Patent application title: TREATMENT OF MUSCULAR DYSTROPHIES AND RELATED DISORDERS
Inventors:
Justin R. Fallon (Providence, RI, US)
Michael Rafii (San Diego, CA, US)
Mark A. Bowe (Damascus, MD, US)
Beth A. Mckechnie (North Attleboro, MA, US)
Alison R. Amenta (Pawtucket, RI, US)
Mary Lynn Mercado (Robbinsville, NJ, US)
Hiroki Hagiwara (Tokyo, JP)
Hiroki Hagiwara (Tokyo, JP)
IPC8 Class: AA61K3817FI
USPC Class:
Class name:
Publication date: 2015-07-02
Patent application number: 20150182589
Abstract:
The invention provides, among other aspects, compositions and methods for
treating, preventing, and diagnosing diseases or conditions associated
with an abnormal level or activity of biglycan; diseases or conditions
associated with an abnormal level or activity of collagen VI; disorders
associated with an unstable cytoplasmic membrane, due, e.g., to an
unstable dystrophin associated protein complex (DAPC); and disorders
associated with abnormal synapses or neuromuscular junctions, including
those resulting from an abnormal MuSK activation or acetylcholine
receptor (AChR) aggregation.Claims:
1. A method for stabilizing collagen VI-deficient dystrophin-associated
protein complexes (DAPCs) on the surface of a cell, comprising contacting
the cell with an effective amount of a biglycan therapeutic, such that
the collagen VI-deficient DAPCs are stabilized.
2. The method of claim 1, wherein the biglycan therapeutic is a polypeptide including a biglycan amino acid sequence which is at least about 90% identical to SEQ ID No. 9, or a portion thereof.
3. The method of claim 2, wherein the biglycan therapeutic binds to MuSK.
4. The method of claim 2, wherein the biglycan therapeutic binds to a α-sarcoglycan and/or γ-sarcoglycan.
5. The method of claim 2, wherein the biglycan therapeutic binds to a collagen VI polypeptide.
6. The method of claim 2, wherein the biglycan therapeutic induces phosphorylation of sarcoglycans.
7. The method of claim 2, wherein the biglycan therapeutic upregulates utrophin levels.
8. The method of claim 2, wherein the biglycan amino acid sequence includes one or more LLRs of human biglycan having SEQ ID NO: 9.
9. The method of claim 2, wherein the polypeptide is derivatized with one or more glycosaminoglycan (GAG) side chains.
10. The method of claim 2, wherein the biglycan amino acid sequence is at least about 90% identical to amino acids 38-365 of SEQ ID NO: 9.
11. The method of claim 2, wherein the biglycan amino acid sequence is at least about 95% identical to amino acids 38-365 of SEQ ID NO: 9.
12. The method of claim 2, wherein the biglycan amino acid sequence is encoded by a nucleic acid which hybridizes to SEQ ID NO: 8.
13. The method of claim 1, wherein the cell is a muscle cell.
14. A method for treating or preventing a condition associated with a collagen VI deficiency, comprising administering to the subject a pharmaceutically effective amount of biglycan therapeutic.
15-21. (canceled)
22. A method for treating or preventing a condition associated with an abnormal dystrophin-associated complex (DAPC) in cells of a subject, comprising administering a pharmaceutically effective amount of a collagen VI therapeutic.
23-28. (canceled)
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent application Ser. No. 12/786,294 filed May 24, 2010 and now U.S. Pat. No. 8,822,418, which is a continuation of U.S. patent application Ser. No. 10/486,678 filed Sep. 1, 2004 and now U.S. Pat. No. 7,759,314, which is a national stage filing under 35 U.S.C. §371 of International Application No. PCT/US2002/026201, filed Aug. 15, 2002, which claims the benefit of U.S. Provisional Application No. 60/312,551, filed Aug. 15, 2001. The specification of each of these applications is hereby incorporated by reference in their entirety. International Application No. PCT/US2002/026201 was published under PCT Article 21(2) in English.
BACKGROUND OF THE INVENTION
[0003] The dystrophin-associated protein complex (DAPC) links the cytoskeleton to the extracellular matrix and is necessary for maintaining the integrity of the muscle cell\plasma membrane. The core DAPC consists of the cytoskeletal scaffolding molecule dystrophin and the dystroglycan and sarcoglycan transmembrane subcomplexes. The DAPC also serves to localize key signaling molecules to the cell surface, at least in part through its associated syntrophins (Brenman, et al. (1996) Cell. 84: 757-767; Bredt, et al. (1998), Proc Natl Acad Sci USA. 95: 14592). Mutations in either dystrophin or any of the sarcoglycans result in muscular dystrophies characterized by breakdown of the muscle cell membrane, loss of myofibers, and fibrosis (Hoffman, et al. 1987. Cell. 51: 919; Straub, and Campbell (1997) Curr Opin Neurol. 10: 168). Moreover, mutations in the extracellular matrix protein laminin-α2, which associates with the DAPC on the cell surface, is the basis of a major congenital muscular dystrophy (Helbling-Leclerc, et al. (1995) Nat Genet. 11: 216).
[0004] The α-/β-dystroglycan subcomplex forms a critical structural link in the DAPC. The transmembrane β-dystroglycan and the wholly extracellular α-dystroglycan arise by proteolytic cleavage of a common precursor (Ibraghimov, et al. (1992) Nature 355: 696; Bowe, et al. (1994) Neuron 12: 1173). The cytoplasmic tail of β-dystroglycan binds dystrophin, while the highly glycosylated, mucin-like α-dystroglycan binds to several ECM elements including agrin, laminin, and perlecan (Ervasti and Campbell, (1993) J Cell Biol. 122: 809; Bowe, et al. (1994) Neuron. 12: 1173; Gee, et al. (1994) Cell 77: 675; Hemler, (1999) Cell 97: 543). This binding to matrix proteins appears to be essential for assembly of basal lamina, since mice deficient in dystroglycan fail to form these structures and die very early in development (Henry, M. D. and K. P. Campbell. 1998. Cell. 95: 859). β-Dystroglycan can bind the signaling adapter molecule Grb2 and associates indirectly with p125FAK (Yang, et al. (1995) J. Biol. Chem. 270: 11711; Cavaldesi, et al. (1999), J. Neurochem. 72: 01648). Although the significance of these associations remains unknown, these binding properties suggest that dystroglycan may also serve to localize signaling molecules to the cell surface.
[0005] Several lines of evidence suggest that dystroglycan may also function in neuromuscular junction formation, in particular, in postsynaptic differentiation. For purposes of clarity, the components of the neuromuscular junction are summarized here. The major structural features of the neuromuscular junction (NMJ) or nerve-muscle synapse are the pre- and post-synaptic specializations of the motor neuron and muscle, respectively, the intervening synaptic basal lamina, and the specialized Schwann cell cap (Salpeter, et al (1987) The Vertebrate Neuromuscular Junction. New York, Alan R. Liss.). The presynaptic apparatus is marked by ordered arrays of synaptic vesicles, a subset of which are poised to fuse with the plasma membrane at the active zones, and release acethylcholine that is recognized by acetylcholine receptors (AChRs) on the muscle, and ultimately results in electrical activation and contraction of the muscle (Heuser, et al (1981) J. Cell Biol. 88: 564) Immediately across the 50 nm synaptic cleft from these zones are the crests of the postjunctional folds. These crests bristle with Acetylcholine receptors (AChRs), which can reach densities of >10,000 molecules/μm2 (Fertuck, et al (1976) J. Cell. Biol. 69: 144). The localized and tightly regulated secretion of acetylcholine into the narrow synaptic cleft, coupled with the high AChR density in the postsynaptic membrane, ensures rapid and reliable synaptic transmission between neuron and muscle. Perturbations of these specializations, such as the decrease in the number of functional AChRs seen in myasthenia gravis, can lead to debilitating and often fatal clinical outcomes (Oosterhuis, et al (1992) Neurology & Neurosurgery 5: 638).
[0006] The synaptic basal lamina (SBL) is interposed between the pre- and post-synaptic membranes and contains molecules important for the structure, function, and regulation of the neuromuscular junction (Bowe, M. A & Fallon, J. R., (1995) Ann. Rev. Neurosci. 18: 443; Sanes, et al. (1999) Ann. Rev. Neurosci. 22: 389). It consists of a distinct set of extracellular matrix molecules including specialized laminins, proteoglycans and collagens (Hall, et al (1993) Neuron 10: (Suppl.) 99). The SBL also contains molecules essential for the regulation of synaptic structure and function including AChE, neuregulins, and agrin. The SBL thus serves both as a specialized structure for maintaining the localized differentiation of the synapse as well as a repository for essential regulatory molecules.
[0007] The molecular composition of the postsynaptic membrane is known in considerable detail. As noted above, the most abundant membrane protein is the AChR. The cytosolic AChR associated protein rapsyn (formerly known as the 43 kD protein) is present at stoichiometric levels with the receptor and is likely to form a key link between the cytosolic domain of the AChR and the cytoskeleton (Froehner, et al (1995) Nature 377: 195; Gautam, et al. (1995) Nature 377: 232). The postsynaptic membrane is also enriched in erbB2-4, some or all of which serve as neuregulin receptors (Altiok, et al. (1995) EMBO J. 14: 4258; Zhu, et al. (1995) EMBO J. 14: 5842). AChR and other molecules essential for nerve-muscle communication. The cytoskeletal elements can be broadly grouped into two subsets. Dystrophin and utrophin are members of the dystrophin-associated protein complex, or DAPC, and are linked to the synaptic basal lamina via the transmembrane heteromer α-/β-dystroglycan. The postsynaptic cytoskeleton is also enriched in several focal adhesion-associated molecules including α-actinin, vinculin, talin, paxillin, and filamin (Sanes, et al (1999) Ann. Rev. Neurosci. 22: 389). The latter proteins probably communicate, directly or indirectly, with the extracellular matrix through integrins, some of which are enriched at synapses (Martin, et al. (1996) Dev. Biol. 174: 125). Actin is associated with both sets of cytoskeletal molecules (Rybakova et al. (1996) J. Cell Biol. 135: 661; Amann, et al. (1998) J. Biol. Chem. 273: 28419-23; Schoenwaelder et al. (1999) Curr. Opin. Cell. Biol. 11: 274). The functions of these specialized sets of proteins are considered below.
[0008] α-Dystroglycan binds the synapse organizing molecule agrin (Bowe, et al. (1994) Neuron. 12: 1173; Campanelli, et al. (1994) Cell. 77: 663; Gee, et al. (1994) Cell. 77: 675; Sugiyama, et al. (1994) Neuron. 13: 103; O'Toole, et al. (1996) Proc Natl Acad Sci USA. 93: 7369) (reviewed in Fallon and Hall, (1994) Trends Neurosci. 17: 469), and β-dystroglycan binds to the AChR-associated protein rapsyn (Cartaud, et al. (1998) J Biol Chem. 273: 11321). Further, agrin-induced AChR clustering on the postsynaptic membrane is markedly decreased in muscle cells expressing reduced levels of dystroglycan (Montanaro, et al. (1998) J Neurosci. 18: 1250). The precise role of dystroglycan in this process is unknown. Currently available evidence suggests that dystroglycan is not part of the primary agrin receptor, but rather may play a structural role in the organization of postsynaptic specializations (Gesemann, et al. (1995) Biol. 128: 625; Glass, et al. (1996) Cell. 85: 513; Jacobson, et al. (1998) J Neurosci. 18: 6340).
[0009] Another molecule that plays an important role in neuromuscular junction formation is the tyrosine kinase receptor MuSK, which becomes phosphorylated in response to agrin. However, agrin does not bind to MuSK and it is unclear how agrin stimulates MuSK. The existence of a co-receptor had been suggested. Activation of MuSK by antibody cross-linking is sufficient to induce the clustering of AChRs on cultured myotubes (Xie et al. (1997) Nat. Biotechnol. 15:768 and Hopf and Hoch (1998) J Biol. Chem. 273: 6467) and a constitutively active MuSK can induce postsynaptic differentiation in vivo (Jones et al. (1999) J. Neurosci. 19:3376). However, MuSK phosphorylation is necessary but not sufficient for agrin-induced AChR clustering.
[0010] The realm of dystroglycan function ranges far beyond muscle. As noted above, mice defective in dystroglycan die long before muscle differentiation. In a surprising development, α-dystroglycan in non-muscle cells has been shown to function as a receptor for Lassa Fever and choriomeningitis fever viruses (Cao, W., et al., 1998, Science. 282: 2079), and on Schwann cells as a co-receptor for Mycobacterium leprae (Rambukkana, et al. (1998) Science. 282: 2076). Dystroglycan is also abundant in brain, but its function there is not understood (Gorecki, et al. (1994) Hum Mol Genet. 3: 1589; Smalheiser and Kim (1995) J Biol Chem. 270: 15425).
[0011] α-Dystroglycan is comprised of three known domains. An amino-terminal domain folds into an autonomous globular configuration (Brancaccio, et al. (1995) Febs Lett. 368: 139). The middle third of the protein is serine- and threonine-rich, and is highly glycosylated (Brancaccio, et al. (1997) Eur J Biochem. 246: 166). Indeed, the core molecular weight of α-dystroglycan is ˜68 kDa, but the native molecule migrates on SDS-PAGE as a polydisperse band whose size ranges from 120-190 kDa, depending upon the species and tissue source (Ervasti and Campbell (1993) J Cell Biol. 122: 809; Bowe, et al. (1994) Neuron. 12: 1173; Gee, et al. (1994) Cell. 77: 675; Matsumura, et al. (1997) J Biol Chem. 272: 13904). Glycosylation of α-dystroglycan, probably in this middle third, is essential for its laminin- and agrin-binding properties.
[0012] While it is clear that dystroglycan and the DAPC play crucial roles in a variety of processes in muscle as well as in other tissues, the underlying mechanisms remain obscure.
SUMMARY OF THE INVENTION
[0013] In certain aspects, the invention provides methods and compositions for stabilizing dystrophin-associated protein complexes (DAPCs) on the surface of a cell. Stabilizing DAPC complexes on cell membranes allows membranes to be less "leaky" and thus, provides a longer life span to cells. In certain aspects, the invention also provides methods for activating a postynaptic membrane, such as to render the membrane more sensitive to an incoming signal from a neural cell (e.g., at a neuromuscular junction). Activating a postsynaptic membrane may comprise stimulating aggregation of AChR on the cell membrane and/or activating MuSK, such as by phosphorylation. In certain aspects, the invention provides methods for treating a condition associated with a collagen VI abnormality, such as a deficiency or structural disorganization.
[0014] In one embodiment, the method comprises contacting the target cell with a biglycan polypeptide comprising an amino acid sequence which is at least about 90% identical to the biglycan sequence of SEQ ID NO: 9 or a portion thereof. In a preferred method, the biglycan polypeptide binds to α-dystroglycan; collagen VI; α-sarcoglycan and/or γ-sarcoglycan. In an even more preferred embodiment, the biglycan polypeptide stimulates phosphorylation of α-sarcoglycan on a cell membrane. The biglycan polypeptide also preferably potentiates agrin-induced AChR aggregation on the surface of the cell; stimulate the phosphorylation of MuSK on the cell; and/or potentiates agrin-induced phosphorylation of MuSK. In certain preferred embodiments, the biglycan polypeptide interacts with and/or stimulates the expression of collagen VI.
[0015] The biglycan polypeptide may comprise one or more 24 amino acid repeat motifs in the Leucine Rich Repeat (LRR) of human biglycan having SEQ ID NO: 9. In another embodiment, the biglycan polypeptide comprises a cysteine-rich region, e.g., the C-terminal or the N-terminal Cysteine-rich region. The biglycan polypeptide may include one or more glycosaminoglycan (GAG) chains. In an even more preferred embodiment, the biglycan polypeptide comprises an amino acid sequence which is at least about 90% identical to amino acids 20-368 or 38-368 of SEQ ID NO: 9, even more preferably at least 95% identical or 100% identical to amino acids 20-368 or 38-368 of SEQ ID NO: 9. In another embodiment, the biglycan polypeptide is encoded by a nucleic acid which hybridizes to SEQ ID NO: 8. The biglycan polypeptide can be Torpedo DAG-125, or the human biglycan of SEQ ID NO: 9, or a portion thereof having at least one biological activity of biglycan.
[0016] In other embodiments, the biglycan therapeutic is a peptide fragment of the full length protein. Preferably it is a fragment which retains the ability to induce phosphorylation of sarcoglycans and upregulate utrophin activity/expression. For instance, a preferred peptide fragment binds to and activates MuSK. In certain preferred embodiments the peptide fragment has the ability to upregulate collagen VI activity/expression.
[0017] In further embodiments, the method comprises contacting the target cell with a collagen VI polypeptide comprising an amino acid sequence which is at least about 90% identical to a collagen α1(VI) sequence, a collagen α2(VI) sequence or a collagen α3(VI) sequence, exemplified by SEQ ID Nos: 11 and 12, 13 and 14, and 15 and 16, respectively, or a portion thereof. In a preferred method the collagen VI polypeptide is a portion of a mature collagen peptide (e.g. signal sequence is removed). In a preferred method, the collagen VI polypeptide binds to bigycan. In certain embodiments, the method comprises contacting the target cell with a collagen VI therapeutic comprising a collagen VI monomer, the monomer comprising a collagen α1(VI) chain, a collagen α2(VI) chain and a collagen α3(VI) chain in a 1:1:1 ratio. Optionally, the therapeutic comprises multimers of collagen VI monomers.
[0018] In other embodiments, the collagen VI therapeutic is a peptide fragment of a full length collagen VI α1(VI) chain α2(VI) chain or α3(VI) chain. Preferably it is a fragment which retains the ability to bind biglycan.
[0019] In other embodiments, the subject biglycan or collagen VI therapeutics are peptidomimetics of a portion of a biglycan or collagen VI protein, respectively. Peptidomimetics are compounds based on, or derived from, peptides and proteins. The peptidomimetics of the present invention typically can be obtained by structural modification of a known biglycan or collagen VI peptide sequence using unnatural amino acids, conformational restraints, isosteric replacement, and the like. The subject peptidomimetics constitute the continum of structural space between peptides and nonpeptide synthetic structures; biglycan and collagen VI peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent biglycan or collagen VI peptides.
[0020] Moreover, as is apparent from the present disclosure, mimetopes of the subject biglycan and collagen VI peptides can be provided. Such peptidomimetics can have such attributes as being non-hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the corresponding peptide), increased specificity and/or potency, and increased cell permeability for intracellular localization of the peptidomimetic. For illustrative purposes, peptide analogs of the present invention can be generated using, for example, benzodiazepines (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p123), C-7 mimics (Huffman et al. in Peptides: Chemistry and Biologyy, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105), keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71), diaminoketones (Natarajan et al. (1984) Biochem Biophys Res Commun 124:141), and methyleneamino-modifed (Roark et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p134). Also, see generally, Session III: Analytic and synthetic methods, in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988)
[0021] In addition to a variety of sidechain replacements which can be carried out to generate the subject biglycan and collagen VI peptidomimetics, the present invention specifically contemplates the use of conformationally restrained mimics of peptide secondary structure. Numerous surrogates have been developed for the amide bond of peptides. Frequently exploited surrogates for the amide bond include the following groups (i) trans-olefins, (ii) fluoroalkene, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.
##STR00001##
Examples of Surrogates
##STR00002##
[0023] Additionally, peptidomimietics based on more substantial modifications of the backbone of the biglycan or collagen VI peptide can be used. Peptidomimetics which fall in this category include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called peptoids).
##STR00003##
Examples of Analogs
##STR00004##
[0024] Furthermore, the methods of combinatorial chemistry are being brought to bear, c.f. Verdine et al. PCT publication WO9948897, on the development of new peptidomimetics. For example, one embodiment of a so-called "peptide morphing" strategy focuses on the random generation of a library of peptide analogs that comprise a wide range of peptide bond substitutes.
##STR00005##
[0025] In certain embodiments, the invention also provides a method for treating or preventing a condition associated with an abnormal dystrophin-associated protein complex (DAPC) in cells of a subject, comprising administering to the subject a pharmaceutically efficient amount of a biglycan polypeptide, peptide or peptidomimetic or a biglycan agonist (collectively referred to herein as "biglycan therapeutics") which stabilizes the DAPC. In certain embodiments, the invention provides a method for treating or preventing a condition associated with an abnormal dystrophin-associated protein complex (DAPC) in cells of a subject, comprising administering to the subject a pharmaceutically efficient amount of a collagen VI polypeptide, peptide or peptidomimetic or a biglycan agonist (collectively referred to herein as "collagen VI therapeutics") which stabilizes the DAPC. Optionally, the DAPC is of a type that is deficient in collagen VI function. Examples of diseases that can be treated or prevented include muscular dystrophies, such as Duchenne's Muscular Dystrophy, Becker's Muscular Dystrophy, Congenital Muscular Dystrophy, Ullrich Congenital Muscular Dystrophy, Limb-girdle Muscular Dystrophy, and mytonic dystrophy; cardiomyopathies, Bethlem myopathy and Sorsby's fundus dystrophy. In certain embodiments, the invention relates to a combination therapy comprising administering a collagen VI therapeutic and a biglycan therapeutic, optionally as a single combination therapeutic composition.
[0026] In another example, the invention provides a method for treating or preventing a condition characterized by an abnormal neuromuscular junction or synapse in a subject, comprising administering to the subject a pharmaceutically efficient amount of a biglycan therapeutic which binds to, and/or induces phosphorylation of MuSK and/or which induces aggregation of acetylcholine receptors (AChRs), or a collagen VI therapeutic. The condition can be a neuromuscular or neurological disease.
[0027] The invention also provides methods for treating, preventing and diagnosing diseases or disorders that are associated with abnormal levels or activity of biglycan; with unstable cytoplasmic membranes, due in particular, to unstable DAPCs; or abnormal synapses or neuromuscular junctions.
[0028] In yet another example, the invention provides a diagnostic method for determining whether a subject has or is at risk of developing a condition associated with an abnormal DAPC or abnormal synapse or neuromuscular junction, or other disease associated with an abnormal biglycan level or activity, comprising determining the level or activity of biglycan in a tissue of the subject, wherein the presence of an abnormal level and/or activity of biglycan in the tissue of a subject indicates that the subject has or is at risk of developing a condition associated with an abnormal DAPC or abnormal synapse or neuromuscular junction or other disease associated with an abnormal biglycan level or activity.
[0029] In further embodiments, the invention provides screening methods for identifying agents with inhibit or potentiate the activity of biglycan, such as a human biglycan or Torpedo DAG-125, such as agents which potentiate or inhibit biglycan binding to another molecule, such as a member of a DAPC or MuSK. Agents identified in these assays can be used, e.g., in therapeutic methods, as biglycan therapeutics. Screening methods for identifying agents which modulate phosphorylation induced by biglycan are also within the scope of the invention.
[0030] In additional embodiments, the invention relates to screening methods for identifying agents with inhibit or potentiate the activity of collagen VI, such as a human collagen VI, such as agents which potentiate or inhibit collagen VI binding to biglycan. Agents identified in these assays can be used, e.g., in therapeutic methods, as collagen VI therapeutics.
[0031] Other aspects of the invention are described below or will be apparent to those skilled in the art in light of the present disclosure.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 is a diagram of the interaction between DAG-125 or biglycan with an example of a DAPC.
[0033] FIG. 2 shows the results of a ligand blot overlay assay, in which filters with various extracts (as indicated) were incubated with portions of α-dystroglycan.
[0034] FIG. 3A shows DAG-125 incubated with goat anti-mouse Ig-conjugated agarose beads in the presence or absence of in vitro translated dystroglycan polypeptide (DG345-750) and/or anti-dystroglycan monoclonal antibody (NCL-β-DG; Novocastra, Newcastle-on-Tyne, UK).
[0035] FIG. 3B shows DAG-125 incubated with glutathione-sepharose beads that had been pre-incubated with either bacterially produced GST or a bacterially produced GST-dystroglycan fusion protein (GST-DG345-653). A fusion protein of glutathione S-transferase (GST) and amino acids 345-653 of dystroglycan was produced by using PCR-based subcloning to introduce dystroglycan coding sequence into the bacterial protein expression vector pGEX-1 T (Pharmacia, Piscataway, N.J.).
[0036] FIG. 3C shows DAG-125 and native α-dystroglycan. Alkaline extracts of Torpedo electric organ membranes contain both DAG-125 and α-dystroglycan. This extract was applied to agarose columns conjugated to either control antibody or to an anti-Torpedo dystroglycan monoclonal antibody (MAb3B3; Bowe, M. A., et al. (1994) Neuron. 12: 1173).
[0037] FIG. 4 is a diagram showing portions of dystroglycan used in a blot overlay assays and the presence (+) or absence (-) of binding.
[0038] FIG. 5A shows a blot overlay assay in which a filter with synaptic membranes, input or elute from a column was incubated with a portion of alphα-dystroglycan.
[0039] FIG. 5B shows the sequence alignment between the Torpedo DAG-125 sequences (SEQ ID NOs: 1-3) and human biglycan (SEQ ID NOs: 4-6).
[0040] FIG. 5C is a diagram of the structure of biglycan: the prepro-region, which is absent in the mature biglycan corresponds to amino acids 1-37 of SEQ ID NO: 9; the N-terminal cysteine-rich region corresponds to amino acids 38-80 of SEQ ID NO: 9; the LLR region corresponds to about amino acids 81-314 of SEQ ID NO: 9; and the C-terminal cysteine-rich region corresponds to amino acids 315-368 of SEQ ID NO: 9. Circles represent chondroitin sulfate side chains. "S-S" denotes intrachain disulfide binding.
[0041] FIG. 6 shows the results of an analysis of Torpedo DAG-125 glycosylation.
[0042] FIG. 7 shows that the binding of dystroglycan to biglycan is dependent upon specific chondroitin sulfate side chains. QE-Bgn is bacterially expressed biglycan core. AC stands for articular cartilage.
[0043] FIG. 8A shows an overlay assay blot containing biglycan proteoglycan (BGN-PG), biglycan core (BGN), a biglycan-decorin hybrid (Hybrid), decorin proteoglycan (DEC-PG), decorin (DEC), bacterially produced biglycan (QE-BIG), and Torpedo electric organ membrane fraction (TEOM), which was incubated with 35S labeled α-sarcoglycan.
[0044] FIG. 8B shows an overlay assay blot containing biglycan proteoglycan (BGN-PG), biglycan core (BGN), a biglycan-decorin hybrid (Hybrid), decorin proteoglycan (DEC-PG), decorin (DEC), bacterially produced biglycan (QE-BIG), and Torpedo electric organ membrane fraction (TEOM), which was incubated with γ-sarcoglycan.
[0045] FIG. 8C shows an overlay assay blot containing biglycan proteoglycan (BGN-PG), biglycan core (BGN), a biglycan-decorin hybrid (Hybrid), decorin proteoglycan (DEC-PG), decorin (DEC), bacterially produced biglycan (QE-BIG), and Torpedo electric organ membrane fraction (TEOM), which was incubated with delta-sarcoglycan.
[0046] FIG. 9 shows biglycan expression at the neuromuscular junction.
[0047] FIG. 10 shows the upregulation of biglycan expression in wild type (wt) and dystrophic (mdx) muscle.
[0048] FIG. 11 shows the results of a co-immunoprecipitation of biglycan with recombinant MuSK-Fc.
[0049] FIG. 12 is a Western blot containing cell extracts of cells incubated with or without agrin and with biglycan proteoglycan (BGNPG) or decoring proteoglycan (DECPG) incubated with anti-phosphotyrosine antibody.
[0050] FIG. 13A shows a genotype analysis. PCR genotyping was performed on genomic DNA using primer pairs specific for mutant and wild type biglycan alleles (Xu et al. 1998). PCR products from a wild type (male; +/o), a heterozygote (female; +/-), and a knockout (male; -/o) are shown. Size of PCR products is indicated on left.
[0051] FIG. 13B shows defective agrin-induced AChR clustering in myotubes cultured from biglycan null mice and its rescue by addition of exogneous biglycan. A Bgn female (+/-) was mated to a Bgn male (+/o) and primary cultures were established from each male pup in the resulting litter. The genotype of each pup was determined as shown in FIG. 13A. Myotube cultures derived from each mouse were then treated either with or without recombinant agrin4,8 for 18 hours. Myotubes were then labeled with rhodamine-a-bungarotoxin to visualize AChRs. Wild type myotubes show a robust AChR clustering response to agrin, while myotubes from biglycan-/o mice fail to cluster AChR in response to agrin. Exogenous biglycan (1.4 nM) restores the agrin-induced AChR clustering response.
[0052] FIG. 13C shows quantification of AChR clustering. AChR clusters and myotubes were counted in a minimum of 10 fields for cultures treated either with (AGRIN) or without (Con) recombinant agrin4,8 in the presence of biglycan (1.4 nM) as indicated. A similar deficit in agrin-induced AChR clustering was observed in two other experiments.
[0053] FIG. 14 shows the level of serum creatine kinase in wild type and biglycan knock out mice.
[0054] FIG. 15. Exogenous biglycan induces α-sarcoglycan phosphorylation in a MuSK dependent manner. Wild type C2C12 myotubes (lanes 1, 2, and 6) and MuSK null myotubes (lanes 3-5) were treated for thirty minutes as follows: lanes 1, 3, and 6, unstimulated; lanes 2 and 5, stimulated with a mixture of recombinant proteoglycan and core biglycan (produced in osteosarcoma cells; 1 mg/mL); lane 4, stimulated with agrin 12.4.8. The cultures were detergent extracted and α-sarcoglycan was immunoprecipitated, separated by SDS-PAGE, blotted, and probed with anti-phosphotyrosine antibody (lanes 1-5) or MIgG (lane 6). The addition of biglycan induced tyrosine phosphorylation of α-sarcoglycan and p35 in wild type C2C12 cells but not in MuSK knockout cells.
[0055] FIG. 16A. Sarcoglycan binding to synaptic membrane fractions from Torpedo electric organ (TEOM). TEOM were separated on SDS-PAGE gels, blotted onto nitrocellulose and probed with either 35S-methionine-labelled in vitro translated α-dystroglycan or sarcoglycans (α, β, γ, or δ) as indicated and analyzed by autoradiography. α-Dystroglycan as well as α- and γ-sarcoglycan bound to a polydisperse band whose center of migration. was. ˜125 kD. In previous work a polypeptide with identical mobility and appearance was purified from these fractions and shown to be the proteoglycan biglycan (Bowe et al., 2000). No binding of β- or δ-sarcoglycan to this or any other polypeptide in these fractions was detected.
[0056] FIG. 16B. Binding of α-dystroglycan and sarcoglycans to purified recombinant biglycan proteoglycan. Biglycan was separated on SDS-PAGE and either stained with silver or blotted onto nitrocellulose (`Overlay`) and probed as described in above. α-Dystroglycan and α- and γ-sarcoglycan bind to this recombinant, GAG-containing biglycan proteoglycan while no binding of β- or δ-sarcoglycan is detected.
[0057] FIG. 16C. The biglycan core polypeptide is sufficient for sarcoglycan binding. Purified recombinant biglycan core polypeptide was separated by SDS-PAGE and either silver stained or blotted and probed as described above. α-Dystroglycan did not bind to this GAG-free biglycan. In contrast, both α- and γ-sarcoglycan bind to the biglycan core polypeptide.
[0058] FIG. 17A. Co-immunoprecipitation of purified recombinant biglycan to recombinant sarcoglycan. His-tagged biglycan core polypeptide was incubated with the indicated 35S-methionine labelled in vitro translated sarcoglycan for 1 hr followed by either anti-biglycan, antipoly-His or normal rabbit Ig. Immune complexes were then precipitated with protein G beads and analyzed by SDS-PAGE and autoradiography. Note that both α- and γ-sarcoglycan co-immunoprecipitate with biglycan, while β- or δ-sarcoglycan do not. The labelling of the various sarcoglycans is shown by direct autoradiography of SDS-PAGE-separated in vitro translated polypeptides (`Input`).
[0059] FIG. 17B. Co-immunoprecipitation of biglycan with native sarcoglycans. Purified recombinant biglycan core was incubated with detergent extracts from cultured C2C12 muscle cells. The resulting complexes were then incubated with the indicated anti-sarcoglycan antibodies and western blots of the resulting immunoprecipitates were probed with anti-biglycan antisera. Native α- and γ-sarcoglycan, but not β- or δ-sarcoglycan, co-immunoprecipitate with biglycan. Control experiments showed that each of the anti-sarcoglycan antibodies immunoprecipitated their cognate antigens under these conditions (not shown).
[0060] FIG. 18A. Predicted domain structure of biglycan, decorin and a biglycan-decorin chimera. The location of the pre-pro peptide (`prepro`), 6-His tag, cysteine-rich amino- and carboyxldomains, LRRS (numbered 1-10; some scheme predicts an 11th) and GAG attachment sites (asterisks) are indicated: Note that these sites are present in the proteins used in this experiment, but they are not substituted with GAGs.
[0061] FIG. 18B. Binding of sarcoglycans to biglycan, decorin and a chimera. One microgram of each of the purified recombinant proteins was separated by SDS-PAGE and either directly stained (`silver`) or blotted and probed with 35S-methionine-labelled, in vitro translated sarcoglycans as indicated. Both α- and γ-sarcoglycan bind to the immobilized biglycan core but not to decorin core. In contrast only α-sarcoglycan binds to the biglycan-decorin chimeric protein. Thus the first 30 amino acids of biglycan is involved in binding to α-sarcoglycan. Neither β- nor δ-sarcoglycan bind to either biglycan, decorin or the chimera. These results indicate that the binding sites for α- and γ-sarcoglycan on biglycan are distinct.
[0062] FIG. 19A. PCR genotyping was performed on genomic DNA using primer pairs specific for mutant and wild type biglycan alleles. Shown are results from a wild type male (+/o), heterozygote female (+/-) and null male (-/o).
[0063] FIG. 19B. KCI-washed membranes from skeletal muscle of Bgn null and littermate controls were prepared as described in Methods. Each preparation was separated by SDS-PAGE and either stained for total protein (Coomassie) or transferred to nitrocellulose and probed with rabbit anti-biglycan or normal rabbit serum. In wild type muscle the anti-biglycan recognized polypeptides of ˜37 kD and ˜105 kD which are likely to correspond to the core and proteoglycan form of biglycan, respectively (see Results). Neither polypeptide was detected in membrane fractions from Bgn null mice.
[0064] FIG. 20A. Serum Creatine Kiriase from Bgn null and wildtype littermate controls was measured in mice from 8-12 weeks old were assayed (Sigma). CK levels from biglycan null mice are ˜10 fold greater than wildtype and decorin null mice.
[0065] FIG. 20B. EBD uptake. Mice were injected intravenously with EBD and then returned to their cage for 6 hr. Dye uptake into muscle was assessed by fluorescence microscopy. In bgn null mice some muscle fibers exhibited complete permeation by dye, while in other cells the uptake was limited to a perimembranous distribution. No uptake was observed in muscle from normal animals, while virtually all fibers in mdx mice showed complete permeation.
[0066] FIG. 21. Histopathology of muscle from biglycan null mice. Haematoxylin and eosin stained fresh-frozen sections of skeletal muscle (quadraceps femoris, 8 um thick) from wildtype and BGN-10 mice (AGE). Bgn null mice exhibit groups muscle fibers with centrally nucleated fibers, which are characteristic of muscle fibers that have regenerated in the adult animal. virtually all myofibers show central nuclei in mdx muscle, while such profiles are rarely detected in normal muscle
[0067] FIG. 22. Reduced collagen VI expression in biglycan null mice. Frozen sections from biglycan null mice and wild type littermate controls were immunolabelled with the indicated antibodies. The expression of dystrophin (and. several other DAPC components, see Table I) is similar in muscles from mice of both genotypes. The level of collagen VI is reduced in biglycan null mice relative to controls. The expression levels of decorin are unaffected in biglycan null mice. All comparisons are from tissue prepared, sectioned and immunostained in the same experiment. Images were acquired under identical conditions for each set.
