of diabetes and/or obesity. Preferably, this method comprises administering to the patient a substance (compound) in an effective amount which blocks or inhibits expression of iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta mass or activity.

[0134]In another embodiment, the present invention is directed to a method of modulating fatty acid utilization in a patient. In an aspect, the patient is a living human patient. In this aspect, the method comprises increasing or decreasing iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta activity in the patient. Patients in need of such treatment include those patients suffering from one of diabetes and/or obesity. Preferably, this method comprises administering to the patient a substance (compound) in an effective amount which enhances expression of iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta mass or activity.

[0135]In an aspect a method of identifying an agent which changes the ratio of transacylase to lipase activity in a living mammal by administering a compound to a mammal and determining if the transacylase to lipase activity ratio was changed by lipid analysis and if the ratio was changed then determining that the drug is an anti-obesity drug.

[0136]In an aspect, the invention comprises a method for ameliorating at least one symptom of a symptomatology comprising obesity and clinical manifestation of type 2 metabolic syndrome in a living human which comprises treating a human cell expressing iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta in a pharmacologically effective manner with a pharmacologically effective amount of an iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression or enzymatic inhibitor.

[0137]A method of treating at least one of an overweight and obese disorders, the method comprises administering to a subject (in need of such treatment) a therapeutically effective amount of composition comprising an inhibitor of human iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta.

[0138]In an aspect, a method of identifying an anti-obesity drug which comprises administering a drug to an animal and determining if there has been any change in iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression, hydrolytic activity, phospholipase A₂ activity, transesterification activity, or metabolic futile cycling and if so determining that the drug is an anti-obesity drug.

[0139]A method of practicing medicine which comprises administering a therapeutic amount of a drug to a patient at risk for obesity or being obese, the drug being an inhibitor of human iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta.

[0140]A method of providing therapy to a patient in need thereof which comprises administering a drug to a patient at risk for obesity, the drug being an inhibitor of the expressing of human iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta.

[0141]A method of providing therapy to a patient in need thereof which comprises administering a drug to a patient at risk for obesity, the drug being an activator of the expressing of human iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta.

[0142]A method for treating a diabetic which comprises administering a drug in an effective amount to modulate iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression whereby the insulin requirement of the patient is decreased.

[0143]A method of treating diabetes which comprises administering a drug in an effective amount to modulate iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression whereby the insulin requirement of the patient is decreased.

[0144]In an aspect, the present discovery encompasses genetically engineered cells capable of identifying substances which modulate iPLA₂zeta expression in a living cell. In an aspect, such cells comprise a promoter operably linked to iPLA₂zeta gene and a reporter gene. This reporter gene preferably encodes an enzyme capable of being detected by at least one of a suitable radiometric, fluorometric or luminometric assay such as, for example, a reporter sequence encoding a luciferase. In an aspect, the promoter sequence is a baculovirus promoter sequence and the cells are Sf9 cells.

[0145]In an aspect, the present discovery encompasses genetically engineered cells capable of identifying substances which modulate iPLA₂eta expression in a living cell. In an aspect, such cells comprise a promoter operably linked to iPLA₂eta gene and a reporter gene. This reporter gene preferably encodes an enzyme capable of being detected by at least one of a suitable radiometric, fluorometric or luminometric assay such as, for example, a reporter sequence encoding a luciferase. In an aspect, the promoter sequence is a baculovirus promoter sequence and the cells are Sf9 cells.

[0146]In an aspect, as an example of its utility, the invention comprises a method for prioritizing the therapeutic capability of drugs putative efficacy against obesity, comprising administering drugs to a living animal system which is actively expressing iPLA₂zeta, measuring any modulation of the iPLA₂zeta expression by a TAG or FFA's/glycerol analysis of an effect and determining if the modulation was an increase or a decrease or no change in iPLA₂zeta expression level. If the modulation is determined to be a decrease then determining that the drug was effective in inhibiting iPLA₂zeta, a value is assigned to that modulation and is thereafter compared to the modulation of other drugs. In an aspect, a prioritization can be set up by comprising the magnitudes of the various respective modulations and a hierarchy of drugs can be established. From this, it is possible to establish a priority of work on the drugs.

[0147]In an aspect, as an example of its utility, the invention comprises a method for prioritizing the therapeutic capability of drugs putative efficacy against obesity, comprising administering drugs to a living animal system which is actively expressing iPLA₂eta, measuring any modulation of the iPLA₂eta expression by a TAG or FFA's/glycerol analysis of an effect and determining if the modulation was an increase or a decrease or no change in iPLA₂eta expression level. If the modulation is determined to be a decrease then determining that the drug was effective in inhibiting iPLA₂eta, a value is assigned to that modulation and is thereafter compared to the modulation of other drugs. In an aspect, a prioritization can be set up by comprising the magnitudes of the various respective modulations and a hierarchy of drugs can be established. From this, it is possible to establish a priority of work on the drugs.

[0148]As an example of its utility, the present invention includes a method and research tool for identifying substances which modulate iPLA₂zeta expression. In an aspect, the screening method and research tool comprises a screening method contacting a candidate substance with cells capably expressing iPLA₂zeta or a fragment thereof, and measuring the expression of iPLA₂zeta or a fragment thereof by the cells by an analysis of an effluent for the TAG content.

[0149]As an example of its utility, the present invention includes a method and research tool for identifying substances which modulate iPLA₂eta expression. In an aspect, the screening method and research tool comprises a screening method contacting a candidate substance with cells capably expressing iPLA₂eta or a fragment thereof, and measuring the expression of iPLA₂eta or a fragment thereof by the cells by an analysis of an effluent for the TAG content.

[0150]Traditionally, futile cycling has been envisaged as a mechanism to facilitate the rapid response of biochemical pathways to external perturbations. Thus, TAG futile cycling may enable the rapid and immediate response to changing metabolic conditions. Triglyceride futile cycling provides a continuously mobile pool of fatty acids so that the residence time of any given fatty acid on the glycerol backbone is time-limited, thereby reflecting the recent metabolic and dietary history of that cell. Furthermore, the presence of enzymes which can potentially alter their relative anabolic (transacylation) vs. catabolic (lipase) activities provides a potential mechanism to rapidly switch cellular metabolic balance horn energy storage to mobilization. In this regard, the upregulation of adiponutrin during feeding suggests that it may serve an anabolic function facilitating the flow of fatty acids into TAG through acylglycerol transacylation (thereby effectively decreasing systemic fatty acid release) or alternatively, adiponutrin may prevent excessive TAG accumulation in the adipocyte.

[0151]In the terminal step of acyl-CoA dependent triacylglycerol synthesis, diacylglycerol is acylated by one of two acyl-CoA:diacylgiycerol acyltransferases (DGATs) to produce TAG. While mice lacking acyl-CoA:diacylglycerol acyltransferase-1 (DGAT-1) have been demonstrated to have essentially normal adipose tissue and circulating TAG levels (42,43), DGAT-2 knockout mice have dramatically reduced tissue and serum triglyceride levels (severe lipopenia) which is lethal within 24 hours after birth (44). Clearly, these results illustrate the importance of DGAT-2 catalyzed TAG synthesis in post-natal survival. To our knowledge, acyl-CoA independent TAG synthesis in adipocytes has not been previously documented, which may be due in part to the presence of high monoacylglycerol and diacylglycerol lipase (HSL) activities present in in vitro assay systems. Rat intestinal enterocytes contain a 50-52 kDa acyl-CoA independent sn-1,2(2,3)-diacylglycerol transacylase of unknown primary sequence which utilizes diolein or mono-olein acyl donors for transacylation of diolein or mono-olein acyl acceptors to form triolein (24). Since no amino acid sequence data was published on the identity of this partially purified protein, it is unknown whether this protein was adiponutrin, TTS-2.2, or another protein entirely. Although heparin-releasable hepatic lipase has been shown to catalyze acyl transfer from the 1(3)-position of neutral glycerides to various lipid acceptors, to our knowledge, iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta represent the first intracellular mammalian acylglycerol transacylases to be identified at the molecular level.

[0152]GS2 (termed "gene sequence 2") was originally cloned in 1994 as the second gene present within a CpG island-rich contig of the distal short arm of human X chromosome (45). Located midway between the steroid sulfatase (STS) and Kallman syndrome (KALI) loci, the GS2 gene is comprised of 7 exons which are distributed over 26 kb (45). GS2 mRNA transcripts of varying size are highly expressed in liver, brain, and skeletal muscle with lower amounts present in lung, placenta, kidney, and pancreas (45). Expression levels of GS2 mRNA in adipose tissue have not been examined to our knowledge. Deletion of GS2 appears to be non-lethal, although the precise phenotype is not clear since patients with X-linked ichthyosis who have the deleted GS2 (iPLA₂eta) in addition to the steroid sulfatase gene (STS) are indistinguishable from those with mutations within the STS gene alone at the current level of discrimination (46).