[0068] FIG. 23. An exemplary DAPC comprising collagen VI.
DETAILED DESCRIPTION OF THE INVENTION
I. Overview
[0069] Certain embodiments of the invention are based in part on the observation that biglycan interacts with, and regulates and/or induces modification of the dystrophin-associated protein complex (DAPC), as well as activates components playing an important role in neuromuscular junction formation. In particular, biglycan is shown to interact with α-dystroglycan, an extracellular component of the DAPC, as well as with α-sarcoglycan and γ-sarcoglycan, which are components of the sarcoglycan complex of the DAPC. Biglycan is also shown to induce phosphorylation of α-sarcoglycan, showing that biglycan does not solely interact with components of the DAPC, but also causes modification of the components. The proteoglycan of the invention has been found to be overexpressed in an animal model of muscular dystrophy that is characterized by the absence of dystrophin. The integrity of the DAPC and its association with the extracellular matrix (ECM) are essential for muscle cell viability. Accordingly, biglycan is believed to stabilize the DAPC complex at the surface of cells, in particular, muscle cells, and can be part of a compensatory mechanism that allows survival of dystrophin negative fibers.
[0070] It has also been shown herein that biglycan is involved in neuromuscular junction formation, e.g., induced by agrin. Agrin, which is an extracellular matrix protein present in the synaptic basal lamina, is secreted by the nerve terminal and triggers neuromuscular junction formation by activating the receptor tyrosine kinase MuSK, thereby inducing phosphorylation and clustering of AChR. It had not previously been known how agrin activates the receptor MuSK, since agrin does not bind directly to this receptor. As described below, activation of the receptor MuSK by agrin is actually potentiated by biglycan. This discovery is based at least in part on the finding that biglycan binds directly to the MuSK receptor; biglycan directly induces the tyrosine phosphorylation of MuSK; biglycan potentiates agrin-induced phosphorylation of MuSK; and biglycan potentiates agrin-induced clustering of AChRs. In addition, the appended examples demonstrate that myotubes from biglycan deficient mice show a defective response to agrin, in particular the cells are defective in agrin-induced AChR clustering, which was further shown to be corrected by the addition of biglycan to the culture media of the myotubes. Thus, it is clearly shown that the absence of biglycan in cells results in a deficiency in agrin-induced AChR clustering, which can be corrected by the ectopic addition of biglycan to the cells. The role of biglycan in mediating neuromuscular junction formation, in particular, postynaptic differentiation, is further supported by the fact biglycan binds to α-dystroglycan (shown herein), and that α- and β-dystroglycans interact with components of the postsynaptic membrane. For example, agrin binds to α-dystroglycan (see FIG. 1) and β-dystroglycan binds to the AChR-associated protein rapsyn. In addition, agrin-induced AChR clustering is markedly decreased in muscle cells expressing reduced levels of dystroglycan, further demonstrating the role of dystroglycan in postsynaptic membranes. Thus, it was demonstrated herein that biglycan plays an important role in the formation of neuromuscular junctions both by interacting with the agrin receptor MuSK and by interacting with α-dystroglycan. It is contemplated that biglycan plays both functional and structural roles in the organization of the postsynaptic specializations.
[0071] Moreover, as described further below, biglycan also regulates utrophin expression and localization. Agrin can cause an upregulation of utrophin expression and direct it to be localized to specific domains on the cell surface. The signaling receptor for agrin is the receptor tyrosine kinase MuSK. Agrin also induces the tyrosine phosphorylation of α- and γ-sarcoglycan in cultured myotubes. Biglycan can also regulate the tyrosine phosphorylation of α- and γ-sarcoglycan. Moreover, the receptor tyrosine kinase MuSK is required for this biglycan-induced tyrosine phosphorylation of these proteins. These observations indicate that biglycan can act directly to organize the DAPC, including utrophin, on the muscle cell surface.
[0072] Furthermore, since DAPCs are also found in brain, agrin has been found in senile plaques in brains of subjects with Alzheimer's disease, and peripheral and central neural deficiencies are present in some patients lacking dystrophin, biglycan is also believed to be involved in formation of synapses.
[0073] Thus, the results described herein indicate that biglycan plays an important role in maintaining the integrity of muscle cell plasma membrane, at least in part by interacting with α-dystroglycan and the sarcoglycans in the DAPC; in neuromuscular junction formation, at least in part by mediating agrin-induced AChR clustering and MuSK activation; and also probably in synapse formation. Based at least on these findings, the invention provides compositions and methods for diagnosing, treating and/or preventing diseases or conditions associated with a dysfunctional DAPC, an unstable cellular structure, a defect in neuromuscular junctions or synapses. Such diseases include, in particular, muscular dystrophies, such as Duchenne, Limb-girdle, other myopathies, such as Bethlem myopathy, neuromuscular disorders, and neurological disorders.
[0074] Furthermore, in view of the wide tissue distribution of DAPCs and dystroglycans, biglycan is likely to play a role in regulating signaling through the cytoplasmic membrane and/or maintaining the integrity of cytoplasmic membranes of cells other than muscle cells. For example, dystroglycan or other DAPC components are abundant in brain, kidney, and heart. Thus, the invention provides, more generally, compositions, diagnostic and therapeutic methods for diseases or disorders associated with an abnormality of a membrane protein complex with which the protein of the invention interacts, e.g., the DAPC, or MuSK receptor.
[0075] Based at least on the fact that dystroglycan is known to be a receptor used by microorganisms for entering cells, e.g., Lassa Fever and choriomeningitis fever viruses, the compositions of the invention, particularly biglycan therapeutics, can be used for treating and/or preventing infections by such microorganisms. Without wanting to be limited to a specific mechanism of action, biglycan therapeutics may hinder or inhibit binding of the microorganism to dystroglycan.
[0076] Both human biglycan (described, e.g., in Fischer et al. as "bone small proteoglycan" J. Biol. Chem. 264: 4571 (1996); GenBank Accession No. J04599; SEQ ID NO: 9) and DAG-125 isolated from Torpedo electric organ have been shown to interact with DAPC components. Based on sequence homologies between the two proteins and similar biological activities (further described herein), it is believed that the human biglycan (SEQ ID NO: 9) may be the human ortholog of the Torpedo DAG-125. Alternatively, the human ortholog of the Torpedo DAG-125 may be a protein that is highly related to human biglycan. For purposes of clarity, the term "biglycan" as used herein is intended to include the human biglycan (SEQ ID NO: 9) and Torpedo DAG-125, as well as homologs of these proteoglycans.
[0077] In addition, it is shown herein that a biglycan deficiency leads to a decrease in collagen VI in the extracellular matrix, revealing a surprising collagen VI-based mechanism for DAPC association with the extracellular matrix and providing an explanation for the role of collagen VI in muscle. Mutations in the genes encoding this heterotrimeric collagen are the basis for Bethlem myopathy. This myopathy is characterized by dystrophic changes that are most pronounced in infants and children but typically resolve as the affected individual ages. Targeted mutation of the αX(VI) chain results in mice that show elevated EBD uptake and centrally located nuclei. Interestingly, neither these collagen VI mutant mice nor the Bethlem patients show elevated serum creatine kinase levels. The collagen VI-based matrix association is mechanistically and functionally distinct from the well established dystrophin/β-dystroglycan/α-dystroglycan/basal lamina axis (FIG. 24). α-Dystroglycan binds three G-domain containing basal lamina proteins--laminin-2, perlecan and agrin. These interactions generally involve α-dystroglycan glycosylation and involve a different domain than that mediating biglycan interaction. Further, the α-dystroglycan-basal lamina complex persists in the absence of sarcoglycans. Collagen VI is a microfibrillar collagen that is not a basal lamina component. On the other hand, β-dystroglycan, dystrophin and laminin persist in biglycan null mice while collagen VI expression is reduced. Potential cytoskeletal elements of the sarcoglycan-biglycan axis may include filamin-C, which binds to δ- and γ-sarcoglycan. Thus the DAPC has at least two partially independent paths for matrix interaction.
[0078] Accordingly, it is disclosed herein that biglycans may be used to treat disorders related to a deficiency in collagen VI, and, furthermore, that collagen VI is a component of certain DAPCs, and may be used to stabilize certain DAPCs. Collagen VI, as it occurs in the healthy human body, is a polymer composed primarily of collagen VI monomers, wherein each monomer is a complex formed from the α1(VI), α2(VI) and α3(VI) polypeptide chains. A deficiency in collagen VI, as the term is used herein, is intended to include any situation where there is less collagen VI than is typical for the relevant tissue or cell type as well as any situation where there is less functionally active or functionally arranged (e.g. assembled into a functional matrix) collagen VI.
II. Definitions
[0079] For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below.
[0080] "GAGs" refers to glycosaminoglycans, which is used interchangeably herein with "mucopolysaccharides," are long, unbranched polysaccharide chains composed of repeating disaccharide units. One of the two sugars is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine). Glycosaminoglycans are covalently linked to a serine residue of a core protein, to form a proteoglycan molecule.
[0081] The term "glycan" is used interchangeably herein with the term "polysaccharide" and "oligosaccharide."
[0082] The term "glycoprotein" refers to a protein which contains one or more carbohydrate groups covalently attached to the polypeptide chain. Typically, a glycoprotein contains from 1% to 60% carbohydrate by weight in the form of numerous, relatively short, branched oligosaccharide chains of variable composition. In contrast to glycoproteins, proteoglycans are much larger (up to millions of daltons), and they contain 90% to 95% carbohydrate by weight in the form of may long, unbranched glycosaminoglycan chains.
[0083] The term "proteoglycan of the invention" refers to a proteoglycan molecule having one or more of the characteristics and biological activities of biglycan. Accordingly, a preferred proteoglycan of the invention includes a proteoglycan having one or more of the following characteristics: a molecular weight between 100 and 150 kDa, or an apparent mobility of 125 kDa, as determined on an SDS acrylamide gel; one or more glycosaminoglycan side chain; a molecular weight of the core between 35 and 40 kDa, preferably around 37 kDa; an amino acid sequence selected from SEQ ID NO: 1-6 and 9 or variant thereof; one of more biological activities of biglycan, as listed infra, under the corresponding definition. In one embodiment, the proteoglycan of the invention is a SLRP, e.g., human biglycan. A preferred proteoglycan of the invention is Torpedo DAG-125 or a mammalian, preferably human, ortholog thereof. Another preferred proteoglycan of the invention is biglycan, e.g., human biglycan having SEQ ID NO: 9. The term "proteoglycan of the invention" further includes portions of the wildtype proteoglycan, provided that these portions have at least one biological activity of a biglycan protein. Accordingly, the term "proteoglycan of the invention" includes molecules that consist only of the core (i.e., protein part of the molecule), or of the GAG side chains, portions thereof and/or combinations thereof.
[0084] The term "biglycan" refers to proteoglycans having at least one biological activity of human biglycan or Torpedo DAG-125. Preferred biglycans include Torpedo DAG-125 (comprising SEQ ID NO: 1-3), human biglycan (SEQ ID NO: 9), as well as homologs and fragments thereof. Preferred homologs are proteoglycans or proteins or peptides having at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, and even more preferably, at least about 98 or 99% identity. Even more preferred homologs are those which have a certain percentage of homology (or identity) with human biglycan or Torpedo DAG-125 and have at least one biological activity of these proteoglycans. The term biglycan is not limited to the full length biglycan, but includes also portions having at least one activity of biglycan.
[0085] The term "human biglycan" refers to the proteoglycan described in Fischer et al. J. Biol. Chem. 264: 4571 (1989), having GenBank Accession No. J04599, and the amino acid sequence set forth in SEQ ID NO: 9. A cDNA sequence encoding the human biglycan protein is set forth in SEQ ID NO: 7, and the open reading frame thereof as SEQ ID NO: 8.
[0086] The term "biglycan core" refers to a biglycan that does not include GAG chains.
[0087] The term "biglycan proteoglycan" or "biglycan PG" refers to a biglycan having at least one GAG chain.
[0088] The term "biglycan nucleic acid" refers to a nucleic acid encoding a biglycan proteoglycan, e.g., a nucleic acid encoding a protein having SEQ ID NO: 9.
[0089] A "biological activity of biglycan" is intended to refer to one or more of: the ability to maintain the integrity of a plasma membrane; the ability to stabilize DAPCs on plasma membranes; the ability to bind to one or more components of DAPCs; e.g., binding to α-dystroglycan, binding to a sarcoglycan component, such as α-sarcoglycan or γ-sarcoglycan; binding to MuSK; stimulating the formation of neuromuscluar junctions, such as by stimulating postsynaptic differentiation; potentiation of AChR aggregation, e.g., agrin-induced AChR aggregation; phosphorylation of DAPC components, e.g., sarcoglycans; stimulation MuSK phosphorylation or potentiating agrin-induced MuSK phosphorylation.
[0090] A "biglycan therapeutic" is a compound which can be used for treating or preventing a disease that is associated with an abnormal cytoplasmic membrane, e.g., an unstable membrane; an abnormal DAPC; abnormal neuromuscular junction; abnormal synapse; abnormal AChR aggregation; or abnormal MuSK activation. A biglycan therapeutic can be an agonist or an antagonist of one or more of the biological activities of biglycan. A therapeutic can be any type of compound, including a protein or derivative thereof, e.g., a proteoglycan, a nucleic acid, a glycan, or a small organic or synthetic molecule.
[0091] "Collagen VI" is used to describe the collagen VI monomer, which is a complex formed from the α1(VI), α2(VI) and α3(VI) polypeptide chains, as well as multimers comprising more than one collagen VI monomer. For example, collagen VI is frequently found in vivo as part of a network of beaded filaments. A "collagen VI polypeptide" includes any of the complete α1(VI), α2(VI) and α3(VI) polypeptide chains as well as fragments that are recognizably derived from the α1(VI), α2(VI) and α3(VI) polypeptide chains.
[0092] A "biological activity of collagen VI" is intended to refer to one or more of: the ability to multimerize with collagen VI monomers and the ability to interact with biglycan.
[0093] A "collagen VI therapeutic" is a compound which can be used for treating or preventing a disease that is associated with an abnormal cytoplasmic membrane, e.g., an unstable membrane; an abnormal DAPC; abnormal neuromuscular junction; abnormal synapse; abnormal biglycan deficiency; abnormal AChR aggregation; or abnormal MuSK activation. A collagen VI therapeutic can be an agonist or an antagonist of one or more of the biological activities of collagen VI. A therapeutic can be any type of compound, including a protein or derivative thereof, e.g., a proteoglycan, a nucleic acid, a glycan, or a small organic or synthetic molecule.
[0094] The term "abnormal" is used interchangeably herein with "aberrant" and refers to a molecule, or activity with differs from the wild type or normal molecule or activity.
[0095] The term "DAPC" refers to "dystrophin-associated protein complex", a membrane complex of the type set forth in FIG. 1, which comprises dystrophin and one or more of the following: α- and betα-dystroglycans, the sarcoglycan transmembrane complex and collagen VI. A DAPC that is deficient for a component, such as collagen VI, is a DAPC that has less of the component or less of an active form of the component than is typical or healthy.
[0096] "Sarcoglycans" exit in different forms including α-, beta-, γ-, delta-, and epsilon-sarcoglycans. Certain sarcoglycans are specific for certain tissues, e.g., alpha and delta-sarcoglycans are skeletal muscle specific.
[0097] "Dystrophin-associated proteins" includes proteins or glycoproteins, such as alphα-dystroglycan, dystrobrevin, sarcospan and the syntrophins.
[0098] The term "AChR" refers to acetylcholine receptor.
[0099] The term "SLRP" refers to small leucine rich repeat proteoglycan.
[0100] The term "MASC" refers to muscle cell-associated specificity component.
[0101] The term "RATL" refers to rapsyn-associated transmembrane linker.
[0102] The term "HSPG" refers to heparan sulfate proteoglycans.
[0103] The term "MuSK" used interchangeably herein with "muscle specific kinase," refers to a protein tyrosine kinase, that is expressed in normal and denervated muscle, as well as other tissues including heart, spleen, ovary or retina (See Valenzuela, D., et al., 1995, Neuron 15: 573-584). The tyrosine kinase has alternatively been referred to as "Dmk" for "denervated muscle kinase." Thus, the terms MuSK and Dmk may be used interchangeably. The protein appears to be related to the Trk family of tyrosine kinases, and is further described in U.S. Pat. No. 5,814,478.
[0104] The term "MuSK activating molecule" as used herein refers to a molecule which is capable of inducing phosphorylation of the MuSK receptor in the context of a differentiated muscle cell. One such activating molecule is agrin as described in the Examples set forth herein.
[0105] The term "or" is used herein interchangeably with the term "and/or", unless context clearly indicates otherwise.
[0106] The following terms are used to describe the sequence relationships between two or more polynucleotides: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", and "substantial identity". A "reference sequence" is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing, such as a polynucleotide sequence of SEQ ID NO: 7 or 8, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length. Since two polynucleotides may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window", as used herein, refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. The reference sequence may be a subset of a larger sequence, for example, as a segment of the full-length human biglycan polynucleotide sequence.
[0107] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
[0108] "Small molecule" as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention to identify compounds that modulate the bioactivity of a proteoglycan of the invention.
[0109] A "myoblast" is a cell, that by fusion with other myoblasts, gives rise to myotubes that eventually develop into skeletal muscle fibres. The term is sometimes used for all the cells recognisable as immediate precursors of skeletal muscle fibres. Alternatively, the term is reserved for those post-mitotic cells capable of fusion, others being referred to as presumptive myoblasts.
[0110] The term "including" is used to mean, and interchangeably with, the phrase "including but not limited to".
[0111] "Myofibril" is a long cylindrical organelle of striated muscle, composed of regular arrays of thick and thin filaments, and constituting the contractile apparatus.
[0112] A "myotube" is an elongated multinucleate cells (three or more nuclei) that contain some peripherally located myofibrils. They are formed in vivo or in vitro by the fusion of myoblasts and eventually develop into mature muscle fibres that have peripherally located nuclei and most of their cytoplasm filled with myofibrils. In fact, there is no very clear distinction between myotubes and muscle fibers proper.
[0113] "Utrophin" (dystrophin associated protein) is an autosomal homologue of dystrophin (of size 395 kD) localised near the neuromuscular junction in adult muscle, though in the absence of dystrophin (i.e. in Duchenne muscular dystrophy) utrophin is also located on the cytoplasmic face of the sarcolemma.
[0114] As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. The term "transduction" is generally used herein when the transfection with a nucleic acid is by viral delivery of the nucleic acid. "Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of a polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the recombinant protein is disrupted.
[0115] As used herein, the term "transgene" refers to a nucleic acid sequence which has been introduced into a cell. Daughter cells deriving from a cell in which a transgene has been introduced are also said to contain the transgene (unless it has been deleted). A transgene can encode, e.g., a polypeptide, partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene). Alternatively, a transgene can also be present in an episome. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, (e.g. intron), that may be necessary for optimal expression of a selected coding sequence.
[0116] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
[0117] "Derived from" as that phrase is used herein indicates a peptide or nucleotide sequence selected from within a given sequence. A peptide or nucleotide sequence derived from a named sequence may contain a small number of modifications relative to the parent sequence, in most cases representing deletion, replacement or insertion of less than about 15%, preferably less than about 10%, and in many cases less than about 5%, of amino acid residues or base pairs present in the parent sequence. In the case of DNAs, one DNA molecule is also considered to be derived from another if the two are capable of selectively hybridizing to one another.
[0118] The terms "chimeric", "fusion" and "composite" are used to denote a protein, peptide domain or nucleotide sequence or molecule containing at least two component portions which are mutually heterologous in the sense that they are not, otherwise, found directly (covalently) linked in nature. More specifically, the component portions are not found in the same continuous polypeptide or gene in nature, at least not in the same order or orientation or with the same spacing present in the chimeric protein or composite domain. Such materials contain components derived from at least two different proteins or genes or from at least two non-adjacent portions of the same protein or gene. Composite proteins, and DNA sequences which encode them, are recombinant in the sense that they contain at least two constituent portions which are not otherwise found directly linked (covalently) together in nature.
[0119] The term "modulate" refers to inhibiting or stimulating.
[0120] The terms "activating a postsynaptic membrane" refers to the stimulation of the transfer of a signal at neuromuscular junction, generally, from a nerve cell to a mucle cell. Activation usually includes the stimulation of aggregation of AChR on the cell membrane at the neuromuscular junction; and/or the phosphorylation of MuSK. Activation results in induction of postsynaptic differentiation.
[0121] The term "treating" with regard to a subject, refers to improving at least one symptom of the subject's disease or disorder. Treating can be curing the disease or condition or improving it.
III. Compounds of the Invention
[0122] One aspect of the invention provides biglycan therapeutics for use in maintaining the integrity of plasma cell membranes, in particular, biglycan therapeutics which stabilize dystrophin associated protein complexes (DAPC) in these membranes, thereby preventing the disintegration of the membranes. In further aspects, the invention also provides biglycan therapeutics which stimulate neuromuscular junction formation, such as by stimulating postsynaptic membrane differentiation, and more generally compounds which stimulate synapse formation. In certain aspects, the invention provides biglycan therapeutics for use in modulating collagen VI expression or activity, and optionally, biglycan therapeutics may be used to treat or prevent a disorder that involves a collagen VI-deficiency. In certain aspects, the invention provides collagen VI therapeutics, and such therapeutics may be used to stabilize DAPCs.
[0123] In a particular embodiment, the biglycan therapeutics bind to one or more components of the DAPC. The compound preferably binds to α-dystroglycan and/or to a sarcoglycan component, such as α-sarcoglycan. In an even more preferred embodiment, the compound of the invention binds both to α-dystroglycan and to a component of the sarcoglycan complex, e.g., selected from the group consisting of α-sarcoglycan, γ-sarcoglycan and δ-sarcoglycan. The component of the sarcoglycan to which the compound of the invention binds is preferably α-sarcoglycan. Generally, the compound of the invention contacts one or more components of the DAPC, e.g., to thereby stabilize the complex and reduce destabilization of the plasma membrane resulting from an abnormal DAPC complex, such as those seen in muscular dystrophies.
[0124] In certain embodiments, the biglycan binds to collagen VI or upregulates production or proper organization of collagen VI.
[0125] Yet in an even more preferred embodiment, the compound of the invention binds to a region of α-dystroglycan which is different from the region to which agrin, laminin and perlecan bind (see FIG. 1). Binding of the compounds of the invention do not require the presence of glycosyl side chains on α-dystroglycan. More preferably, the compounds of the invention bind to the C-terminal part of α-dystroglycan, preferably to about amino acids 345 to 891, more preferably to about amino acids 1-750, about amino acids 30-654, about amino acids 345-653, or about amino acids 494-653 of human alphα-dystroglycan. Thus, a preferred compound of the invention binds to a region consisting essentially of the C-terminal 150 amino acids of α-dystroglycan, i.e., amino acids 494-653.
[0126] Other biglycan therapeutics of the invention bind to the receptor tyrosine kinase MuSK. Such compounds can bind to MuSK and/or α-dystroglycan and/or a component of the sarcoglycan complex, e.g., α-sarcoglycan. In preferred embodiments, the biglycan therapeutic activates MuSK and induces phosphorylation of α and/or γ-sarcoglycan.
[0127] The subject biglycan therapeutics preferably bind specifically to one or more of the above-cited molecules, i.e., they do not significantly or at a detectable level bind to other molecules to produce an undesirable effect in the cell or extracellular space. The compounds preferably bind with a dissociation constant of 10-6 or less, and even more preferably with a dissociation constant of 10-7, 10-8, 10-9, 10-10, 10-11, 10-12, or 10-13 M or less. The dissociation constant can be determined according to methods well known in the art.
[0128] Binding assays for determining the level of binding of a compound to a component of the DAPC or to MuSK or for identifying members of, e.g., a library of compounds which bind to these molecules are known in the art and are also further described herein. Methods for preparing DAPC components or MuSK for use in such assays are also known. Such components can be isolated from tissue or, when they are proteins, can be prepared recombinantly or synthetically. Their nucleotide and amino acid sequences are publicly available, e.g., from GenBank, or from publications.
[0129] Other preferred biglycan therapeutics of the invention have one or more biological activities of biglycan, in addition to, or instead of, being able to bind one or more components of the DAPC and/or MuSK. For example, a biglycan therapeutic of the invention can stimulate neuromuscular junction formation, in particular, postsynatic membrane differentiation, including inducing aggregation of AChRs and/or stimulating or stimulating agrin-induced tyrosine phorphorylation of MusK.
[0130] The biglycan therapeutic of the invention can be a protein or derivative thereof, in particular a proteoglycan, a nucleic acid, such as a nucleic acid encoding a proteoglycan of the invention, a glycan, a peptidomimetic or derivative thereof, or a small organic molecule. Generally, the compound can be any type of molecule provided that the compound has the required characteristics, e.g., binding to α-sarcoglycan and/or other DAPC components.
[0131] In a preferred embodiment, the biglycan therapeutic of the invention is a proteoglycan having a molecular weight from about 100 kDa to about 150 kDa, preferably from about 110 kDa to about 140 kDa, and most preferably from about 120 to about 130 kDa, as determined, e.g., by migration on an SDS acrylamide gel. The core of the proteoglycan of the invention has a molecular weight from about 25 to about 45 kDa, preferably from about 30 to about 40 kDa and most preferably around 37 kDa. Fragments or portions of these proteoglycans are also within the scope of the invention. The proteoglycan preferably contains one or more glycosaminoglycan side chains, such as a mucopolysaccharide side chain, e.g., heparan, chondroitin, or dermatan. Preferred side chains consist of chonddroitin sulfate, e.g., 4-sulfate (chondroitin sulfate type A) and 6-sulfate (chondroitin sulfate type C). Any side chain can be used in the invention, provided that the proteoglycan has at least one bioactivity of biglycan.
[0132] In an even more preferred embodiment, the proteoglycan biglycan therapeutic of the invention comprises one or more of the following amino acid sequence in its core: IQAIEFEDL (SEQ ID NO: 1); LGLGFNEIR (SEQ ID NO: 2); and TSYHGISLFNNPVNYWDVL (SEQ ID NO: 3), or amino acid sequences related thereto, such as amino acid sequences from the mammalian ortholog of the Torpedo protein from which these amino acid sequences were obtained. The proteoglycan preferably contains all three of these sequences or sequences related thereto. For example, the proteoglycan of the invention can comprise one or more of the following amino acid sequences, which are part of human biglycan: IQAIELEDL (SEQ ID NO: 4); LGLGHNQIR (SEQ ID NO: 5); and AYYNGISLFNNPVPYWEVQ (SEQ ID NO: 6).
[0133] Although compositions including, and methods using, Torpedo DAG-125 are within the scope of the invention, preferred compositions and methods are those relating to mammalian, including vertebrate, homologs of Torpedo DAG-125, referred to herein as orthologs of Torpedo DAG-125. Preferred orthologs of Torpedo DAG-125 are human, rodent, murine, canine, feline, ovine, and bovine orthologs. As shown herein, it is highly likely that the mammalian DAG-125 is biglycan, however, it may also be a molecule that is related to biglycan, and, e.g., also to decorin (see below), but is actually a not previously described protein. Thus, the invention also provides compositions comprising the mammalian ortholog of Torpedo DAG-125, such as the human ortholog of Torpedo DAG-125.
[0134] A mammalian ortholog of Torpedo DAG-125 can be isolated by screening libraries with probes containing nucleotide sequences encoding one or more of SEQ ID NOs 1-3. Numerous other methods are available for cloning the mammalian ortholog of Torpedo DAG-125. For example, antibodies to Torpedo DAG-125 can be produced and used to screen mammalian expression libraries. The identification of the cloned proteins as mammalian ortholgogs of Torpedo DAG-125 can be established by performing the same biological assays as those described in the Examples employing Torpedo DAG-125.
[0135] Thus, the proteoglycan of the invention can also be a member of the family of small leucine-rich proteoglycans (SLRP), also referred to as "nonaggreagating or small dermatan-sulfate proteoglycans because of their inability to interact with hyaluronan, or because of their type of glycosaminoglycans, respectively. SLRPs are organized into three classes based on their protein and genomic organization. All SLRPs are characterized by a central domain containing leucine rich repeats (LRR) flanked at either side by small cysteine clusters. The SLRPs are described, e.g., in Iozzo et al. (1998) Ann. Rev. Biochem. 67:609, specifically incorporated herein by reference.
[0136] SLRP protein cores range from ˜35-45 kD with one or two GAG chains attached at the extreme N-terminus. The general structure of the SLRP protein core consists of a tandem array of 6-10 leucine-rich repeats (LRR) flanked by domains with conserved, disulfide-bonded cysteines (FIG. 5C). Depending upon the extent of glycosylation and number of GAG chains, the native molecular weight ranges from ˜100-250 kD. On the basis of their sequence homology, Iozzo, supra, has proposed that SLRPs be grouped into three classes consisting of: 1) biglycan and decorin; 2) fibromodulin, lumican, keratocan, PREPLP, and osteoadherin; and 3) epiphycan and osteoglycin. The most compelling feature of the SLRP protein core are the LRRs. Such repeats (24aa each in the SLRPs) mediate protein-protein interactions in a wide variety of intracellular, transmembrane, and extracellular contexts (Kobe & Deisenhofer, (1994) Trends Biochem. Sci. 19: 415-21). The neurotrophin binding site on trkB, for example, is an LRR (Windisch et al., (1995) Biochemistry 34: 11256-63). The repeats are thought to have a general structure of an α-helix followed by beta-sheet in an anti-parallel array, although sequence analysis has suggested that this order might be reversed in the SLRPs (Hocking et al., (1998) Matrix Biol. 17: 1-19). It is likely that the conserved residues of each repeat dictate their secondary structure, while the intervening amino acids determine specificity of ligand binding.
[0137] Preferred SLRPs for use in the invention include Class I SLRPs, such as biglycan and decorin. The partial amino acid sequences of DAG-125, the Torpedo proteoglycan which was shown to bind to alphα-dystroglycan (see Examples) shows strong homology to human biglycan (see FIG. 5B): a 78% identity was found in a total of 37 amino acid long sequence. Biglycan from rodent, pig and human are >95% identical. Decorin and biglycan from human are only 55% identical. Such homology is consistent with decorin and biglycan having both shared and unique functions. Thus, although Torpedo DAG-125 has amino acid sequence that more closely resemble that of human biglycan, based on the similarity of structure and function between biglycan and decorin, the latter proteoglycan and derivatives thereof may also be used to practice the invention.
[0138] Nucleotide and amino acid sequences of biglycan and decorin genes and proteins from various species are publically available, such as in GenBank. For example, human biglycan can be found under GenBank Accession No. J04599 (human hPGI encoding bone small proteoglycan I (biglycan), described in Fisher et al. (1989) J. Biol. Chem. 264: 4571; SEQ ID Nos: 7-9) and M65154; cow biglycan can be found under GenBank Accession No. L07953; rat biglycan can be found under GenBank Accession No. U17834, mouse biglycan can be found under GenBank Accession No. L20276 and X53928; ovis biglycan can be found under GenBank Accession No. AF034842; human decorin can be found at GenBank Accession No. M14219; rabbit decorin can be found at GenBank Accession No. 147020; chick decorin can be found at GenBank Accession No. P28675; Equus decorin can be found at GenBank Accession No. AF038; bovine decorin can be found at GenBank Accession No. P21793; ovis decorin can be found at GenBank Accession No. AF125041; and rat decorin can be found at GenBank Accession No. Q01129. Sequences of biglycan and decorin and other SLRPs can be found in GenBank.
[0139] Decorin and biglycan have one and two glycosaminoglycan (GAG) chains, respectively. Their composition is tissue specific and can be regulated at a number of levels (Hocking et al., (1998) Matrix Biol 17: 1-19). For example, the biglycan GAG from skin and cartilage is predominantly dermatan sulfate, while biglycan synthesized in bone is a chondroitin sulfate proteoglycan. Heparan sulfate side chains have not been reported. Both the protein core and the cell type contribute to the distinct glycosylation of these SLRPs.
[0140] Other proteoglycans or cores thereof of the invention include fusion proteins. For example, biglycan or a portion thereof can be fused to an immunoglobulin portion. Alternatively, the fusion protein is a combination between two or more portions of proteoglycans of the invention, e.g., a portion of a biglycan molecule fused to a portion of a decorin molecule (see examples).
[0141] Portions and fragments of the proteoglycans of the invention are also within the scope of the invention. A portion is typically at least five, 10, 15, or 20 amino acids long. Preferred portions are those which are sufficient for exerting a biological activity, such as interacting with a DAPC component. Portions can comprise or consist of one or more specific domain of a protein. Domains of biglycan and decorin include two cysteine-rich regions (included in the N- and C-terminal 40-50 amino acids of mature biglycan) and leucine-rich repeats (LRRs). The "LRR region" refers to the region of biglycan containing the repeats, and consists essentially of amino acids 81-314. Each individual repeat is referred to herein as an "LRR." LRRs are believed to mediate protein: protein interactions and may thus be sufficient for stabilizing DAPCs and postsynaptic membranes. Based at least on the observation that both decorin and biglycan bind to MuSK and that the LLR region in both of these proteins is very similar, it is believed that the LRRs are involved in mediating the interaction of biglycan (and decorin) with MuSK and may be involved in mediating MuSK phosphorylation.
[0142] Another preferred biglycan of the invention consists of a portion of biglycan that is capable of binding to a sarcoglycan. It has been shown that the α-sarcoglycan binding domain of human biglycan is located in the N-terminal domain of the mature biglycan protein, i.e., amino acids 38-80, and more specifically, amino acids 38-58 of SEQ ID NO: 9. The GAG chains are not necessary for binding to α-sarcgoglycan. It has also been shown that the C-terminal cysteine-rich domain mediates interaction with γ-sarcoglycan. Accordingly, preferred biglycans of the invention include portions of biglycan consisting of the N-terminal or the C-terminal cysteine-rich domain, i.e., amino acids 38-80 and 315-368 of SEQ ID NO: 9. Combinations of certain domains of biglycan are also within the scope of the invention.
[0143] Thus, preferred fragments consist of at least about 30 amino acids, at least about 40 amino acids, 50, 60, 70, 80, 90, 100, 150, or 200 amino acids. Short portions of the proteoglycans of the invention are termed "mini-proteoglycan of the invention." For example, a biglycan core fragment of about 20, 30 or 40 amino acids is referred to as a "mini-biglycan."
[0144] Human biglycan consists of 368 amino acids (SEQ ID NO: 9), of which amino acids 1-19 constitute a signal peptide (GenBank Accession No. NP--001702 and Fisher et al., supra). Thus biglycan without a signal peptide consists of amino acids 20-368 of SEQ ID NO: 9. The mature biglycan protein consists of amino acids 38-368 of SEQ ID NO: 9, since amino acids 1-37, being a pre-propeptide, are cleaved during processing. Amino acids 38-80 correspond to the N-terminal cysteine-rich region. About amino acids 81-314 corresponds to the leucine rich repeat region, containing 10 repeats of about 24 or 23 amino acids. The open reading frame in the cDNA encoding human biglycan corresponds to nucleotides 121-1227 of SEQ ID NO: 7 and is represented as SEQ ID NO: 8. The nucleotide sequence encoding a mature form of biglycan consists in nucleotides 232-1227 of SEQ ID NO: 7.
[0145] In addition to agonists, the invention also provides antagonists of biglycan. An antagonist can be, e.g., a portion of the wild type proteoglycan of the invention which inhibits the action of the wild type proteoglycan, such as by competitively inhibiting the binding of the wild type proteoglycan to a target protein such as a component of a DAPC. Thus, an antagonist can be a dominant negative mutant.
[0146] The proteoglycan can be a mature form of the proteoglycan core, i.e., deprived of the signal peptide, or the full length proteoglycan with the signal peptide.
[0147] Preferred proteoglycans of the invention are encoded by nucleotide sequences which are at least about 70%, preferably at least about 80%, even more preferably at least about 85%, at least about 90%, at least about 95%, at least about 98%, and even more preferably at least about 99% identical to the nucleotide sequence of an SLRP, e.g., biglycan, or ortholog thereof, or portion thereof.
[0148] Preferred nucleic acids of the invention include those encoding a polypeptide comprising an amino acid sequence which is at least about 70%, preferably at least about 80%, even more preferably at least about 85%, at least about 90%, at least about 95%, at least about 98%, and even more preferably at least about 99% identical to the nucleotide sequence of an SLRP, e.g., biglycan (e.g., SEQ ID NO: 7 or 8 encoding human biglycan) or DAG-125 or ortholog thereof, portion thereof. In one embodiment, the nucleic acid encodes a polypeptide containing one or more of SEQ ID NOs: 1-3 or SEQ ID NOs: 4-6 or 9.
[0149] Another aspect of the invention provides a nucleic acid which hybridizes under stringent conditions to a nucleic acid encoding biglycan, e.g., having one or more of SEQ ID NOS: 1 to 6 or 9, or complement thereof. Appropriate stringency conditions which promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature of salt concentration may be held constant while the other variable is changed. In a preferred embodiment, a nucleic acid of the present invention will bind to one of SEQ ID NOS 1 to 6 or complement thereof or nucleic acid encoding a SLRP under moderately stringent conditions, for example at about 2.0×SSC and about 40° C. In a particularly preferred embodiment, a nucleic acid of the present invention will hybridize to a nucleotide sequence encoding one of SEQ ID NOS: 1 to 6 or 9, such as a nucleic acid having SEQ ID NO: 7 or 8, or a complement thereof under high stringency conditions.
[0150] In a further aspect, the invention provides collagen VI therapeutics for use in subject methods, such as for stabilizing dystrophin associated protein complexes (DAPCs). Optionally, the DAPCs to be stabilized are collagen VI-deficient DAPCs.
[0151] In a particular embodiment, the collagen VI therapeutics binds to one or more components of the DAPC. The compound preferably binds to biglycan. Generally, the compound of the invention contacts one or more components of the DAPC, e.g., to thereby stabilize the complex and reduce destabilization of the plasma membrane resulting from an abnormal DAPC complex, such as those seen in muscular dystrophies. Methods for assessing the interaction between collagen VI and biglycan are described, for example, in Wiberg et al. (2001) J. Biol. Chem. 276:18947-18952.
[0152] The subject collagen VI therapeutics preferably bind specifically to one or more of the above-cited molecules, i.e., they do not significantly or at a detectable level bind to other molecules to produce an undesirable effect in the cell. The compounds preferably bind with a dissociation constant of 10-6 or less, and even more preferably with a dissociation constant of 10-7, 10-8, 10-9, 10-10, 10-11, 10-12, or 10-13 M or less. The dissociation constant can be determined according to methods well known in the art.
[0153] Other preferred compounds of the invention have one or more biological activities of collagen VI, such as the ability to form collagen VI monomers with endogenous collagen VI subunits or the ability to form collagen VI polymers.
[0154] In certain embodiments a collagen VI therapeutic comprises a polypeptide comprising an amino acid sequence which is at least about 90% identical to a collagen α1(VI) sequence, such as shown in SEQ ID No: 11 (an example of a human precursor sequence) and SEQ ID No: 12 (an example of a human mature chain). In certain embodiments a collagen VI therapeutic comprises a polypeptide comprising an amino acid sequence which is at least about 90% identical to a collagen α1(VI) sequence, such as shown in SEQ ID No: 11 (an example of a human precursor sequence) and SEQ ID No: 12 (an example of a human mature chain). In certain embodiments a collagen VI therapeutic comprises a polypeptide comprising an amino acid sequence which is at least about 90% identical to a collagen α2(VI) sequence, such as shown in SEQ ID No: 13 (an example of a human precursor sequence) and SEQ ID No: 14 (an example of a human mature chain). In certain embodiments a collagen VI therapeutic comprises a polypeptide comprising an amino acid sequence which is at least about 90% identical to a collagen α3(VI) sequence, such as shown in SEQ ID No: 15 (an example of a human precursor sequence) and SEQ ID No: 16 (an example of a human mature chain). In preferred embodiments, the collagen VI polypeptide is a portion of a mature collagen peptide (e.g. signal sequence is removed). Optionally, the collagen VI polypeptide binds to bigycan. In certain embodiments, a collagen VI therapeutic comprises more than one collagen VI polypeptide. For example, a collagen VI therapeutic may comprise a collagen VI monomer, the monomer comprising a collagen α1(VI) chain, a collagen α2(VI) chain and a collagen α3(VI) chain in a 1:1:1 ratio. Optionally, the therapeutic comprises multimers of collagen VI monomers. Exemplary collagen VI polypeptide and nucleic acid sequences are shown in Tables 1 and 2, respectively.
TABLE-US-00001 TABLE 1 Examples of Collagen VI Polypeptides Name Amino Acid Sequence Human mraarallpl llqacwtaaq depetprava fqdcpvdlff vldtsesval α1(VI) rlkpygalvd kvksftkrfi dnlrdryyrc drnlvwnaga lhysdeveii precursor qgltrmpggr dalkssvdav kyfgkgtytd caikkgleql lvggshlken Chain kylivvtdgh plegykepcg gledavneak hlgvkvfsva itpdhleprl (gi:13878903) siiatdhtyr rnftaadwgq srdaeeaisq tidtivdmik nnveqvccsf (SEQ ID ecqpargppg lrgdpgfege rgkpglpgek geagdpgrpg dlgpvgyqgm NO: 11) kgekgsrgek gsrgpkgykg ekgkrgidgv dgvkgemgyp glpgckgspg fdgiqgppgp kgdpgafglk gekgepgadg eagrpgargp sgdegpagep gppgekgeag degnpgpdga pgerggpger gprgtpgprg prgdpgeagp qgdqgregpv gvpgdpgeag pigpkgyrgd egppgsegar gapgpagppg dpglmgerge dgpagngteg fpgfpgypgn rgapgingtk gypglkgdeg eagdpgddnn diaprgvkga kgyrgpegpq gppghqgppg pdeceildii mkmcscceck cgpidllfvl dssesiglqn feiakdfvvk vidrlsrdel vkfepgqsya gvvqyshsqm qehvslrsps irnvqelkea ikslqwmagg tftgealqyt rdqllppspn nrialvitdg rsdtqrdttp lnvlcspgiq vvsvgikdvf dfipgsdqln viscqglaps qgrpglslvk enyaelleda flknvtaqic idkkcpdytc pitfsspadi tilldgsasv gshnfdttkr fakrlaerfl tagrtdpand vrvavvqysg tgqqrperas lqflqnytal asavdamdfi ndatdvndal gyvtrfyrea ssgaakkrll lfsdgnsqga tpaaiekavq eaqragieif vvvvgrqvne phirvlvtgk taeydvpyge shlfrvpsyq allrgvfhqt vsrkvalg Human qdepetprava fqdcpvdlff vldtsesval rlkpygalvd kvksftkrfi α1(VI) dnlrdryyrc drnlvwnaga lhysdeveii qgltrmpggr dalkssvdav mature chain kyfgkgtytd caikkgleql lvggshlken kylivvtdgh plegykepcg (SEQ ID gledavneak hlgvkvfsva itpdhleprl siiatdhtyr rnftaadwgq NO: 12) srdaeeaisq tidtivdmik nnveqvccsf ecqpargppg lrgdpgfege rgkpglpgek geagdpgrpg dlgpvgyqgm kgekgsrgek gsrgpkgykg ekgkrgidgv dgvkgemgyp glpgckgspg fdgiqgppgp kgdpgafglk gekgepgadg eagrpgargp sgdegpagep gppgekgeag degnpgpdga pgerggpger gprgtpgprg prgdpgeagp qgdqgregpv gvpgdpgeag pigpkgyrgd egppgsegar gapgpagppg dpglmgerge dgpagngteg fpgfpgypgn rgapgingtk gypglkgdeg eagdpgddnn diaprgvkga kgyrgpegpq gppghqgppg pdeceildii mkmcscceck cgpidllfvl dssesiglqn feiakdfvvk vidrlsrdel vkfepgqsya gvvqyshsqm qehvslrsps irnvqelkea ikslqwmagg tftgealqyt rdqllppspn nrialvitdg rsdtqrdttp lnvlcspgiq vvsvgikdvf dfipgsdqln viscqglaps qgrpglslvk enyaelleda flknvtaqic idkkcpdytc pitfsspadi tilldgsasv gshnfdttkr fakrlaerfl tagrtdpand vrvavvqysg tgqqrperas lqflqnytal asavdamdfi ndatdvndal gyvtrfyrea ssgaakkrll lfsdgnsqga tpaaiekavq eaqragieif vvvvgrqvne phirvlvtgk taeydvpyge shlfrvpsyq allrgvfhqt vsrkvalg Human mlqgtcsvll lwgilgaiqa qqqevispdt ternnncpek tdcpihvyfv α2(VI) ldtsesvtmq sptdillfhm kqfvpqfisq lqnefyldqv alswrygglh precursor fsdqvevfsp pgsdrasfik nlqgissfrr gtftdcalan mteqirqdrs Chain kgtvhfavvi tdghvtgspc ggiklqaera reegirlfav apnqnlkeqg (gi:13603394) lrdiastphe lyrndyatml pdsteinqdt inriikvmkh eaygecykvs (SEQ ID cleipgpsgp kgyrgqkgak gnmgepgepg qkgrqgdpgi egpigfpgpk NO: 13) gvpgfkgekg efgadgrkga pglagkngtd gqkgklgrig ppgckgdpgn rgpdgypgea gspgergdqg gkgdpgrpgr rgppgeigak gskgyqgnng apgspgvkga kggpgprgpk gepgrrgdpg tkgspgsdgp kgekgdpgpe gprglagevg nkgakgdrgl pgprgpqgal gepgkqgsrg dpgdagprgd sgqpgpkgdp grpgfsypgp rgapgekgep gprgpeggrg dfglkgepgr kgekgepadp gppgepgprg prgvpgpege pgppgdpglt ecdvmtyvre tcgccdcekr cgaldvvfvi dssesigytn ftleknfvin vvnrlgaiak dpksetgtry gvvqyshegt feaiqlddeh idslssfkea vknlewiagg twtpsalkfa ydrlikesrr qktrvfavvi tdgrhdprdd dlnlralcdr dvtvtaigig dmfhekhese nlysiacdkp qqvrnmtlfs dlvaekfidd medvlcpdpq ivcpdlpcqt elsvaqctqr pvdivflldg serlgeqnfh karrfveqva rrltlarrdd dpinarvall qfggpgeqqv afplshnita ihealettqy lnsfshvgag vvhainaivr sprggarrha elsfvfltdg vtgndslhes ahsmrnenvv ptvlalgsdv dmdvlttlsl gdraavfhek dydslaqpgf fdrfirwic Human qqqevispdt ternnncpek tdcpihvyfv ldtsesvtmq sptdillfhm α2(VI) kqfvpqfisq lqnefyldqv alswrygglh fsdqvevfsp pgsdrasfik mature chain nlqgissfrr gtftdcalan mteqirqdrs kgtvhfavvi tdghvtgspc (SEQ ID ggiklqaera reegirlfav apnqnlkeqg lrdiastphe lyrndyatml NO: 14) pdsteinqdt inriikvmkh eaygecykvs cleipgpsgp kgyrgqkgak gnmgepgepg qkgrqgdpgi egpigfpgpk gvpgfkgekg efgadgrkga pglagkngtd gqkgklgrig ppgckgdpgn rgpdgypgea gspgergdqg gkgdpgrpgr rgppgeigak gskgyqgnng apgspgvkga kggpgprgpk gepgrrgdpg tkgspgsdgp kgekgdpgpe gprglagevg nkgakgdrgl pgprgpqgal gepgkqgsrg dpgdagprgd sgqpgpkgdp grpgfsypgp rgapgekgep gprgpeggrg dfglkgepgr kgekgepadp gppgepgprg prgvpgpege pgppgdpglt ecdvmtyvre tcgccdcekr cgaldvvfvi dssesigytn ftleknfvin vvnrlgaiak dpksetgtry gvvqyshegt feaiqlddeh idslssfkea vknlewiagg twtpsalkfa ydrlikesrr qktrvfavvi tdgrhdprdd dlnlralcdr dvtvtaigig dmfhekhese nlysiacdkp qqvrnmtlfs dlvaekfidd medvlcpdpq ivcpdlpcqt elsvaqctqr pvdivflldg serlgeqnfh karrfveqva rrltlarrdd dpinarvall qfggpgeqqv afplshnita ihealettqy lnsfshvgag vvhainaivr sprggarrha elsfvfltdg vtgndslhes ahsmrnenvv ptvlalgsdv dmdvlttlsl gdraavfhek dydslaqpgf fdrfirwic Human mrkhrhlplv avfclflsgf ptthaqqqqa dvkngaaadi iflvdsswti α3(VI) geehfqlvre flydvvksla vgendfhfal vqfngnphte fllntyrtkq precursor evlshisnms yiggtnqtgk gleyimqshl tkaagsragd gvpqvivvlt Chain dghskdglal psaelksadv nvfaigveda degalkeias epinmhmfnl (gi:5921193) enftslhdiv gnlvscvhss vsperagdte tlkditaqds adiiflidgs (SEQ ID nntgsvnfav ildflvnlle klpigtqqir vgvvqfsdep rtmfsldtys NO: 15) tkaqvlgavk algfaggela niglaldfvv enhftraggs rveegvpqvl vlisagpssd eirygvvalk qasvfsfglg aqaasraelq hiatddnlvf tvpefrsfgd lqekllpyiv gvaqrhivlk pptivtqvie vnkrdivflv dgssalglan fnairdfiak viqrleigqd liqvavaqya dtvrpefyfn thptkrevit avrkmkpldg salytgsald fvrnnlftss agyraaegip kllvlitggk sldeisqpaq elkrssimaf aignkgadqa eleeiafdss lvfipaefra aplqgmlpgl laplrtlsgt pevhsnkrdi iflldgsanv gktnfpyvrd fvmnlvnsld igndnirvgl vqfsdtpvte fslntyqtks dilghlrqlq lqggsglntg salsyvyanh fteaggsrir ehvpqlllll tagqsedsyl qaanaltrag iltfcvgasq ankaeleqia fnpslvylmd dfsslpalpq gliqplttyv sggveevpla qpeskrdilf lfdgsanlvg qfpvvrdfly kiidelnvkp egtriavaqy sddvkvesrf dehqskpeil nlvkrmkikt gkalnlgyal dyaqryifvk sagsriedgv lqflvllvag rssdrvdgpa snlkqsgvvp fifqaknadp aeleqivlsp afilaaeslp kigdlhpqiv nllksvhnga papvsgekdv vflldgsegv rsgfpllkef vqrvvesldv gqdrvrvavv qysdrtrpef ylnsymnkqd vvnavrqltl lggptpntga alefvlrnil vssagsrite gvpqllivlt adrsgddvrn psvvvkrgga vpigigigna ditemqtisf ipdfavaipt frqlgtvqqv iservtqltr eelsrlqpvl qplpspgvgg krdvvflidg sqsagpefqy vrtlierlvd yldvgfdttr vaviqfsddp kaefllnahs skdevqnavq rlrpkggrqi nvgnaleyvs rnifkrplgs rieegvpqfl vlissgksdd evvvpavelk qfgvapftia rnadqeelvk islspeyvfs vstfrelpsl eqklltpitt ltseqiqkll astrypppav esdaadivfl idssegvrpd gfahirdfvs rivrrinigp skvrvgvvqf sndvfpefyl ktyrsqapvl dairrlrlrg gspintgkal efvarnlfvk sagsriedgv pqhlvlvlgg ksqddvsrfa qvirssgivs lgvgdrnidr telqtitndp rlvftvrefr elpnieerim nsfgpsaatp appgvdtppp srpekkkadi vflldgsinf rrdsfqevlr fvseivdtvy edgdsiqvgl vqynsdptde fflkdfstkr qiidainkvv ykggrhantk vglehlrvnh fvpeagsrld qrvpqiafvi tggksvedaq dvslaltqrg vkvfavgvrn idseevgkia snsatafrvg nvqelselse qvletlhdam heticpgvtd aakacnldvi lgfdgsrdqn vfvaqkgfes kvdailnris qmhrvscsgg rsptvrvsvv antpsgpvea fdfdeyqpem lekfrnmrsq hpyvltedtl kvylnkfrqs spdsvkvvih ftdgadgdla dlhrasenlr qegvralilv glervvnler lmhlefgrgf mydrplrinl ldldyelaeq ldniaekacc gvpckcsgqr gdrgpigsig pkgipgedgy rgypgdeggp gergppgvng tqgfqgcpgq rgvkgsrgfp gekgevgeig ldgldgedgd kglpgssgek gnpgrrgdkg prgekgergd vgirgdpgnp gqdsqergpk getgdlgpmg vpgrdgvpgg pgetgknggf grrgppgakg nkggpgqpgf egeqgtrgaq gpagpagppg ligeqgisgp rgsggargap gergrtgplg rkgepgepgp kggignpgpr getgddgrdg vgsegrrgkk gergfpgypg pkgnpgepgl ngttgpkgir grrgnsgppg ivgqkgrpgy pgpagprgnr gdsidqcali qsikdkcpcc ygplecpvfp telafaldts egvnqdtfgr mrdvvlsivn vltiaesncp tgarvavvty nnevtteirf adskrksvll dkiknlqval tskqqsleta msfvarntfk rvrngflmrk vavffsntpt raspqlreav lklsdagitp lfltrqedrq linalqinnt avghalvlpa grdltdflen vltchvcldi cnidpscgfg swrpsfrdrr aagsdvdidm afildsaett tlfqfnemkk yiaylvrqld mspdpkasqh farvavvqha psesvdnasm ppvkvefslt dygskeklvd flsrgmtqlq gtralgsaie ytienvfesa pnprdlkivv lmltgevpeq qleeaqrvil qakckgyffv vlgigrkvni kevytfasep ndvffklvdk stelneeplm rfgrllpsfv ssenafylsp dirkqcdwfq gdqptknlvk fghkqvnvpn nvtssptsnp vtttkpvttt kpvttttkpv ttttkpvtii nqpsvkpaaa kpapakpvaa kpvatktatv rppvavkpat aakpvaakpa avrppaaaak pvatkpevpr pqaakpaatk pattkpvvkm lrevqvfeit ensaklhwer peppgpyfyd ltvtsandqs lvlkqnitvt drviggllag qtyhvavvcy lrsqvratyh gsfstkksqp pppqparsas sstinlmvst eplaltetdi cklpkdegtc rdfilkwyyd pntkscarfw yggcggnenk fgsqkecekv capvlakpgv isvmgt Human qqqqa dvkngaaadi iflvdsswti geehfqlvre flydvvksla α3(VI) vgendfhfal vqfngnphte fllntyrtkq evlshisnms yiggtnqtgk mature chain gleyimqshl tkaagsragd gvpqvivvlt dghskdglal psaelksadv (SEQ ID nvfaigveda degalkeias epinmhmfnl enftslhdiv gnlvscvhss NO: 16) vsperagdte tlkditaqds adiiflidgs nntgsvnfav ildflvnlle klpigtqqir vgvvqfsdep rtmfsldtys tkaqvlgavk algfaggela niglaldfvv enhftraggs rveegvpqvl vlisagpssd eirygvvalk qasvfsfglg aqaasraelq hiatddnlvf tvpefrsfgd lqekllpyiv gvaqrhivlk pptivtqvie vnkrdivflv dgssalglan fnairdfiak viqrleigqd liqvavaqya dtvrpefyfn thptkrevit avrkmkpldg salytgsald fvrnnlftss agyraaegip kllvlitggk sldeisqpaq elkrssimaf aignkgadqa eleeiafdss lvfipaefra aplqgmlpgl laplrtlsgt pevhsnkrdi iflldgsanv gktnfpyvrd fvmnlvnsld igndnirvgl vqfsdtpvte fslntyqtks dilghlrqlq lqggsglntg salsyvyanh fteaggsrir ehvpqlllll tagqsedsyl qaanaltrag iltfcvgasq ankaeleqia fnpslvylmd dfsslpalpq gliqplttyv sggveevpla qpeskrdilf lfdgsanlvg qfpvvrdfly kiidelnvkp egtriavaqy sddvkvesrf dehqskpeil nlvkrmkikt gkalnlgyal dyaqryifvk sagsriedgv lqflvllvag rssdrvdgpa snlkqsgvvp fifqaknadp aeleqivlsp afilaaeslp kigdlhpqiv nllksvhnga papvsgekdv vflldgsegv rsgfpllkef vqrvvesldv gqdrvrvavv qysdrtrpef ylnsymnkqd vvnavrqltl lggptpntga alefvlrnil vssagsrite gvpqllivlt adrsgddvrn psvvvkrgga vpigigigna ditemqtisf ipdfavaipt frqlgtvqqv iservtqltr eelsrlqpvl qplpspgvgg krdvvflidg sqsagpefqy vrtlierlvd yldvgfdttr vaviqfsddp kaefllnahs skdevqnavq rlrpkggrqi nvgnaleyvs rnifkrplgs rieegvpqfl vlissgksdd evvvpavelk qfgvapftia rnadqeelvk islspeyvfs vstfrelpsl eqklltpitt ltseqiqkll astrypppav esdaadivfl idssegvrpd gfahirdfvs rivrrinigp skvrvgvvqf sndvfpefyl ktyrsqapvl dairrlrlrg gspintgkal efvarnlfvk sagsriedgv pqhlvlvlgg ksqddvsrfa qvirssgivs lgvgdrnidr telqtitndp rlvftvrefr elpnieerim nsfgpsaatp appgvdtppp srpekkkadi vflldgsinf rrdsfqevlr fvseivdtvy edgdsiqvgl vqynsdptde fflkdfstkr qiidainkvv ykggrhantk vglehlrvnh fvpeagsrld qrvpqiafvi tggksvedaq dvslaltqrg vkvfavgvrn idseevgkia snsatafrvg nvqelselse qvletlhdam heticpgvtd aakacnldvi lgfdgsrdqn vfvaqkgfes kvdailnris qmhrvscsgg rsptvrvsvv antpsgpvea fdfdeyqpem lekfrnmrsq hpyvltedtl kvylnkfrqs spdsvkvvih ftdgadgdla dlhrasenlr qegvralilv glervvnler lmhlefgrgf mydrplrinl ldldyelaeq ldniaekacc gvpckcsgqr gdrgpigsig pkgipgedgy rgypgdeggp gergppgvng tqgfqgcpgq rgvkgsrgfp gekgevgeig ldgldgedgd kglpgssgek gnpgrrgdkg prgekgergd vgirgdpgnp gqdsqergpk getgdlgpmg vpgrdgvpgg pgetgknggf grrgppgakg nkggpgqpgf egeqgtrgaq gpagpagppg ligeqgisgp rgsggargap gergrtgplg rkgepgepgp kggignpgpr getgddgrdg vgsegrrgkk gergfpgypg pkgnpgepgl ngttgpkgir grrgnsgppg ivgqkgrpgy pgpagprgnr gdsidqcali qsikdkcpcc ygplecpvfp telafaldts egvnqdtfgr mrdvvlsivn vltiaesncp tgarvavvty nnevtteirf adskrksvll dkiknlqval tskqqsleta msfvarntfk rvrngflmrk vavffsntpt raspqlreav lklsdagitp lfltrqedrq linalqinnt avghalvlpa grdltdflen vltchvcldi cnidpscgfg swrpsfrdrr aagsdvdidm afildsaett tlfqfnemkk yiaylvrqld mspdpkasqh farvavvqha psesvdnasm ppvkvefslt dygskeklvd flsrgmtqlq gtralgsaie ytienvfesa pnprdlkivv lmltgevpeq qleeaqrvil qakckgyffv vlgigrkvni kevytfasep ndvffklvdk stelneeplm rfgrllpsfv ssenafylsp dirkqcdwfq gdqptknlvk fghkqvnvpn nvtssptsnp vtttkpvttt kpvttttkpv ttttkpvtii nqpsvkpaaa kpapakpvaa kpvatktatv rppvavkpat aakpvaakpa avrppaaaak pvatkpevpr pqaakpaatk pattkpvvkm lrevqvfeit ensaklhwer peppgpyfyd ltvtsandqs lvlkqnitvt drviggllag qtyhvavvcy lrsqvratyh gsfstkksqp pppqparsas sstinlmvst eplaltetdi cklpkdegtc rdfilkwyyd pntkscarfw yggcggnenk fgsqkecekv capvlakpgv isvmgt
TABLE-US-00002 TABLE 2 Examples of Nucleic Acids Encoding Collagen VI Polypeptides Name Nucleic Acid Sequences (mRNAs and cDNAs) Human cactctggct gggagcagaa ggcagcctcg gtctctgggc ggcggcggcg α1(VI) gccctctctg ccctggccgc gctgtgtggt gaccgcaggc ccgagacatg precursor agggcggccc gtgctctgct gcccctgctg ctgcaggcct gctggacagc chain cgcgcaggat gagccggaga ccccgagggc cgtggccttc caggactgcc (gi:15011912) ccgtggacct gttctttgtg ctggacacct ctgagagcgt ggccctgagg (SEQ ID ctgaagccct acggggccct cgtggacaaa gtcaagtcct tcaccaagcg NO: 17) cttcatcgac aacctgaggg acaggtacta ccgctgtgac cgaaacctgg tgtggaacgc aggcgcgctg cactacagtg acgaggtgga gatcatccaa ggcctcacgc gcatgcctgg cggccgcgac gcactcaaaa gcagcgtgga cgcggtcaag tactttggga agggcaccta caccgactgc gctatcaaga aggggctgga gcagctcctc gtggggggct cccacctgaa ggagaataag tacctgattg tggtgaccga cgggcacccc ctggagggct acaaggaacc ctgtgggggg ctggaggatg ctgtgaacga ggccaagcac ctgggcgtca aagtcttctc ggtggccatc acacccgacc acctggagcc gcgtctgagc atcatcgcca cggaccacac gtaccggcgc aacttcacgg cggctgactg gggccagagc cgcgacgcag aggaggccat cagccagacc atcgacacca tcgtggacat gatcaaaaat aacgttgagc aagtgtgctg ctccttcgaa tgccagcctg caagaggacc tccgggcctc cggggcgacc ccggctttga gggagaacga ggcaagccgg ggctcccagg agagaaggga gaagccggag atcctggaag acccggggac ctcggacctg ttgggtacca gggaatgaag ggagaaaaag ggagccgtgg ggagaagggc tccaggggac caaagggcta caagggagag aagggcaagc gtggcatcga cggggtggac ggcgtgaagg gggagatggg gtacccaggc ctgccaggct gcaagggctc gccgggtttt gacggcattc aaggaccccc tggccccaag ggagaccccg gcgcctttgg actgaaagga gaaaagggcg agcctggagc tgacggggag gccgggagac caggagctcg gggaccatct ggagacgagg ggccagccgg agagcctggg ccccccggag agaaaggaga ggcgggcgac gaggggaacc caggacctga cggtgccccc ggggagcggg gtggccctgg agagagagga ccacggggga ccccaggccc gcggggacca agaggagacc ctggtgaagc tggcccgcag ggtgatcagg gaagagaagg gcccgttggt gtccctggag acccgggcga ggctggccct atcggaccta aaggctaccg aggcgatgag ggtcccccag ggtccgaggg tgccagagga gccccaggac ctgccggacc ccctggagac ccggggctga tgggagaaag gggagaagac ggccccgctg gaaatggcac cgagggcttc cccggcttcc ccgggtatcc cgggaacagg ggcgctcccg ggataaacgg cacgaagggc taccccggcc tcaaggggga cgagggagaa gccggggacc ccggagacga taacaacgac attgcacccc gaggagtcaa aggagcaaag gggtaccggg gtcccgaggg cccccaggga cccccaggac accaaggacc gcctgggccg gacgaatgcg agattttgga catcatcatg aaaatgtgct cttgctgtga atgcaagtgc ggccccatcg acctcctgtt cgtgctggac agctcagaga gcattggcct gcagaacttc gagattgcca aggacttcgt cgtcaaggtc atcgaccggc tgagccggga cgagctggtc aagttcgagc cagggcagtc gtacgcgggt gtggtgcagt acagccacag ccagatgcag gagcacgtga gcctgcgcag ccccagcatc cggaacgtgc aggagctcaa ggaagccatc aagagcctgc agtggatggc gggcggcacc ttcacggggg aggccctgca gtacacgcgg gaccagctgc tgccgcccag cccgaacaac cgcatcgccc tggtcatcac tgacgggcgc tcagacactc agagggacac cacaccgctc aacgtgctct gcagccccgg catccaggtg gtctccgtgg gcatcaaaga cgtgtttgac ttcatcccag gctcagacca gctcaatgtc atttcttgcc aaggcctggc accatcccag ggccggcccg gcctctcgct ggtcaaggag aactatgcag agctgctgga ggatgccttc ctgaagaatg tcaccgccca gatctgcata gacaagaagt gtccagatta cacctgcccc atcacgttct cctccccggc tgacatcacc atcctgctgg acggctccgc cagcgtgggc agccacaact ttgacaccac caagcgcttc gccaagcgcc tggccgagcg cttcctcaca gcgggcagga cggaccccgc ccacgacgtg cgggtggcgg tggtgcagta cagcggcacg ggccagcagc gcccagagcg ggcgtcgctg cagttcctgc agaactacac ggccctggcc agtgccgtcg atgccatgga ctttatcaac gacgccaccg acgtcaacga tgccctgggc tatgtgaccc gcttctaccg cgaggcctcg tccggcgctg ccaagaagag gctgctgctc ttctcagatg gcaactcgca gggcgccacg cccgctgcca tcgagaaggc cgtgcaggaa gcccagcggg caggcatcga gatcttcgtg gtggtcgtgg gccgccaggt gaatgagccc cacatccgcg tcctggtcac cggcaagacg gccgagtacg acgtggccta cggcgagagc cacctgttcc gtgtccccag ctaccaggcc ctgctccgcg gtgtcttcca ccagacagtc tccaggaagg tggcgctggg ctagcccacc ctgcacgccg gcaccaaacc ctgtcctccc acccctcccc actcatcact aaacagagcc caagcttgga aagccaggac acaacgctgc tgcctgcttt gtgcagggtc ctccggggct cagccctgag ttggcatcac ctgcgcaggg ccctctgggg ctcagctctg agctagtgtc acctgcacag ggccctctga ggctcagccc tgagctggcg tcacctgtgc agggccctct ggggctcagc cctgagctgg cctcacctgg gttccccacc ccgggctctc ctgccctgcc ctcctgcccg ccctccctcc tgcctgcgca gctccttccc taggcacctc tgtgctgcat cccaccagcc tgagcaagac gcctctcggg gcctgtgccg cactagcctc cctctcctct gtccccatag ctggtttttc ccaccaatcc tcacctaaca gttactttac aattaaactc aaagcaagct cttctcctca gcttggggca gccattggcc tctgtctcgt tttgggaaac caaggtcagg aggccgttgc agacataaat ctcggcgact cggccccgtc tcctgagggt cctgctggtg accggcctgg accttggccc tacagccctg gaggccgctg ctgaccagca ctgaccccga cctcagagag tactcgcagg ggcgctggct gcactcaaga ccctcgagat taacggtgct aaccccgtct gctcctccct cccgcagaga ctggggcctg gactggacat gagagcccct tggtgccaca gagggctgtg tcttactaga aacaacgcaa acctctcctt cctcagaata gtgatgtgtt cgacgtttta tcaaaggccc cctttctatg ttcatgttag ttttgctcct tctgtgtttt tttctgaacc atatccatgt tgctgacttt tccaaataaa ggttttcact cctc Human agggccacag gtgctgccaa gatgctccag ggcacctgct ccgtgctcct α2(VI) gctctgggga atcctggggg ccatccaggc ccagcagcag gaggtcatct precursor cgccggacac taccgagaga aacaacaact gcccagagaa gaccgactgc chain cccatccacg tgtacttcgt gctggacacc tcggagagcg tcaccatgca (gi: 13603393) gtcccccacg gacatcctgc tcttccacat gaagcagttc gtgccgcagt (SEQ ID tcatcagcca gctgcagaac gagttctacc tggaccaggt ggcgctgagc NO: 18) tggcgctacg gcggcctgca cttctctgac caggtggagg tgttcagccc accgggcagc gaccgggcct ccttcatcaa gaacctgcag ggcatcagct ccttccgccg cggcaccttc accgactgcg cgctggccaa catgacggag cagatccggc aggaccgcag caagggcacc gtccacttcg ccgtggtcat caccgacggc cacgtcaccg gcagcccctg cgggggcatc aagctgcagg ccgagcgggc ccgcgaggag ggcatccggc tcttcgccgt ggcccccaac cagaacctga aggagcaggg cctgcgggac atcgccagca cgccgcacga gctctaccgc aacgactacg ccaccatgct gcccgactcc accgagatca accaggacac catcaaccgc atcatcaagg tcatgaaaca cgaagcctac ggagagtgct acaaggtgag ctgcctggaa atccctgggc cctctgggcc caagggctac cgtggacaga agggtgccaa gggcaacatg ggtgagccgg gagagcctgg ccagaaggga agacagggag acccgggcat cgaaggcccc attggattcc caggacccaa gggcgttcct ggcttcaaag gagagaaggg tgaatttgga gccgacggtc gcaagggggc ccctggcctg gctggcaaga acgggaccga tggacagaag ggcaagctgg ggcgcatcgg acctcctggc tgcaagggag accctggaaa ccggggcccc gacggttacc cgggggaagc agggagtcca ggggagcgag gagaccaagg cggcaagggg gaccctggcc gcccaggacg cagagggccc ccgggagaaa tcggggccaa gggaagcaag gggtatcaag gcaacaatgg agccccagga agtcctggtg tgaaaggagc caagggcggg cctgggcccc gcggacccaa aggcgagccg gggcgcaggg gagaccccgg caccaagggc agcccaggca gcgatggccc caagggggag aagggggacc ctggccctga gggcccccgc ggcctggctg gagaggttgg caacaaagga gccaagggag accgaggctt gcctggaccc agaggccccc agggagctct tggggagccc ggaaagcagg gatctcgggg agaccccggt gatgcaggac cccgtggaga ctcaggacag ccaggcccca agggagaccc cggcaggcct ggattcagct acccaggacc ccgaggagca cccggagaaa aaggcgagcc cggcccacgc ggccccgagg gaggccgagg cgactttggc ttgaaaggag aacctgggag gaaaggagag aaaggagagc ctgcggatcc tggtccccct ggtgagccag gccctcgggg gccaagagga gtcccaggac ccgagggtga gcccggcccc cctggagacc ccggtctcac ggagtgtgac gtcatgacct acgtgaggga gacctgcggg tgctgcgact gtgagaagcg ctgtggcgcc ctggacgtgg tcttcgtcat cgacagctcc gagagcattg ggtacaccaa cttcacactg gagaagaact tcgtcatcaa cgtggtcaac aggctgggtg ccatcgctaa ggaccccaag tccgagacag ggacgcgtgt gggcgtggtg cagtacagcc acgagggcac ctttgaggcc atccagctgg acgacgaaca tatcgactcc ctgtcgagct tcaaggaggc tgtcaagaac ctcgagtgga ttgcgggcgg cacctggaca ccctcagccc tcaagtttgc ctacgaccgc ctcatcaagg agagccggcg ccagaagaca cgtgtgtttg cggtggtcat cacggacggg cgccacgacc ctcgggacga tgacctcaac ttgcgggcgc tgtgcgatcg cgacgtcaca gtgacggcca tcggcatcgg ggacatgttc cacgagaagc acgagagtga aaacctctac tccatcgcct gcgacaagcc acagcaggtg cgcaacatga cgctgttctc cgacctggtc gctgagaagt tcatcgatga catggaggac gtcctctgcc cggaccctca gatcgtgtgc ccagaccttc cctgccaaac agagctgtcc gtggcacagt gcacgcagcg gcccgtggac atcgtcttcc tgctggacgg ctccgagcgg ctgggtgagc agaacttcca caaggcccgg cgcttcgtgg agcaggtggc gcggcggctg acgctggccc ggagggacga cgaccctctc aacgcacgcg tggcgctgct gcagtttggt ggccccggcg agcagcaggt ggccttcccg ctgagccaca acctcactgc catccacgag gcgctggaga ccacacaata cctgaactcc ttctcgcacg tgggcgcagg cgtggtgcac gccatcaatg ccatcgtgcg cagcccgcgt ggcggggccc ggaggcacgc agagctgtcc ttcgtgttcc tcacggacgg cgtcacgggc aacgacagtc tgcacgagtc ggcgcactcc atgcgcaacg agaacgtggt acccaccgtc ctggccttgg gcagcgacgt ggacatggac gtgctcacca cgctcagcct gggtgaccgc gccgccgtgt tccacgagaa ggactatgac agcctggcgc aacccggctt cttcgaccgc ttcatccgct ggatctgcta gcgccgccgc ccgggccccg cagtcgaggg tcgtgagccc accccgtcca tggtgctaag cgggcccggg tcccacacgg ccagcaccgc tgctcactcg gacgacgccc tgggcctgca cctctccagc tcctcccacg gggtccccgt agccccggcc cccgcccagc cccaggtctc cccaggccct ccgcaggctg cccggcctcc ctccccctgc agccatccca aggctcctga cctacctggc ccctgagctc tggagcaagc cctgacccaa taaaggcttt gaacccaaaa aaaaaaa Human cagtttggag ctcagtcttc caccaaaggc cgttcagttc tcctgggctc α3(VI) cagcctcctg caaggactgc aagagttttc ctccgcagct ctgagtctcc precursor acttttttgg tggagaaagg ctgcaaaaag aaaaagagac gcagtgagtg chain ggaaaagtat gcatcctatt caaacctaat tgaatcgagg agcccaggga (gi:3127925) cacacgcctt caggtttgct caggggttca tatttggtgc ttagacaaat (SEQ ID tcaaaatgag gaaacatcgg cacttgccct tagtggccgt cttttgcctc NO: 19) tttctctcag gctttcctac aactcatgcc cagcagcagc aagcagatgt caaaaatggt gcggctgctg atataatatt tctagtggat tcctcttgga ccattggaga ggaacatttc caacttgttc gagagtttct atatgatgtt gtaaaatcct tagctgtggg agaaaatgat ttccattttg ctctggtcca gttcaacgga aacccacata ccgagttcct gttaaatacg tatcgtacta aacaagaagt cctttctcat atttccaaca tgtcttatat tgggggaacc aatcagactg gaaaaggatt agaatacata atgcaaagcc acctcaccaa ggctgctgga agccgggccg gtgacggagt ccctcaggtt atcgtagtgt taactgatgg acactcgaag gatggccttg ctctgccctc agcggaactt aagtctgctg atgttaacgt gtttgcaatt ggagttgagg atgcagatga aggagcgtta aaagaaatag caagtgaacc gctcaatatg catatgttca acctagagaa ttttacctca cttcatgaca tagtaggaaa cttagtgtcc tgtgtgcatt catccgtgag tccagaaagg gctggggaca cggaaaccct taaagacatc acagcacaag actctgctga cattattttc cttattgatg gatcaaacaa caccggaagt gtcaatttcg cagtcattct cgacttcctt gtaaatctcc ttgagaaact cccaattgga actcagcaga tccgagtggg ggtggtccag tttagcgatg agcccagaac catgttttcc ttggacacct actccaccaa ggcccaggtt ctgggtgcag tgaaagccct cgggtttgct ggtggggagt tggccaatat cggcctcgcc cttgatttcg tggtggagaa ccacttcacc cgggcagggg gcagccgcgt ggaggaaggg gttccccagg tgctggtcct cataagtgcc gggccttcta gtgacgagat tcgctacggg gtggtagcac tgaagcaggc tagcgtgttc tcattcggcc ttggagccca ggccgcctcc agggcagagc ttcagcacat agctaccgat gacaacttgg tgtttactgt cccggaattc cgtagctttg gggacctcca ggagaaatta ctgccgtaca ttgttggcgt ggcccaaagg cacattgtct tgaaaccgcc aaccattgtc acacaagtca ttgaagtcaa caagagagac atagtcttcc tggtggatgg ctcatctgca ctgggactgg ccaacttcaa tgccatccga gacttcattg ctaaagtcat ccagaggctg gaaatcggac aggatcttat ccaggtggca gtggcccagt atgcagacac tgtgaggcct gaattttatt tcaataccca tccaacaaaa agggaagtca taaccgctgt gcggaaaatg aagcccctgg acggctcggc cctgtacacg ggctctgctc tagactttgt tcgtaacaac ctattcacga gttcagccgg ctaccgggct gccgagggga ttcctaagct tttggtgctg atcacaggtg gtaagtccct agatgaaatc agccagcctg cccaggagct gaagagaagc agcataatgg cctttgccat tgggaacaag ggtgccgatc aggctgagct ggaagagatc gctttcgact cctccctggt gttcatccca gctgagttcc gagccgcccc attgcaaggc atgctgcctg gcttgctggc acctctcagg accctctctg gaacccctga agttcactca aacaaaagag atatcatctt tcttttggat ggatcagcca acgttggaaa aaccaatttc ccttatgtgc gcgactttgt aatgaaccta gttaacagcc ttgatattgg aaatgacaat attcgtgttg gtttagtgca atttagtgac actcctgtaa cggagttctc tttaaacaca taccagacca agtcagatat ccttggtcat ctgaggcagc tgcagctcca gggaggttcg ggcctgaaca caggctcagc cctaagctat gtctatgcca accacttcac ggaagctggc ggcagcagga tccgtgaaca cgtgccgcag ctcctgcttc tgctcacagc tgggcagtct gaggactcct atttgcaagc tgccaacgcc ttgacacgcg cgggcatcct gactttttgt gtgggagcta gccaggcgaa taaggcagag cttgagcaga ttgcttttaa cccaagcctg gtgtatctca tggatgattt cagctccctg ccagctttgc ctcagcagct gattcagccc ctaaccacat atgttagtgg aggtgtggag gaagtaccac tcgctcagcc agagagcaag cgagacattc tgttcctctt tgacggctca gccaatcttg tgggccagtt ccctgttgtc cgtgactttc tctacaagat tatcgatgag ctcaatgtga agccagaggg gacccgaatt gcggtggctc agtacagcga tgatgtcaag gtggagtccc gttttgatga gcaccagagt aagcctgaga tcctgaatct tgtgaagaga atgaagatca agacgggcaa agccctcaac ctgggctacg cgctggacta tgcacagagg tacatttttg tgaagtctgc tggcagccgg atcgaggatg gagtgcttca gttcctggtg ctgctggtcg caggaaggtc atctgaccgt gtggatgggc cagcaagtaa cctgaagcag agtggggttg tgcctttcat cttccaagcc aagaacgcag accctgctga gttagagcag atcgtgctgt ctccagcgtt tatcctggct gcagagtcgc ttcccaagat tggagatctt catccacaga tagtgaatct cttaaaatca gtgcacaacg gagcaccagc accagtttca ggtgaaaagg acgtggtgtt tctgcttgat ggctctgagg gcgtcaggag cggcttccct ctgttgaaag agtttgtcca gagagtggtg gaaagcctgg atgtgggcca ggaccgggtc cgcgtggccg tggtgcagta cagcgaccgg accaggcccg agttctacct gaattcatac atgaacaagc aggacgtcgt caacgctgtc cgccagctga ccctgctggg agggccgacc cccaacaccg gggccgccct ggagtttgtc ctgaggaaca tcctggtcag ctctgcggga agcaggataa cagaaggtgt gccccagctg ctgatcgtcc tcacggccga caggtctggg gatgatgtgc ggaacccctc cgtggtcgtg aagaggggtg gggctgtgcc cattggcatt ggcatcggga acgctgacat cacagagatg cagaccatct ccttcatccc ggactttgcc gtggccattc ccacctttcg ccagctgggg accgtccaac aggtcatctc tgagagggtg acccagctca cccgcgagga gctgagcagg ctgcagccgg tgttgcagcc tctaccgagc ccaggtgttg gtggcaagag ggacgtggtc tttctcatcg atgggtccca aagtgccggg cctgagttcc agtacgttcg caccctcata gagaggctgg ttgactacct ggacgtgggc tttgacacca cccgggtggc tgtcatccag ttcagcgatg accccaaggc ggagttcctg ctgaacgccc attccagcaa ggatgaagtg cagaacgcgg tgcagcggct gaggcccaag ggagggcggc agatcaacgt gggcaatgcc ctggagtacg tgtccaggaa catcttcaag aggcccctgg ggagccgcat tgaagagggc gtcccacagt tcctggtcct catctcgtct ggaaagtctg acgatgaggt ggtcgtcccg gcggtggagc tcaagcagtt tggcgtggcc cctttcacga tcgccaggaa cgcagaccag gaggagctgg tgaagatctc gctgagcccc gaatatgtgt tctcggtgag caccttccgg gagctgccca gcctggagca gaaactgctg acgcccatca cgaccctgac ctcagagcag