[0153]The metabolic pathways which contribute to the elevated levels of circulating non-esterified fatty acids in obese individuals are unclear. Clinically, high serum free fatty acid levels are treated by administration of thiazolidinediones (TZDs) which mediate their effects through PPARgamma. One mechanism through which TZDs are believed to decrease serum non-esterified fatty acids is through the induction of glycerol kinase (9) and phosphoenolpyruvate carboxykinase (47) which provide glycerol-3-phosphate for re-esterification of fatty acyl equivalents (TAG synthesis) in the adipocyte. Clearly, the potential significance of the relative triglyceride lipase activities of iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta in comparison to their transacylase activities in contributing to serum free fatty acid levels in individuals with metabolic syndrome X and type 2 diabetes is of great interest. Although in vitro activity measurements demonstrate that the TAG lipase activities of each iPLA₂ isoform are greater than their respective transacylase activities, past-translational modifications (e.g. phosphorylation), protein-protein interactions, and/or specific lipid droplet microenvironments may alter the transacylasellipase activity ratios of iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta in vivo. Intriguingly, treatment of differentiating 3T3-L1 adipocytes with troglitazone down-regulates adiponutrin (iPLA₂epsilon) mRNA levels (27). Thus, the up-regulation of adiponutrin, which has been observed to occur in obese rats (26), may contribute to either the high basal levels of circulating non-esterified fatty acids (due to TAG lipase activity) or to adipocyte hypertrophy (due to acylglycerol transacylation) observed in obese individuals.

[0154]We have identified three novel human triacylglycerol lipases/transacylases which are related to the iPLA₂ family of enzymes by virtue of their dual signature nucleotide binding and lipase consensus motifs. Moreover, iPLA₂zeta (TTS-2.2), like iPLA₂epsilon (adiponutrin) (26), is upregulated during 3T3-L1 adipocyte differentiation and all three of the novel iPLA₂ isoforms are present in a human liposarcoma cell line. Collectively, these results provide a new foundation to increase understanding of triglyceride homeostasis in adipocytes. Of particular interest will be studies identifying the effects of altering iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta expression levels and determination of the diversity of biologically relevant isoforms and their specific effects on cellular lipid homeostasis. Multiple mechanisms controlling iPLA₂epsilon, iPLA₂zeta, and iPLA₂eta protein mass (via transcription and/or translation) and lipase/transacylase activity (via post-translational modification and/or protein-protein interactions) likely contribute to the regulation of the anabolic and catabolic fluxes of acyl equivalents in adipocytes.

[0155]In another aspect of this invention, a method of medically treating a living mammal comprises administering to a living mammal or to a cell thereof a therapeutically effective amount of a compound (drug or pharmaceutical) which inhibits iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta expression to the mammal or result in a different isoform expression or different enzymatic activity or posttranslational modification.

[0156]Compounds shown to inhibit iPLA₂epsilon, iPLA₂zeta, and/or iPLA₂eta can be utilized as diagnostics, therapeutics, and as research reagents and provided in kits. They can be utilized in pharmaceutical compositions by adding an effective amount of an oligonucleotide of the invention to a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism can be contacted with an oligonucleotide of the invention having a sequence that is capable of specifically hybridizing with a strand of target nucleic acid that codes for the undesirable protein.

[0157]The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. In general, for therapeutics, a patient in need of such therapy is administered an oligomer in accordance with the invention, commonly in a pharmaceutically acceptable carrier, depending on the age of the patient and the severity of the disease state being treated. Further, the treatment may be a single dose or may be a regimen that may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient, and may extend from once daily to once every 20 years. Following treatment, the patient is monitored for changes in his/her condition and for alleviation of the symptoms of the disease state. The dosage of the oligomer may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, or if the disease state has been ablated.

[0158]Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligomer is administered in maintenance doses, ranging from 0.01 microgram to 100 g per kg of body weight, once or more daily, to once every several years.

[0159]The pharmaceutical compositions of the present invention may be effectively administered in a number of ways to mammals depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraparitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0160]Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0161]Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0162]Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[0163]Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

[0164]Key Words: Adiponutrin, TTS-2.2, GS2, Lipase, Transacylase, Phospholipase A₂, Triacylglycerol.

Abbreviations

[0165]BEL--(E)-6-(bromomethy 1 ene)-3-(1-naphthalenyl)-2H-tetrahydropyran-₂-one.

[0166]iPLA₂-Calcium-independent phospholipase A₂.

[0167]DAG--Diacylglycerol.

[0168]DOG--Diolein.

[0169]DGAT--Acyl:CoA:diacylglycerol acyltransferase.

[0170]GS2--Gene sequence 2.

[0171]MAG--Monoacylglycerol.

[0172]MOG--Mono-olein.

[0173]TAG--Triacylglycerol.

[0174]TOG--Triolein.

[0175]TTS-2,2--Transport secretion protein-2.2.

Key of Symbols

[0176]α=alpha

[0177]β=beta

[0178]γ=gamma

[0179]Δ=delta

[0180]ε=epsilon

[0181]ξ=zeta

[0182]η=eta

[0183]μ=micro

REFERENCES

[0184]1. Kopelman, P. G. (2000) Nature 404, 635-643. [0185]2. Mokdad, A. H., Bowman, B. A., Ford, E. S., Vinicor, F., Marks, J. S., and Koplan, J. P. (2001) Jama 286, 1195-1200. [0186]3. James, P. T., Leach, R., Kalamara, E., and Shayeghi, M. (2001) Obes Res 9 Suppl 4, 228S-233S. [0187]4. Pi-Sunyer, F. X. (2002) Obes Res 10 Suppl 2, 97S-104S. [0188]5. Boden, G., and Shulman, G. I. (2002) Eur J Clin Invest [0189]32 Suppl 3, 14-23. [0190]6. McGarry, J. D., and Dobbins, R. L. (1999) Diabetologia 42, 128-138 [0191]7. Unger, R, H., and Orci, L. (2001) Faseb J15, 312-321. [0192]8. Schaffer, J. E. (2003) Curr Opin Lipidol 14, 281-287. [0193]9. Guan, H. P., Li, Y., Jensen, M. V., Newgard, C. B., Steppan, C. M., and Lazar, M. A. (2002) Nat Med 8, 1122-1128 [0194]10. Reshef, L., Olswang, Y., Cassuto, H., Blum, B., Croniger, C. M., Kalhan, S. C., Tilghrnan, S. M., and Hanson, R. W. (2003) J Biol Chem 278, 30413-30416. [0195]11. Lehner, R., and Kuksis, A. (1996) Prog Lipid Res 35, 169-201. [0196]12. Coleman, R. A., and Lee, D. P. (2004) Prog Lipid Res 43, 134-176. [0197]13. Holm, C., Kirchgessner, T. G., Svenson, K. L., Fredrikson, G., Nilsson, S., Miller, C. G., Shively, J. E., Heinunann, C., Sparkes, R. S., Mohandas, T., and et al. (1988) Science 241, 1503-1506. [0198]14. Yeaman, S. J. (2004) Biochem J379, 11-22. [0199]15. Kraemer, F. B., and Shen, W. J. (2002) J Lipid Res 43, 1585-1594. [0200]16. Fredrikson, G., Stralfors, P., Nilsson, N. O., and Belfrage, P. (1981) J Biol Chem 256, 6311-6320. [0201]17. Shen, W. J., Patel, S., Natu, V., and Kraemer, F. B. (1998) Biochemisdtry 37, 8973-8979. [0202]18. Anthonsen, M, W., Ronnstrand, L., Wernstedt, C., Degeman, E., and Holm, C. (1998) J Biol Chem 273, 215-221. [0203]19. Greenberg, A. S., Shen, W. J., Muliro, K, Patel, S., Souza, S. C., Roth, R. A., and Kraemer, F. B. (2001) J Biol Chem 276, 45456-45461. [0204]20. Haernmerle, G., Zirnmemann, R., Hayn, M., Theussl, C., Waeg, G., Wagner, E., Sattler, W., Magin, T. M., Wagner, E. F., and Zechner, R. (2002) J Biol Chem 277, 4806-4815. [0205]21. Osuga, J., Ishibashi, S., Oka, T., Yagyu, H., Tozawa, R., Fujimoto, A., Shionoiri, F., Yahagi, N., Kraemer, F. B, Tsutsumi, O., and Yamada, N. (2000) Proc Natl Acad Sci USA 97, 787-792. [0206]22. Wang, S. P., Laurin, N., Himms-Hagen, J., Rudnidci, M. A., Levy, E., Robert, M. F., Pan, L., Oligny, L., and Mitchell, G. A. (2001) Obes Res 9, 119-128. [0207]23. Okazaki, H., Osuga, J., Tamura, Y., Yahagi, N., Tomita, S., Shionoiri, F., Jizuka, Y., Ohashi, K., Harada, K., Kimura, S., Gotoda, T., Shimano, H., Yamada, N., and Ishibashi, S. (2002) Diabetes 51, 3368-3375. [0208]24. Lehner, R., and Kuksis, A. (1993) J Biol Chem 268, 8781-8786. [0209]25. Buhman, K. K., Smith, S. J., Stone, S. J., Repa, J. J., Wong, J. S., Knapp, F. F., Jr., Burri, B. J., Hamilton, R. L., Aburnrad, N. A., and Farese, R. V., Jr. (2002) J Biol Chem 277, 25474-25479. [0210]26. Baulande, S., Lasnier, F., Lucas, M., and Pairault, J. (2001) J Biol Chem 276, 33336-33344. [0211]27. Poison, D., and Thompson, M, (2003) Horm Metab Res 35, 508-510. [0212]28. Poison, D. A., and Thompson, M. P. (2003) Biochem Biophys Res Commun 301, 261-266. [0213]29. Poison, D. A., and Thompson, M. P. (2004) J Nutr Biochem 15, 242-246. [0214]30. Liu, P., Ying, Y., Zhao, Y., Mundy, D. I., Zhu, M, and Anderson, R. G. (2004) J Biol Chem 279, 3787-3792. [0215]31. Frost, S. C., and Lane, M. D. (1985) J Biol Chem 260, 2646-2652. [0216]32. Izem, L., and Morton, R. E. (2001) J Biol Chem 276, 26534-26541. [0217]33. Laemmli, U. K. (1970) Nature 227, 680-685. [0218]34. Gailiard, T. (1971) Biochem J 121, 379-390. [0219]35. Andrews, D. L., Beames, B., Summers, M. D., and Park, W. D. (1988) Biochem J252, 199-206. [0220]36. Pinsirodom, P., and Parkin, K. L. (2000) J Agric Food Chem 48, 155-160. [0221]37. Hazen, S. L., Zupan, L. A., Weiss, R. H., Getman, D. P., and Gross, R. W. (1991) J Biol Chem 266, 7227-7232. [0222]38. Zupan, L. A., Weiss, R. H., Hazen, S. L., Parnas, B. L., Aston, K. W., Lemon, P. J., Getman, D. P., and Gross, R. W. (1993) J Med Chem 36, 95-100. [0223]39. Mancuso, D. J., Jenkins, C. M., and Gross, R. W. (2000) J Biol Chem 275, 9937-9945. [0224]40. Gauthier, A., Vassiliou, G., Benoist, F., and McPherson, R. (2003) J Biol Chem 278, 11945-11953. [0225]41. Vassiliou, G., Benoist, F., Lau, P., Kavaslar, G. N., and McPherson, R. (2001) J Biol Chem 276, 48823-48830. [0226]42. Smith, S. J., Cases, S., Jensen, D. R., Chen, H. C., Sande, E., Tow, B., Sanan, D. A., Raber, J., Eckel, R. H., and Farese, R. V., Jr. (2000) Nat Genet 25, 87-90 [0227]43. Chen, H. C., Smith, S. J., Ladha, Z, Jensen, D. R, Ferreira, L. D., Pulawa, L, K, McGuire, J. G., Pitas, R. E., Eckel, R. H., and Farese, R. V., Jr. (2002) JClin Invest 109, 1049-1055. [0228]44. Stone, S. J., Myers, H. M., Watkins, S. Ma, Brown, B. E., Feingold, K. R., Elias, P. M., and Farese, R. V., Jr. (2004) J Biol Chem 279, 11767-11776. [0229]45. Lee, W. C., Salido, E., and Yen, P. H. (1994) Genomics 22, 372-376. [0230]46. Shapiro, L. J., Yen, P., Pomerantz, D., Martin, E., Rolewic, L., and Mohandas, T. (1989) Proc Natl Acad Sci USA 86, 8477-8481. [0231]47. Tordjman, J., Chauvet, G., Quette, J., Beale, E. G., Forest, C., and Antoine, B. (2003) J Bid Chem 278, 18785-18790. [0232]48. Corpet, F. (1988) Nucleic Acids Res 16, 10881-10890. [0233]Elia, M., Zed, C., Neale, G., and Livesey, G. (1987) Metabolism 36, 251-255 [0234]Van Harmelen, V., Reynisdottir, S., Cianflone, K., Degerman, E., Hoffstedt, J., Nilsell, K., Sniderman, A., and Amer, P. (1999) J Biol Chem 274, 18243-18251 [0235]Jensen, M. D., Ekberg, K., and Landau, B. R. (2001) Am J Physiol Endocrinol Metab 281, E789-793 [0236]Gibbons, G. F., Islam, K., and Pease, R. J. (2000) Biochim Biophys Acta 1483, 37-57 [0237]Raclot, T. (2003) Prog Lipid Res 42, 257-288 [0238]Tang, J., Kriz, R. W., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S. S. (1997) J Biol Chem 272, 8567-8575 [0239]van Tienhoven, M., Atkins, J., Li, Y., and Glynn, P. (2002) J Biol Chem 277, 20942-20948 [0240]Waite, M., and Sisson, P. (1973) J Biol Chem 248, 7985-7992 [0241]Hulsmann, W. C., Oerlemans, M. C., and Jansen, H. (1980) Biochim Biophys Acta 618, 364-369 [0242]Glorian, M., Duplus, E., Beale, E. G., Scott, D. K., Granner, D. K., and Forest, C. (2001) Biochimie 83, 933-943