atccagaagc tcttagccag cactcgctat ccacctccag cagttgagag tgatgctgca gacattgtct ttctgatcga cagctctgag ggagttaggc cagatggctt tgcacatatt cgagattttg ttagcaggat tgttcgaaga ctcaacatcg gccccagtaa agtgagagtt ggggtcgtgc agttcagcaa tgatgtcttc ccagaattct atctgaaaac ctacagatcc caggccccgg tgctggacgc catacggcgc ctgaggctca gaggggggtc cccactgaac actggcaagg ctctcgaatt tgtggcaaga aacctctttg ttaagtctgc ggggagtcgc atagaagacg gggtgcccca acacctggtc ctggtcctgg gtggaaaatc ccaggacgat gtgtccaggt tcgcccaggt gatccgttcc tcgggcattg tgagtttagg ggtaggagac cggaacatcg acagaacaga gctgcagacc atcaccaatg accccagact ggtcttcaca gtgcgagagt tcagagagct tcccaacata gaagaaagaa tcatgaactc gtttggaccc tccgcagcca ctcctgcacc tccaggggtg gacacccctc ctccttcacg gccagagaag aagaaagcag acattgtgtt cctgttggat ggttccatca acttcaggag ggacagtttc caggaagtgc ttcgttttgt gtctgaaata gtggacacag tttatgaaga tggcgactcc atccaagtgg ggcttgtcca gtacaactct gaccccactg acgaattctt cctgaaggac ttctctacca agaggcagat tattgacgcc atcaacaaag tggtctacaa agggggaaga cacgccaaca ctaaggtggg ccttgagcac ctgcgggtaa accactttgt gcctgaggca ggcagccgcc tggaccagcg ggtccctcag attgcctttg tgatcacggg aggaaagtcg gtggaagatg cacaggatgt gagcctggcc ctcacccaga ggggggtcaa agtgtttgct gttggagtga ggaatatcga ctcggaggag gttggaaaga tagcgtccaa cagcgccaca gcgttccgcg tgggcaacgt ccaggagctg tccgaactga gcgagcaagt tttggaaact ttgcatgatg cgatgcatga aaccctttgc cctggtgtaa ctgatgctgc caaagcttgt aatctggatg tgattctggg gtttgatggt tctagagacc agaatgtttt tgtggcccag aagggcttcg agtccaaggt ggacgccatc ttgaacagaa tcagccagat gcacagggtc agctgcagcg gtggccgctc gcccaccgtg cgtgtgtcag tggtggccaa cacgccctcg ggcccggtgg aggcctttga ctttgacgag taccagccag agatgctcga gaagttccgg aacatgcgca gccagcaccc ctacgtcctc acggaggaca ccctgaaggt ctacctgaac aagttcagac agtcctcgcc ggacagcgtg aaggtggtca ttcattttac tgatggagca gacggagatc tggctgattt acacagagca tctgagaacc tccgccaaga aggagtccgt gccttgatcc tggtgggcct tgaacgagtg gtcaacttgg agcggctaat gcatctggag tttgggcgag ggtttatgta tgacaggccc ctgaggctta acttgctgga cttggattat gaactagcgg agcagcttga caacattgcc gagaaagctt gctgtggggt tccctgcaag tgctctgggc agaggggaga ccgcgggccc atcggcagca tcgggccaaa gggtattcct ggagaagacg gctaccgagg ctatcctggt gatgagggtg gacccggtga gcgtggtccg cctggtgtga acggcactca aggtttccag ggctgcccgg gccagagagg agtaaagggc tctcggggat tcccaggaga gaagggcgaa gtaggagaaa ttggactgga tggtctggat ggtgaagatg gagacaaagg attgcctggt tcttctggag agaaagggaa tcctggaaga aggggtgata aaggacctcg aggagagaaa ggagaaagag gagatgttgg gattcgaggg gacccgggta acccaggaca agacagccag gagagaggac ccaaaggaga aaccggtgac ctcggcccca tgggtgtccc agggagagat ggagtacctg gaggacctgg agaaactggg aagaatggtg gctttggccg aaggggaccc cccggagcta agggcaacaa gggcggtcct ggccagccgg gctttgaggg agagcagggg accagaggtg cacagggccc agctggtcct gctggtcctc cagggctgat aggagaacaa ggcatttctg gacctagggg aagcggaggt gcccgtggcg ctcctggaga acgaggcaga accggtccac tgggaagaaa gggtgagccc ggagagccag gaccaaaagg aggaatcggg aacccgggcc ctcgtgggga gacgggagat gacgggagag acggagttgg cagtgaagga cgcagaggca aaaaaggaga aagaggattt cctggatacc caggaccaaa gggtaaccca ggtgaacctg ggctaaatgg aacaacagga cccaaaggca tcagaggccg aaggggaaat tcgggacctc cagggatagt tggacagaag gggagacctg gctacccagg accagctggt ccaaggggca acaggggcga ctccatcgat caatgtgccc tcatccaaag catcaaagat aaatgccctt gctgttacgg gcccctggag tgccccgtct tcccaacaga actagccttt gctttagaca cctctgaggg agtcaaccaa gacactttcg gccggatgcg agatgtggtc ttgagtattg tgaatgtcct gaccattgct gagagcaact gcccgacggg ggcccgggtg gctgtggtca cctacaacaa cgaggtgacc acggagatcc ggtttgctga ctccaagagg aagtcggtcc tcctggacaa gattaagaac cttcaggtgg ctctgacatc caaacagcag agtctggaga ctgccatgtc gtttgtggcc aggaacacat ttaagcgtgt gaggaacgga ttcctaatga ggaaagtggc tgttttcttc agcaacacac ccacaagagc atccccacag ctcagagagg ctgtgctcaa actctcagat gcggggatca cccccttgtt ccttacaagg caggaagacc ggcagctcat caacgctttg cagatcaata acacagcagt ggggcatgcg cttgtcctgc ctgcagggag agacctcaca gacttcctgg agaatgtcct cacgtgtcat gtttgcttgg acatctgcaa catcgaccca tcctgtggat ttggcagttg gaggccttcc ttcagggaca ggagagcggc agggagtgat gtggacatcg acatggcttt catcttagac agcgctgaga ccaccaccct gttccagttc aatgagatga agaagtacat agcgtacctg gtcagacaac tggacatgag cccagatccc aaggcctccc agcacttcgc cagagtggca gttgtgcagc acgcgccctc tgagtccgtg gacaatgcca gcatgccacc tgtgaaggtg gaattctccc tgactgacta tggctccaag gagaagctgg tggacttcct cagcagggga atgacacagt tgcagggaac cagggcctta ggcagtgcca ttgaatacac catagagaat gtctttgaaa gtgccccaaa cccacgggac ctgaaaattg tggtcctgat gctgacgggc gaggtgccgg agcagcagct ggaggaggcc cagagagtca tcctgcaggc caaatgcaag ggctacttct tcgtggtcct gggcattggc aggaaggtga acatcaagga ggtatacacc ttcgccagtg agccaaacga cgtcttcttc aaattagtgg acaagtccac cgagctcaac gaggagcctt tgatgcgctt cgggaggctg ttgccgtcct tcgtcagcag tgaaaatgct ttttacttgt ccccagatat caggaaacag tgtgattggt tccaagggga ccaacccaca aagaaccttg tgaagtttgg tcacaaacaa gtaaatgttc cgaataacgt tacttcaagt cctacatcca acccagtgac gacaacgaag ccggtgacta cgacgaagcc ggtgaccacc acaacaaagc ctgtaaccac cacaacaaag cctgtgacta ttataaatca gccatctgtg aagccagccg ctgcaaagcc ggcccctgcg aaacctgtgg ctgccaagcc tgtggccaca aagacggcca ctgttagacc cccagtggcg gtgaagccag caacagcagc gaagcctgta gcagcaaagc cagcagctgt aagacccccc gctgctgctg caaaaccagt ggcgaccaag cctgaggtcc ctaggccaca ggcagccaaa ccagctgcca ccaagccagc caccactaag cccgtggtta agatgctccg tgaagtccag gtgtttgaga taacagagaa cagcgccaaa ctccactggg agaggcctga gccccccggt ccttattttt atgacctcac cgtcacctca gcccatgatc agtccctggt tctgaagcag aacctcacgg tcacggaccg cgtcattgga ggcctgctcg ctgggcagac ataccatgtg gctgtggtct gctacctgag gtctcaggtc agagccacct accacggaag tttcagtaca aagaaatctc agcccccacc tccacagcca gcaaggtcag cttctagttc aaccatcaat ctaatggtga gcacagaacc attggctctc actgaaacag atatatgcaa gttgccgaaa gacgaaggaa cttgcaggga tttcatatta aaatggtact atgatccaaa caccaaaagc tgtgcaagat tctggtatgg aggttgtggt ggaaacgaaa acaaatttgg atcacagaaa gaatgtgaaa aggtttgcgc tcctgtgctc gccaaacccg gagtcatcag tgtgatggga acctaagcgt gggtggccaa catcatatac ctcttgaaga agaaggagtc agccatcgcc aacttgtctc tgtagaagct ccgggtgtag attcccttgc actgtatcat ttcatgcttt gatttacact cgaactcggg agggaacatc ctgctgcatg acctatcagt atggtgctaa tgtgtctgtg gaccctcgct ctctgtctcc agcagttctc tcgaatactt tgaatgttgt gtaacagtta gccactgctg gtgtttatgt gaacattcct atcaatccaa attccctctg gagtttcatg ttatgcctgt tgcaggcaaa tgtaaagtct agaaaataat gcaaatgtca cggctactct atatactttt gcttggttca ttttttttcc cttttagtta agcatgactt tagatgggaa gcctgtgtat cgtggagaaa caagagacca actttttcat tccctgcccc caatttccca gactagattt caagctaatt ttctttttct gaagcctcta acaaatgatc tagttcagaa ggaagcaaaa tcccttaatc tatgtgcacc gttgggacca atgccttaat taaagaattt aaaaaagttg taatagagaa tatttttggc attcctctca atgttgtgtg tttttttttt ttgtgtgctg gagggagggg atttaatttt aattttaaaa tgtttaggaa atttatacaa agaaactttt taataaagta tattgaaagt ttaaaaaaaa aaaaaaaa
[0155] Although compositions including, and methods using, a collagen VI polypeptide from any organism are within the scope of the invention, preferred compositions and methods are those relating to mammalian, including vertebrate, collagen VI polypeptides. Preferred collagen VI polypeptides are human, rodent, murine, canine, feline, ovine, and bovine orthologs, and include naturally occurring variants thereof. Nucleotide and amino acid sequences of collagen VI genes and proteins from various species are publically available, such as in GenBank (see Tables 1 and 2 for examples of Genbank numbers).
[0156] In certain embodiments, a collagen VI therapeutic comprises a collagen VI polypeptide fusion protein. For example, a collagen VI polypeptide or a portion thereof can be fused to an immunoglobulin portion, such as an IgG heavy chain or Fc portion.
[0157] Portions and fragments of a collagen VI polypeptide of the invention are also within the scope of the invention. A portion is typically at least five, 10, 15, or 20 amino acids long. Preferred portions are those which are sufficient for exerting a biological activity, such as interacting with a DAPC component (e.g. biglycan) or forming collagen VI monomers or polymers. Portions can comprise or consist of one or more specific domain of a protein. Optionally, fragments of collagen VI polypeptides consist of at least about 30 amino acids, at least about 40 amino acids, 50, 60, 70, 80, 90, 100, 150, or 200 amino acids.
[0158] In certain embodiments, collagen VI polypeptides of the invention are encoded by nucleotide sequences which are at least about 70%, preferably at least about 80%, even more preferably at least about 85%, at least about 90%, at least about 95%, at least about 98%, and even more preferably at least about 99% identical to the nucleotide sequence of a naturally-occurring collagen VI coding sequence, such as the human coding sequences shown in SEQ ID Nos: 17-19.
[0159] Preferred collagen VI nucleic acids of the invention include those encoding a polypeptide comprising an amino acid sequence which is at least about 70%, preferably at least about 80%, even more preferably at least about 85%, at least about 90%, at least about 95%, at least about 98%, and even more preferably at least about 99% identical to the nucleotide sequence of a human collagen VI coding sequence as shown in SEQ ID Nos: 17-19.
[0160] Another aspect of the invention provides a nucleic acid which hybridizes under stringent conditions to a nucleic acid encoding a collagen VI polypeptide, e.g., encoding one or more of SEQ ID NOS: 10-16, or complement thereof. Appropriate stringency conditions which promote DNA hybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from a low stringency of about 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. In addition, the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22° C., to high stringency conditions at about 65° C. Both temperature and salt may be varied, or temperature of salt concentration may be held constant while the other variable is changed.
[0161] Methods for preparing compounds of the invention are well known in the art. For a compound of the invention which is a protein or a derivative thereof, the compound can be isolated from a tissue or the compound can be recombinantly or synthetically produced. Isolation of protein from a tissue is described in the Examples. The proteins or proteoglycans of the invention isolated from tissue are preferably at least about 70%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% and most preferably, at least about 99% pure. Accordingly, preferred compounds contain less than about 1%, and even more preferably less than about 0.1% of material from which the compound was extracted.
[0162] The protein of the invention can also be produced recombinantly, according to methods well known in the art. Typically, a gene encoding the protein is inserted into a plasmid or vector, and the resulting construct is then transfected into appropriate cells, in which the protein is then expressed, and from which the protein is ultimately purified.
[0163] Accordingly, the present invention further pertains to methods of producing the subject proteins. For example, a host cell transfected with an expression vector encoding a protein of interest can be cultured under appropriate conditions to allow expression of the protein to occur. The protein may be secreted, by inclusion of a secretion signal sequence, and isolated from a mixture of cells and medium containing the protein. Alternatively, the protein may be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art. The proteins can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification with antibodies specific for particular epitopes of the protein.
[0164] Thus, a coding sequence for a protein of the present invention can be used to produce a recombinant form of the protein via microbial or eukaryotic cellular processes. Ligating the polynucleotide sequence into a gene construct, such as an expression vector, and transforming or transfecting into hosts, either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells), are standard procedures.
[0165] Expression vehicles for production of a recombinant protein include plasmids and other vectors. For instance, suitable vectors for the expression of the instant fusion proteins include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
[0166] A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al., (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used.
[0167] The protein can be produced either in eukaryotic cells, e.g., mammalian cells, yeast cells, insect cell (baculovirus system) or in prokaryotic cells. However, if the protein is a proteoglycan, it is preferable to express it in a cell of the same type as that which normally produces that particular proteoglycan. This assures that the correct types of glucose side chain(s) are attached to the core (i.e., protein) of the proteoglycan. In particular, when biglycan is used in the invention, it is preferable that biglycan contains the appropriate GAG side chains. For example, when biglycan is used in the context of muscle cells, it is preferable to produce biglycan in muscle cells, e.g., C2 muscle cells. The biglycan can also be produced in Torpedo cells, e.g., cells from the electric organ of Torpedo.
[0168] Cells that can be used for producing a compound of the invention, e.g., a proteoglycan can further be modified to increase the level and/or activity of an enzyme that catalyzes posttranslational modifications, e.g., glycosylations or sulfonations. For example, a cell can be transformed or cotransfected with an expression construct encoding a sulfotransferase, e.g., a chondroitin sulfotransferase, e.g., a chondroitin-6-sulfotransferase (C6ST; Fukuta et al. (1995) J. Biol. Chem. 270: 18575), or a nervous system involved sulfotransferase (NSIST), described in Nastuk et al. (1998) J. Neuroscience 18: 7167.
[0169] Alternatively, a protein core of a proteoglycan can be produced in a prokaryote, which results in a protein without glucose side chains, and the appropriate side chains can be added later, such as by synthetic chemistry. In yet another embodiment, a proteoglycan is produced in one type of eukaryotic cell and the protein can be stripped of its side chains, prior to adding the appropriate side chains. Methods for synthetically adding glycan side chains to a protein are known in the art.
[0170] In a preferred embodiment, a recombinant protein of the invention, such as biglycan, a collagen VI polypeptide or decorin, is produced using a vaccinia-based system, as described in Krishnan et al. (1999) J. Biol. Chem. 294: 10945 and in Hocking et al. (1996) J. Biol. Chem. 271:19571. Infection of muscle cells with this vector encoding biglycan, a collagen VI polypeptide or decorin for example, results in the production of protein having muscle specific GAG chains. Biophysical studies, such as far UV circular dichroism showed that these recombinant proteins retain their native structure. In an even more preferred embodiment, these recombinant proteins are epitope-tagged, as further described herein, which facilitates co-immunoprecipitation and binding studies.
[0171] For example, a proteoglycan of the invention can be produced in a eukaryotic cell using the vaccinia virus/T7 bacteriophage expression system. A recombinant vaccinia virus, vBGN4 encoding the proteoglycan of the invention, e.g., mature biglycan protein, can be expressed as a polyhistidine fusion protein under control of the T7 phage promoter and expressed, e.g., in HT-1080 cells and UMR106 cells, as described in Hocking et al. (1996) J Biol Chem 271: 19571-7.
[0172] Immortalized cell lines, e.g., muscle cell lines, such as biglycan negative cell lines, can be obtained as described in Jat et al., PNAS (1991) 88: 5096-100; Noble et al., (1992) Brain Pathology 2: 39-46. In one embodiment, a H-2Kb/tsA58 transgenic mouse is used. This mouse is a heterozygote harboring a thermolabile immortalizing gene (the tsA58 mutant of SV40 large T antigen) under the control of an interferon-inducible promoter (this mouse is available at Charles River). When cells containing this gene are cultured, they proliferate indefinitely at 33° C. in the presence of interferon. However, when the temperature is raised to 39° C. (at which temperature the tsA58 antigen is non-functional) and interferon is removed, the cells cease dividing.
[0173] This method has been used for growing a wide variety of cell types, including astrocytes, osteoclasts, trabecular network, and colon epithelial cells (Chambers et al., (1993) PNAS 90: 5578-82; Groves et al., (1993) Dev. Biol. 159: 87-104; Whitehead et al., (1993) PNAS 90: 587-91; Noble et al., (1995) Transgenic Res. 4: 215-25; Tamm et al., (1999) Invest. Ophtamol. Vis. Sci. 40: 1392-403. This technique is well suited for the production of muscle cell lines. For example, in one study alone 65 separate muscle cell lines were derived from animals ranging in age from neonates to four weeks (Morgan et al., (1994) Dev. Biol. 162 486-98). These lines were maintained for upwards of 80 generations. Remarkably, they not only formed myotubes when shifted to non-permissive conditions in culture, but also formed muscle when implanted into host mice. The H-2Kb/tsA58 transgenic method was also used by D. Glass and colleagues to produce a MuSK.sup.-/- muscle cell line (Sugiyama et al., (1997) J. Cell Biol. 139: 181-91).
[0174] To produce conditionally immortalized cell lines, mice having a specific mutation, e.g., a deficiency in biglycan or MuSK, can be crossed with heterozygote H-2Kb/tsA58 transgenic mice. The crosses are straightforward since only one copy of the gene is required for full activity. Muscle cells from neonatal animals can then be plated out and grown under permissive conditions (33° C. with interferon). Proliferating cells can then be cloned and samples from each line shifted to the non-permissive temperature and tested for their ability to form myotubes. Wild type; decorin.sup.-/-; biglycan.sup.-/o; and decorin.sup.-/- biglycan.sup.-/o cell lines are examples of cell lines which can be obtained using this technique.
[0175] In a further embodiment, the compound of the invention is a glycan or polyssacharide. In fact, in certain applications, it may be that in certain cases, the core of a proteoglycan may not be necessary for the desired activity, such as for stabilizing the DAPC by contacting one or more components thereof. For example, it has been shown herein that the GAG side chains of biglycan are necessary for its interaction with α-dystroglycan, indicating that the interaction is likely to be mediated by the GAG side chains.
[0176] The compounds of the invention can also be peptidomimetics or small organic molecules, which can be prepared, e.g., based on the structure of the proteoglyan.
[0177] Although the preferred method for treating subjects with a biglycan or collagen VI is by administration of the agent to the subject (based, for example, on the efficiency of the agent when added to cell cultures), the proteoglycans of the invention can also be produced in a subject, by gene therapy techniques. Thus, e.g., a subject can receive an injection in a muscle (e.g., where the subject has a muscle dystrophy) of a vector encoding a protein or proteoglycan of the invention, such that the vector is capable of entering muscle cells and being expressed therein. Alternatively, the vector can be a viral vector, which is provided with the viral capside and the virus infects the cells, e.g., muscle cells and thereby deliver the vector. Methods and vectors for gene therapy are well known in the art. Illustrative methods are set forth below.
[0178] The preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant fusion proteins by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the -gal containing pBlueBac III).
[0179] In yet other embodiments, the subject expression constructs are derived by insertion of the subject gene into viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. As described in greater detail below, such embodiments of the subject expression constructs are specifically contemplated for use in various in vivo and ex vivo gene therapy protocols.
[0180] Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding a fusion protein of the present invention rendering the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F. M. et al., (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include SYMBOL 121 \f "Symbol"Crip, SYMBOL 121 \f "Symbol"Cre, SYMBOL 121 \f "Symbol"2 and SYMBOL 121 \f "Symbol"Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis et al., (1985) Science 230:1395-1398; Danos and Mulligan, (1988) PNAS USA 85:6460-6464; Wilson et al., (1988) PNAS USA 85:3014-3018; Armentano et al., (1990) PNAS USA 87:6141-6145; Huber et al., (1991) PNAS USA 88:8039-8043; Ferry et al., (1991) PNAS USA 88:8377-8381; Chowdhury et al., (1991) Science 254:1802-1805; van Beusechem et al., (1992) PNAS USA 89:7640-7644; Kay et al., (1992) Human Gene Therapy 3:641-647; Dai et al., (1992) PNAS USA 89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).
[0181] Furthermore, it has been shown that it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al., (1989) PNAS USA 86:9079-9083; Julan et al., (1992) J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology 163:251-254); or coupling cell surface ligands to the viral env proteins (Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.
[0182] Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivate in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., (1988) BioTechniques 6:616; Rosenfeld et al., (1991) Science 252:431-434; and Rosenfeld et al., (1992) Cell 68:143-155). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al., (1992) cited supra), endothelial cells (Lemarchand et al., (1992) PNAS USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993) PNAS USA 90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA 89:2581-2584). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkner et al., supra; and Graham et al., in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127). Expression of the inserted chimeric gene can be under control of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the viral E3 promoter, or exogenously added promoter sequences.
[0183] Yet another viral vector system useful for delivery of the subject chimeric genes is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review, see Muzyczka et al., Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al., (1989) J. Virol. 63:3822-3828; and McLaughlin et al., (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al., (1984) PNAS USA 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; and Flotte et al., (1993) J. Biol. Chem. 268:3781-3790).
[0184] Other viral vector systems that may have application in gene therapy have been derived from herpes virus, vaccinia virus, and several RNA viruses. In particular, herpes virus vectors may provide a unique strategy for persistence of the recombinant gene in cells of the central nervous system and ocular tissue (Pepose et al., (1994) Invest Ophthalmol Vis Sci 35:2662-2666).
[0185] In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a protein in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
[0186] In a representative embodiment, a gene encoding a protein of interest can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al., (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075). For example, lipofection of muscle, neural or cardiac cells can be carried out using liposomes tagged with monoclonal antibodies against specific tissue-associated antigens (Mizuno et al., (1992) Neurol. Med. Chir. 32:873-876).
[0187] In yet another illustrative embodiment, the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as poly-lysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For example, any of the subject gene constructs can be used to transfect specific cells in vivo using a soluble polynucleotide carrier comprising an antibody conjugated to a polycation, e.g. poly-lysine (see U.S. Pat. No. 5,166,320). It will also be appreciated that effective delivery of the subject nucleic acid constructs via-mediated endocytosis can be improved using agents which enhance escape of the gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al., (1993) Science 260-926; Wagner et al., (1992) PNAS USA 89:7934; and Christiano et al., (1993) PNAS USA 90:2122).
[0188] Nucleic acids encoding biglycan or collagen VI proteins can also be administered to a subject as "naked" DNA, as described, e.g., in U.S. Pat. No. 5,679,647 and related patents by Carson et al., in WO 90/11092 and Felgner et al. (1990) Science 247: 1465.
[0189] In clinical settings, the gene delivery systems can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the construct in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al., (1994) PNAS USA 91: 3054-3057).
[0190] A gene encoding a proteoglycan or collagen VI of the invention can be under the control of a constitutive, or inducible promoter. These are well known in the art.
[0191] Methods for determining whether a compound has a biological activity of a biglycan protein are described in the Examples. A biological activity of a biglycan protein is intended to refer to one or more of: the ability to maintain the integrity of a plasma membrane; the ability to stabilize DAPCs on plasma membranes; the ability to bind to one or more components of DAPCs; e.g., binding to α-dystroglycan, binding to a sarcoglycan component, such as α-sarcoglycan; phosphorylation of α-sarcoglycan; binding to MuSK; stimulating the formation of neuromuscular junctions, such as by stimulating postsynaptic differentiation; stimulating AChR aggregation; stimulation of MuSK phosphorylation and potentiation of agrin-induced MuSK phosphorylation. Such methods can further be adapted for screening libraries of compounds for identifying compounds having one or more of the above-described activities.
[0192] Breakdown of cytoplasmic membranes, e.g., the presence of "leaky membranes" can be determined by assays which measure the release of creatine kinase or the absorption of Evans Blue dye, as described, e.g., in Tinsley et al. (1996) Nature 384: 349 and Straub et al. (1997) J. Cell Biol. 139: 375).
[0193] The compounds of the invention can also be tested in a variety of animal models, in particular the mdx mice, which are dystrophin negative (see Examples).
IV. Methods of Treatment
[0194] In certain aspects, the invention provides therapeutic and prophylactic methods of treatment of disorders including muscular, neuromuscular, and neurological disorders. Therapeutic methods are intended to eliminate or at least reduce at least one symptom of a disease or disorder, and preferably cure the disease or disorder. Prophylactic methods include those intended to prevent the appearance of a disease or disorder, i.e., a method which is intended to combat the appearance of the disease or disorder.
[0195] As described herein, biglycan was shown to bind to α-dystroglycan and to sarocoglycans, and thereby functions as a link between various components of DAPCs. Furthermore, biglycan levels were found to be high in muscle cells of mice lacking dystrophin (mdx mice, which are a model of muscular dystrophy). Since the absence of dystrophin in muscle cells is known to destabilize the cytoplasmic membrane, the upregulation of biglycan in dystrophin negative muscle cells may be a compensatory mechanism for the absence of dystrophin. Accordingly, the invention provides for methods for preventing and treating diseases or disorders that are associated with plasma membrane instability or organization, in particular, an instability resulting from an abnormal DAPC on the plasma membrane. Since the DAPC is found on the membrane of muscle cells, diseases that can be treated according to the invention include diseases of the muscle, such as muscular dystrophies and muscle atrophy.
[0196] In that regard, one promising path for treatment and potentially a cure for muscular dystrophy the activation of an endogenous compensatory mechanism based upon the regulated expression of utrophin. Utrophin is a homolog of dystrophin which shares numerous structural and functional properties with it. However, in both normal and in Duchenne's muscle utrophin is only expressed at a fraction of the muscle membrane: the neuromuscular junction and the myotendinous junction. The bulk of the membrane has no utrophin. However, in animal models it has been shown that forced expression of utrophin in muscle lacking dystrophin leads to restoration of the DAPC in the muscle membrane and to rescue of the dystrophic phenotype. Since the utrophin gene is normal in Duchenne patients, a method to activate its expression in muscle and/or to target it to the muscle membrane could serve to restore the DAPC to the membrane and thus promote the health of the muscle cells.
[0197] Several lines of evidence, many of them arising from observations made by the inventors indicate that the small leucine-rich repeat proteoglycan biglycan could be a method for regulating utrophin expression and localization. It has been demonstrated that the protein agrin can cause an upregulation of utrophin expression and direct it to be localized to specific domains on the cell surface. The signaling receptor for agrin is the receptor tyrosine kinase MuSK. It has been observed that agrin can also induce the tyrosine phosphorylation of α- and γ-sarcoglycan in cultured myotubes. It was also observed that biglycan can also regulate the tyrosine phosphorylation of α- and γ-sarcoglycan. Moreover, the receptor tyrosine kinase MuSK is required for this biglycan-induced tyrosine phosphorylation of these proteins. Further, biglycan can bind to MuSK. These observations indicate that biglycan can act directly to organize the DAPC, including utrophin, on the muscle cell surface.
[0198] Thus the present invention contemplates the treatment of these disorders with biglycan therapeutics which upregulate utrophin, activate MuSK and/or induce phosphorylation of sarcoglycans.
[0199] Furthermore, as disclosed herein, biglycan affects collagen VI production and collagen VI presence in DAPCs, and the invention contemplates treatment of collagen VI-related disorders with a biglycan therapeutic. In addition, in certain aspects the invention provides methods for stabilizing DAPCs, particularly collagen-VI deficient DAPCs, by administering a collagen VI therapeutic.
[0200] Merely to illustrate, biglycan polypeptides, peptides or peptidomimetics can be delivered to patients with muscular dystrophy or other conditions where muscle atrophies to upregulate the endogenous utrophin gene expression and/or to promote the localization of utrophin to the muscle membrane. In such embodiments, the biglycan polypeptide may be delivered in the form of a polypeptide in and of itself, or as part of a fusion protein, e.g., fused to a humanized antibody sequence or similar carrier entity. Biglycan polypeptides can be delivered by nucleic acid-based methods including as plasmid DNA, in viral vectors, or other modalities where the nucleic acid sequences encoding biglycan are introduced into patients. The delivery of a biglycan therapeutic can serve to heal the muscle fibers from within by directing the increased expression and regulated localization of utrophin to the muscle cell surface with concomitant restoration of the remainder of the dystrophin-associated protein complex.
[0201] However, the present invention also contemplates the use of agents which act upstream of biglycan, e.g., which induce the expression of native biglycan genes. Treatment with such agents as angotensin II, sodium salicylate, forskolin and 8-bromo-cAMP, for example, results in significant increases in expression of biglycan and can be used as part of a treatment protocol for such disorders.
[0202] Furthermore, since DAPCs are also found on other cell types, the invention also provides methods for treating diseases associated with any abnormal DAPC. For example, DAPC are present in the brain, and since, in addition, agrin has been found in senile plaques in patients with Alzheimers's disease, neurological diseases can also be treated or prevented according to the methods of the invention. A further indication that neurological disorders can be treated or prevented according to the methods described herein is based on the observation that patients with muscular dystrophy often also suffer from peripheral and central nervous system disorder. Accordingly, about one third of patients with Duchenne Muscular Dystrophy have a mental affliction, in particular, mental retardation. Thus, dystrophin, and hence, DAPCs, are believed to play a role in the nervous system.
[0203] Patients with Duchenne's Muscular Dystrophy also have diaphragm problems, indicating a role for dystrophin, and possibly DAPCs in diaphragms. Thus, therapeutics of the invention would also find an application in disorders associated with diaphragm abnormalities.
[0204] It should be noted that diseases that can be treated or prevented include not only those in which biglycan is abnormal, but more generally any disease or condition that is associated with a defect that can be improved or cured by biglycan. In particular, diseases that are characterized by a defect or an abnormality in any component of the DAPC or component associated therewith, thereby resulting, e.g., in an unstable plasma membrane, can be treated or prevented according to the methods of the invention, provided that the proteoglycan of the invention can at least partially cure the defect resulting from the deficient component. In particular, diseases that can be treated according to the method of the invention include any disease associated with an unstable DAPC, which can be rendered more stable by the presence of a proteoglycan of the invention.
[0205] Furthermore, since biglycan was shown to bind to, and phosphorylates MuSK, a receptor which is known for mediating agrin-induced stimulation of neuromuscular junction formation, in particular postsynaptic membrane differentiation, to potentiateagrin-induced AChR aggregation, and to correct a defective agrin-induced AChR aggregation in myotubes of biglycan negative mice by its addition to the myotubes, the invention also provides methods for preventing and treating diseases or disorders of neuromuscular junctions, such as neuromuscular disorders. Most interestingly, exogenously added biglycan was shown to be able to correct a defective agrin-induced AChR aggregation in myotubes of biglycan negative mice.
[0206] A. Exemplary Diseases and Disorders:
[0207] Diseases or disorders that are characterized by a destabilization or improper organization of the plasma membrane of specific cell types include muscular dystrophies (MDs), a group of genetic degenerative myopathies characterized by weakness and muscle atrophy without nervous system involvement. The three main types are pseudohypertrophic (Duchenne, Becker), limb-girdle, and facioscapulohumeral. For example, muscular dystrophies and muscular atrophies are characterized by a breakdown of the muscle cell membrane, i.e., they are characterized by leaky membranes, which are believed to result from a mutation in a component of the DAPC., i.e., dystrophin. Mutations in the sarcoglycans are also known to result in muscular dystrophies and leaky membranes. Accordingly, the invention provides for methods for treating or preventing diseases associated with mutations in dystrophin and/or in sarcoglycans or other component of DAPCs, in particular muscular dystrophies.
[0208] Dystrophin abnormalities are responsible for both the milder Becker's Muscular Dystrophy (BMD) and the severe Duchenne's Muscular Dystrophy (DMD). In BMD dystrophin is made, but it is abnormal in either size and/or amount. The patient is mild to moderately weak. In DMD no protein is made and the patient is wheelchair-bound by age 13 and usually dies by age 20.
[0209] Another type of dystrophy that can be treated according to the methods of the invention includes congenital muscular dystrophy (CMD), a very disabling muscle disease of early clinical onset, is the most frequent cause of severe neonatal hypotonia. Its manifestations are noticed at birth or in the first months of life and consist of muscle hypotonia, often associated with delayed motor milestones, severe and early contractures and joint deformities. Serum creatine kinase is raised, up to 30 times the normal values, in the early stage of the disease, and then rapidly decreases. The histological changes in the muscle biopsies consist of large variation in the size of muscle fibers, a few necrotic and regenerating fibers, marked increase in endomysial collagen tissue, and no specific ultrastructural features. The diagnosis of CMD has been based on the clinical picture and the morphological changes in the muscle biopsy, but it cannot be made with certainty, as other muscle disorders may present with similar clinico-pathological features. Within the group of diseases classified as CMD, various forms have been individualized. The two more common forms are the occidental and the Japanese, the latter being associated with severe mental disturbances, and usually referred to as Fukuyama congenital muscular dystrophy (FCMD).
[0210] One form of congenital muscular dystrophy (CMD) has recently been characterized as being caused by mutations in the laminin alpha 2-chain gene. Laminin is a protein that associates with DAPCs. Thus, the invention also provides methods for treating diseases that are associated with abnormal molecules which normally associate with DAPCs.
[0211] Other muscular dystrophies within the scope of the invention include limb-girdle muscular dystrophy (LGMD), which represents a clinically and genetically heterogeneous class of disorders. These dystrophies are inherited as either autosomal dominant or recessive traits. An autosomal dominant form, LGMD1A, was mapped to 5q31-q33 (Speer, M. C. et al., Am. J. Hum. Genet. 50:1211, 1992; Yamaoka, L. Y. et al., Neuromusc. Disord. 4:471, 1994), while six genes involved in the autosomal recessive forms were mapped to 15q15.1 (LGMD2A) (Beckmann, J. S. et al., C. R. Acad. Sci. Paris 312:141, 1991), 2p16-p13 (LGMD2B) (Bashir, R. et al., Hum. Mol. Genet. 3:455, 1994), 13q12 (LGMD2C) (Ben Othmane, K. et al., Nature Genet. 2:315, 1992; Azibi, K. et al., Hum. Mol. Genet. 2:1423, 1993), 17q12-q21.33 (LGMD2D) (Roberds, S. L. et al., Cell 78:625, 1994; McNally, E. M., et. al., Proc. Nat. Acad. Sci. U.S.A. 91:9690, 1994), 4q12 (LG1MD2E) (Lim, L. E., et. al., Nat. Genet. 11:257, 1994; Bonnemann, C. G. et al. Nat. Genet. 11:266, 1995), and most recently to 5q33-q34 (LGMD2F) (Passos-Bueno, M. R., et. al., Hum. Mol. Genet. 5:815, 1996). Patients with LGMD2C, 2D and 2E have a deficiency of components of the sarcoglycan complex resulting from mutations in the genes encoding gamma-, alpha-, and beta-sarcoglycan, respectively. The gene responsible for LGMD2A has been identified as the muscle-specific calpain, whereas the genes responsible for LGMD1A, 2B and 2F are still unknown.
[0212] Yet other types of muscular dystrophies that can be treated according to the methods of the invention include Welander distal myopathy (WDM), which is an autosomal dominant myopathy with late-adult onset characterized by slow progression of distal muscle weakness. The disorder is considered a model disease for hereditary distal myopathies. The disease is linked to chromosome 2p13. Another muscular dystrophy is Miyoshi myopathya, which is a distal muscular dystrophy that is caused by mutations in the recently cloned gene dysferlin, gene symbol DYSF (Weiler et al. (1999) Hum Mol Genet 8: 871-7). Yet other dystrophies include Hereditary Distal Myopathy, Benign Congenital Hypotonia, Central Core disease, Nemaline Myopathy, and Myotubular (centronuclear) myopathy.
[0213] Other diseases that can be treated or prevented according to the methods of the invention include those characterized by tissue atrophy, e.g., muscle atrophy, other than muscle atrophy resulting from muscular dystrophies, provided that the atrophy is stopped or slowed down upon treatment with a therapeutic of the invention. Furthermore, the invention also provides methods for reversing tissue atrophies, e.g., muscle atrophies. This can be achieved, e.g., by providing to the atrophied tissue a therapeutic of the invention, such as DAG-125 or mammalian ortholog thereof, or biglycan.
[0214] Muscle atrophies can result from denervation (loss of contact by the muscle with its nerve) due to nerve trauma; degenerative, metabolic or inflammatory neuropathy (e.g., GuillianBarre syndrome), peripheral neuropathy, or damage to nerves caused by environmental toxins or drugs. In another embodiment, the muscle atrophy results from denervation due to a motor neuronopathy. Such motor neuronopathies include, but are not limited to: adult motor neuron disease, including Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's disease); infantile and juvenile spinal muscular atrophies, and autoimmune motor neuropathy with multifocal conduction block. In another embodiment, the muscle atrophy results from chronic disuse. Such disuse atrophy may stem from conditions including, but not limited to: paralysis due to stroke, spinal cord injury; skeletal immobilization due to trauma (such as fracture, sprain or dislocation) or prolonged bed rest. In yet another embodiment, the muscle atrophy results from metabolic stress or nutritional insufficiency, including, but not limited to, the cachexia of cancer and other chronic illnesses, fasting or rhabdomyolysis, endocrine disorders such as, but not limited to, disorders of the thyroid gland and diabetes.
[0215] Since muscle tissue atrophy and necrosis are often accompanied by fibrosis of the affected tissue, the reversal or the inhibition of atrophy or necrosis can also result in an inhibition or reversal of fibrosis.
[0216] In addition, the therapeutics of the invention may be of use in the treatment of acquired (toxic or inflammatory) myopathies. Myopathies which occur as a consequence of an inflammatory disease of muscle, include, but not limited to polymyositis and dermatomyositis. Toxic myopathies may be due to agents, including, but are not limited to adiodarone, chloroquine, clofibrate, colchicine, doxorubicin, ethanol, hydroxychloroquine, organophosphates, perihexiline, and vincristine.
[0217] Neuromuscular dystrophies within the scope of the invention include myotonic dystrophy. Myotonic dystrophy (DM; or Steinert's disease) is an autosomal dominant neuromuscular disease which is the most common form of muscular dystrophy affecting adults. The clinical picture in DM is well established but exceptionally variable (Harper, P. S., Myotonic Dystrophy, 2nd ed., W. B. Saunders Co., London, 1989). Although generally considered a disease of muscle, with myotonia, progressive weakness and wasting, DM is characterized by abnormalities in a variety of other systems. DM patients often suffer from cardiac conduction defects, smooth muscle involvement, hypersomnia, cataracts, abnormal glucose response, and, in males, premature balding and testicular atrophy (Harper, P. S., Myotonic Dystrophy, 2nd ed., W. B. Saunders Co., London, 1989). The mildest form, which is occasionally difficult to diagnose, is seen in middle or old age and is characterized by cataracts with little or no muscle involvement. The classical form, showing myotonia and muscle weakness, most frequently has onset in early adult life and in adolescence. The most severe form, which occurs congenitally, is associated with generalized muscular hypoplasia, mental retardation, and high neonatal mortality. This disease and the gene affected is further described in U.S. Pat. No. 5,955,265.
[0218] Another neuromuscular disease is spinal muscular atrophy ("SMA"), which is the second most common neuromuscular disease in children after Duchenne muscular dystrophy. SMA refers to a debilitating neuromuscular disorder which primarily affects infants and young children. This disorder is caused by degeneration of the lower motor neurons, also known as the anterior horn cells of the spinal cord. Normal lower motor neurons stimulate muscles to contract. Neuronal degeneration reduces stimulation which causes muscle tissue to atrophy (see, e.g., U.S. Pat. No. 5,882,868).
[0219] The above-described muscular dystrophies and myopathies are skeletal muscle disorders. However, the invention also pertains to disorders of smooth muscles, e.g., cardiac myopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy and restrictive cardiomyopathy. At least certain smooth muscles, e.g., cardiac muscle, are rich in sarcoglycans. Mutations in sarcoglycans can result in sarcolemmal instability at the myocardial level (see, e.g., Melacini (1999) Muscle Nerve 22: 473). For example, animal models in which a sarcoglycan is mutated show cardiac creatine kinase elevation. In particular, it has been shown that delta-sarcoglycan (Sgcd) null mice develop cardiomyopathy with focal areas of necrosis as the histological hallmark in cardiac and skeletal muscle. The animals also showed an absence of the sarcoglycan-sarcospan (SG-SSPN) complex in skeletal and cardiac membranes. Loss of vascular smooth muscle SG-SSPN complex was associated with irregularities of the coronary vasculature. Thus, disruption of the SG-SSPN complex in vascular smooth muscle perturbs vascular function, which initiates cardiomyopathy and exacerbates muscular dystrophy (Coral-Vazquez et al. (1999) Cell 98: 465).
[0220] Similarly to delta-sarcoglycan negative mice, mice lacking γ-sarcoglycan showed pronounced dystrophic muscle changes in early life (Hack et al. (1998) J Cell Biol 142: 1279). By 20 wk of age, these mice developed cardiomyopathy and died prematurely. Furthermore, apoptotic myonuclei were abundant in skeletal muscle lacking γ-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking γ-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. It was also shown that the loss of γ-sarcoglycan produced secondary reduction of beta- and delta-sarcoglycan with partial retention of α- and epsilon-sarcoglycan, indicating that beta-, γ-, and delta-sarcoglycan function as a unit. Since the other components of the cytoplasmic membrane complex were functional, the complex could be stabilized by the presence of a therapeutic of the invention.
[0221] In addition to animal models, certain cardiomyopathies in humans have been linked to mutations in dystrophin, dystroglycans or sarcoglycans. For example, dystrophin has been identified as the gene responsible for X-linked dilated cardiomyopathy (Towbin J. A. (1998) Curr Opin Cell Biol 10: 131, and references therein). In this case, the dystrophin gene contained a 5'-mutation which results in cardiomyopathy without clinically-apparent skeletal myopathy (Bies et al. (1997) J Mol Cell Cardiol 29: 3175.
[0222] Furthermore, cardiomyopathy was also found in subjects having Duchenne's Muscular Dystrophy (associated with a mutated dystrophin), or other types of muscular dystrophies, such as Limb Girdle Muscular Dystrophy. For example, dilated cardiomyopathy was present in one autosomal dominant case and in three advanced autosomal recessive or sporadic patients, of whom two were found to have alpha sarcoglycan deficiency. Two of these three patients and three other cases showed ECG abnormalities known to be characteristic of the dystrophinopathies. A strong association between the absence of alpha sarcoglycan and the presence of dilated cardiomyopathy was found. In six autosomal dominant cases there were atrioventricular (AV) conduction disturbances, increasing in severity with age and in concomitant presence of muscle weakness. Pacemaker implantation was necessary in certain of these patients (see van der Kooi (1998) Heart 79: 73).
[0223] Therapeutics of the invention can also be used to treat or prevent cardiomyopathy, e.g., dilated cardiomyopathy, of viral origin, e.g., resulting from an enterovirus infection, e.g., a Coxsackievirus B3. It has been shown that purified Coxsackievirus protease 2A cleaves dystrophin in vitro and during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function (Badorff et al. (1999) Nat Med 5: 320. Cleavage of dystrophin results in disruption of the dystrophin-associated glycoproteins α-sarcoglycan and betα-dystroglycan. Thus, cardiomyopathy could be prevented or reversed by administration of a therapeutic of the invention to a subject having been infected with a virus causing cardiomyopathy, e.g., by disruption of dystrophin or a protein associated therewith. Administration of the therapeutic could restabilize or reorganize the cytoplasmic membrane of affected cardiac cells.
[0224] Thus, the therapeutics of the invention can also be used to prevent or to treat smooth muscle disorders, such as cardiac myopathies, and to stop atrophy and/or necrosis of cardiac smooth muscle tissue. The treatment can also be used to promote survival of myocytes.
[0225] Neurological disorders that can be treated according to the methods of the invention include polymyositis, and neurogenic disorders. Another neurological disease that can be treated is Alzheimers' disease.
[0226] Other diseases that can be treated according to the methods of the invention include those in which the proteoglycan of the invention is present at abnormal levels, or has an abnormal activity, relative to that in normal subjects. For example, a disease or disorder could be caused by a lower level of biglycan, resulting in, e.g., unstable cytoplasmic membranes. Alternatively, a disease or disorder could result from an abnormally high level or activity of biglycan, resulting in, e.g., overstimulation of MuSK or over-aggregation of AChRs (see below).
[0227] Other diseases that may be treated according to methods disclosed herein are collagen VI-related disorders. For example, Bethlem's myopathy is caused, at least in part, by mutations in collagen VI genes. Collagen VI function is also compromised in Ullrich Congenital Muscular Dystrophy and Sorsby's fundus dystrophy. In certain embodiments, a collagen VI-related disorder may be treated by administering a biglycan therapeutic. In certain embodiments, a collagen VI-related disorder may be treated by administering a therapeutic comprising a polypeptide of a DAPC, such as a utrophin, a sarcoglycan or a portion thereof.
[0228] Yet other diseases or disorders that are within the scope of the invention include those that are associated with an abnormal interaction between a proteoglycan of the invention and another molecule (other than those of the DAPC or MuSK), e.g., a complement factor, such as C1q. For example, it has been shown that C1q interacts with biglycan (Hocking et al. (1996) J. Biol. Chem. 271: 19571). It is also known that binding of C1q to cell surfaces mediates a number of biological activities including enhancement of phagocytosis and stimulation of superoxide production. Thus, since biglycan binds to C1q, biglycan or another proteoglycan or core thereof, of the invention could be used to inhibit the binding of C1q to its receptor on cell surfaces to inhibit one or more of such biological activities. In addition, compounds of the invention which inhibit the interaction between C1q or other complement component and a cell surface can also be used to inhibit complement mediated necrosis of the cells and tissues containing such cells.
[0229] Also within the scope of the invention are methods for preventing or inhibiting infections of cells by microorganisms, e.g., viruses. For example, it has been shown that dystroglycan is a receptor via which certain microorganisms enter eukaryotic cells (Science (1998) 282: 2079). Thus, by administrating to a subject a therapeutic of the invention which occupies the site on dystroglycan molecules to which the microorganism binds, entering of the microorganism into the cell can be inhibited. This method can be used, e.g., to prevent or inhibit Lassa Fever virus and lymphocytic choriomeningitis virus (LCMV) infection, as well as infection by other arenaviruses, including Oliveros, and Mobala. Soluble alphα-dystroglycan was shown to block both LCMV and LFV infection (Science (1998) 282: 2079).
[0230] In addition to cell cultures, e.g., established from patients having, e.g., a muscular dystrophy, various animal models can be used to select the most appropriate therapeutic for treating a disease. In particular, to identify a therapeutic for use in preventing or treating a muscular dystrophy or cardiomyophaty associated with a mutated or absent DAPC component or, mice having mutated versions of these proteins, or having null mutations in the genes encoding these proteins, can be used. For example, mice having a disrupted sarcoglycan, such as delta-sarcoglycan, can be used. Such mice are described, e.g., Coral-Vazquez et al. (1999) Cell 98: 465. Alternatively, mice deficient in dystrophin (mdx mice), or in α- or γ-sarcoglycans can be used. Such mice have been described herein and in the literature. Additional mice can be made according to known methods in the art. In an illustrative embodiment to identify therapeutics, different therapeutics are administered to delta-sarcoglycan null mice, and the effect of the therapeutics are evaluated by studying cardiac function. Another animal model that can be used for this purpose is the cardiomyopathic hamster that does not express delta-sarcoglycan due to a genomic deletion. This rat is an animal model for autosomal recessive cardiomyopathy., and is further described in Sakamoto et al. FEBS Lett 1999 (1999) 44: 124.
V. Effective Dose and Administration of Therapeutic Compositions
[0231] The above-described diseases or disorders can be treated or ameliorated in a subject by administering to the subject a pharmaceutically efficient amount of a bigylcan therapeutic, collagen VI therapeutic or other therapeutic of the invention. Where the therapeutic is to be a biglycan therapeutic, depending on whether the disease is caused by higher levels or activity or by lower levels or activity of biglycan, an agonist or an antagonist biglycan therapeutic is administered to a subject having the disease. Although a person of skill in the art will be able to predict which therapeutic to administer for treating any of the diseases of the invention, tests can be performed to determine the appropriate therapeutic to administer. Such tests can use, e.g., animal models of the disease. Alternatively, in cases where diseases are due to a mutation in, e.g., biglycan or a collagen VI, in vitro tests can be undertaken to determine the effect of the mutation. This will allow the determination of what type of therapeutic should be administered to a subject having this type of mutation.
[0232] Another manner of administering a therapeutic of the invention to a subject is by preparing cells expressing and secreting the polypeptide or proteoglycan of interest, inserting the cells into a matrix and administering this matrix to the subject at the desired location. Thus, cells engineered in accordance with this invention may also be encapsulated, e.g. using conventional biocompatible materials and methods, prior to implantation into the host organism or patient for the production of a therapeutic protein. See e.g. Hguyen et al, Tissue Implant Systems and Methods for Sustaining viable High Cell Densities within a Host, U.S. Pat. No. 5,314,471 (Baxter International, Inc.); Uludag and Sefton, 1993, J Biomed. Mater. Res. 27(10):1213-24 (HepG2 cells/hydroxyethyl methacrylate-methyl methacrylate membranes); Chang et al, 1993, Hum Gene Ther 4(4):433-40 (mouse Ltk-cells expressing hGH/immunoprotective perm-selective alginate microcapsules; Reddy et al, 1993, J Infect Dis 168(4):1082-3 (alginate); Tai and Sun, 1993, FASEB J 7(11):1061-9 (mouse fibroblasts expressing hGH/alginate-poly-L-lysine-alginate membrane); Ao et al, 1995, Transplantation Proc. 27(6):3349, 3350 (alginate); Rajotte et al, 1995, Transplantation Proc. 27(6):3389 (alginate); Lakey et al, 1995, Transplantation Proc. 27(6):3266 (alginate); Korbutt et al, 1995, Transplantation Proc. 27(6):3212 (alginate); Dorian et al, U.S. Pat. No. 5,429,821 (alginate); Emerich et al, 1993, Exp Neurol 122(1):37-47 (polymer-encapsulated PC12 cells); Sagen et al, 1993, J Neurosci 13(6):2415-23 (bovine chromaffin cells encapsulated in semipermeable polymer membrane and implanted into rat spinal subarachnoid space); Aebischer et al, 1994, Exp Neurol 126(2):151-8 (polymer-encapsulated rat PC12 cells implanted into monkeys; see also Aebischer, WO 92/19595); Savelkoul et al, 1994, J Immunol Methods 170(2):185-96 (encapsulated hybridomas producing antibodies; encapsulated transfected cell lines expressing various cytokines); Winn et al, 1994, PNAS USA 91(6):2324-8 (engineered BHK cells expressing human nerve growth factor encapsulated in an immunoisolation polymeric device and transplanted into rats); Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry 18(5):935-46 (polymer-encapsulated PC12 cells implanted into rats); Kordower et al, 1994, PNAS USA 91(23):10898-902 (polymer-encapsulated engineered BHK cells expressing hNGF implanted into monkeys) and Butler et al WO 95/04521 (encapsulated device). The cells may then be introduced in encapsulated form into an animal host, preferably a mammal and more preferably a human subject in need thereof. Preferably the encapsulating material is semipermeable, permitting release into the host of secreted proteins produced by the encapsulated cells. In many embodiments the semipermeable encapsulation renders the encapsulated cells immunologically isolated from the host organism in which the encapsulated cells are introduced. In those embodiments the cells to be encapsulated may express one or more proteoglycans of the host species and/or from viral proteins or proteins from species other than the host species.
[0233] Alternatively, the therapeutic is a nucleic acid encoding the core of a suitable proteoglycan or a polypeptide disclosed herein. Thus, a subject in need thereof, may receive a dose of viral vector encoding the protein of interest, which may be specifically targeted to a specific tissue, e.g., a dystrophic tissue. The vector can be administered in naked form, or it can be administered as a viral particle (further described herein). For this purpose, various techniques have been developed for modification of target tissue and cells in vivo. A number of viral vectors have been developed, such as described above, which allow for transfection and, in some cases, integration of the virus into the host. See, for example, Dubensky et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science 243, 375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 and Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. The vector may be administered by injection, e.g. intravascularly or intramuscularly, inhalation, or other parenteral mode. Non-viral delivery methods such as administration of the DNA via complexes with liposomes or by injection, catheter or biolistics may also be used.
[0234] In yet another embodiment, cells are obtained from a subject, modified ex vivo, and introduced into the same or a different subject. Additional methods of administration of the therapeutic compounds are set forth below.
[0235] A. Toxicity:
[0236] Toxicity and therapeutic efficacy of compounds of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining. The Ld50 (The Dose Lethal To 50% Of The Population) And The Ed50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
[0237] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. In particular, where the therapeutic is administered for potentiating AChR aggregation, it is desirable to establish the dose that will result in stimulation, if desired, or inhibition, if desired. Tests can then be continued in medical tests. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
[0238] B. Pharmaceutical Compositions:
[0239] Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
[0240] For such therapy, the compounds of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
[0241] For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
[0242] Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0243] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0244] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
[0245] In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0246] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. in addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.
[0247] In clinical settings, a gene delivery system for the therapeutic gene encoding a proteoglycan of the invention can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3054-3057). A gene encoding a proteoglycan of the invention can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).
[0248] A preferred mode of delivering DNA to muscle cells include using recombinant adeno-associated virus vectors, such as those described in U.S. Pat. No. 5,858,351. Alternatively, genes have been delivered to muscle by direct injection of plasmid DNA, such as described by Wolff et al. (1990) Science 247:1465-1468; Acsadi et al. (1991) Nature 352:815-818; Barr and Leiden (1991) Science 254:1507-1509. However, this mode of administration generally results in sustained but generally low levels of expression. Low but sustained expression levels are expected to be effective for practicing the methods of the invention.
[0249] The pharmaceutical preparation of the gene therapy construct or compound of the invention can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle or compound is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
[0250] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
VI. Screening Methods
[0251] The invention further provides methods for identifying agents, e.g., bigylcan therapeutics or collagen VI therapeutics, which optionally modulate membrane integrity, in particular, by modulating DAPC stability, and agents which modulate neuromuscular junction formation, such as by modulating postsynaptic differentiation. Thus, in certain embodiments, the invention provides methods for identifying agents which modulate the activity of a biglycan or collagen VI, and preferably agents that modulate the interaction (whether direct or indirect) between collagen VI and other DAPC components.
[0252] Accordingly, the invention provides screening methods for identifying therapeutics. A therapeutic of the invention can be any type of compound, including a protein, a peptide, a proteoglycan, a polysaccharide, a peptidomimetic, a small molecule, and a nucleic acid. A nucleic acid can be, e.g., a gene, an antisense nucleic acid, a ribozyme, or a triplex molecule.
[0253] Preferred agonists include compounds which mimic at least one biological activity of a biglycan or collagen VI or other DAPC component, e.g., the capability to bind to one or more components of a DAPC, such as alphα-dystroglycan, biglycan or collagen VI, or the capability to stimulate MuSK phosphorylation and/or AChR aggregation. Other preferred agonists include compounds which are capable of increasing the production of the proteoglycan of the invention in a cell, e.g., compounds capable of upregulating the expression of the gene encoding the proteoglycan, and compounds which are capable of enhancing an activity of a proteoglycan of the invention, and/or the interaction of a proteoglycan of the invention with another molecule, such as a component of a DAPC or MuSK.
[0254] Preferred antagonists include compounds which are dominant negative proteins, which, e.g., are capable of binding to α-sarcoglycan, but not to stabilize DAPCs, such as by competing with the endogenous proteoglycan of the invention. Other preferred antagonists include compounds which decrease or inhibit the production of a proteoglycan of the invention in a cell and compounds which are capable of downregulating expression of a gene encoding a proteoglycan of the invention, and compounds which are capable of downregulating an activity of a proteoglycan of the invention and/or its interaction with another molecule, such as α-sarcoglycan. In another preferred embodiment, an antagonist is a modified form of an alphα-dystroglycan or other molecule capable of binding to the wildtype proteoglycan of the invention, which is capable of interacting with the proteoglycan of the invention, but which does not have biological activity, e.g., which does not stabilize DAPCs.
[0255] The invention also provides screening methods for identifying therapeutics which are capable of binding to a proteoglycan of the invention, e.g., a wild-type proteoglycan of the invention or a mutated form thereof, and thereby modulate the a biological activity of a proteoglycan of the invention, or degrades, or causes the proteoglycan of the invention to be degraded. For example, such a therapeutic can be an antibody or derivative thereof which interacts specifically with a proteoglycan of the invention (either wild-type or mutated).
[0256] Thus, the invention provides screening methods for identifying agonist and antagonist compounds, comprising selecting compounds which are capable of interacting with a proteoglycan of the invention or with a molecule interacting with a proteoglycan of the invention, such a component of a DAPC or MuSK, and/or compounds which are capable of modulating the interaction of an a proteoglycan of the invention with another molecule, such as a component of a DAPC or MuSK. In general, a molecule which is capable of interacting with a proteoglycan or collagen VI of the invention is referred to herein as a "candidate therapeutic binding partner" or "CT-binding partner" and can be a component of a DAPC, e.g., a dystroglycan or a sarcoglycan, or MuSK.
[0257] The compounds of the invention can be identified using various assays depending on the type of compound and activity of the compound that is desired. Set forth below are at least some assays that can be used for identifying therapeutics of the invention. It is within the skill of the art to design additional assays for identifying therapeutics.
[0258] A. Cell-Free Assays
[0259] Cell-free assays can be used to identify compounds which are capable of interacting with a proteoglycan of the invention or binding partner thereof, to thereby modify the activity of the proteoglycan of the invention or binding partner thereof. Such a compound can, e.g., modify the structure of a proteoglycan of the invention or binding partner thereof and thereby affect its activity. Cell-free assays can also be used to identify compounds which modulate the interaction between a proteoglycan of the invention and a PT-binding partner, such as a component of a DAPC. In a preferred embodiment, cell-free assays for identifying such compounds consist essentially in a reaction mixture containing a proteoglycan of the invention, and a test compound or a library of test compounds with or without a binding partner. A test compound can be, e.g., a derivative of a CT-binding partner, e.g., an biologically inactive target peptide, or a small molecule.
[0260] These assays can be performed with a complete proteoglycan molecule of the invention. Alternatively, the screening assays can be performed with potions thereof, such as the core only, one or more LLR domains, the glycosamino glycan chains only, or portions thereof, or combinations of these portions. These can be prepared as set forth supra.
[0261] Accordingly, one exemplary screening assay of the present invention includes the steps of contacting a biglycan or collagen VI polypeptide of the invention or functional fragment thereof or a binding partner with a test compound or library of test compounds and detecting the formation of complexes. For detection purposes, the molecule can be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker. Interaction of a test compound with a proteoglycan of the invention or fragment thereof or CT-binding partner can then be detected by determining the level of the two labels after an incubation step and a washing step. The presence of two labels after the washing step is indicative of an interaction.
[0262] An interaction between molecules can also be identified by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon. Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants. In one embodiment, a library of test compounds can be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell. A solution containing the proteoglycan of the invention, functional fragment thereof, analog or CT-binding partner is then flown continuously over the sensor surface. A change in the resonance angle as shown on a signal recording, indicates that an interaction has occurred. This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia.
[0263] Another exemplary screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) a proteoglycan of the invention, (ii) a CT-binding partner (e.g., α-sarcoglycan), and (iii) a test compound; and (b) detecting interaction of the proteoglycan of the invention and the CT-binding protein. The proteoglycan of the invention and CT-binding partner can be produced recombinantly, purified from a source, e.g., plasma, or chemically synthesized, as described herein. A statistically significant change (potentiation or inhibition) in the interaction of the proteoglycan of the invention and CT-binding protein in the presence of the test compound, relative to the interaction in the absence of the test compound, indicates a potential agonist (mimetic or potentiator) or antagonist (inhibitor) of a bioactivity for the test compound. The compounds of this assay can be contacted simultaneously. Alternatively, a proteoglycan of the invention can first be contacted with a test compound for an appropriate amount of time, following which the CT-binding partner is added to the reaction mixture. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. In the control assay, isolated and purified proteoglycan of the invention or binding partner is added to a composition containing the CT-binding partner or proteoglycan of the invention, and the formation of a complex is quantitated in the absence of the test compound.
[0264] Complex formation between a proteoglycan of the invention and a CT-binding partner may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled proteoglycans of the invention or CT-binding partners, by immunoassay, or by chromatographic detection.
[0265] Typically, it will be desirable to immobilize either the proteoglycan of the invention or its binding partner to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a proteoglycan of the invention to a CT-binding partner, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase/ACE-2 (GST/proteoglycan of the invention) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the PT-inding partner, e.g. an 35S-labeled CT-binding partner, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of proteoglycan of the invention or CT-binding partner found in the bead fraction is quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
[0266] Other techniques for immobilizing proteins on matrices are also available for use in the subject assay. For instance, either the proteoglycan of the invention or its cognate binding partner can be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated proteoglycan molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the proteoglycan of the invention can be derivatized to the wells of the plate, and the proteoglycan of the invention trapped in the wells by antibody conjugation. As above, preparations of a CT-binding protein and a test compound are incubated in the proteoglycan presenting wells of the plate, and the amount of complex trapped in the well can be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the CT-binding partner, or which are reactive with protein and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity. In the instance of the latter, the enzyme can be chemically conjugated or provided as a fusion protein with the CT-binding partner. To illustrate, the CT-binding partner can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-1-napthol. Likewise, a fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
[0267] For processes which rely on immunodetection for quantitating one of the proteins trapped in the complex, antibodies against the proteoglycan of the invention, can be used. Alternatively, the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the sequence of the core of the proteoglycan of the invention, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, NJ).