[0243]While the discovery has been described in terms of various specific embodiments, those skilled in the art will recognize that the discovery can be practiced with modification within the spirit and scope of the discovery.

Sequence CWU 1

301487PRTHomo sapiens 1Met Tyr Asp Ala Glu Arg Gly Trp Ser Leu Ser Phe Ala Gly Cys Gly1 5 10 15Phe Leu Gly Phe Tyr His Val Gly Ala Thr Arg Cys Leu Ser Glu His20 25 30Ala Pro His Leu Leu Arg Asp Ala Arg Met Leu Phe Gly Ala Ser Ala35 40 45Gly Ala Leu His Cys Val Gly Val Leu Ser Gly Ile Pro Leu Glu Gln50 55 60Thr Leu Gln Val Leu Ser Asp Leu Val Arg Lys Ala Arg Ser Arg Asn65 70 75 80Ile Gly Ile Phe His Pro Ser Phe Asn Leu Ser Lys Phe Leu Arg Gln85 90 95Gly Leu Cys Lys Cys Leu Pro Ala Asn Val His Gln Leu Ile Ser Gly100 105 110Lys Ile Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Leu115 120 125Val Ser Asp Phe Arg Ser Lys Asp Glu Val Val Asp Ala Leu Val Cys130 135 140Ser Cys Phe Ile Pro Phe Tyr Ser Gly Leu Ile Pro Pro Ser Phe Arg145 150 155 160Gly Val Arg Tyr Val Asp Gly Gly Val Ser Asp Asn Val Pro Phe Ile165 170 175Asp Ala Lys Thr Thr Ile Thr Val Ser Pro Phe Tyr Gly Glu Tyr Asp180 185 190Ile Cys Pro Lys Val Lys Ser Thr Asn Phe Leu His Val Asp Ile Thr195 200 205Lys Leu Ser Leu Arg Leu Cys Thr Gly Asn Leu Tyr Leu Leu Ser Arg210 215 220Ala Phe Val Pro Pro Asp Leu Lys Val Leu Gly Glu Ile Cys Leu Arg225 230 235 240Gly Tyr Leu Asp Ala Phe Arg Phe Leu Glu Glu Lys Gly Ile Cys Asn245 250 255Arg Pro Gln Pro Gly Leu Lys Ser Ser Ser Glu Gly Met Asp Pro Glu260 265 270Val Ala Met Pro Ser Trp Ala Asn Met Ser Leu Asp Ser Ser Pro Glu275 280 285Ser Ala Ala Leu Ala Val Arg Leu Glu Gly Asp Glu Leu Leu Asp His290 295 300Leu Arg Leu Ser Ile Leu Pro Trp Asp Glu Ser Ile Leu Asp Thr Leu305 310 315 320Ser Pro Arg Leu Ala Thr Ala Leu Ser Glu Glu Met Lys Asp Lys Gly325 330 335Gly Tyr Met Ser Lys Ile Cys Asn Leu Leu Pro Ile Arg Ile Met Ser340 345 350Tyr Val Met Leu Pro Cys Thr Leu Pro Val Glu Ser Ala Ile Ala Ile355 360 365Val Gln Arg Leu Val Thr Trp Leu Pro Asp Met Pro Asp Asp Val Leu370 375 380Trp Leu Gln Trp Val Thr Ser Gln Val Phe Thr Arg Val Leu Met Cys385 390 395 400Leu Leu Pro Ala Ser Arg Ser Gln Met Pro Val Ser Ser Gln Gln Ala405 410 415Ser Pro Cys Thr Pro Glu Gln Asp Trp Pro Cys Trp Thr Pro Cys Ser420 425 430Pro Glu Gly Cys Pro Ala Glu Thr Lys Ala Glu Ala Thr Pro Arg Ser435 440 445Ile Leu Arg Ser Ser Leu Asn Phe Phe Leu Gly Asn Lys Val Pro Ala450 455 460Gly Ala Glu Gly Leu Ser Thr Phe Pro Ser Phe Ser Leu Glu Lys Ser465 470 475 480Leu His His His His His His4852510PRTHomo sapiens 2Met Phe Pro Arg Glu Lys Thr Trp Asn Ile Ser Phe Ala Gly Cys Gly1 5 10 15Phe Leu Gly Val Tyr Tyr Val Gly Val Ala Ser Cys Leu Arg Glu His20 25 30Ala Pro Phe Leu Val Ala Asn Ala Thr His Ile Tyr Gly Ala Ser Ala35 40 45Gly Ala Leu Thr Ala Thr Ala Leu Val Thr Gly Val Cys Leu Gly Glu50 55 60Ala Gly Ala Lys Phe Ile Glu Val Ser Lys Glu Ala Arg Lys Arg Phe65 70 75 80Leu Gly Pro Leu His Pro Ser Phe Asn Leu Val Lys Ile Ile Arg Ser85 90 95Phe Leu Leu Lys Val Leu Pro Ala Asp Ser His Glu His Ala Ser Gly100 105 110Arg Leu Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Ile115 120 125Ile Ser His Phe Asn Ser Lys Asp Glu Leu Ile Gln Ala Asn Val Cys130 135 140Ser Gly Phe Ile Pro Val Tyr Cys Gly Leu Ile Pro Pro Ser Leu Gln145 150 155 160Gly Val Arg Tyr Val Asp Gly Gly Ile Ser Asp Asn Leu Pro Leu Tyr165 170 175Glu Leu Lys Asn Thr Ile Thr Val Ser Pro Phe Ser Gly Glu Ser Asp180 185 190Ile Cys Pro Gln Asp Ser Ser Thr Asn Ile His Glu Leu Arg Val Thr195 200 205Asn Thr Ser Ile Gln Phe Asn Leu Arg Asn Leu Tyr Arg Leu Ser Lys210 215 220Ala Leu Phe Pro Pro Glu Pro Leu Val Leu Arg Glu Met Cys Lys Gln225 230 235 240Gly Tyr Arg Asp Gly Leu Arg Phe Leu Gln Arg Asn Gly Leu Leu Asn245 250 255Arg Pro Asn Pro Leu Leu Ala Leu Pro Pro Ala Arg Pro His Gly Pro260 265 270Glu Asp Lys Asp Gln Ala Val Glu Ser Ala Gln Ala Glu Asp Tyr Ser275 280 285Gln Leu Pro Gly Glu Asp His Ile Leu Glu His Leu Pro Ala Arg Leu290 295 300Asn Glu Ala Leu Leu Glu Ala Cys Val Glu Pro Thr Asp Leu Leu Thr305 310 315 320Thr Leu Ser Asn Met Leu Pro Val Arg Leu Ala Thr Ala Met Met Val325 330 335Pro Tyr Thr Leu Pro Leu Glu Ser Ala Leu Ser Phe Thr Ile Arg Leu340 345 350Leu Glu Trp Leu Pro Asp Val Pro Glu Asp Ile Arg Trp Met Lys Glu355 360 365Gln Thr Gly Ser Ile Cys Gln Tyr Leu Val Met Arg Ala Lys Arg Lys370 375 380Leu Gly Arg His Leu Pro Ser Arg Leu Pro Glu Gln Val Glu Leu Arg385 390 395 400Arg Val Gln Ser Leu Pro Ser Val Pro Leu Ser Cys Ala Ala Tyr Arg405 410 415Glu Ala Leu Pro Gly Trp Met Arg Asn Asn Leu Ser Leu Gly Asp Ala420 425 430Leu Ala Lys Trp Glu Glu Cys Gln Arg Gln Leu Leu Leu Gly Leu Phe435 440 445Cys Thr Asn Val Ala Phe Pro Pro Glu Ala Leu Arg Met Arg Ala Pro450 455 460Ala Asp Pro Ala Pro Ala Pro Ala Asp Pro Ala Ser Pro Gln His Gln465 470 475 480Pro Ala Gly Pro Ala Pro Leu Leu Ser Thr Pro Ala Pro Glu Ala Arg485 490 495Pro Val Ile Gly Ala Leu Gly Leu His His His His His His500 505 5103267PRTHomo sapiens 3Met Lys His Ile Asn Leu Ser Phe Ala Ala Cys Gly Phe Leu Gly Ile1 5 10 15Tyr His Leu Gly Ala Ala Ser Ala Leu Cys Arg His Gly Lys Lys Leu20 25 30Val Lys Asp Val Lys Ala Phe Ala Gly Ala Ser Ala Gly Phe Thr Ser35 40 45Leu Val Ala Ser Val Leu Leu Thr Ala Pro Glu Lys Ile Glu Glu Cys50 55 60Asn Gln Phe Thr Tyr Lys Phe Ala Glu Glu Ile Arg Arg Gln Ser Phe65 70 75 80Gly Ala Val Thr Pro Gly Tyr Asp Phe Met Ala Arg Leu Arg Ser Gly85 90 95Met Glu Ser Ile Leu Pro Pro Ser Ala His Phe Thr Glu Leu Ala Gln100 105 110Asn Arg Leu His Val Ser Ile Thr Asn Ala Lys Thr Arg Glu Asn His115 120 125Leu Val Ser Thr Phe Ser Ser Arg Glu Asp Leu Ile Lys Val Leu Leu130 135 140Ala Ser Ser Phe Val Pro Ile Tyr Ala Gly Leu Lys Leu Val Glu Tyr145 150 155 160Lys Gly Gln Lys Trp Val Asp Phe Thr Gly Gly Leu Thr Asn Ala Leu165 170 175Pro Ile Leu Pro Val Gly Arg Thr Val Thr Ile Ser Pro Phe Ser Gly180 185 190Arg Leu Asp Ile Ser Pro Gln Asp Lys Gly Gln Leu Asp Leu Tyr Val195 200 205Asn Ile Ala Lys Gln Asp Ile Met Leu Ser Leu Ala Asn Leu Val Arg210 215 220Leu Asn Gln Ala Phe Thr Leu Phe Pro Pro Ser Lys Arg Lys Met Glu225 230 235 240Ser Leu Tyr Gln Cys Gly Phe Asp Asp Thr Val Lys Phe Leu Leu Lys245 250 255Glu Asn Trp Phe Glu His His His His His His260 26541515DNAHomo sapiensCDS(1)..