[0268] Cell-free assays can also be used to identify compounds which interact with a proteoglycan of the invention and modulate an activity of a proteoglycan of the invention. Accordingly, in one embodiment, a proteoglycan of the invention is contacted with a test compound and the catalytic activity of the proteoglycan of the invention is monitored. In one embodiment, the ability of the proteoglycan of the invention to bind to a binding partner is determined. The binding affinity of a proteoglycan of the invention to a binding partner can be determined according to methods known in the art.
[0269] B. Cell Based Assays
[0270] Cell based assays can be used, in particular, to identify compounds which modulate expression of a gene encoding a proteoglycan of the invention, modulate translation of the mRNA encoding a proteoglycan of the invention, modulate the posttranslational modification of the core protein of the proteoglycan, or which modulate the stability of the mRNA or protein. Accordingly, in one embodiment, a cell which is capable of producing a proteoglycan of the invention, e.g., a muscle cell, is incubated with a test compound and the amount of proteoglycan of the invention produced in the cell medium is measured and compared to that produced from a cell which has not been contacted with the test compound. The specificity of the compound vis a vis the proteoglycan of the invention can be confirmed by various control analysis, e.g., measuring the expression of one or more control genes.
[0271] Cell based assays can also rely on a reporter gene system detecting whether two molecules interact or not, e.g., the classic two hybrid system, that can be conducted in yeast or in mammalian cells.
[0272] Compounds which can be tested include small molecules, proteins, and nucleic acids. In particular, this assay can be used to determine the efficacity of antisense molecules or ribozymes that bind to RNA encoding the proteoglycan of the invention.
[0273] In another embodiment, the effect of a test compound on transcription of a gene encoding a proteoglycan is determined by transfection experiments using a reporter gene operatively linked to at least a portion of the promoter of a gene encoding a proteoglycan of the invention. A promoter region of a gene can be isolated, e.g., from a genomic library according to methods known in the art. Promoters of genes encoding proteoglycans, e.g., biglycan, are publically available, e.g, from GenBank. The reporter gene can be any gene encoding a protein which is readily quantifiable, e.g, the luciferase or CAT gene, well known in the art.
[0274] This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
[0275] C. Assays for Identifying Compounds which Modulate Phosphorylation
[0276] Biglycan was shown to bind and activate MuSK and induce phosphorylation of α-sarcoglycan. Accordingly, compounds which stimulate phosphorylation of such substrates may exercise at least part of the activity of biglycan in stabilizing muscle cell membranes or of potentiating postsynaptic membranes. Thus, also within the scope of the invention are methods for identifying such compounds. In one embodiment, the method comprises contacting a cell, e.g., a muscle cell, with a compound, and monitoring the level of phosphorylation of a DAPC component, such as α-sarcoglycan, or activation of MuSK, wherein a higher level of phosphorylation relative to that in an untreated cell indicates that the compound stimulates phosphorylation. Such assays can also be conducted in vitro using cell extracts or purified proteins. For example, the method may comprise contacting a purified sarcoglycan or MuSK and a cell extract from biglycan-activated cells (i.e., cells contacted with biglycan) or a kinase in the presence of a test compound, and monitoring whether the presence of the test compound prevents or stimulates phosphorylation.
VII. Kits of the Invention
[0277] The invention provides kits for diagnostic tests or therapeutic purposes.
[0278] Kits for therapeutic or preventive purposes can include a therapeutic and optionally a method for administering the therapeutic or buffer necessary for solubilizing the therapeutic.
[0279] The present invention is further illustrated by the following examples which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications as cited throughout this application are hereby expressly incorporated by reference.
[0280] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
VIII. Examples
Example 1
Characterization of a Dystroglycan-Binding Protein, DAG-125
[0281] This Example describes the identification of a dystroglycan-binding protein, termed DAG-125.
[0282] In order to identify novel dystroglycan binding partners, a ligand blot overlay assay, was developed as follows. Postsynaptic and non-synaptic membrane fractions from Torpedo electric organ were prepared as previously described (Bowe, et al. (1994) Neuron. 12: 1173). All handling of membranes and protein was performed at 4° C.
[0283] Membrane proteins were separated by SDS-PAGE (5-15% gradient gel), and transferred to nitrocellulose. To detect dystroglycan binding proteins, the nitrocellulose was rinsed and blocked for 3 hr in Hank's Balanced Salt Solution containing 1 mM CaCl2, 1 mM MgCl2, 1% bovine serum albumin, 1% Nonfat Dry Milk, 1 mM DTT, 10 mM HEPES, pH 7.4, and was then incubated overnight in the same buffer containing 35S-methionine-labelled dystroglycan fragments produced by in vitro transcription/translation as follows.
[0284] DNA fragments encoding DG1-891 and DG345-891 (human alpha-dystroglycan sequence is described, e.g., in Ibraghimov-BeskrovnayaHum (1993) Mol Genet 2: 1651) were cloned in the in vitro expression vector pMGT developed by A. Ahn (Ahn and Kunkel (1995) J Cell Biol. 128: 363). Additional in vitro expression plasmids used in this study (including DG1-750, DG776-891, and DG345-653) were prepared by PCR-based subcloning of these inserts. The PCR primers included restriction sites for religation into the EcoRI site of pMGT. Dystroglycan protein fragments were generated by in vitro transcription/translation using the Promega TNT T7 coupled reticulocyte system as per the manufacturer's instructions. For protein to be used in ligand blot overlay assay, the reaction mixture contained 35S-methionine (with no unlabeled methionine). After incubation for 2 hr, the reaction mixture was passed over Bio-Spin desalting columns (Bio-Rad, Hercules, Calif.) to remove unincorporated amino acids and salts.
[0285] After incubation of the blots with the in vitro translated proteins, the blots were rinsed and dried and bound dystroglycan fragments were visualized by autoradiography. To detect dystroglycan present in the SDS-PAGE sample, an agrin blot overlay assay was performed essentially as described in O'Toole, et al. (1996) PNAS 93:7369. Briefly, the nitrocellulose was rinsed and blocked for 3 hr in HEPES-buffered Minimum Essential Medium supplemented with 1% bovine serum albumin and 10% horse serum. It was then incubated for 4 hr in this buffer containing recombinant rat agrin (isoform A0B0, prepared as described in O'Toole et al., supra), followed by a second layer containing 1 μg/ml anti-agrin antibody 125I-Mab-131 (Stressgen Laboratories, Victoria, BC). Bound anti-agrin antibody was visualized by autoradiography.
[0286] The results are shown in FIG. 2. Lanes 1 and 2 indicate that certain fragments of dystroglycan bound to an about 125 kD, highly glycosylated polypeptide, which was termed DAG-125 (for "Dystroglycan-Associated Glycoprotein, 125 kDa"). As shown in FIG. 2A, the extracellular domain of dystroglycan (lane 1: DG1-750) bound to DAG-125, while the intracellular portion of dystroglycan (lane 2: DG776-891) did not.
[0287] Lanes 3 and 4 of FIG. 2 show that DAG-125 is enriched in synaptic as compared to non-synaptic membranes.
[0288] To solubilize DAG-125, synaptic membranes were centrifuged at 100,000×g for 1 hour (hr) and resuspended in ddH2O. The pH was adjusted to 11.0 or 12.0 (as indicated) with NaOH and the membranes stirred for 1 hr. Insoluble material was removed by centrifugation at 100,000×g for 1 hr. The alkaline extract was neutralized with 10 mM Tris HCl and adjusted to pH 7.4. DAG-125 remained soluble under these conditions as determined by resistance to pelleting during a second centrifugation. Lanes 5-7 of FIG. 2 show that DAG-125 is a peripheral membrane protein that can be extracted from the synaptic membrane by alkaline treatment. Synaptic membranes were extracted at pH 12 and the insoluble (lane 6) and soluble fraction (lane 7) were analyzed. Greater than 90% of DAG-125 is solubilized by pH 12.0 treatment. Thus, DAG-125 is likely to be a peripheral membrane protein, since it is removed from the membranes by alkaline-treatment.
Example 2
Association Between α-Dystroglycan and DAG-125
[0289] This Example demonstrates that DAG-125 associates with in vitro-translated α-dystroglycan, bacterially produced GST-α-dystroglycan fusion protein and native α-dystroglycan in solution.
[0290] DAG-125 was solubilized by alkaline-treatment, and neutralized, as described above, and incubated with column matrices and recombinant or native dystroglycan as indicated in FIG. 3. The input material and eluates from the beads were analyzed by ligand blot overlay assay for the presence of DAG-125 (35S-DG345-653 as probe) or native α-dystroglycan (agrin overlay, see Example 1).
[0291] FIG. 3A shows DAG-125 incubated with goat anti-mouse Ig-conjugated agarose beads in the presence or absence of in vitro translated dystroglycan polypeptide (DG345-750) and/or anti-dystroglycan monoclonal antibody (NCL-β-DG; Novocastra, Newcastle-on-Tyne, UK). The results indicate that DAG-125 co-precipitated with dystroglycan plus anti-dystroglycan antibody (lane 5), but was not precipitated in the absence of either or both (lanes 2-4). Thus, DAG-125 binds to in vitro translated dystroglycan peptide DG345-750.
[0292] FIG. 3B shows DAG-125 incubated with glutathione-sepharose beads that had been pre-incubated with either bacterially produced GST or a bacterially produced GST-dystroglycan fusion protein (GST-DG345-653). A fusion protein of glutathione S-transferase (GST) and amino acids 345-653 of dystroglycan was produced by using PCR-based subcloning to introduce dystroglycan coding sequence into the bacterial protein expression vector pGEX-1 T (Pharmacia, Piscataway, N.J.). The resulting bacterial expression plasmid, pGST-DG345-653, was then introduced into the E. coli strain BL21 and expressed fusion protein recovered from the cytoplasmic fraction as per manufacturer's instructions. Control protein (GST) was obtained using pGEX-1 T. The results show that DAG-125 was co-precipitated with the dystroglycan fusion protein (lane 3), but not with GST alone (lane 2). Thus, DAG-125 binds to alphα-dystroglycan peptide 345-653 produced in bacteria.
[0293] FIG. 3C shows DAG-125 and native α-dystroglycan. Alkaline extracts of Torpedo electric organ membranes contain both DAG-125 and α-dystroglycan. This extract was applied to agarose columns conjugated to either control antibody or to an anti-Torpedo dystroglycan monoclonal antibody (MAb3B3; Bowe, M. A., et al. (1994) Neuron. 12: 1173). The results show that native α-dystroglycan and DAG-125 were co-precipitated by the anti-Torpedo dystroglycan antibody, Mab3B3, (lanes 3 and 6), but not by control antibody (lanes 2 and 5). Western blots indicate that Mab3B3 does not recognize DAG-125 (see Bowe, M. A., et al., 1994, Neuron. 12: 1173-1180).
[0294] Thus, FIG. 3 shows that DAG-125 co-precipitates with in vitro-translated alphα-dystroglycan, bacterially produced GST-alphα-dystroglycan protein, and with native alphα-dystroglycan.
Example 3
Localization of the DAG-125 Binding Domain of α-Dystroglycan
[0295] This Example describes that the DAG-125 binding domain of α-dystroglycan is contained in an approximately 150 amino acid carboxyl-terminal domain of the protein.
[0296] In order to determine the region of α-dystroglycan that interacts with DAG-125, a panel of dystroglycan fragments were prepared by in vitro translation (FIG. 4) and the ability of each to bind DAG-125 was tested using the ligand blot overlay assay. FIG. 4, which show the results, indicates that DAG-125 binds to the carboxyl-terminal one-third of α-dystroglycan. A small contribution from the middle third of α-dystroglycan is also possible. The ectodomain of β-dystroglycan does not appear to contribute to binding of DAG-125. Moreover, these fragments were produced under conditions in which the polypeptides are not glycosylated. Therefore, carbohydrate side chains on dystroglycan are not necessary for its binding to DAG-125.
[0297] Thus, the major binding domain is contained in about 150 amino acid region of dystroglycan. The location of this domain and the lack of a carbohydrate requirement indicate that α-dystroglycan's binding site for biglycan is distinct from that mediating association with agrin, laminin, and perlecan.
Example 4
Identification of DAG-125 as Biglycan or a Proteoglycan Related Thereto
[0298] This Examples demonstrates that DAG-125 is biglycan or a protein related thereto.
[0299] It was found that DAG-125 co-purified with postsynaptic membranes, but that, however, it was insoluble in all non-ionic detergents tested including Triton X-100 and n-octyl-β-D-glucopyranoside, both of which efficiently extract α/β-dystroglycan from these membranes (Bowe, et al. (1994) Neuron. 12: 1173; Deyst, et al. (1995) J Biol Chem. 270: 25956-9). Even without detergent, about 50% of DAG-125 could be extracted at pH 11 and near-complete solubilization was achieved by a short pH 12 treatment (see FIG. 2A). Importantly, DAG-125 remained soluble when returned to neutral pH. Based upon these properties and the findings that DAG-125 binds to both heparin and chondroitin sulfate columns, the following purification protocol was developed.
[0300] Postsynaptic-rich membrane fractions were first pre-extracted with 25 mM n-octyl- -D-glucopyranoside to remove detergent-soluble proteins. DAG-125 was then solubilized by alkaline extraction (pH 12.0), as described in Example 1. The alkaline extract was diluted in SEN Buffer (20 mM Tris HCl, 100 mM NaCl, 23 μg/ml aprotinin, 0.5 μg/ml leupeptin, 5 mM benzamidine, 0.7 μg/ml pepstatin A, 1 mM phenylmethylsulfonylflouride, 0.02% azide, 0.1% Tween 20, pH 7.6) and recentrifuged to remove any proteins precipitating upon neutralization. The extract remained in SEN Buffer for the remainder of the purification, with only the NaCl concentration changed as indicated. The extract was passed over a MAb3B3 column (Bowe, et al. (1994) Neuron. 12: 1173) to remove α-dystroglycan. The MAb3B3 column flow-through was passed over a combined, non-DAG-125-binding lectin-agarose column (peanut agglutinin and ulex europaeus agglutinin I, Vector Labs, Burlingame, Calif.) as a second pre-clear. The flow-through was next applied to a column of chondroitin sulfate-agarose (CS-agarose). The CS-agarose column was prepared by coupling chondroitin sulfate B (Sigma, St. Louis, Mo.; #C-3788) to -aminohexyl-agarose (Sigma) activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (Sigma). After incubation with the lectin column flow-through, the CS column was washed extensively and eluted with a 0.1-2.0 M NaCl gradient. DAG-125 eluted in 0.3-0.65 M NaCl. These fractions were pooled, diluted to 0.3 M NaCl, and applied to a heparin-agarose column (Sigma #H-0402). The column was washed and eluted with a 0.3-2 M NaCl gradient. DAG-125 eluted in 0.6-0.85 M NaCl. These fractions were pooled, concentrated by ethanol precipitation (final purity of DAG-125 of about 30%), redissolved in SDS-PAGE sample buffer, separated on a 5-15% gradient gel, and transferred to a PVDF membrane. A portion of the PVDF membrane was analyzed for DAG-125 by blot overlay and the remainder was transiently stained with Ponceau S. Two regions ("U" and "L"; see FIG. 5A) of the DAG-125 band on the Ponceau stained membrane were excised and digested with trypsin. The released peptides were analyzed by HPLC using a C8 column and UV detection. The column profiles were virtually identical, indicating that the polydisperse band is due to the presence of a single, heterogeneously glycosylated protein.
[0301] Three peptides from the trypsin digest were collected as fractions from the HPLC analysis and subjected to automated Edman degradation, as described previously (Bowe, et al. (1994) Neuron. 12: 1173). The sequences obtained were compared to public databases. The alignment of the Torpedo DAG-125 peptides to the deduced sequence of human biglycan (amino acids 241-249; 258-266; and 330-348) is shown in FIG. 5B. Human biglycan is described in Fisher et al. (1989), infra) and its amino acid sequence is set fort in SEQ ID NO: 9. All DAG-125 peptide fragments were highly homologous to mammalian biglycan, with an overall 76% identity (FIG. 5B). Thus, DAG-125 is a Torpedo orthologue of mammalian biglycan or a close homolog thereof.
[0302] Human biglycan, produced in the vaccinia system, as described below, was also shown to bind to α-dystroglycan. The binding was less strong than with Torpedo DAG-125, probably reflecting the fact that the biglycan produced in this system is a mixture of core biglycan and proteoglycan biglycan. However, this further supports that Torpedo orthologue of mammalian biglycan or a close homolog thereof.
[0303] The domain structure of human biglycan is shown in FIG. 5C. Biglycan is one of a family of small leucine-rich repeat proteins (Hocking et al. (1998) Matrix Biol. 17: 1). It consists of a pre-pro-peptide that is not present in the mature polypeptide. This domain is followed by a short unique sequence with two chondroitin sulfate attachment sites (shown as stacked beads in the Figure). There are two pairs and one pair of disulfide-linked cysteines at the amino and carboxyl-terminal domains, respectively. Finally, the bulk of the protein is comprised of 10 (or 11 depending upon the classification of the region within the carboxyl-terminal cysteine pair) leucine-rich repeats. The position of the three Torpedo peptides relative to the human sequence is indicated by horizontal lines.
Example 5
Chondroitin Sulfate Chains of Biglycan are Necessary for Binding of Biglycan to α-Dystroglycan
[0304] Mammalian biglycan is often substituted with chondroitin sulfate. To determine if Torpedo biglycan is also a chondroitin sulfate proteoglycan and whether glycosylation is important for its binding to α-dystroglycan, DAG-125 was digested with various glycosidases and glycosaminoglycanases and the products were analyzed by α-dystroglycan ligand blot overlay with 35S-DG345-653.
[0305] Enzyme treatments were carried out on alkaline-extracted Torpedo electric organ synaptic membrane proteins at 37° C. overnight. Enzymes, final concentration, supplier and catalog numbers are listed in Table I. All reactions were performed in the protease inhibitors present in SEN Buffer, with the addition of 1 mM EDTA, 10 mM N-ethylmaleimide, and 0.8% mouse serum albumin. Chondroitinases (all forms) were buffered with 100 mM Tris-acetate (pH 8.0). Hyaluronidase and keratanase were buffered with 50 mM sodium acetate (pH 5.0). Heparinases (I, II, and III), chondro-4-sulfatase and chondro-6-sulfatase were buffered with 10 mM NaPO4 (pH 7.4). N-Glycanase, O-glycanase, neuraminidase, α-N-acetylgalactosaminidase, β-N-acetylglucoasaminidase were buffered with 50 mM Tris HCl (pH 7.3). Control treatments included buffers and protease inhibitors without added enzymes.
[0306] The results, are shown in FIG. 6 and in Table I.
TABLE-US-00003 TABLE I Inhibit Enzyme Conc Enzyme Binding? (Units/mL) Source Cat. # Chondroitinase ABC + 0.5 Sigma C-2905 Chondroitinase ABC + - 0.5 Sigma C-2905 5 mM ZnCl2 Chondroitinase ABC, + 0.5 Sigma C-3667 Protease-free Chondroitinase ABC, + 0.5 Roche 1080717 Protease-free Chondroitinase AC + 0.5 Sigma C-2780 Chondroitinase B +/- 25 Sigma C-8058 Heparinase I - 25 Sigma H-2519 Heparinase II - 5 Sigma H-3812 Heparinase III - 5 Sigma H-8891 (Heparitinase) Chondro-4-sulfatase +/- 0.5 Sigma C-2655 Chondro-6-sulfatase - 0.5 Sigma C-2655 Keratanase - 0.02 Roche 982954 α-N-acetyl- - 2 Sigma A-9763 galactosaminidase β-N-acetyl- - 8 Sigma A-2264 glucoasaminidase N-Glycanase - 15 Genzyme N-Gly-1 O-Glycanase - 0.03 Genzyme B2950 Neuraminidase - 1 Genzyme NSS-1
[0307] The results indicate that removal of chondroitin sulfate side chains abolished the binding to α-dystroglycan. Chondroitinase B (specific for dermatan sulfate) had a much smaller effect compared to chondroitinases which removed chondroitin sulfate A and C. No other glycosidase or glycosaminoglycanase treatment had a detectable effect on α-dystroglycan binding (see Table I). Several lines of evidence indicate that the effects of chondroitinase digestion are due to chondroitinase activity and not to contaminating proteases: 1) the digestions were performed in a cocktail of protease inhibitors; 2) the same result was seen with four different preparations of chondroitinase, including two which had been affinity purified to remove proteases; and 3) the effect was prevented by addition of 5 mM Zn2+, an inhibitor of chondroitinase but not of proteases.
[0308] To further investigate the binding properties of biglycan, the binding of α-dystroglycan to biglycan derived from a variety of sources, as well as to decorin, a small leucine-rich proteoglycan that is about 50% identical to biglycan, were investigated.
[0309] Biglycan (or decorin) were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining for protein (lanes 1-5 of FIG. 7) or blot overlay assay for dystroglycan binding (lanes 6-10 of FIG. 7): lanes 1, 6: alkaline extract of Torpedo synaptic membranes (1 μg total protein, of which biglycan is estimated to be <2%); lanes 2, 7: lysate of non-induced bacteria; lanes 3, 8: lysate of induced bacteria expressing recombinant human biglycan (QE-Bgn; prominent band at ˜37 kD--arrow); lanes 4, 9: biglycan purified from bovine articular cartilage (4 μg; Sigma); lanes 5, 10: decorin purified from bovine articular cartilage (4 μg; Sigma). The results indicate that biglycan present in electric organ binds dystroglycan much more strongly then biglycan or decorin purified from articular cartilage (compare Coomassie staining to dystroglycan overlay).
[0310] The recombinant human biglycan was produced as follows. P16, a cloning plasmid consisting of Bluescript containing a cDNA encoding human biglycan (SEQ ID NO: 9) was provide by Larry Fisher (National Institute of Dental Research, National Institutes of Health) (Fisher et al. (1989), supra). The sequence encoding the mature secreted peptide (amino acids 1-343) was amplified by PCR and subcloned into the bacterial expression vector pQE9 (Qiagen, Valencia, Calif.). The resulting plasmid, pQE-biglycan, adds the sequence MRGSHHHHHHGS (SEQ ID NO: 10) to the amino terminus Recombinant protein was produced in E. coli strain M15[pREP4]. Uninduced bacteria provide control protein. Induced or non-induced bacteria were isolated by centrifugation and resuspended in SDS-PAGE sample buffer for analysis by ligand blot overlay. Thus, bacterially-expressed biglycan, which contains no chondroitin sulfate side chains, did not bind α-dystroglycan (FIG. 7), consistent with a requirement for chondroitin sulfate chains. Biglycan purified from articular cartilage bound α-dystroglycan poorly, even at >100-fold higher loading than that used for Torpedo biglycan analysis. These findings indicate that specific chondroitin sulfate chains are required to mediate α-dystroglycan binding to biglycan.
[0311] Thus, biglycan from Torpedo synaptic membranes is substituted with chondroitin sulfate chains, which are predominantly chondroitin sulfate A and/or C, and chondroitin sulfate substitution of biglycan is necessary for binding to dystroglycan.
Example 6
Biglycan Binds to Sarcoglycan Components
[0312] This Example describes that biglycan core binds to α- and to gamma sarcoglycans and that biglycan proteoglycan also binds to γ-sarcoglycan, and that decorin failed to bind to any of the sarcoglycans (no detectable level of binding was observed).
[0313] The binding of biglycan and decorin to the different components of sarcoglycan of the DAPC was investigated by overlay assay using recombinantly produced human sarcoglycans, on biglycan proteoglycan (core and side chains), biglycan core (no side chains), decorin proteoglycan (core and side chains), decorin core (no side chains), a hybrid between biglycan and decorin core (the "hybrid" with side chains), and Torpedo electric organ membrane fraction (TEOM). The hybrid contained the first 30 amino acids of human biglycan (cysteine rich domain) and the remaining portion of the biglycan molecule was swapped with that of decorin. The sarcoglycans were produced by in vitro transcription and translation using a Promega TNT kit, as described in Ahn and Kunkel (1995) J. Cell Biol. 128: 363. The biglycan and decorin core polypeptide and proteoglycan were produced recombinantly by vaccinia-virus infection of rat osteosarcoma cells, as described in Hocking et al. (1996) J. Biol. Chem. 271:19571. Briefly, the cDNA sequence encoding the mature core protein of human biglycan ligated to a polyhistidine fusion cassette under the control of T7 promoter was inserted into the pBGN4 vector. An encephalomyocarditis virus untranslated region was inserted downstream of the T7 promoter to facilitate cap-independent ribosome binding and thereby increases translation efficiency up to 10-fold. The fusion cassette encodes the canine insulin signal sequence (INS), six consecutive histidine residues (POLYHIS), and the factor Xa recognition site (Xa). A recombinant vaccina virus, vBGNA, encoding the T7 regulated BGN4 construct, was generated by a homologus recombination event between wild-type vaccinia virus and thymidine kinase flanking sequences in the plasmid, pBGN4. There are two extra amino acids between the polyhistidine sequence and the Factor Xa site and two extra amino acids between the Factor Xa site and the start of the mature core protein sequence of biglycan. Thus, the vector contains from 5' to 3': EMC UTR-INS-POLYHIS-[Glu-Ser]-Xa-[Leu-Glu]-mature biglycan devoid of the biglycan signal sequence and propeptide sequence). The biglycan that is produced from this system is a mixture containing proteoglycan biglycan and biglycan devoid of glycaosaminoglycan chains ("core biglycan").
[0314] The overlay assays were preformed as described above for DAG-125.
[0315] The results, which are shown as FIGS. 8 A-C, indicate the following: α-sarcoglycan binds to biglycan core and to the hybrid; γ-sarcoglycan binds to biglycan core, to biglycan proteoglycan and very weakly to the hybrid; and δ-sarcoglycan binds to biglycan core very weakly.
[0316] Thus, biglycan binds to -sarcoglycan via its core peptide. Furthermore, since the hybrid binds to -sarcoglycan, but that decorin does not bind to it, binding of biglycan to α-sarcoglycan occurs through the N-terminal 30 amino acids of biglycan, i.e., the region that includes the cysteine-rich region, but no leucine-rich repeats. In addition, the results indicate that glycosylation of sarcoglycan is not necessary for its binding to biglycan.
[0317] Human biglycan was also shown to bind to native α- and γ-sarcoglycan in solution. This was demonstrated by isolating native human α- and γ-sarcoglycan by detergent extraction of cultured mouse myotubes, incubating the extracts with recombinant human core biglycan prepared as described above, and then immumoprecipitating the resulting complexes were then immunoprecipitated with antibodies to α-sarcoglycan (vector laboratories). The immunoprecipitates were then resolved by sds-polyacrylamide gel electrophoresis and western blotted with antibodies to biglycan. The anti-biglycan antibody was raised against a bacterially-produced biglycan fusion protein. The results, which are shown in FIG. 8D, show that native sarcoglycans alpha and gamma bind to biglycan.
Example 7
Biglycan is Expressed at Synaptic and Non-Synaptic Regions and is Up-Regulated in Dystrophic Muscle
[0318] Previous reports have shown that biglycan mRNA and protein are expressed in muscle (Bianco, et al. (1990) J. Histochem Cytochem. 38: 1549; Bosse, et al. (1993) J. Histochem. Cytochem. 41: 13). Since the biglycan that was used in the above-described Examples was obtained from synaptic membranes, it was investigated whether biglycan is also expressed at the neuromuscular junction.
[0319] Frozen sections of normal adult mouse muscle were double-labeled with α-bungarotoxin (Bgtx; to localize AChRs) and antibodies to biglycan. Cryostat sections (10 μm) of leg muscle from fresh-frozen wild-type (C57 BL) mice were mounted on slides, fixed, and treated with chondroitinase essentially as described in (Bianco, P., et al., 1990, J Histochem Cytochem. 38:1549). Primary antibodies were anti-biglycan (LF-106; generously provided by L. Fisher) diluted in PBS containing 5% BSA, 1% normal goat or horse serum, and 0.1% Triton X-100. Incubation in primary antibodies or non-immune control serum proceeded overnight at 4° C. Except where noted, all subsequent steps were performed at room temperature. Bound antibodies were detected with Cy3-labelled anti-rabbit Ig (Jackson Laboratories, West Grove, Pa.). For double-labelling, sections were first fixed for 5 min in 1% formaldehyde, rinsed and incubated in fluorescein-conjugated α-bungarotoxin (Molecular Probes, Eugene Oreg.) for 1 hr. The sections were then washed, fixed, treated with chondroitinase and stained for biglycan as described above. Sections were air-dried, mounted in Citifluor (Ted Pella, Redding, Calif.) and examined on a Nikon Eclipse microscope. Images were acquired on a cooled CCD camera using IP Lab Spectrum software and then imported to Adobe Photoshop.
[0320] The results, which are shown in FIG. 9, indicate that biglycan immunoreactivity is distributed over the entire periphery of the myofibers and synapses, and that it is also concentrated at some neuromuscular junctions.
[0321] Since biglycan binds to a component of the DAPC, it was investigated whether or not its expression was altered in a mouse model of muscular dystrophy in which dystrophin is absent, i.e., the mdx mouse. Adult mice, which contain almost exclusively regenerated muscle fibers that survive due to utrophin compensation were investigated (Grady, et al. (1997) Cell 90: 729). Frozen sections of normal and mdx muscle from 6 wk old mice were mounted on the same slides and immunostained for biglycan as described above. Immunostaining revealed that the level of biglycan expressed in mdx muscle is elevated compared to control animals (FIG. 10). These observations raise the possibility that biglycan could be part of the compensatory mechanism that allows survival of dystrophin negative muscle fibers.
Example 8
Biglycan Binds to the MuSK Ectodomain
[0322] This Example demonstrates that biglycan binds to other components of the synaptic membrane, in particular, the MuSK ectodomain.
[0323] Torpedo biglycan (DAG-125) was solubilized by alkaline extraction and neutralized, as described in Example 1, and incubated with protein A-agarose beads and with either human IgG (HIgG) or with human Fc fusion proteins containing the ectodomains of recombinant human MuSK (Glass et al. (1996) Cell; and Donzuela et al. (1995) Neuron), TIE-2, or TRK for co-precipitations. The results, which are shown in FIG. 11, indicate that Torpedo biglycan binds to the MuSK ectodomain, but not to IgG, nor to the two unrelated receptor tyrosine kinase ectodomains TIE-2 and TRK. It was also shown that MuSK solubilized from muscle membranes binds to Torpedo biglycan. Decorin was also shown to bind to MuSK.
[0324] Thus, DAG-125 binds to MuSK.
Example 9
Biglycan Preparations Potentiate Agrin-Induced AChR Clustering on Myotubes
[0325] This Example demonstrates that biglycan potentiates agrin-induced AChR clustering.
[0326] Primary chick myotubes were incubated for 20 hours with recombinant biglycan core (no GAG) with or without the addition of 1 unit (about 10 pM) of recombinant rat agrin isoform 12-4-8. Cultures incubated in 1 nM biglycan+agrin increased AChR clustering by an average of 50% over cultures incubated in 1 unit of agrin only. Higher concentrations of biglycan had no effect or possibly inhibited agrin-induced clustering. In another example, exogenous biglycan-enriched preparations (about 30% pure) were also found to potentiate agrin-induced AChR clustering when applied to cultured chick myotubes.
[0327] Thus, biglycan potentiaties (50% increase) agrin-induced AChR clustering when present at about 10-9 M (i.e., about 1.4 nM). At higher concentrations (10-8 M, 10-7 M, i.e., about 140 nM) biglycan inhibits agrin-induced AChR clustering. This was demonstrated on wild-type chick myotubes, which were prepared as described in Nastuk et al., 1991 (Neuron 7: 807-818), using either core or proteoglycan human recombinant biglycan, produced by the vaccinia system, described above. Thus, there is a biphasic effect of biglycan on agrin-induced AChR clustering.
Example 10
Biglycan and Decorin Induce Tyrosine Phosphorylation of MuSK
[0328] The culture of chick myotubes with agrin resulted, as expected, in the stimulation of phosphorylation of MuSK. It was observed that the stimulation of chick myotubes with human biglycan proteoglycan, decorin-proteoglycan, biglycan core and decorin core (separately) also induce tyrosine phosphorylation of MusK on muscle cells. Phosphorylation was determined by immunoprecipitation and Western blot using an anti-phosphotyrosine antibody. The biglycan and decorin proteoglycan and core were produced by the vaccinia system described above. The results are shown in FIG. 12.
[0329] Similarly to agrin-induced AChR clustering, agrin-induced MuSK phosphorylation was also shown to be biphasic: human biglycan core can either potentiate (at 1.4 nM) or inhibit (at 140 nM) agrin-induced MuSK phosphorylation in cultured C2C12 myotubes.
Example 11
Myotubes Cultured from Biglycan.sup.-/o Mice Show a Defective Response to Agrin
[0330] The role of biglycan in mediating agrin-induced AChR clustering was further proved by using biglycan knockout mice (biglycan.sup.-/o male mice).
[0331] Biglycan.sup.-/o mice were generated by Marian Young at the NIH. PCR genotyping of the mice was performed on genomic DNA using primer pairs specific for mutant and wild type biglycan alleles (Xu et al. (1998) Nat. Genet. 20:78). PCR products from a wild type (male; +/o), a heterozygote (female; +/-), and a knockout (male; -/o) are shown in FIG. 13A.
[0332] A Bgn female (+/-) was mated to a Bgn male (+/o) and primary cultures were established from each male pup in the resulting litter. The genotype of each pup was determined as described in the previous paragraph. Myotube cultures derived from each mouse were then treated either with or without recombinant agrin 4,8 for 18 hours. Agrin 4,8 is an alternatively spliced variant, having a four amino acid insert at site Y and an eight amino acid insert at site Z (see, e.g., Iozzo R. I (1998) Ann. Rev. Biochem. 67:609, and Firns et al. (1993) Neuron 11:491). Myotubes were then labeled with rhodamine- -bungarotoxin to visualize AChRs. As shown in FIG. 13B, the agrin-induced AChR clustering on the biglycan.sup.-/o myotubes is greatly reduced compared to those from wild type littermate controls. These results thus provide strong and direct evidence for a role of biglycan in agrin-induced AChR clustering.
[0333] FIG. 13C shows a quantitation of AChR clustering. AChR clusters and myotubes were counted in a minimum of 10 fields for cultures treated either with (AGRIN) or without (Con) recombinant agrin 4,8.
Example 12
Recovery of Response to Agrin in Biglycan.sup.-/o Mice by the Addition of Recombinant Biglycan
[0334] This example shows that the defective response of AChR aggregation in biglycan.sup.-/o mice in response to agrin can be rescued by the addition of exogenous recombinant humanbiglycan core.
[0335] This was demonstrated by adding 1.4 nM (0.05 micrograms/ml) of recombinant core human biglycan, produced in the vaccinia system described above, to the cultures of biglycan.sup.-/o myotubes described in Example 11. AchR clustering was measured as determined in Example 11.
[0336] The results, which are presented in FIG. 13B, indicate that the addition of biglycan core restores the response of biglycan.sup.-/o myotubes to agrin.
[0337] Thus, this experiment proves the importance of biglycan in agrin-induced AChR clustering. In addition, since this example was performed with core biglycan, i.e., with no proteoglycan side chains, this example demonstrates that the core is particularly important for the agrin-induced postsynaptic differentiation. This further demonstrates that biglycan affects a cell simply by contacting the cell with biglycan.
Example 13
Serum Creatine Kinase is Elevated in Biglycan Knockout Mice
[0338] Serum creating kinase (CK) levels from four mice (two male, two female) ages 16 weeks old were assayed. As shown in FIG. 15, CK levels from biglycan knockout mice are about 10 fold greater than wild types. Sera from three other wild type female mice had similar CK levels as these wild type males.
[0339] Thus, although biglycan.sup.-/o mice do not show gross abnormalities (Xu et al. (1998) Nat. Genet. 20:78), the expression of dystrophin and utrophin are not grossly abnormal, and the synapses also appear grossly normal, they have an abnormally high CK level, relative to wildtype animals. Such elevations are a hallmark of muscle cell damage, such as that seen in muscular dystrophy (Emery (1993) Duchenne Muscular Dystrophy Oxford Monographs on Medical Genetics. Oxford: New York. Oxford Univ. Press). In addition, these mice have leaky membranes, as judged by Evans Blue uptake, and show signs of muscle cell death and regeneration as judged by the presence of myofibers with centrally-located nuclei in the adult. Thus, these results indicate that the muscle cell plasma membrane is likely to be compromised in these animals. These observations, together with the restoration of agrin-induced AChR clustering in myotubes from biglycan.sup.-/o mice by the addition of biglycan, strongly suggest that the absence of biglycan or the presence of a defective biglycan results in defective muscle and/or nerve plasma membrane which can be restored by the addition of exogenous biglycan.
[0340] The observation that plasma membrane integrity is compromised in biglycan null mice indicated that there may be muscle fiber death and regeneration in these animals. To test this, the histology of muscle from biglycan null mice and littermate controls was examined. As shown in FIG. 22, we observed that approximately 15% of myofibers in biglycan null mice had centrally located nuclei. Such a nuclear disposition is characteristic of regenerating myofibers. A similar percentage of fibers was observed at all ages examined (1, 3 and 6 months). We did not observe any indication of mononuclear cell infiltration, nor was there any evidence of fibrosis. Taken together, these results indicated that biglycan null mice display a distinct, relatively mild muscular dystrophy phenotype.
[0341] Immunofluorescence analysis of frozen sections from biglycan null mice showed that the level of dystrophin, α-, β-, γ- and δ-sarcoglycan and β-dystroglycan at the muscle cell is similar in biglycan null mice and littermate controls. However, analysis of collagen VI expression revealed a striking difference. In wild-type littermate controls collagen VI is expressed in the endomysium and the perimysium. In contrast, the levels of collagen VI are reduced in the endomysium of the biglycan null mice. Notably, the expression of decorin, which can also bind this collagen is not affected in the mutant mice. Thus collagen VI expression is selectively reduced in mice lacking biglycan.
Example 14
Biglycan Core Stimulates MuKD Dependent Tyrosine Phosphorylation of α-Sarcoglycan and a 35 kD DAPC Component in Myobtubes
[0342] This example demonstrates that biglycan induces tyrosine phosphorylation of DAPC components and has therefore a signaling function.
[0343] Human biglycan was prepared using the vaccina system described above Wildtype myotubes or MuSK null myotubes were incubated for 30 minutes in the presence of 1 microgram/ml (27 nM) of a mixture of core and proteoglycan forms of human biglycan. The cultures were detergent extracted and α-sarcoglycan was immunoprecipitated, separated by SDS-PAGE, blotted, and probed with anti-phosphotyrosine antibody or MIgG. The results, which are presented in FIG. 15, show that the tyrosine phosphorylation of α-sarcoglycan is increased in the presence of biglycan in wild type cells, but not in MuSK null myotubes. In addition, it was observed that an unidentified 35 kD DAPC component was also phosphorylated in wild type cells but not in MuSK null myotubes In addition, the results show that biglycan is capable of a signaling function, in the absence of agrin.
EQUIVALENTS
[0344] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence CWU
1
1
1919PRTTorpedo sp. 1Ile Gln Ala Ile Glu Phe Glu Asp Leu 1
529PRTTorpedo sp. 2Leu Gly Leu Gly Phe Asn Glu Ile Arg 1
5319PRTTorpedo sp. 3Thr Ser Tyr His Gly Ile Ser Leu Phe Asn Asn Pro Val
Asn Tyr Trp 1 5 10 15Asp
Val Leu49PRTHomo sapiens 4Ile Gln Ala Ile Glu Leu Glu Asp Leu 1
559PRTHomo sapiens 5Leu Gly Leu Gly His Asn Gln Ile Arg 1
5619PRTHomo sapiens 6Ala Tyr Tyr Asn Gly Ile Ser Leu Phe Asn Asn Pro
Val Pro Tyr Trp 1 5 10
15Glu Val Gln71685DNAHomo sapiens 7gagtagctgc tttcggtccg ccggacacac
cggacagata gacgtgcgga cggcccacca 60ccccagcccg ccaactagtc agcctgcgcc
tggcgcctcc cctctccagg tccatccgcc 120atgtggcccc tgtggcgcct cgtgtctctg
ctggccctga gccaggccct gccctttgag 180cagagaggct tctgggactt caccctggac
gatgggccat tcatgatgaa cgatgaggaa 240gcttcgggcg ctgacacctc aggcgtcctg
gacccggact ctgtcacacc cacctacagc 300gccatgtgtc ctttcggctg ccactgccac
ctgcgggtgg ttcagtgctc cgacctgggt 360ctgaagtctg tgcccaaaga gatctcccct
gacaccacgc tgctggacct gcagaacaac 420gacatctccg agctccgcaa ggatgacttc
aagggtctcc agcacctcta cgccctcgtc 480ctggtgaaca acaagatctc caagatccat
gagaaggcct tcagcccact gcggaagctg 540cagaagctct acatctccaa gaaccacctg
gtggagatcc cgcccaacct acccagctcc 600ctggtggagc tccgcatcca cgacaaccgc
atccgcaagg tgcccaaggg agtgttcagc 660gggctccgga acatgaactg catcgagatg
ggcgggaacc cactggagaa cagtggcttt 720gaacctggag ccttcgatgg cctgaagctc
aactacctgc gcatctcaga ggccaagctg 780actggcatcc ccaaagacct ccctgagacc
ctgaatgaac tccacctaga ccacaacaaa 840atccaggcca tcgaactgga ggacctgctt
cgctactcca agctgtacag gctgggccta 900ggccacaacc agatcaggat gatcgagaac
gggagcctga gcttcctgcc caccctccgg 960gagctccact tggacaacaa caagttggcc
agggtgccct cagggctccc agacctcaag 1020ctcctccagg tggtctatct gcactccaac
aacatcacca aagtgggtgt caacgacttc 1080tgtcccatgg gcttcggggt gaagcgggcc
tactacaacg gcatcagcct cttcaacaac 1140cccgtgccct actgggaggt gcagccggcc
actttccgct gcgtcactga ccgcctggcc 1200atccagtttg gcaactacaa aaagtagagg
cagctgcagc caccgcgggg cctcagtggg 1260ggtctctggg gaacacagcc agacatcctg
atggggaggc agagccagga agctaagcca 1320gggcccagct gcgtccaacc cagcccccca
cctcaggtcc ctgaccccag ctcgatgccc 1380catcaccgcc tctccctggc tcccaagggt
gcaggtgggc gcaaggcccg gcccccatca 1440catgttccct tggcctcaga gctgcccctg
ctctcccacc acagccaccc agaggcaccc 1500catgaagctt ttttctcgtt cactcccaaa
cccaagtgtc caaagctcca gtcctaggag 1560aacagtccct gggtcagcag ccaggaggcg
gtccataaga atggggacag tgggctctgc 1620cagggctgcc gcacctgtcc agaacaacat
gttctgttcc tcctcctcat gcatttccag 1680ccttg
168581104DNAHomo sapiens 8atgtggcccc
tgtggcgcct cgtgtctctg caggccctga gccaggccct gccctttgag 60cagagaggct
tctgggactt caccctggac gatgggccat tcatgatgaa cgatgaggaa 120gcttcgggcg
ctgacacctc aggcgtcctg gacccggact ctgtcacacc cacctacagc 180gccatgtgtc
ctttcggctg ccactgccac ctgcgggtgg ttcagtgctc cgacctgggt 240ctgaagtctg
tgcccaaaga gatctcccct gacaccacgc tgctggacct gcagaacaac 300gacatctccg
agctccgcaa ggatgacttc aagggtctcc agcacctcta cgccctcgtc 360ctggtgaaca
acaagatctc caagatccat gagaaggcct tcagcccact gcggaagctg 420cagaagctct
acatctccaa gaaccacctg gtggagatcc cgcccaacct acccagctcc 480ctggtggagc
tccgcatcca cgacaaccgc atccgcaagg tgcccaaggg agtgttcagc 540gggctccgga
acatgaactg catcgagatg ggcgggaacc cactggagaa cagtggcttt 600gaacctggag
ccttcgatgg cctgaagctc aactacctgc gcatctcaga ggccaagctg 660actggcatcc
ccaaagacct ccctgagacc ctgaatgaac tccacctaga ccacaacaaa 720atccaggcca
tcgaactgga ggacctgctt cgctactcca agctgtacag gctgggccta 780ggccacaacc
agatcaggat gatcgagaac gggagcctga gcttcctgcc caccctccgg 840gagctccact
tggacaacaa caagttggcc agggtgccct cagggctccc agacctcaag 900ctcctccagg
tggtctatct gcactccaac aacatcacca aagtgggtgt caacgacttc 960tgtcccatgg
gcttcggggt gaagcgggcc tactacaacg gcatcagcct cttcaacaac 1020cccgtgccct
actgggaggt gcagccggcc actttccgct gcgtcactga ccgcctggcc 1080atccagtttg
gcaactacaa aaag 11049373PRTHomo
sapiens 9Met Trp Pro Leu Trp Arg Leu Val Ser Leu Leu Ala Leu Ser Gln Ala
1 5 10 15Leu Pro Phe Glu
Gln Arg Gly Phe Trp Asp Phe Thr Leu Asp Asp Gly 20
25 30Pro Phe Met Met Asn Asp Glu Glu Ala Ser Gly
Ala Asp Thr Ser Gly 35 40 45Val
Leu Asp Pro Asp Ser Val Thr Pro Thr Tyr Ser Ala Met Cys Pro 50
55 60Phe Gly Tyr Cys His Cys His Leu Arg Val
Val Gln Cys Ser Asp Leu65 70 75
80Gly Leu Lys Ser Val Pro Lys Gly Ile Ser Pro Asp Thr Thr Leu
Leu 85 90 95Asp Leu Gln
Asn Asn Asp Ile Ser Glu Leu Arg Lys Asp Asp Phe Lys 100
105 110Gly Leu Gly Asn His Leu Tyr Ala Leu Val
Leu Val Asn Asn Lys Ile 115 120
125Ser Lys Ile His Glu Lys Ala Phe Ser Pro Leu Arg Lys Leu Gln Lys 130
135 140Leu Tyr Ile Ser Lys Asn His Leu
Val Glu Ile Pro Pro Asn Leu Pro145 150
155 160Ser Ser Leu Val Glu Leu Arg Ile His Asp Asn Arg
Ile Arg Lys Val 165 170
175Pro Lys Gly Val Phe Ser Gly Leu Arg Asn Met Asn Cys Ile Glu Met
180 185 190Gly Gly Asn Pro Leu Glu
Asn Ser Gly Phe Glu Pro Gly Ala Phe Asp 195 200
205Gly Leu Lys Leu Asn Tyr Leu Arg Ile Ser Glu Ala Lys Leu
Thr Gly 210 215 220Ile Pro Lys Asp Leu
Pro Glu Thr Leu Asn Glu Leu His Leu Asp His225 230
235 240Asn Lys Ile Gln Ala Ile Glu Leu Glu Asp
Leu Leu Arg Tyr Ser Lys 245 250
255Leu Tyr Arg Leu Gly Leu Gly His Asn Gln Ile Glu Arg Met Ile Glu
260 265 270Asn Gly Ser Leu Ser
Phe Leu Pro Thr Leu Arg Glu Leu His Leu Asp 275
280 285Asn Asn Lys Leu Ala Arg Val Pro Ser Gly Leu Pro
Asp Leu Lys Leu 290 295 300Leu Gln Val
Val Tyr Leu His Ser Asn Asn Ile Thr Lys Val Gly Val305
310 315 320Asn Asp Phe Cys Pro Met Gly
Phe Gly Val Lys Arg Ala Tyr Tyr Asn 325
330 335Gly Ile Ser Leu Phe Asn Asn Pro Val Pro Tyr Trp
Glu Val Gln Pro 340 345 350Ala
Thr Phe Arg Cys Val Thr Asp Arg Leu Ala Leu Leu Glu Gln Phe 355
360 365Gly Asn Tyr Lys Lys
3701012PRTArtificial SequencePlasmid pQE-biglycan 10Met Arg Gly Ser His
His His His His His Gly Ser 1 5
10111028PRTHomo sapiens 11Met Arg Ala Ala Arg Ala Leu Leu Pro Leu Leu Leu
Gln Ala Cys Trp 1 5 10
15Thr Ala Ala Gln Asp Glu Pro Glu Thr Pro Arg Ala Val Ala Phe Gln
20 25 30Asp Cys Pro Val Asp Leu Phe
Phe Val Leu Asp Thr Ser Glu Ser Val 35 40
45Ala Leu Arg Leu Lys Pro Tyr Gly Ala Leu Val Asp Lys Val Lys
Ser 50 55 60Phe Thr Lys Arg Phe Ile
Asp Asn Leu Arg Asp Arg Tyr Tyr Arg Cys65 70
75 80Asp Arg Asn Leu Val Trp Asn Ala Gly Ala Leu
His Tyr Ser Asp Glu 85 90
95Val Glu Ile Ile Gln Gly Leu Thr Arg Met Pro Gly Gly Arg Asp Ala
100 105 110Leu Lys Ser Ser Val Asp
Ala Val Lys Tyr Phe Gly Lys Gly Thr Tyr 115 120
125Thr Asp Cys Ala Ile Lys Lys Gly Leu Glu Gln Leu Leu Val
Gly Gly 130 135 140Ser His Leu Lys Glu
Asn Lys Tyr Leu Ile Val Val Thr Asp Gly His145 150
155 160Pro Leu Glu Gly Tyr Lys Glu Pro Cys Gly
Gly Leu Glu Asp Ala Val 165 170
175Asn Glu Ala Lys His Leu Gly Val Lys Val Phe Ser Val Ala Ile Thr
180 185 190Pro Asp His Leu Glu
Pro Arg Leu Ser Ile Ile Ala Thr Asp His Thr 195
200 205Tyr Arg Arg Asn Phe Thr Ala Ala Asp Trp Gly Gln
Ser Arg Asp Ala 210 215 220Glu Glu Ala
Ile Ser Gln Thr Ile Asp Thr Ile Val Asp Met Ile Lys225
230 235 240Asn Asn Val Glu Gln Val Cys
Cys Ser Phe Glu Cys Gln Pro Ala Arg 245
250 255Gly Pro Pro Gly Leu Arg Gly Asp Pro Gly Phe Glu
Gly Glu Arg Gly 260 265 270Lys
Pro Gly Leu Pro Gly Glu Lys Gly Glu Ala Gly Asp Pro Gly Arg 275
280 285Pro Gly Asp Leu Gly Pro Val Gly Tyr
Gln Gly Met Lys Gly Glu Lys 290 295
300Gly Ser Arg Gly Glu Lys Gly Ser Arg Gly Pro Lys Gly Tyr Lys Gly305
310 315 320Glu Lys Gly Lys
Arg Gly Ile Asp Gly Val Asp Gly Val Lys Gly Glu 325
330 335Met Gly Tyr Pro Gly Leu Pro Gly Cys Lys
Gly Ser Pro Gly Phe Asp 340 345
350Gly Ile Gln Gly Pro Pro Gly Pro Lys Gly Asp Pro Gly Ala Phe Gly
355 360 365Leu Lys Gly Glu Lys Gly Glu
Pro Gly Ala Asp Gly Glu Ala Gly Arg 370 375
380Pro Gly Ala Arg Gly Pro Ser Gly Asp Glu Gly Pro Ala Gly Glu
Pro385 390 395 400Gly Pro
Pro Gly Glu Lys Gly Glu Ala Gly Asp Glu Gly Asn Pro Gly
405 410 415Pro Asp Gly Ala Pro Gly Glu
Arg Gly Gly Pro Gly Glu Arg Gly Pro 420 425
430Arg Gly Thr Pro Gly Pro Arg Gly Pro Arg Gly Asp Pro Gly
Glu Ala 435 440 445Gly Pro Gln Gly
Asp Gln Gly Arg Glu Gly Pro Val Gly Val Pro Gly 450
455 460Asp Pro Gly Glu Ala Gly Pro Ile Gly Pro Lys Gly
Tyr Arg Gly Asp465 470 475
480Glu Gly Pro Pro Gly Ser Glu Gly Ala Arg Gly Ala Pro Gly Pro Ala
485 490 495Gly Pro Pro Gly Asp
Pro Gly Leu Met Gly Glu Arg Gly Glu Asp Gly 500
505 510Pro Ala Gly Asn Gly Thr Glu Gly Phe Pro Gly Phe
Pro Gly Tyr Pro 515 520 525Gly Asn
Arg Gly Ala Pro Gly Ile Asn Gly Thr Lys Gly Tyr Pro Gly 530
535 540Leu Lys Gly Asp Glu Gly Glu Ala Gly Asp Pro
Gly Asp Asp Asn Asn545 550 555
560Asp Ile Ala Pro Arg Gly Val Lys Gly Ala Lys Gly Tyr Arg Gly Pro
565 570 575Glu Gly Pro Gln
Gly Pro Pro Gly His Gln Gly Pro Pro Gly Pro Asp 580
585 590Glu Cys Glu Ile Leu Asp Ile Ile Met Lys Met
Cys Ser Cys Cys Glu 595 600 605Cys
Lys Cys Gly Pro Ile Asp Leu Leu Phe Val Leu Asp Ser Ser Glu 610
615 620Ser Ile Gly Leu Gln Asn Phe Glu Ile Ala
Lys Asp Phe Val Val Lys625 630 635
640Val Ile Asp Arg Leu Ser Arg Asp Glu Leu Val Lys Phe Glu Pro
Gly 645 650 655Gln Ser Tyr
Ala Gly Val Val Gln Tyr Ser His Ser Gln Met Gln Glu 660
665 670His Val Ser Leu Arg Ser Pro Ser Ile Arg
Asn Val Gln Glu Leu Lys 675 680
685Glu Ala Ile Lys Ser Leu Gln Trp Met Ala Gly Gly Thr Phe Thr Gly 690
695 700Glu Ala Leu Gln Tyr Thr Arg Asp
Gln Leu Leu Pro Pro Ser Pro Asn705 710
715 720Asn Arg Ile Ala Leu Val Ile Thr Asp Gly Arg Ser
Asp Thr Gln Arg 725 730
735Asp Thr Thr Pro Leu Asn Val Leu Cys Ser Pro Gly Ile Gln Val Val
740 745 750Ser Val Gly Ile Lys Asp
Val Phe Asp Phe Ile Pro Gly Ser Asp Gln 755 760
765Leu Asn Val Ile Ser Cys Gln Gly Leu Ala Pro Ser Gln Gly
Arg Pro 770 775 780Gly Leu Ser Leu Val
Lys Glu Asn Tyr Ala Glu Leu Leu Glu Asp Ala785 790
795 800Phe Leu Lys Asn Val Thr Ala Gln Ile Cys
Ile Asp Lys Lys Cys Pro 805 810
815Asp Tyr Thr Cys Pro Ile Thr Phe Ser Ser Pro Ala Asp Ile Thr Ile
820 825 830Leu Leu Asp Gly Ser
Ala Ser Val Gly Ser His Asn Phe Asp Thr Thr 835
840 845Lys Arg Phe Ala Lys Arg Leu Ala Glu Arg Phe Leu
Thr Ala Gly Arg 850 855 860Thr Asp Pro
Ala His Asp Val Arg Val Ala Val Val Gln Tyr Ser Gly865
870 875 880Thr Gly Gln Gln Arg Pro Glu
Arg Ala Ser Leu Gln Phe Leu Gln Asn 885
890 895Tyr Thr Ala Leu Ala Ser Ala Val Asp Ala Met Asp
Phe Ile Asn Asp 900 905 910Ala
Thr Asp Val Asn Asp Ala Leu Gly Tyr Val Thr Arg Phe Tyr Arg 915
920 925Glu Ala Ser Ser Gly Ala Ala Lys Lys
Arg Leu Leu Leu Phe Ser Asp 930 935
940Gly Asn Ser Gln Gly Ala Thr Pro Ala Ala Ile Glu Lys Ala Val Gln945
950 955 960Glu Ala Gln Arg
Ala Gly Ile Glu Ile Phe Val Val Val Val Gly Arg 965
970 975Gln Val Asn Glu Pro His Ile Arg Val Leu
Val Thr Gly Lys Thr Ala 980 985
990Glu Tyr Asp Val Pro Tyr Gly Glu Ser His Leu Phe Arg Val Pro Ser
995 1000 1005Tyr Gln Ala Leu Leu Arg Gly
Val Phe His Gln Thr Val Ser Arg Lys 1010 1015
1020Val Ala Leu Gly1025121009PRTHomo sapiens 12Gln Asp Glu Pro Glu
Thr Pro Arg Ala Val Ala Phe Gln Asp Cys Pro 1 5
10 15Val Asp Leu Phe Phe Val Leu Asp Thr Ser Glu
Ser Val Ala Leu Arg 20 25
30Leu Lys Pro Tyr Gly Ala Leu Val Asp Lys Val Lys Ser Phe Thr Lys
35 40 45Arg Phe Ile Asp Asn Leu Arg Asp
Arg Tyr Tyr Arg Cys Asp Arg Asn 50 55
60Leu Val Trp Asn Ala Gly Ala Leu His Tyr Ser Asp Glu Val Glu Ile65
70 75 80Ile Gln Gly Leu Thr
Arg Met Pro Gly Gly Arg Asp Ala Leu Lys Ser 85
90 95Ser Val Asp Ala Val Lys Tyr Phe Gly Lys Gly
Thr Tyr Thr Asp Cys 100 105
110Ala Ile Lys Lys Gly Leu Glu Gln Leu Leu Val Gly Gly Ser His Leu
115 120 125Lys Glu Asn Lys Tyr Leu Ile
Val Val Thr Asp Gly His Pro Leu Glu 130 135
140Gly Tyr Lys Glu Pro Cys Gly Gly Leu Glu Asp Ala Val Asn Glu
Ala145 150 155 160Lys His
Leu Gly Val Lys Val Phe Ser Val Ala Ile Thr Pro Asp His
165 170 175Leu Glu Pro Arg Leu Ser Ile
Ile Ala Thr Asp His Thr Tyr Arg Arg 180 185
190Asn Phe Thr Ala Ala Asp Trp Gly Gln Ser Arg Asp Ala Glu
Glu Ala 195 200 205Ile Ser Gln Thr
Ile Asp Thr Ile Val Asp Met Ile Lys Asn Asn Val 210
215 220Glu Gln Val Cys Cys Ser Phe Glu Cys Gln Pro Ala
Arg Gly Pro Pro225 230 235
240Gly Leu Arg Gly Asp Pro Gly Phe Glu Gly Glu Arg Gly Lys Pro Gly
245 250 255Leu Pro Gly Glu Lys
Gly Glu Ala Gly Asp Pro Gly Arg Pro Gly Asp 260
265 270Leu Gly Pro Val Gly Tyr Gln Gly Met Lys Gly Glu
Lys Gly Ser Arg 275 280 285Gly Glu
Lys Gly Ser Arg Gly Pro Lys Gly Tyr Lys Gly Glu Lys Gly 290
295 300Lys Arg Gly Ile Asp Gly Val Asp Gly Val Lys
Gly Glu Met Gly Tyr305 310 315
320Pro Gly Leu Pro Gly Cys Lys Gly Ser Pro Gly Phe Asp Gly Ile Gln
325 330 335Gly Pro Pro Gly
Pro Lys Gly Asp Pro Gly Ala Phe Gly Leu Lys Gly 340
345 350Glu Lys Gly Glu Pro Gly Ala Asp Gly Glu Ala
Gly Arg Pro Gly Ala 355 360 365Arg
Gly Pro Ser Gly Asp Glu Gly Pro Ala Gly Glu Pro Gly Pro Pro 370
375 380Gly Glu Lys Gly Glu Ala Gly Asp Glu Gly
Asn Pro Gly Pro Asp Gly385 390 395
400Ala Pro Gly Glu Arg Gly Gly Pro Gly Glu Arg Gly Pro Arg Gly
Thr 405 410 415Pro Gly Pro
Arg Gly Pro Arg Gly Asp Pro Gly Glu Ala Gly Pro Gln 420
425 430Gly Asp Gln Gly Arg Glu Gly Pro Val Gly
Val Pro Gly Asp Pro Gly 435 440
445Glu Ala Gly Pro Ile Gly Pro Lys Gly Tyr Arg Gly Asp Glu Gly Pro 450
455 460Pro Gly Ser Glu Gly Ala Arg Gly
Ala Pro Gly Pro Ala Gly Pro Pro465 470
475 480Gly Asp Pro Gly Leu Met Gly Glu Arg Gly Glu Asp
Gly Pro Ala Gly 485 490
495Asn Gly Thr Glu Gly Phe Pro Gly Phe Pro Gly Tyr Pro Gly Asn Arg
500 505 510Gly Ala Pro Gly Ile Asn
Gly Thr Lys Gly Tyr Pro Gly Leu Lys Gly 515 520
525Asp Glu Gly Glu Ala Gly Asp Pro Gly Asp Asp Asn Asn Asp
Ile Ala 530 535 540Pro Arg Gly Val Lys
Gly Ala Lys Gly Tyr Arg Gly Pro Glu Gly Pro545 550
555 560Gln Gly Pro Pro Gly His Gln Gly Pro Pro
Gly Pro Asp Glu Cys Glu 565 570
575Ile Leu Asp Ile Ile Met Lys Met Cys Ser Cys Cys Glu Cys Lys Cys
580 585 590Gly Pro Ile Asp Leu
Leu Phe Val Leu Asp Ser Ser Glu Ser Ile Gly 595
600 605Leu Gln Asn Phe Glu Ile Ala Lys Asp Phe Val Val
Lys Val Ile Asp 610 615 620Arg Leu Ser
Arg Asp Glu Leu Val Lys Phe Glu Pro Gly Gln Ser Tyr625
630 635 640Ala Gly Val Val Gln Tyr Ser
His Ser Gln Met Gln Glu His Val Ser 645
650 655Leu Arg Ser Pro Ser Ile Arg Asn Val Gln Glu Leu
Lys Glu Ala Ile 660 665 670Lys
Ser Leu Gln Trp Met Ala Gly Gly Thr Phe Thr Gly Glu Ala Leu 675
680 685Gln Tyr Thr Arg Asp Gln Leu Leu Pro
Pro Ser Pro Asn Asn Arg Ile 690 695
700Ala Leu Val Ile Thr Asp Gly Arg Ser Asp Thr Gln Arg Asp Thr Thr705
710 715 720Pro Leu Asn Val
Leu Cys Ser Pro Gly Ile Gln Val Val Ser Val Gly 725
730 735Ile Lys Asp Val Phe Asp Phe Ile Pro Gly
Ser Asp Gln Leu Asn Val 740 745
750Ile Ser Cys Gln Gly Leu Ala Pro Ser Gln Gly Arg Pro Gly Leu Ser
755 760 765Leu Val Lys Glu Asn Tyr Ala
Glu Leu Leu Glu Asp Ala Phe Leu Lys 770 775
780Asn Val Thr Ala Gln Ile Cys Ile Asp Lys Lys Cys Pro Asp Tyr
Thr785 790 795 800Cys Pro
Ile Thr Phe Ser Ser Pro Ala Asp Ile Thr Ile Leu Leu Asp
805 810 815Gly Ser Ala Ser Val Gly Ser
His Asn Phe Asp Thr Thr Lys Arg Phe 820 825
830Ala Lys Arg Leu Ala Glu Arg Phe Leu Thr Ala Gly Arg Thr
Asp Pro 835 840 845Ala His Asp Val
Arg Val Ala Val Val Gln Tyr Ser Gly Thr Gly Gln 850
855 860Gln Arg Pro Glu Arg Ala Ser Leu Gln Phe Leu Gln
Asn Tyr Thr Ala865 870 875
880Leu Ala Ser Ala Val Asp Ala Met Asp Phe Ile Asn Asp Ala Thr Asp
885 890 895Val Asn Asp Ala Leu
Gly Tyr Val Thr Arg Phe Tyr Arg Glu Ala Ser 900
905 910Ser Gly Ala Ala Lys Lys Arg Leu Leu Leu Phe Ser
Asp Gly Asn Ser 915 920 925Gln Gly
Ala Thr Pro Ala Ala Ile Glu Lys Ala Val Gln Glu Ala Gln 930
935 940Arg Ala Gly Ile Glu Ile Phe Val Val Val Val
Gly Arg Gln Val Asn945 950 955
960Glu Pro His Ile Arg Val Leu Val Thr Gly Lys Thr Ala Glu Tyr Asp
965 970 975Val Pro Tyr Gly
Glu Ser His Leu Phe Arg Val Pro Ser Tyr Gln Ala 980
985 990Leu Leu Arg Gly Val Phe His Gln Thr Val Ser
Arg Lys Val Ala Leu 995 1000
1005Gly131019PRTHomo sapiens 13Met Leu Gln Gly Thr Cys Ser Val Leu Leu
Leu Trp Gly Ile Leu Gly 1 5 10
15Ala Ile Gln Ala Gln Gln Gln Glu Val Ile Ser Pro Asp Thr Thr Glu
20 25 30Arg Asn Asn Asn Cys Pro
Glu Lys Thr Asp Cys Pro Ile His Val Tyr 35 40
45Phe Val Leu Asp Thr Ser Glu Ser Val Thr Met Gln Ser Pro
Thr Asp 50 55 60Ile Leu Leu Phe His
Met Lys Gln Phe Val Pro Gln Phe Ile Ser Gln65 70
75 80Leu Gln Asn Glu Phe Tyr Leu Asp Gln Val
Ala Leu Ser Trp Arg Tyr 85 90
95Gly Gly Leu His Phe Ser Asp Gln Val Glu Val Phe Ser Pro Pro Gly
100 105 110Ser Asp Arg Ala Ser
Phe Ile Lys Asn Leu Gln Gly Ile Ser Ser Phe 115
120 125Arg Arg Gly Thr Phe Thr Asp Cys Ala Leu Ala Asn
Met Thr Glu Gln 130 135 140Ile Arg Gln
Asp Arg Ser Lys Gly Thr Val His Phe Ala Val Val Ile145
150 155 160Thr Asp Gly His Val Thr Gly
Ser Pro Cys Gly Gly Ile Lys Leu Gln 165
170 175Ala Glu Arg Ala Arg Glu Glu Gly Ile Arg Leu Phe
Ala Val Ala Pro 180 185 190Asn
Gln Asn Leu Lys Glu Gln Gly Leu Arg Asp Ile Ala Ser Thr Pro 195
200 205His Glu Leu Tyr Arg Asn Asp Tyr Ala
Thr Met Leu Pro Asp Ser Thr 210 215
220Glu Ile Asn Gln Asp Thr Ile Asn Arg Ile Ile Lys Val Met Lys His225
230 235 240Glu Ala Tyr Gly
Glu Cys Tyr Lys Val Ser Cys Leu Glu Ile Pro Gly 245
250 255Pro Ser Gly Pro Lys Gly Tyr Arg Gly Gln
Lys Gly Ala Lys Gly Asn 260 265
270Met Gly Glu Pro Gly Glu Pro Gly Gln Lys Gly Arg Gln Gly Asp Pro
275 280 285Gly Ile Glu Gly Pro Ile Gly
Phe Pro Gly Pro Lys Gly Val Pro Gly 290 295
300Phe Lys Gly Glu Lys Gly Glu Phe Gly Ala Asp Gly Arg Lys Gly
Ala305 310 315 320Pro Gly
Leu Ala Gly Lys Asn Gly Thr Asp Gly Gln Lys Gly Lys Leu
325 330 335Gly Arg Ile Gly Pro Pro Gly
Cys Lys Gly Asp Pro Gly Asn Arg Gly 340 345
350Pro Asp Gly Tyr Pro Gly Glu Ala Gly Ser Pro Gly Glu Arg
Gly Asp 355 360 365Gln Gly Gly Lys
Gly Asp Pro Gly Arg Pro Gly Arg Arg Gly Pro Pro 370
375 380Gly Glu Ile Gly Ala Lys Gly Ser Lys Gly Tyr Gln
Gly Asn Asn Gly385 390 395
400Ala Pro Gly Ser Pro Gly Val Lys Gly Ala Lys Gly Gly Pro Gly Pro
405 410 415Arg Gly Pro Lys Gly
Glu Pro Gly Arg Arg Gly Asp Pro Gly Thr Lys 420
425 430Gly Ser Pro Gly Ser Asp Gly Pro Lys Gly Glu Lys
Gly Asp Pro Gly 435 440 445Pro Glu
Gly Pro Arg Gly Leu Ala Gly Glu Val Gly Asn Lys Gly Ala 450
455 460Lys Gly Asp Arg Gly Leu Pro Gly Pro Arg Gly
Pro Gln Gly Ala Leu465 470 475
480Gly Glu Pro Gly Lys Gln Gly Ser Arg Gly Asp Pro Gly Asp Ala Gly
485 490 495Pro Arg Gly Asp
Ser Gly Gln Pro Gly Pro Lys Gly Asp Pro Gly Arg 500
505 510Pro Gly Phe Ser Tyr Pro Gly Pro Arg Gly Ala
Pro Gly Glu Lys Gly 515 520 525Glu
Pro Gly Pro Arg Gly Pro Glu Gly Gly Arg Gly Asp Phe Gly Leu 530
535 540Lys Gly Glu Pro Gly Arg Lys Gly Glu Lys
Gly Glu Pro Ala Asp Pro545 550 555
560Gly Pro Pro Gly Glu Pro Gly Pro Arg Gly Pro Arg Gly Val Pro
Gly 565 570 575Pro Glu Gly
Glu Pro Gly Pro Pro Gly Asp Pro Gly Leu Thr Glu Cys 580
585 590Asp Val Met Thr Tyr Val Arg Glu Thr Cys
Gly Cys Cys Asp Cys Glu 595 600