(1512) 4atg ttt ccc cgc gag aag acg tgg aac atc tcg ttc gcg ggc tgc ggc 48Met Phe Pro Arg Glu Lys Thr Trp Asn Ile Ser Phe Ala Gly Cys Gly1 5 10 15ttc ctc ggc gtc tac tac gtc ggc gtg gcc tcc tgc ctc cgc gag cac 96Phe Leu Gly Val Tyr Tyr Val Gly Val Ala Ser Cys Leu Arg Glu His20 25 30gcg ccc ttc ctg gtg gcc aac gcc acg cac atc tac ggc gcc tcg gcc 144Ala Pro Phe Leu Val Ala Asn Ala Thr His Ile Tyr Gly Ala Ser Ala35 40 45ggg gcg ctc acg gcc acg gcg ctg gtc acc ggg gtc tgc ctg ggt gag 192Gly Ala Leu Thr Ala Thr Ala Leu Val Thr Gly Val Cys Leu Gly Glu50 55 60gct ggt gcc aag ttc att gag gta tct aaa gag gcc cgg aag cgg ttc 240Ala Gly Ala Lys Phe Ile Glu Val Ser Lys Glu Ala Arg Lys Arg Phe65 70 75 80ctg ggc ccc ctg cac ccc tcc ttc aac ctg gta aag atc atc cgc agt 288Leu Gly Pro Leu His Pro Ser Phe Asn Leu Val Lys Ile Ile Arg Ser85 90 95ttc ctg ctg aag gtc ctg cct gct gat agc cat gag cat gcc agt ggg 336Phe Leu Leu Lys Val Leu Pro Ala Asp Ser His Glu His Ala Ser Gly100 105 110cgc ctg ggc atc tcc ctg acc cgc gtg tca gac ggc gag aat gtc att 384Arg Leu Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Ile115 120 125ata tcc cac ttc aac tcc aag gac gag ctc atc cag gcc aat gtc tgc 432Ile Ser His Phe Asn Ser Lys Asp Glu Leu Ile Gln Ala Asn Val Cys130 135 140agc ggt ttc atc ccc gtg tac tgt ggg ctc atc cct ccc tcc ctc cag 480Ser Gly Phe Ile Pro Val Tyr Cys Gly Leu Ile Pro Pro Ser Leu Gln145 150 155 160ggg gtg cgc tac gtg gat ggt ggc att tca gac aac ctg cca ctc tat 528Gly Val Arg Tyr Val Asp Gly Gly Ile Ser Asp Asn Leu Pro Leu Tyr165 170 175gag ctt aag aac acc atc aca gtg tcc ccc ttc tcg ggc gag agt gac 576Glu Leu Lys Asn Thr Ile Thr Val Ser Pro Phe Ser Gly Glu Ser Asp180 185 190atc tgt ccg cag gac agc tcc acc aac atc cac gag ctg cgg gtc acc 624Ile Cys Pro Gln Asp Ser Ser Thr Asn Ile His Glu Leu Arg Val Thr195 200 205aac acc agc atc cag ttc aac ctg cgc aac ctc tac cgc ctc tcc aag 672Asn Thr Ser Ile Gln Phe Asn Leu Arg Asn Leu Tyr Arg Leu Ser Lys210 215 220gcc ctc ttc ccg ccg gag ccc ctg gtg ctg cga gag atg tgc aag cag 720Ala Leu Phe Pro Pro Glu Pro Leu Val Leu Arg Glu Met Cys Lys Gln225 230 235 240gga tac cgg gat ggc ctg cgc ttt ctg cag cgg aac ggc ctc ctg aac 768Gly Tyr Arg Asp Gly Leu Arg Phe Leu Gln Arg Asn Gly Leu Leu Asn245 250 255cgg ccc aac ccc ttg ctg gcg ttg ccc ccc gcc cgc ccc cac ggc cca 816Arg Pro Asn Pro Leu Leu Ala Leu Pro Pro Ala Arg Pro His Gly Pro260 265 270gag gac aag gac cag gca gtg gag agc gcc caa gcg gag gat tac tcg 864Glu Asp Lys Asp Gln Ala Val Glu Ser Ala Gln Ala Glu Asp Tyr Ser275 280 285cag ctg ccc gga gaa gat cac atc ctg gag cac ctg ccc gcc cgg ctc 912Gln Leu Pro Gly Glu Asp His Ile Leu Glu His Leu Pro Ala Arg Leu290 295 300aat gag gcc ctg ctg gag gcc tgc gtg gag ccc acg gac ctg ctg acc 960Asn Glu Ala Leu Leu Glu Ala Cys Val Glu Pro Thr Asp Leu Leu Thr305 310 315 320acc ctc tcc aac atg ctg cct gtg cgt ctg gcc acg gcc atg atg gtg 1008Thr Leu Ser Asn Met Leu Pro Val Arg Leu Ala Thr Ala Met Met Val325 330 335ccc tac acg ctg ccg ctg gag agc gct ctg tcc ttc acc atc cgc ttg 1056Pro Tyr Thr Leu Pro Leu Glu Ser Ala Leu Ser Phe Thr Ile Arg Leu340 345 350ctg gag tgg ctg ccc gac gtt ccc gag gac atc cgg tgg atg aag gag 1104Leu Glu Trp Leu Pro Asp Val Pro Glu Asp Ile Arg Trp Met Lys Glu355 360 365cag acg ggc agc atc tgc cag tac ctg gtg atg cgc gcc aag agg aag 1152Gln Thr Gly Ser Ile Cys Gln Tyr Leu Val Met Arg Ala Lys Arg Lys370 375 380ctg ggc agg cac ctg ccc tcc agg ctg ccg gag cag gtg gag ctg cgc 1200Leu Gly Arg His Leu Pro Ser Arg Leu Pro Glu Gln Val Glu Leu Arg385 390 395 400cgc gtc cag tcg ctg ccg tcc gtg ccg ctg tcc tgc gcc gcc tac aga 1248Arg Val Gln Ser Leu Pro Ser Val Pro Leu Ser Cys Ala Ala Tyr Arg405 410 415gag gca ctg ccc ggc tgg atg cgc aac aac ctc tcg ctg ggg gac gcg 1296Glu Ala Leu Pro Gly Trp Met Arg Asn Asn Leu Ser Leu Gly Asp Ala420 425 430ctg gcc aag tgg gag gag tgc cag cgc cag ctg ctg ctc ggc ctc ttc 1344Leu Ala Lys Trp Glu Glu Cys Gln Arg Gln Leu Leu Leu Gly Leu Phe435 440 445tgc acc aac gtg gcc ttc ccg ccc gaa gct ctg cgc atg cgc gca ccc 1392Cys Thr Asn Val Ala Phe Pro Pro Glu Ala Leu Arg Met Arg Ala Pro450 455 460gcc gac ccg gct ccc gcc ccc gcg gac cca gca tcc ccg cag cac cag 1440Ala Asp Pro Ala Pro Ala Pro Ala Asp Pro Ala Ser Pro Gln His Gln465 470 475 480ccg gcc ggg cct gcc ccc ttg ctg agc acc cct gct ccc gag gcc cgg 1488Pro Ala Gly Pro Ala Pro Leu Leu Ser Thr Pro Ala Pro Glu Ala Arg485 490 495ccc gtg atc ggg gcc ctg ggg ctg tga 1515Pro Val Ile Gly Ala Leu Gly Leu5005762DNAHomo sapiensCDS(1)..(759) 5atg aag cac atc aac cta tca ttt gca gcg tgt gga ttt ctg ggc att 48Met Lys His Ile Asn Leu Ser Phe Ala Ala Cys Gly Phe Leu Gly Ile 1 5 10 15tac cac ttg ggg gca gca tct gca ctt tgc aga cat ggc aaa aaa ctt 96Tyr His Leu Gly Ala Ala Ser Ala Leu Cys Arg His Gly Lys Lys Leu20 25 30gtg aag gat gtc aaa gcc ttc gct ggg gcg tct gcg gga tcg ttg gtt 144Val Lys Asp Val Lys Ala Phe Ala Gly Ala Ser Ala Gly Ser Leu Val35 40 45gct tct gtt ctg cta aca gca cca gaa aaa ata gag gaa tgt aac caa 192Ala Ser Val Leu Leu Thr Ala Pro Glu Lys Ile Glu Glu Cys Asn Gln50 55 60ttt acc tac aag ttt gcc gaa gaa atc aga agg cag tct ttc ggg gca 240Phe Thr Tyr Lys Phe Ala Glu Glu Ile Arg Arg Gln Ser Phe Gly Ala65 70 75 80gta acg ccc ggt tat gac ttc atg gcc cga cta aga agt ggg atg gag 288Val Thr Pro Gly Tyr Asp Phe Met Ala Arg Leu Arg Ser Gly Met Glu85 90 95tcg att ctt cct ccc agc gct cac gag ctg gcc cag aac cga ctg cac 336Ser Ile Leu Pro Pro Ser Ala His Glu Leu Ala Gln Asn Arg Leu His100 105 110gta tcc atc acc aac gcc aaa acc aga gaa aat cac tta gtc tcc act 384Val Ser Ile Thr Asn Ala Lys Thr Arg Glu Asn His Leu Val Ser Thr115 120 125ttt tcc tcc agg gag gac ctc att aag gtc ctc cta gcc agc agt ttt 432Phe Ser Ser Arg Glu Asp Leu Ile Lys Val Leu Leu Ala Ser Ser Phe130 135 140gtg ccc att tat gca gga ctg aag cta gtg gaa tac aaa ggg cag aag 480Val Pro Ile Tyr Ala Gly Leu Lys Leu Val Glu Tyr Lys Gly Gln Lys145 150 155 160tgg gtg gac gga ggc ctc acc aac gct ctt ccc atc ctg ccc gtc ggc 528Trp Val Asp Gly Gly Leu Thr Asn Ala Leu Pro Ile Leu Pro Val Gly165 170 175cgg aca gta acc atc tcc ccc ttc agt gga cga ctg gac atc tcc ccg 576Arg Thr Val Thr Ile Ser Pro Phe Ser Gly Arg Leu Asp Ile Ser Pro180 185 190cag gac aaa ggg cag cta gat ctg tat gtt aat atc gcc aag cag gat 624Gln Asp Lys Gly Gln Leu Asp Leu Tyr Val Asn Ile Ala Lys Gln Asp195 200 205atc atg ttg tcc ctg gca aac ctg gtg aga ctc aac caa gcc ctt ttt 672Ile Met Leu Ser Leu Ala Asn Leu Val Arg Leu Asn Gln Ala Leu Phe210 215 220ccc cca agc aag agg aaa atg gaa tct ttg tat cag tgt ggt ttt gat 720Pro Pro Ser Lys Arg Lys Met Glu Ser Leu Tyr Gln Cys Gly Phe Asp225 230 235 240gac act gtt aag ttt tta ctt aaa gaa aat tgg ttt gaa taa 762Asp Thr Val Lys Phe Leu Leu Lys Glu Asn Trp Phe Glu245 2506504PRTHomo sapiens 6Met Phe Pro Arg Glu Lys Thr Trp Asn Ile Ser Phe Ala Gly Cys Gly 1 5 10 15Phe Leu Gly Val Tyr Tyr Val Gly Val Ala Ser Cys Leu Arg Glu His20 25 30Ala Pro Phe Leu Val Ala Asn Ala Thr His Ile Tyr Gly Ala Ser Ala35 40 45Gly Ala Leu Thr Ala Thr Ala Leu Val Thr Gly Val Cys Leu Gly Glu50 55 60Ala Gly Ala Lys Phe Ile Glu Val Ser Lys Glu Ala Arg Lys Arg Phe65 70 75 80Leu Gly Pro Leu His Pro Ser Phe Asn Leu Val Lys Ile Ile Arg Ser85 90 95Phe Leu Leu Lys Val Leu Pro Ala Asp Ser His Glu His Ala Ser Gly100 105 110Arg Leu Gly Ile Ser Leu Thr Arg Val

Ser Asp Gly Glu Asn Val Ile115 120 125Ile Ser His Phe Asn Ser Lys Asp Glu Leu Ile Gln Ala Asn Val Cys130 135 140Ser Gly Phe Ile Pro Val Tyr Cys Gly Leu Ile Pro Pro Ser Leu Gln145 150 155 160Gly Val Arg Tyr Val Asp Gly Gly Ile Ser Asp Asn Leu Pro Leu Tyr165 170 175Glu Leu Lys Asn Thr Ile Thr Val Ser Pro Phe Ser Gly Glu Ser Asp180 185 190Ile Cys Pro Gln Asp Ser Ser Thr Asn Ile His Glu Leu Arg Val Thr195 200 205Asn Thr Ser Ile Gln Phe Asn Leu Arg Asn Leu Tyr Arg Leu Ser Lys210 215 220Ala Leu Phe Pro Pro Glu Pro Leu Val Leu Arg Glu Met Cys Lys Gln225 230 235 240Gly Tyr Arg Asp Gly Leu Arg Phe Leu Gln Arg Asn Gly Leu Leu Asn245 250 255Arg Pro Asn Pro Leu Leu Ala Leu Pro Pro Ala Arg Pro His Gly Pro260 265 270Glu Asp Lys Asp Gln Ala Val Glu Ser Ala Gln Ala Glu Asp Tyr Ser275 280 285Gln Leu Pro Gly Glu Asp His Ile Leu Glu His Leu Pro Ala Arg Leu290 295 300Asn Glu Ala Leu Leu Glu Ala Cys Val Glu Pro Thr Asp Leu Leu Thr305 310 315 320Thr Leu Ser Asn Met Leu Pro Val Arg Leu Ala Thr Ala Met Met Val325 330 335Pro Tyr Thr Leu Pro Leu Glu Ser Ala Leu Ser Phe Thr Ile Arg Leu340 345 350Leu Glu Trp Leu Pro Asp Val Pro Glu Asp Ile Arg Trp Met Lys Glu355 360 365Gln Thr Gly Ser Ile Cys Gln Tyr Leu Val Met Arg Ala Lys Arg Lys370 375 380Leu Gly Arg His Leu Pro Ser Arg Leu Pro Glu Gln Val Glu Leu Arg385 390 395 400Arg Val Gln Ser Leu Pro Ser Val Pro Leu Ser Cys Ala Ala Tyr Arg405 410 415Glu Ala Leu Pro Gly Trp Met Arg Asn Asn Leu Ser Leu Gly Asp Ala420 425 430Leu Ala Lys Trp Glu Glu Cys Gln Arg Gln Leu Leu Leu Gly Leu Phe435 440 445Cys Thr Asn Val Ala Phe Pro Pro Glu Ala Leu Arg Met Arg Ala Pro450 455 460Ala Asp Pro Ala Pro Ala Pro Ala Asp Pro Ala Ser Pro Gln His Gln465 470 475 480Pro Ala Gly Pro Ala Pro Leu Leu Ser Thr Pro Ala Pro Glu Ala Arg485 490 495Pro Val Ile Gly Ala Leu Gly Leu5007253PRTHomo sapiens 7Met Lys His Ile Asn Leu Ser Phe Ala Ala Cys Gly Phe Leu Gly Ile 1 5 10 15Tyr His Leu Gly Ala Ala Ser Ala Leu Cys Arg His Gly Lys Lys Leu20 25 30Val Lys Asp Val Lys Ala Phe Ala Gly Ala Ser Ala Gly Ser Leu Val35 40 45Ala Ser Val Leu Leu Thr Ala Pro Glu Lys Ile Glu Glu Cys Asn Gln50 55 60Phe Thr Tyr Lys Phe Ala Glu Glu Ile Arg Arg Gln Ser Phe Gly Ala65 70 75 80Val Thr Pro Gly Tyr Asp Phe Met Ala Arg Leu Arg Ser Gly Met Glu85 90 95Ser Ile Leu Pro Pro Ser Ala His Glu Leu Ala Gln Asn Arg Leu His100 105 110Val Ser Ile Thr Asn Ala Lys Thr Arg Glu Asn His Leu Val Ser Thr115 120 125Phe Ser Ser Arg Glu Asp Leu Ile Lys Val Leu Leu Ala Ser Ser Phe130 135 140Val Pro Ile Tyr Ala Gly Leu Lys Leu Val Glu Tyr Lys Gly Gln Lys145 150 155 160Trp Val Asp Gly Gly Leu Thr Asn Ala Leu Pro Ile Leu Pro Val Gly165 170 175Arg Thr Val Thr Ile Ser Pro Phe Ser Gly Arg Leu Asp Ile Ser Pro180 185 190Gln Asp Lys Gly Gln Leu Asp Leu Tyr Val Asn Ile Ala Lys Gln Asp195 200 205Ile Met Leu Ser Leu Ala Asn Leu Val Arg Leu Asn Gln Ala Leu Phe210 215 220Pro Pro Ser Lys Arg Lys Met Glu Ser Leu Tyr Gln Cys Gly Phe Asp225 230 235 240Asp Thr Val Lys Phe Leu Leu Lys Glu Asn Trp Phe Glu245 25081446DNAHomo sapiensCDS(1)..(1443) 8atg tac gac gca gag cgc ggc tgg agc ttg tcc ttc gcg ggc tgc ggc 48Met Tyr Asp Ala Glu Arg Gly Trp Ser Leu Ser Phe Ala Gly Cys Gly 1 5 10 15ttc ctg ggc ttc tac cac gtc ggg gcg acc cgc tgc ctg agc gag cac 96Phe Leu Gly Phe Tyr His Val Gly Ala Thr Arg Cys Leu Ser Glu His20 25 30gcc ccg cac ctc ctc cgc gac gcg cgc atg ttg ttc ggc gct tcg gcc 144Ala Pro His Leu Leu Arg Asp Ala Arg Met Leu Phe Gly Ala Ser Ala35 40 45ggg gcg ttg cac tgc gtc ggc gtc ctc tcc ggt atc ccg ctg gag cag 192Gly Ala Leu His Cys Val Gly Val Leu Ser Gly Ile Pro Leu Glu Gln50 55 60act ctg cag gtc ctc tca gat ctt gtg cgg aag gcc agg agt cgg aac 240Thr Leu Gln Val Leu Ser Asp Leu Val Arg Lys Ala Arg Ser Arg Asn65 70 75 80att ggc atc ttc cat cca tcc ttc aac tta agc aag ttc ctc cga cag 288Ile Gly Ile Phe His Pro Ser Phe Asn Leu Ser Lys Phe Leu Arg Gln85 90 95ggt ctc tgc aaa tgc ctc ccg gcc aat gtc cac cag ctc atc tcc ggc 336Gly Leu Cys Lys Cys Leu Pro Ala Asn Val His Gln Leu Ile Ser Gly100 105 110aaa ata ggc atc tct ctt acc aga gtg tct gat ggg gaa aac gtt ctg 384Lys Ile Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Leu115 120 125gtg tct gac ttt cgg tcc aaa gac gaa gtc gtg gat gcc ttg gta tgt 432Val Ser Asp Phe Arg Ser Lys Asp Glu Val Val Asp Ala Leu Val Cys130 135 140tcc tgc ttc atc ccc ttc tac agt ggc ctt atc cct cct tcc ttc aga 480Ser Cys Phe Ile Pro Phe Tyr Ser Gly Leu Ile Pro Pro Ser Phe Arg145 150 155 160ggc gtg cga tat gtg gat gga gga gtg agt gac aac gta ccc ttc att 528Gly Val Arg Tyr Val Asp Gly Gly Val Ser Asp Asn Val Pro Phe Ile165 170 175gat gcc aaa aca acc atc acc gtg tcc ccc ttc tat ggg gag tac gac 576Asp Ala Lys Thr Thr Ile Thr Val Ser Pro Phe Tyr Gly Glu Tyr Asp180 185 190atc tgc cct aaa gtc aag tcc acg aac ttt ctt cat gtg gac atc acc 624Ile Cys Pro Lys Val Lys Ser Thr Asn Phe Leu His Val Asp Ile Thr195 200 205aag ctc agt cta cgc ctc tgc aca ggg aac ctc tac ctt ctc tcg aga 672Lys Leu Ser Leu Arg Leu Cys Thr Gly Asn Leu Tyr Leu Leu Ser Arg210 215 220gct ttt gtc ccc ccg gat ctc aag gtg ctg gga gag ata tgc ctt cga 720Ala Phe Val Pro Pro Asp Leu Lys Val Leu Gly Glu Ile Cys Leu Arg225 230 235 240gga tat ttg gat gca ttc agg ttc ttg gaa gag aag ggc atc tgc aac 768Gly Tyr Leu Asp Ala Phe Arg Phe Leu Glu Glu Lys Gly Ile Cys Asn245 250 255agg ccc cag cca ggc ctg aag tca tcc tca gaa ggg atg gat cct gag 816Arg Pro Gln Pro Gly Leu Lys Ser Ser Ser Glu Gly Met Asp Pro Glu260 265 270gtc gcc atg ccc agc tgg gca aac atg agt ctg gat tct tcc ccg gag 864Val Ala Met Pro Ser Trp Ala Asn Met Ser Leu Asp Ser Ser Pro Glu275 280 285tcg gct gcc ttg gct gtg agg ctg gag gga gat gag ctg cta gac cac 912Ser Ala Ala Leu Ala Val Arg Leu Glu Gly Asp Glu Leu Leu Asp His290 295 300ctg cgt ctc agc atc ctg ccc tgg gat gag agc atc ctg gac acc ctc 960Leu Arg Leu Ser Ile Leu Pro Trp Asp Glu Ser Ile Leu Asp Thr Leu305 310 315 320tcg ccc agg ctc gct aca gca ctg agt gaa gaa atg aaa gac aaa ggt 1008Ser Pro Arg Leu Ala Thr Ala Leu Ser Glu Glu Met Lys Asp Lys Gly325 330 335gga tac atg agc aag att tgc aac ttg cta ccc att agg ata atg tct 1056Gly Tyr Met Ser Lys Ile Cys Asn Leu Leu Pro Ile Arg Ile Met Ser340 345 350tat gta atg ctg ccc tgt acc ctg cct gtg gaa tct gcc att gcg att 1104Tyr Val Met Leu Pro Cys Thr Leu Pro Val Glu Ser Ala Ile Ala Ile355 360 365gtc cag aga ctg gtg aca tgg ctt cca gat atg ccc gac gat gtc ctg 1152Val Gln Arg Leu Val Thr Trp Leu Pro Asp Met Pro Asp Asp Val Leu370 375 380tgg ttg cag tgg gtg acc tca cag gtg ttc act cga gtg ctg atg tgt 1200Trp Leu Gln Trp Val Thr Ser Gln Val Phe Thr Arg Val Leu Met Cys385 390 395 400ctg ctc ccc gcc tcc agg tcc caa atg cca gtg agc agc caa cag gcc 1248Leu Leu Pro Ala Ser Arg Ser Gln Met Pro Val Ser Ser Gln Gln Ala405 410 415tcc cca tgc aca cct gag cag gac tgg ccc tgc tgg act ccc tgc tcc 1296Ser Pro Cys Thr Pro Glu Gln Asp Trp Pro Cys Trp Thr Pro Cys Ser420 425 430ccc gag ggc tgt cca gca gag acc aaa gca gag gcc acc ccg cgg tcc 1344Pro Glu Gly Cys Pro Ala Glu Thr Lys Ala Glu Ala Thr Pro Arg Ser435 440 445atc ctc agg tcc agc ctg aac ttc ttc ttg ggc aat aaa gta cct gct 1392Ile Leu Arg Ser Ser Leu Asn Phe Phe Leu Gly Asn Lys Val Pro Ala450 455 460ggt gct gag ggg ctc tcc acc ttt ccc agt ttt tca cta gag aag agt 1440Gly Ala Glu Gly Leu Ser Thr Phe Pro Ser Phe Ser Leu Glu Lys Ser465 470 475 480ctg tga 1446Leu9481PRTHomo sapiens 9Met Tyr Asp Ala Glu Arg Gly Trp Ser Leu Ser Phe Ala Gly Cys Gly 1 5 10 15Phe Leu Gly Phe Tyr His Val Gly Ala Thr Arg Cys Leu Ser Glu His20 25 30Ala Pro His Leu Leu Arg Asp Ala Arg Met Leu Phe Gly Ala Ser Ala35 40 45Gly Ala Leu His Cys Val Gly Val Leu Ser Gly Ile Pro Leu Glu Gln50 55 60Thr Leu Gln Val Leu Ser Asp Leu Val Arg Lys Ala Arg Ser Arg Asn65 70 75 80Ile Gly Ile Phe His Pro Ser Phe Asn Leu Ser Lys Phe Leu Arg Gln85 90 95Gly Leu Cys Lys Cys Leu Pro Ala Asn Val His Gln Leu Ile Ser Gly100 105 110Lys Ile Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Leu115 120 125Val Ser Asp Phe Arg Ser Lys Asp Glu Val Val Asp Ala Leu Val Cys130 135 140Ser Cys Phe Ile Pro Phe Tyr Ser Gly Leu Ile Pro Pro Ser Phe Arg145 150 155 160Gly Val Arg Tyr Val Asp Gly Gly Val Ser Asp Asn Val Pro Phe Ile165 170 175Asp Ala Lys Thr Thr Ile Thr Val Ser Pro Phe Tyr Gly Glu Tyr Asp180 185 190Ile Cys Pro Lys Val Lys Ser Thr Asn Phe Leu His Val Asp Ile Thr195 200 205Lys Leu Ser Leu Arg Leu Cys Thr Gly Asn Leu Tyr Leu Leu Ser Arg210 215 220Ala Phe Val Pro Pro Asp Leu Lys Val Leu Gly Glu Ile Cys Leu Arg225 230 235 240Gly Tyr Leu Asp Ala Phe Arg Phe Leu Glu Glu Lys Gly Ile Cys Asn245 250 255Arg Pro Gln Pro Gly Leu Lys Ser Ser Ser Glu Gly Met Asp Pro Glu260 265 270Val Ala Met Pro Ser Trp Ala Asn Met Ser Leu Asp Ser Ser Pro Glu275 280 285Ser Ala Ala Leu Ala Val Arg Leu Glu Gly Asp Glu Leu Leu Asp His290 295 300Leu Arg Leu Ser Ile Leu Pro Trp Asp Glu Ser Ile Leu Asp Thr Leu305 310 315 320Ser Pro Arg Leu Ala Thr Ala Leu Ser Glu Glu Met Lys Asp Lys Gly325 330 335Gly Tyr Met Ser Lys Ile Cys Asn Leu Leu Pro Ile Arg Ile Met Ser340 345 350Tyr Val Met Leu Pro Cys Thr Leu Pro Val Glu Ser Ala Ile Ala Ile355 360 365Val Gln Arg Leu Val Thr Trp Leu Pro Asp Met Pro Asp Asp Val Leu370 375 380Trp Leu Gln Trp Val Thr Ser Gln Val Phe Thr Arg Val Leu Met Cys385 390 395 400Leu Leu Pro Ala Ser Arg Ser Gln Met Pro Val Ser Ser Gln Gln Ala405 410 415Ser Pro Cys Thr Pro Glu Gln Asp Trp Pro Cys Trp Thr Pro Cys Ser420 425 430Pro Glu Gly Cys Pro Ala Glu Thr Lys Ala Glu Ala Thr Pro Arg Ser435 440 445Ile Leu Arg Ser Ser Leu Asn Phe Phe Leu Gly Asn Lys Val Pro Ala450 455 460Gly Ala Glu Gly Leu Ser Thr Phe Pro Ser Phe Ser Leu Glu Lys Ser465 470 475 480Leu101446DNAHomo sapiensCDS(1)..(1443) 10atg tac gac gca gag cgc ggc tgg agc ttg tcc ttc gcg ggc tgc ggc 48Met Tyr Asp Ala Glu Arg Gly Trp Ser Leu Ser Phe Ala Gly Cys Gly 1 5 10 15ttc ctg ggc ttc tac cac gtc ggg gcg acc cgc tgc ctg agc gag cac 96Phe Leu Gly Phe Tyr His Val Gly Ala Thr Arg Cys Leu Ser Glu His20 25 30gcc ccg cac ctc ctc cgc gac gcg cgc atg ttg ttc ggc gct tcg gcc 144Ala Pro His Leu Leu Arg Asp Ala Arg Met Leu Phe Gly Ala Ser Ala35 40 45ggg gcg ttg cac tgc gtc ggc gtc ctc tcc ggt atc ccg ctg gag cag 192Gly Ala Leu His Cys Val Gly Val Leu Ser Gly Ile Pro Leu Glu Gln50 55 60act ctg cag gtc ctc tca gat ctt gtg cgg aag gcc agg agt cgg aac 240Thr Leu Gln Val Leu Ser Asp Leu Val Arg Lys Ala Arg Ser Arg Asn65 70 75 80att ggc atc ttc cat cca tcc ttc aac tta agc aag ttc ctc cga cag 288Ile Gly Ile Phe His Pro Ser Phe Asn Leu Ser Lys Phe Leu Arg Gln85 90 95ggt ctc tgc aaa tgc ctc ccg gcc aat gtc cac cag ctc atc tcc ggc 336Gly Leu Cys Lys Cys Leu Pro Ala Asn Val His Gln Leu Ile Ser Gly100 105 110aaa ata ggc atc tct ctt acc aga gtg tct gat ggg gaa aac gtt ctg 384Lys Ile Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Leu115 120 125gtg tct gac ttt cgg tcc aaa gac gaa gtc gtg gat gcc ttg gta tgt 432Val Ser Asp Phe Arg Ser Lys Asp Glu Val Val Asp Ala Leu Val Cys130 135 140tcc tgc ttc atc ccc ttc tac agt ggc ctt atc cct cct tcc ttc aga 480Ser Cys Phe Ile Pro Phe Tyr Ser Gly Leu Ile Pro Pro Ser Phe Arg145 150 155 160ggc gtg cga tat gtg gat gga gga gtg agt gac aac gta ccc ttc att 528Gly Val Arg Tyr Val Asp Gly Gly Val Ser Asp Asn Val Pro Phe Ile165 170 175gat gcc aaa aca acc atc acc gtg tcc ccc ttc tat ggg gag tac gac 576Asp Ala Lys Thr Thr Ile Thr Val Ser Pro Phe Tyr Gly Glu Tyr Asp180 185 190atc tgc cct aaa gtc aag tcc acg aac ttt ctt cat gtg gac atc acc 624Ile Cys Pro Lys Val Lys Ser Thr Asn Phe Leu His Val Asp Ile Thr195 200 205aag ctc agt cta cgc ctc tgc aca ggg aac ctc tac ctt ctc tcg aga 672Lys Leu Ser Leu Arg Leu Cys Thr Gly Asn Leu Tyr Leu Leu Ser Arg210 215 220gct ttt gtc ccc ccg gat ctc aag gtg ctg gga gag ata tgc ctt cga 720Ala Phe Val Pro Pro Asp Leu Lys Val Leu Gly Glu Ile Cys Leu Arg225 230 235 240gga tat ttg gat gca ttc agg ttc ttg gaa gag aag ggc atc tgc aac 768Gly Tyr Leu Asp Ala Phe Arg Phe Leu Glu Glu Lys Gly Ile Cys Asn245 250 255agg ccc cag cca ggc ctg aag tca tcc tca gaa ggg atg gat cct gag 816Arg Pro Gln Pro Gly Leu Lys Ser Ser Ser Glu Gly Met Asp Pro Glu260 265 270gtc gcc atg ccc agc tgg gca aac atg agt ctg gat tct tcc ccg gag 864Val Ala Met Pro Ser Trp Ala Asn Met Ser Leu Asp Ser Ser Pro Glu275 280 285tcg gct gcc ttg gct gtg agg ctg gag gga gat gag ctg cta gac cac 912Ser Ala Ala Leu Ala Val Arg Leu Glu Gly Asp Glu Leu Leu Asp His290 295 300ctg cgt ctc agc atc ctg ccc tgg gat gag agc atc ctg gac acc ctc 960Leu Arg Leu Ser Ile Leu Pro Trp Asp Glu Ser Ile Leu Asp Thr Leu305 310 315 320tcg ccc agg ctc gct aca gca ctg agt gaa gaa atg aaa gac aaa ggt 1008Ser Pro Arg Leu Ala Thr Ala Leu Ser Glu Glu Met Lys Asp Lys Gly325 330 335gga tac atg agc aag att tgc aac ttg cta ccc att agg ata atg tct 1056Gly Tyr Met Ser Lys Ile Cys Asn Leu Leu Pro Ile Arg Ile Met Ser340 345 350tat gta atg ctg ccc tgt acc ctg cct gtg gaa tct gcc att gcg att 1104Tyr Val Met Leu Pro Cys Thr Leu Pro Val Glu Ser Ala Ile Ala Ile355 360 365gtc cag aga ctg gtg aca tgg ctt cca gat atg ccc gac gat gtc ctg 1152Val Gln Arg Leu Val Thr Trp Leu Pro Asp Met Pro Asp Asp Val Leu370 375 380tgg ttg cag tgg gtg acc tca cag gtg ttc act cga gtg ctg atg tgt 1200Trp Leu Gln Trp Val Thr Ser Gln Val Phe Thr Arg Val Leu Met Cys385 390 395 400ctg ctc ccc gcc tcc agg tcc caa atg cca gtg agc agc caa cag gcc 1248Leu Leu Pro Ala Ser Arg Ser Gln Met Pro Val Ser Ser Gln Gln Ala405 410 415tcc cca tgc aca cct gag cag gac tgg ccc tgc tgg act ccc tgc tcc 1296Ser Pro Cys Thr Pro Glu Gln Asp Trp Pro Cys Trp Thr Pro Cys Ser420 425 430ccc

aag ggc tgt cca gca gag acc aaa gca gag gcc acc ccg cgg tcc 1344Pro Lys Gly Cys Pro Ala Glu Thr Lys Ala Glu Ala Thr Pro Arg Ser435 440 445atc ctc agg tcc agc ctg aac ttc ttc ttg ggc aat aaa gta cct gct 1392Ile Leu Arg Ser Ser Leu Asn Phe Phe Leu Gly Asn Lys Val Pro Ala450 455 460ggt gct gag ggg ctc tcc acc ttt ccc agt ttt tca cta gag aag agt 1440Gly Ala Glu Gly Leu Ser Thr Phe Pro Ser Phe Ser Leu Glu Lys Ser465 470 475 480ctg tga 1446Leu11481PRTHomo sapiens 11Met Tyr Asp Ala Glu Arg Gly Trp Ser Leu Ser Phe Ala Gly Cys Gly 1 5 10 15Phe Leu Gly Phe Tyr His Val Gly Ala Thr Arg Cys Leu Ser Glu His20 25 30Ala Pro His Leu Leu Arg Asp Ala Arg Met Leu Phe Gly Ala Ser Ala35 40 45Gly Ala Leu His Cys Val Gly Val Leu Ser Gly Ile Pro Leu Glu Gln50 55 60Thr Leu Gln Val Leu Ser Asp Leu Val Arg Lys Ala Arg Ser Arg Asn65 70 75 80Ile Gly Ile Phe His Pro Ser Phe Asn Leu Ser Lys Phe Leu Arg Gln85 90 95Gly Leu Cys Lys Cys Leu Pro Ala Asn Val His Gln Leu Ile Ser Gly100 105 110Lys Ile Gly Ile Ser Leu Thr Arg Val Ser Asp Gly Glu Asn Val Leu115 120 125Val Ser Asp Phe Arg Ser Lys Asp Glu Val Val Asp Ala Leu Val Cys130 135 140Ser Cys Phe Ile Pro Phe Tyr Ser Gly Leu Ile Pro Pro Ser Phe Arg145 150 155 160Gly Val Arg Tyr Val Asp Gly Gly Val Ser Asp Asn Val Pro Phe Ile165 170 175Asp Ala Lys Thr Thr Ile Thr Val Ser Pro Phe Tyr Gly Glu Tyr Asp180 185 190Ile Cys Pro Lys Val Lys Ser Thr Asn Phe Leu His Val Asp Ile Thr195 200 205Lys Leu Ser Leu Arg Leu Cys Thr Gly Asn Leu Tyr Leu Leu Ser Arg210 215 220Ala Phe Val Pro Pro Asp Leu Lys Val Leu Gly Glu Ile Cys Leu Arg225 230 235 240Gly Tyr Leu Asp Ala Phe Arg Phe Leu Glu Glu Lys Gly Ile Cys Asn245 250 255Arg Pro Gln Pro Gly Leu Lys Ser Ser Ser Glu Gly Met Asp Pro Glu260 265 270Val Ala Met Pro Ser Trp Ala Asn Met Ser Leu Asp Ser Ser Pro Glu275 280 285Ser Ala Ala Leu Ala Val Arg Leu Glu Gly Asp Glu Leu Leu Asp His290 295 300Leu Arg Leu Ser Ile Leu Pro Trp Asp Glu Ser Ile Leu Asp Thr Leu305 310 315 320Ser Pro Arg Leu Ala Thr Ala Leu Ser Glu Glu Met Lys Asp Lys Gly325 330 335Gly Tyr Met Ser Lys Ile Cys Asn Leu Leu Pro Ile Arg Ile Met Ser340 345 350Tyr Val Met Leu Pro Cys Thr Leu Pro Val Glu Ser Ala Ile Ala Ile355 360 365Val Gln Arg Leu Val Thr Trp Leu Pro Asp Met Pro Asp Asp Val Leu370 375 380Trp Leu Gln Trp Val Thr Ser Gln Val Phe Thr Arg Val Leu Met Cys385 390 395 400Leu Leu Pro Ala Ser Arg Ser Gln Met Pro Val Ser Ser Gln Gln Ala405 410 415Ser Pro Cys Thr Pro Glu Gln Asp Trp Pro Cys Trp Thr Pro Cys Ser420 425 430Pro Lys Gly Cys Pro Ala Glu Thr Lys Ala Glu Ala Thr Pro Arg Ser435 440 445Ile Leu Arg Ser Ser Leu Asn Phe Phe Leu Gly Asn Lys Val Pro Ala450 455 460Gly Ala Glu Gly Leu Ser Thr Phe Pro Ser Phe Ser Leu Glu Lys Ser465 470 475 480Leu126PRTHomo sapiens 12Gly Cys Gly Phe Leu Gly1 5135PRTHomo sapiens 13Gly Ala Ser Ala Gly1 51424DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 14ggcaaaatag gcatctctct tacc 241522DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 15ggagggataa ggccactgta ga 221628DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 16aacataccaa ggcatccacg acttcgtc 281716DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17ccacggcgct ggtcac 161821DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 18gcaggacctt cagcaggaaa c 211925DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 19tggcaccagc ctcacccagg cagac 252026DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 20gcacagaaaa tgaggattat taaagg 262122DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 21cgctgcaaat gataggttga tg 222232DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 22tgcttcattc tagctgtagc actgcgagca ac 322318DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 23actgcacgcg gtcacctt 182421DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 24cacgaggtcc atgaggatct c 212528DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 25tgtgcagtct ccctctcggc cgtataat 282618DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 26gccacagcgc tggtcact 182723DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 27cctccttgga cacctcaata atg 232822DNAArtificial SequenceDescription of Artificial Sequence Synthetic probe 28cctgcctggg tgaagcaggt gc 22296PRTArtificial SequenceDescription of Artificial Sequence Synthetic 6xHis tag 29His His His His His His1 53012PRTArtificial SequenceDescription of Artificial Sequence Synthetic His tag 30His His His His His His His His His His His His1 5 10

Claims

1. An isolated, purified, and characterized human calcium-independent phospholipase A₂ (iPLA₂) protein comprising a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 1.

2. The protein of claim 1 wherein the polypeptide has a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:11.

3. The protein of claim 2 wherein the polypeptide has a histidine tag.

4. The protein of claim 3 wherein the histidine tag comprises from 4 to 12 histidine residues.

5. The protein of claim 1 wherein the polypeptide is an iPLA₂epsilon polypeptide having a sequence shown in SEQ ID NO: 1.

6. The protein of claim 1 wherein the polypeptide is an iPLA₂zeta polypeptide having a sequence shown in SEQ ID NO: 2.

7. The protein of claim 1 wherein the polypeptide is an iPLA₂eta polypeptide having a sequence shown in SEQ ID NO: 3.

8. An isolated and characterized human gene comprising a calcium-independent phospholipase A₂ (iPLA₂) polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 8, and SEQ ID NO: 10.

9. The gene of claim 8 wherein the polynucleotide is an iPLA₂zeta polynucleotide having a sequence shown in SEQ ID NO: 4.

10. The gene of claim 8 wherein the polynucleotide is an iPLA₂eta polynucleotide having a sequence shown in SEQ ID NO: 5.

11. The gene of claim 8 wherein the polynucleotide is an iPLA₂epsilon polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10.

12. A genetically engineered expression vector comprising the gene of claim 8.

13. A genetically engineered cell capable of identifying substances which modulate iPLA₂zeta expression, the genetically engineered cell comprising the gene of claim 9.

14. The genetically engineered cell of claim 13 further comprising a promoter operably linked to the gene of claim 9 and a reporter gene.

15. The genetically engineered cell of claim 13 wherein the cell is an Sf9 cell.

16. A genetically engineered cell capable of identifying substances which modulate iPLA₂eta expression in a living cell, the genetically engineered cell comprising the gene of claim 10.

17. The genetically engineered cell of claim 16 further comprising a promoter operably linked to the gene of claim 10 and a reporter gene.

18. The genetically engineered cell of claim 16 wherein the cell is an Sf9 cell.

19. A genetically engineered cell capable of identifying substances which modulate iPLA₂epsilon expression in a living cell, the genetically engineered cell comprising the gene of claim 11.

20. The genetically engineered cell of claim 19 wherein the cell is an Sf9 cell.

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