605Lys Arg Cys Gly Ala Leu Asp Val Val Phe Val Ile Asp Ser Ser Glu 610
615 620Ser Ile Gly Tyr Thr Asn Phe Thr
Leu Glu Lys Asn Phe Val Ile Asn625 630
635 640Val Val Asn Arg Leu Gly Ala Ile Ala Lys Asp Pro
Lys Ser Glu Thr 645 650
655Gly Thr Arg Val Gly Val Val Gln Tyr Ser His Glu Gly Thr Phe Glu
660 665 670Ala Ile Gln Leu Asp Asp
Glu His Ile Asp Ser Leu Ser Ser Phe Lys 675 680
685Glu Ala Val Lys Asn Leu Glu Trp Ile Ala Gly Gly Thr Trp
Thr Pro 690 695 700Ser Ala Leu Lys Phe
Ala Tyr Asp Arg Leu Ile Lys Glu Ser Arg Arg705 710
715 720Gln Lys Thr Arg Val Phe Ala Val Val Ile
Thr Asp Gly Arg His Asp 725 730
735Pro Arg Asp Asp Asp Leu Asn Leu Arg Ala Leu Cys Asp Arg Asp Val
740 745 750Thr Val Thr Ala Ile
Gly Ile Gly Asp Met Phe His Glu Lys His Glu 755
760 765Ser Glu Asn Leu Tyr Ser Ile Ala Cys Asp Lys Pro
Gln Gln Val Arg 770 775 780Asn Met Thr
Leu Phe Ser Asp Leu Val Ala Glu Lys Phe Ile Asp Asp785
790 795 800Met Glu Asp Val Leu Cys Pro
Asp Pro Gln Ile Val Cys Pro Asp Leu 805
810 815Pro Cys Gln Thr Glu Leu Ser Val Ala Gln Cys Thr
Gln Arg Pro Val 820 825 830Asp
Ile Val Phe Leu Leu Asp Gly Ser Glu Arg Leu Gly Glu Gln Asn 835
840 845Phe His Lys Ala Arg Arg Phe Val Glu
Gln Val Ala Arg Arg Leu Thr 850 855
860Leu Ala Arg Arg Asp Asp Asp Pro Leu Asn Ala Arg Val Ala Leu Leu865
870 875 880Gln Phe Gly Gly
Pro Gly Glu Gln Gln Val Ala Phe Pro Leu Ser His 885
890 895Asn Leu Thr Ala Ile His Glu Ala Leu Glu
Thr Thr Gln Tyr Leu Asn 900 905
910Ser Phe Ser His Val Gly Ala Gly Val Val His Ala Ile Asn Ala Ile
915 920 925Val Arg Ser Pro Arg Gly Gly
Ala Arg Arg His Ala Glu Leu Ser Phe 930 935
940Val Phe Leu Thr Asp Gly Val Thr Gly Asn Asp Ser Leu His Glu
Ser945 950 955 960Ala His
Ser Met Arg Asn Glu Asn Val Val Pro Thr Val Leu Ala Leu
965 970 975Gly Ser Asp Val Asp Met Asp
Val Leu Thr Thr Leu Ser Leu Gly Asp 980 985
990Arg Ala Ala Val Phe His Glu Lys Asp Tyr Asp Ser Leu Ala
Gln Pro 995 1000 1005Gly Phe Phe
Asp Arg Phe Ile Arg Trp Ile Cys 1010 101514999PRTHomo
sapiens 14Gln Gln Gln Glu Val Ile Ser Pro Asp Thr Thr Glu Arg Asn Asn Asn
1 5 10 15Cys Pro Glu Lys
Thr Asp Cys Pro Ile His Val Tyr Phe Val Leu Asp 20
25 30Thr Ser Glu Ser Val Thr Met Gln Ser Pro Thr
Asp Ile Leu Leu Phe 35 40 45His
Met Lys Gln Phe Val Pro Gln Phe Ile Ser Gln Leu Gln Asn Glu 50
55 60Phe Tyr Leu Asp Gln Val Ala Leu Ser Trp
Arg Tyr Gly Gly Leu His65 70 75
80Phe Ser Asp Gln Val Glu Val Phe Ser Pro Pro Gly Ser Asp Arg
Ala 85 90 95Ser Phe Ile
Lys Asn Leu Gln Gly Ile Ser Ser Phe Arg Arg Gly Thr 100
105 110Phe Thr Asp Cys Ala Leu Ala Asn Met Thr
Glu Gln Ile Arg Gln Asp 115 120
125Arg Ser Lys Gly Thr Val His Phe Ala Val Val Ile Thr Asp Gly His 130
135 140Val Thr Gly Ser Pro Cys Gly Gly
Ile Lys Leu Gln Ala Glu Arg Ala145 150
155 160Arg Glu Glu Gly Ile Arg Leu Phe Ala Val Ala Pro
Asn Gln Asn Leu 165 170
175Lys Glu Gln Gly Leu Arg Asp Ile Ala Ser Thr Pro His Glu Leu Tyr
180 185 190Arg Asn Asp Tyr Ala Thr
Met Leu Pro Asp Ser Thr Glu Ile Asn Gln 195 200
205Asp Thr Ile Asn Arg Ile Ile Lys Val Met Lys His Glu Ala
Tyr Gly 210 215 220Glu Cys Tyr Lys Val
Ser Cys Leu Glu Ile Pro Gly Pro Ser Gly Pro225 230
235 240Lys Gly Tyr Arg Gly Gln Lys Gly Ala Lys
Gly Asn Met Gly Glu Pro 245 250
255Gly Glu Pro Gly Gln Lys Gly Arg Gln Gly Asp Pro Gly Ile Glu Gly
260 265 270Pro Ile Gly Phe Pro
Gly Pro Lys Gly Val Pro Gly Phe Lys Gly Glu 275
280 285Lys Gly Glu Phe Gly Ala Asp Gly Arg Lys Gly Ala
Pro Gly Leu Ala 290 295 300Gly Lys Asn
Gly Thr Asp Gly Gln Lys Gly Lys Leu Gly Arg Ile Gly305
310 315 320Pro Pro Gly Cys Lys Gly Asp
Pro Gly Asn Arg Gly Pro Asp Gly Tyr 325
330 335Pro Gly Glu Ala Gly Ser Pro Gly Glu Arg Gly Asp
Gln Gly Gly Lys 340 345 350Gly
Asp Pro Gly Arg Pro Gly Arg Arg Gly Pro Pro Gly Glu Ile Gly 355
360 365Ala Lys Gly Ser Lys Gly Tyr Gln Gly
Asn Asn Gly Ala Pro Gly Ser 370 375
380Pro Gly Val Lys Gly Ala Lys Gly Gly Pro Gly Pro Arg Gly Pro Lys385
390 395 400Gly Glu Pro Gly
Arg Arg Gly Asp Pro Gly Thr Lys Gly Ser Pro Gly 405
410 415Ser Asp Gly Pro Lys Gly Glu Lys Gly Asp
Pro Gly Pro Glu Gly Pro 420 425
430Arg Gly Leu Ala Gly Glu Val Gly Asn Lys Gly Ala Lys Gly Asp Arg
435 440 445Gly Leu Pro Gly Pro Arg Gly
Pro Gln Gly Ala Leu Gly Glu Pro Gly 450 455
460Lys Gln Gly Ser Arg Gly Asp Pro Gly Asp Ala Gly Pro Arg Gly
Asp465 470 475 480Ser Gly
Gln Pro Gly Pro Lys Gly Asp Pro Gly Arg Pro Gly Phe Ser
485 490 495Tyr Pro Gly Pro Arg Gly Ala
Pro Gly Glu Lys Gly Glu Pro Gly Pro 500 505
510Arg Gly Pro Glu Gly Gly Arg Gly Asp Phe Gly Leu Lys Gly
Glu Pro 515 520 525Gly Arg Lys Gly
Glu Lys Gly Glu Pro Ala Asp Pro Gly Pro Pro Gly 530
535 540Glu Pro Gly Pro Arg Gly Pro Arg Gly Val Pro Gly
Pro Glu Gly Glu545 550 555
560Pro Gly Pro Pro Gly Asp Pro Gly Leu Thr Glu Cys Asp Val Met Thr
565 570 575Tyr Val Arg Glu Thr
Cys Gly Cys Cys Asp Cys Glu Lys Arg Cys Gly 580
585 590Ala Leu Asp Val Val Phe Val Ile Asp Ser Ser Glu
Ser Ile Gly Tyr 595 600 605Thr Asn
Phe Thr Leu Glu Lys Asn Phe Val Ile Asn Val Val Asn Arg 610
615 620Leu Gly Ala Ile Ala Lys Asp Pro Lys Ser Glu
Thr Gly Thr Arg Val625 630 635
640Gly Val Val Gln Tyr Ser His Glu Gly Thr Phe Glu Ala Ile Gln Leu
645 650 655Asp Asp Glu His
Ile Asp Ser Leu Ser Ser Phe Lys Glu Ala Val Lys 660
665 670Asn Leu Glu Trp Ile Ala Gly Gly Thr Trp Thr
Pro Ser Ala Leu Lys 675 680 685Phe
Ala Tyr Asp Arg Leu Ile Lys Glu Ser Arg Arg Gln Lys Thr Arg 690
695 700Val Phe Ala Val Val Ile Thr Asp Gly Arg
His Asp Pro Arg Asp Asp705 710 715
720Asp Leu Asn Leu Arg Ala Leu Cys Asp Arg Asp Val Thr Val Thr
Ala 725 730 735Ile Gly Ile
Gly Asp Met Phe His Glu Lys His Glu Ser Glu Asn Leu 740
745 750Tyr Ser Ile Ala Cys Asp Lys Pro Gln Gln
Val Arg Asn Met Thr Leu 755 760
765Phe Ser Asp Leu Val Ala Glu Lys Phe Ile Asp Asp Met Glu Asp Val 770
775 780Leu Cys Pro Asp Pro Gln Ile Val
Cys Pro Asp Leu Pro Cys Gln Thr785 790
795 800Glu Leu Ser Val Ala Gln Cys Thr Gln Arg Pro Val
Asp Ile Val Phe 805 810
815Leu Leu Asp Gly Ser Glu Arg Leu Gly Glu Gln Asn Phe His Lys Ala
820 825 830Arg Arg Phe Val Glu Gln
Val Ala Arg Arg Leu Thr Leu Ala Arg Arg 835 840
845Asp Asp Asp Pro Leu Asn Ala Arg Val Ala Leu Leu Gln Phe
Gly Gly 850 855 860Pro Gly Glu Gln Gln
Val Ala Phe Pro Leu Ser His Asn Leu Thr Ala865 870
875 880Ile His Glu Ala Leu Glu Thr Thr Gln Tyr
Leu Asn Ser Phe Ser His 885 890
895Val Gly Ala Gly Val Val His Ala Ile Asn Ala Ile Val Arg Ser Pro
900 905 910Arg Gly Gly Ala Arg
Arg His Ala Glu Leu Ser Phe Val Phe Leu Thr 915
920 925Asp Gly Val Thr Gly Asn Asp Ser Leu His Glu Ser
Ala His Ser Met 930 935 940Arg Asn Glu
Asn Val Val Pro Thr Val Leu Ala Leu Gly Ser Asp Val945
950 955 960Asp Met Asp Val Leu Thr Thr
Leu Ser Leu Gly Asp Arg Ala Ala Val 965
970 975Phe His Glu Lys Asp Tyr Asp Ser Leu Ala Gln Pro
Gly Phe Phe Asp 980 985 990Arg
Phe Ile Arg Trp Ile Cys 995153176PRTHomo sapiens 15Met Arg Lys His
Arg His Leu Pro Leu Val Ala Val Phe Cys Leu Phe 1 5
10 15Leu Ser Gly Phe Pro Thr Thr His Ala Gln
Gln Gln Gln Ala Asp Val 20 25
30Lys Asn Gly Ala Ala Ala Asp Ile Ile Phe Leu Val Asp Ser Ser Trp
35 40 45Thr Ile Gly Glu Glu His Phe Gln
Leu Val Arg Glu Phe Leu Tyr Asp 50 55
60Val Val Lys Ser Leu Ala Val Gly Glu Asn Asp Phe His Phe Ala Leu65
70 75 80Val Gln Phe Asn Gly
Asn Pro His Thr Glu Phe Leu Leu Asn Thr Tyr 85
90 95Arg Thr Lys Gln Glu Val Leu Ser His Ile Ser
Asn Met Ser Tyr Ile 100 105
110Gly Gly Thr Asn Gln Thr Gly Lys Gly Leu Glu Tyr Ile Met Gln Ser
115 120 125His Leu Thr Lys Ala Ala Gly
Ser Arg Ala Gly Asp Gly Val Pro Gln 130 135
140Val Ile Val Val Leu Thr Asp Gly His Ser Lys Asp Gly Leu Ala
Leu145 150 155 160Pro Ser
Ala Glu Leu Lys Ser Ala Asp Val Asn Val Phe Ala Ile Gly
165 170 175Val Glu Asp Ala Asp Glu Gly
Ala Leu Lys Glu Ile Ala Ser Glu Pro 180 185
190Leu Asn Met His Met Phe Asn Leu Glu Asn Phe Thr Ser Leu
His Asp 195 200 205Ile Val Gly Asn
Leu Val Ser Cys Val His Ser Ser Val Ser Pro Glu 210
215 220Arg Ala Gly Asp Thr Glu Thr Leu Lys Asp Ile Thr
Ala Gln Asp Ser225 230 235
240Ala Asp Ile Ile Phe Leu Ile Asp Gly Ser Asn Asn Thr Gly Ser Val
245 250 255Asn Phe Ala Val Ile
Leu Asp Phe Leu Val Asn Leu Leu Glu Lys Leu 260
265 270Pro Ile Gly Thr Gln Gln Ile Arg Val Gly Val Val
Gln Phe Ser Asp 275 280 285Glu Pro
Arg Thr Met Phe Ser Leu Asp Thr Tyr Ser Thr Lys Ala Gln 290
295 300Val Leu Gly Ala Val Lys Ala Leu Gly Phe Ala
Gly Gly Glu Leu Ala305 310 315
320Asn Ile Gly Leu Ala Leu Asp Phe Val Val Glu Asn His Phe Thr Arg
325 330 335Ala Gly Gly Ser
Arg Val Glu Glu Gly Val Pro Gln Val Leu Val Leu 340
345 350Ile Ser Ala Gly Pro Ser Ser Asp Glu Ile Arg
Tyr Gly Val Val Ala 355 360 365Leu
Lys Gln Ala Ser Val Phe Ser Phe Gly Leu Gly Ala Gln Ala Ala 370
375 380Ser Arg Ala Glu Leu Gln His Ile Ala Thr
Asp Asp Asn Leu Val Phe385 390 395
400Thr Val Pro Glu Phe Arg Ser Phe Gly Asp Leu Gln Glu Lys Leu
Leu 405 410 415Pro Tyr Ile
Val Gly Val Ala Gln Arg His Ile Val Leu Lys Pro Pro 420
425 430Thr Ile Val Thr Gln Val Ile Glu Val Asn
Lys Arg Asp Ile Val Phe 435 440
445Leu Val Asp Gly Ser Ser Ala Leu Gly Leu Ala Asn Phe Asn Ala Ile 450
455 460Arg Asp Phe Ile Ala Lys Val Ile
Gln Arg Leu Glu Ile Gly Gln Asp465 470
475 480Leu Ile Gln Val Ala Val Ala Gln Tyr Ala Asp Thr
Val Arg Pro Glu 485 490
495Phe Tyr Phe Asn Thr His Pro Thr Lys Arg Glu Val Ile Thr Ala Val
500 505 510Arg Lys Met Lys Pro Leu
Asp Gly Ser Ala Leu Tyr Thr Gly Ser Ala 515 520
525Leu Asp Phe Val Arg Asn Asn Leu Phe Thr Ser Ser Ala Gly
Tyr Arg 530 535 540Ala Ala Glu Gly Ile
Pro Lys Leu Leu Val Leu Ile Thr Gly Gly Lys545 550
555 560Ser Leu Asp Glu Ile Ser Gln Pro Ala Gln
Glu Leu Lys Arg Ser Ser 565 570
575Ile Met Ala Phe Ala Ile Gly Asn Lys Gly Ala Asp Gln Ala Glu Leu
580 585 590Glu Glu Ile Ala Phe
Asp Ser Ser Leu Val Phe Ile Pro Ala Glu Phe 595
600 605Arg Ala Ala Pro Leu Gln Gly Met Leu Pro Gly Leu
Leu Ala Pro Leu 610 615 620Arg Thr Leu
Ser Gly Thr Pro Glu Val His Ser Asn Lys Arg Asp Ile625
630 635 640Ile Phe Leu Leu Asp Gly Ser
Ala Asn Val Gly Lys Thr Asn Phe Pro 645
650 655Tyr Val Arg Asp Phe Val Met Asn Leu Val Asn Ser
Leu Asp Ile Gly 660 665 670Asn
Asp Asn Ile Arg Val Gly Leu Val Gln Phe Ser Asp Thr Pro Val 675
680 685Thr Glu Phe Ser Leu Asn Thr Tyr Gln
Thr Lys Ser Asp Ile Leu Gly 690 695
700His Leu Arg Gln Leu Gln Leu Gln Gly Gly Ser Gly Leu Asn Thr Gly705
710 715 720Ser Ala Leu Ser
Tyr Val Tyr Ala Asn His Phe Thr Glu Ala Gly Gly 725
730 735Ser Arg Ile Arg Glu His Val Pro Gln Leu
Leu Leu Leu Leu Thr Ala 740 745
750Gly Gln Ser Glu Asp Ser Tyr Leu Gln Ala Ala Asn Ala Leu Thr Arg
755 760 765Ala Gly Ile Leu Thr Phe Cys
Val Gly Ala Ser Gln Ala Asn Lys Ala 770 775
780Glu Leu Glu Gln Ile Ala Phe Asn Pro Ser Leu Val Tyr Leu Met
Asp785 790 795 800Asp Phe
Ser Ser Leu Pro Ala Leu Pro Gln Gln Leu Ile Gln Pro Leu
805 810 815Thr Thr Tyr Val Ser Gly Gly
Val Glu Glu Val Pro Leu Ala Gln Pro 820 825
830Glu Ser Lys Arg Asp Ile Leu Phe Leu Phe Asp Gly Ser Ala
Asn Leu 835 840 845Val Gly Gln Phe
Pro Val Val Arg Asp Phe Leu Tyr Lys Ile Ile Asp 850
855 860Glu Leu Asn Val Lys Pro Glu Gly Thr Arg Ile Ala
Val Ala Gln Tyr865 870 875
880Ser Asp Asp Val Lys Val Glu Ser Arg Phe Asp Glu His Gln Ser Lys
885 890 895Pro Glu Ile Leu Asn
Leu Val Lys Arg Met Lys Ile Lys Thr Gly Lys 900
905 910Ala Leu Asn Leu Gly Tyr Ala Leu Asp Tyr Ala Gln
Arg Tyr Ile Phe 915 920 925Val Lys
Ser Ala Gly Ser Arg Ile Glu Asp Gly Val Leu Gln Phe Leu 930
935 940Val Leu Leu Val Ala Gly Arg Ser Ser Asp Arg
Val Asp Gly Pro Ala945 950 955
960Ser Asn Leu Lys Gln Ser Gly Val Val Pro Phe Ile Phe Gln Ala Lys
965 970 975Asn Ala Asp Pro
Ala Glu Leu Glu Gln Ile Val Leu Ser Pro Ala Phe 980
985 990Ile Leu Ala Ala Glu Ser Leu Pro Lys Ile Gly
Asp Leu His Pro Gln 995 1000
1005Ile Val Asn Leu Leu Lys Ser Val His Asn Gly Ala Pro Ala Pro Val
1010 1015 1020Ser Gly Glu Lys Asp Val Val
Phe Leu Leu Asp Gly Ser Glu Gly Val1025 1030
1035 1040Arg Ser Gly Phe Pro Leu Leu Lys Glu Phe Val Gln
Arg Val Val Glu 1045 1050
1055Ser Leu Asp Val Gly Gln Asp Arg Val Arg Val Ala Val Val Gln Tyr
1060 1065 1070Ser Asp Arg Thr Arg Pro
Glu Phe Tyr Leu Asn Ser Tyr Met Asn Lys 1075 1080
1085Gln Asp Val Val Asn Ala Val Arg Gln Leu Thr Leu Leu Gly
Gly Pro 1090 1095 1100Thr Pro Asn Thr
Gly Ala Ala Leu Glu Phe Val Leu Arg Asn Ile Leu1105 1110
1115 1120Val Ser Ser Ala Gly Ser Arg Ile Thr
Glu Gly Val Pro Gln Leu Leu 1125 1130
1135Ile Val Leu Thr Ala Asp Arg Ser Gly Asp Asp Val Arg Asn Pro
Ser 1140 1145 1150Val Val Val
Lys Arg Gly Gly Ala Val Pro Ile Gly Ile Gly Ile Gly 1155
1160 1165Asn Ala Asp Ile Thr Glu Met Gln Thr Ile Ser
Phe Ile Pro Asp Phe 1170 1175 1180Ala
Val Ala Ile Pro Thr Phe Arg Gln Leu Gly Thr Val Gln Gln Val1185
1190 1195 1200Ile Ser Glu Arg Val Thr
Gln Leu Thr Arg Glu Glu Leu Ser Arg Leu 1205
1210 1215Gln Pro Val Leu Gln Pro Leu Pro Ser Pro Gly Val
Gly Gly Lys Arg 1220 1225
1230Asp Val Val Phe Leu Ile Asp Gly Ser Gln Ser Ala Gly Pro Glu Phe
1235 1240 1245Gln Tyr Val Arg Thr Leu Ile
Glu Arg Leu Val Asp Tyr Leu Asp Val 1250 1255
1260Gly Phe Asp Thr Thr Arg Val Ala Val Ile Gln Phe Ser Asp Asp
Pro1265 1270 1275 1280Lys
Ala Glu Phe Leu Leu Asn Ala His Ser Ser Lys Asp Glu Val Gln
1285 1290 1295Asn Ala Val Gln Arg Leu Arg
Pro Lys Gly Gly Arg Gln Ile Asn Val 1300 1305
1310Gly Asn Ala Leu Glu Tyr Val Ser Arg Asn Ile Phe Lys Arg
Pro Leu 1315 1320 1325Gly Ser Arg
Ile Glu Glu Gly Val Pro Gln Phe Leu Val Leu Ile Ser 1330
1335 1340Ser Gly Lys Ser Asp Asp Glu Val Val Val Pro Ala
Val Glu Leu Lys1345 1350 1355
1360Gln Phe Gly Val Ala Pro Phe Thr Ile Ala Arg Asn Ala Asp Gln Glu
1365 1370 1375Glu Leu Val Lys Ile
Ser Leu Ser Pro Glu Tyr Val Phe Ser Val Ser 1380
1385 1390Thr Phe Arg Glu Leu Pro Ser Leu Glu Gln Lys Leu
Leu Thr Pro Ile 1395 1400 1405Thr
Thr Leu Thr Ser Glu Gln Ile Gln Lys Leu Leu Ala Ser Thr Arg 1410
1415 1420Tyr Pro Pro Pro Ala Val Glu Ser Asp Ala
Ala Asp Ile Val Phe Leu1425 1430 1435
1440Ile Asp Ser Ser Glu Gly Val Arg Pro Asp Gly Phe Ala His Ile
Arg 1445 1450 1455Asp Phe
Val Ser Arg Ile Val Arg Arg Leu Asn Ile Gly Pro Ser Lys 1460
1465 1470Val Arg Val Gly Val Val Gln Phe Ser
Asn Asp Val Phe Pro Glu Phe 1475 1480
1485Tyr Leu Lys Thr Tyr Arg Ser Gln Ala Pro Val Leu Asp Ala Ile Arg
1490 1495 1500Arg Leu Arg Leu Arg Gly Gly
Ser Pro Leu Asn Thr Gly Lys Ala Leu1505 1510
1515 1520Glu Phe Val Ala Arg Asn Leu Phe Val Lys Ser Ala
Gly Ser Arg Ile 1525 1530
1535Glu Asp Gly Val Pro Gln His Leu Val Leu Val Leu Gly Gly Lys Ser
1540 1545 1550Gln Asp Asp Val Ser Arg
Phe Ala Gln Val Ile Arg Ser Ser Gly Ile 1555 1560
1565Val Ser Leu Gly Val Gly Asp Arg Asn Ile Asp Arg Thr Glu
Leu Gln 1570 1575 1580Thr Ile Thr Asn
Asp Pro Arg Leu Val Phe Thr Val Arg Glu Phe Arg1585 1590
1595 1600Glu Leu Pro Asn Ile Glu Glu Arg Ile
Met Asn Ser Phe Gly Pro Ser 1605 1610
1615Ala Ala Thr Pro Ala Pro Pro Gly Val Asp Thr Pro Pro Pro Ser
Arg 1620 1625 1630Pro Glu Lys
Lys Lys Ala Asp Ile Val Phe Leu Leu Asp Gly Ser Ile 1635
1640 1645Asn Phe Arg Arg Asp Ser Phe Gln Glu Val Leu
Arg Phe Val Ser Glu 1650 1655 1660Ile
Val Asp Thr Val Tyr Glu Asp Gly Asp Ser Ile Gln Val Gly Leu1665
1670 1675 1680Val Gln Tyr Asn Ser Asp
Pro Thr Asp Glu Phe Phe Leu Lys Asp Phe 1685
1690 1695Ser Thr Lys Arg Gln Ile Ile Asp Ala Ile Asn Lys
Val Val Tyr Lys 1700 1705
1710Gly Gly Arg His Ala Asn Thr Lys Val Gly Leu Glu His Leu Arg Val
1715 1720 1725Asn His Phe Val Pro Glu Ala
Gly Ser Arg Leu Asp Gln Arg Val Pro 1730 1735
1740Gln Ile Ala Phe Val Ile Thr Gly Gly Lys Ser Val Glu Asp Ala
Gln1745 1750 1755 1760Asp
Val Ser Leu Ala Leu Thr Gln Arg Gly Val Lys Val Phe Ala Val
1765 1770 1775Gly Val Arg Asn Ile Asp Ser
Glu Glu Val Gly Lys Ile Ala Ser Asn 1780 1785
1790Ser Ala Thr Ala Phe Arg Val Gly Asn Val Gln Glu Leu Ser
Glu Leu 1795 1800 1805Ser Glu Gln
Val Leu Glu Thr Leu His Asp Ala Met His Glu Thr Leu 1810
1815 1820Cys Pro Gly Val Thr Asp Ala Ala Lys Ala Cys Asn
Leu Asp Val Ile1825 1830 1835
1840Leu Gly Phe Asp Gly Ser Arg Asp Gln Asn Val Phe Val Ala Gln Lys
1845 1850 1855Gly Phe Glu Ser Lys
Val Asp Ala Ile Leu Asn Arg Ile Ser Gln Met 1860
1865 1870His Arg Val Ser Cys Ser Gly Gly Arg Ser Pro Thr
Val Arg Val Ser 1875 1880 1885Val
Val Ala Asn Thr Pro Ser Gly Pro Val Glu Ala Phe Asp Phe Asp 1890
1895 1900Glu Tyr Gln Pro Glu Met Leu Glu Lys Phe
Arg Asn Met Arg Ser Gln1905 1910 1915
1920His Pro Tyr Val Leu Thr Glu Asp Thr Leu Lys Val Tyr Leu Asn
Lys 1925 1930 1935Phe Arg
Gln Ser Ser Pro Asp Ser Val Lys Val Val Ile His Phe Thr 1940
1945 1950Asp Gly Ala Asp Gly Asp Leu Ala Asp
Leu His Arg Ala Ser Glu Asn 1955 1960
1965Leu Arg Gln Glu Gly Val Arg Ala Leu Ile Leu Val Gly Leu Glu Arg
1970 1975 1980Val Val Asn Leu Glu Arg Leu
Met His Leu Glu Phe Gly Arg Gly Phe1985 1990
1995 2000Met Tyr Asp Arg Pro Leu Arg Leu Asn Leu Leu Asp
Leu Asp Tyr Glu 2005 2010
2015Leu Ala Glu Gln Leu Asp Asn Ile Ala Glu Lys Ala Cys Cys Gly Val
2020 2025 2030Pro Cys Lys Cys Ser Gly
Gln Arg Gly Asp Arg Gly Pro Ile Gly Ser 2035 2040
2045Ile Gly Pro Lys Gly Ile Pro Gly Glu Asp Gly Tyr Arg Gly
Tyr Pro 2050 2055 2060Gly Asp Glu Gly
Gly Pro Gly Glu Arg Gly Pro Pro Gly Val Asn Gly2065 2070
2075 2080Thr Gln Gly Phe Gln Gly Cys Pro Gly
Gln Arg Gly Val Lys Gly Ser 2085 2090
2095Arg Gly Phe Pro Gly Glu Lys Gly Glu Val Gly Glu Ile Gly Leu
Asp 2100 2105 2110Gly Leu Asp
Gly Glu Asp Gly Asp Lys Gly Leu Pro Gly Ser Ser Gly 2115
2120 2125Glu Lys Gly Asn Pro Gly Arg Arg Gly Asp Lys
Gly Pro Arg Gly Glu 2130 2135 2140Lys
Gly Glu Arg Gly Asp Val Gly Ile Arg Gly Asp Pro Gly Asn Pro2145
2150 2155 2160Gly Gln Asp Ser Gln Glu
Arg Gly Pro Lys Gly Glu Thr Gly Asp Leu 2165
2170 2175Gly Pro Met Gly Val Pro Gly Arg Asp Gly Val Pro
Gly Gly Pro Gly 2180 2185
2190Glu Thr Gly Lys Asn Gly Gly Phe Gly Arg Arg Gly Pro Pro Gly Ala
2195 2200 2205Lys Gly Asn Lys Gly Gly Pro
Gly Gln Pro Gly Phe Glu Gly Glu Gln 2210 2215
2220Gly Thr Arg Gly Ala Gln Gly Pro Ala Gly Pro Ala Gly Pro Pro
Gly2225 2230 2235 2240Leu
Ile Gly Glu Gln Gly Ile Ser Gly Pro Arg Gly Ser Gly Gly Ala
2245 2250 2255Arg Gly Ala Pro Gly Glu Arg
Gly Arg Thr Gly Pro Leu Gly Arg Lys 2260 2265
2270Gly Glu Pro Gly Glu Pro Gly Pro Lys Gly Gly Ile Gly Asn
Pro Gly 2275 2280 2285Pro Arg Gly
Glu Thr Gly Asp Asp Gly Arg Asp Gly Val Gly Ser Glu 2290
2295 2300Gly Arg Arg Gly Lys Lys Gly Glu Arg Gly Phe Pro
Gly Tyr Pro Gly2305 2310 2315
2320Pro Lys Gly Asn Pro Gly Glu Pro Gly Leu Asn Gly Thr Thr Gly Pro
2325 2330 2335Lys Gly Ile Arg Gly
Arg Arg Gly Asn Ser Gly Pro Pro Gly Ile Val 2340
2345 2350Gly Gln Lys Gly Arg Pro Gly Tyr Pro Gly Pro Ala
Gly Pro Arg Gly 2355 2360 2365Asn
Arg Gly Asp Ser Ile Asp Gln Cys Ala Leu Ile Gln Ser Ile Lys 2370
2375 2380Asp Lys Cys Pro Cys Cys Tyr Gly Pro Leu
Glu Cys Pro Val Phe Pro2385 2390 2395
2400Thr Glu Leu Ala Phe Ala Leu Asp Thr Ser Glu Gly Val Asn Gln
Asp 2405 2410 2415Thr Phe
Gly Arg Met Arg Asp Val Val Leu Ser Ile Val Asn Val Leu 2420
2425 2430Thr Ile Ala Glu Ser Asn Cys Pro Thr
Gly Ala Arg Val Ala Val Val 2435 2440
2445Thr Tyr Asn Asn Glu Val Thr Thr Glu Ile Arg Phe Ala Asp Ser Lys
2450 2455 2460Arg Lys Ser Val Leu Leu Asp
Lys Ile Lys Asn Leu Gln Val Ala Leu2465 2470
2475 2480Thr Ser Lys Gln Gln Ser Leu Glu Thr Ala Met Ser
Phe Val Ala Arg 2485 2490
2495Asn Thr Phe Lys Arg Val Arg Asn Gly Phe Leu Met Arg Lys Val Ala
2500 2505 2510Val Phe Phe Ser Asn Thr
Pro Thr Arg Ala Ser Pro Gln Leu Arg Glu 2515 2520
2525Ala Val Leu Lys Leu Ser Asp Ala Gly Ile Thr Pro Leu Phe
Leu Thr 2530 2535 2540Arg Gln Glu Asp
Arg Gln Leu Ile Asn Ala Leu Gln Ile Asn Asn Thr2545 2550
2555 2560Ala Val Gly His Ala Leu Val Leu Pro
Ala Gly Arg Asp Leu Thr Asp 2565 2570
2575Phe Leu Glu Asn Val Leu Thr Cys His Val Cys Leu Asp Ile Cys
Asn 2580 2585 2590Ile Asp Pro
Ser Cys Gly Phe Gly Ser Trp Arg Pro Ser Phe Arg Asp 2595
2600 2605Arg Arg Ala Ala Gly Ser Asp Val Asp Ile Asp
Met Ala Phe Ile Leu 2610 2615 2620Asp
Ser Ala Glu Thr Thr Thr Leu Phe Gln Phe Asn Glu Met Lys Lys2625
2630 2635 2640Tyr Ile Ala Tyr Leu Val
Arg Gln Leu Asp Met Ser Pro Asp Pro Lys 2645
2650 2655Ala Ser Gln His Phe Ala Arg Val Ala Val Val Gln
His Ala Pro Ser 2660 2665
2670Glu Ser Val Asp Asn Ala Ser Met Pro Pro Val Lys Val Glu Phe Ser
2675 2680 2685Leu Thr Asp Tyr Gly Ser Lys
Glu Lys Leu Val Asp Phe Leu Ser Arg 2690 2695
2700Gly Met Thr Gln Leu Gln Gly Thr Arg Ala Leu Gly Ser Ala Ile
Glu2705 2710 2715 2720Tyr
Thr Ile Glu Asn Val Phe Glu Ser Ala Pro Asn Pro Arg Asp Leu
2725 2730 2735Lys Ile Val Val Leu Met Leu
Thr Gly Glu Val Pro Glu Gln Gln Leu 2740 2745
2750Glu Glu Ala Gln Arg Val Ile Leu Gln Ala Lys Cys Lys Gly
Tyr Phe 2755 2760 2765Phe Val Val
Leu Gly Ile Gly Arg Lys Val Asn Ile Lys Glu Val Tyr 2770
2775 2780Thr Phe Ala Ser Glu Pro Asn Asp Val Phe Phe Lys
Leu Val Asp Lys2785 2790 2795
2800Ser Thr Glu Leu Asn Glu Glu Pro Leu Met Arg Phe Gly Arg Leu Leu
2805 2810 2815Pro Ser Phe Val Ser
Ser Glu Asn Ala Phe Tyr Leu Ser Pro Asp Ile 2820
2825 2830Arg Lys Gln Cys Asp Trp Phe Gln Gly Asp Gln Pro
Thr Lys Asn Leu 2835 2840 2845Val
Lys Phe Gly His Lys Gln Val Asn Val Pro Asn Asn Val Thr Ser 2850
2855 2860Ser Pro Thr Ser Asn Pro Val Thr Thr Thr
Lys Pro Val Thr Thr Thr2865 2870 2875
2880Lys Pro Val Thr Thr Thr Thr Lys Pro Val Thr Thr Thr Thr Lys
Pro 2885 2890 2895Val Thr
Ile Ile Asn Gln Pro Ser Val Lys Pro Ala Ala Ala Lys Pro 2900
2905 2910Ala Pro Ala Lys Pro Val Ala Ala Lys
Pro Val Ala Thr Lys Thr Ala 2915 2920
2925Thr Val Arg Pro Pro Val Ala Val Lys Pro Ala Thr Ala Ala Lys Pro
2930 2935 2940Val Ala Ala Lys Pro Ala Ala
Val Arg Pro Pro Ala Ala Ala Ala Lys2945 2950
2955 2960Pro Val Ala Thr Lys Pro Glu Val Pro Arg Pro Gln
Ala Ala Lys Pro 2965 2970
2975Ala Ala Thr Lys Pro Ala Thr Thr Lys Pro Val Val Lys Met Leu Arg
2980 2985 2990Glu Val Gln Val Phe Glu
Ile Thr Glu Asn Ser Ala Lys Leu His Trp 2995 3000
3005Glu Arg Pro Glu Pro Pro Gly Pro Tyr Phe Tyr Asp Leu Thr
Val Thr 3010 3015 3020Ser Ala His Asp
Gln Ser Leu Val Leu Lys Gln Asn Leu Thr Val Thr3025 3030
3035 3040Asp Arg Val Ile Gly Gly Leu Leu Ala
Gly Gln Thr Tyr His Val Ala 3045 3050
3055Val Val Cys Tyr Leu Arg Ser Gln Val Arg Ala Thr Tyr His Gly
Ser 3060 3065 3070Phe Ser Thr
Lys Lys Ser Gln Pro Pro Pro Pro Gln Pro Ala Arg Ser 3075
3080 3085Ala Ser Ser Ser Thr Ile Asn Leu Met Val Ser
Thr Glu Pro Leu Ala 3090 3095 3100Leu
Thr Glu Thr Asp Ile Cys Lys Leu Pro Lys Asp Glu Gly Thr Cys3105
3110 3115 3120Arg Asp Phe Ile Leu Lys
Trp Tyr Tyr Asp Pro Asn Thr Lys Ser Cys 3125
3130 3135Ala Arg Phe Trp Tyr Gly Gly Cys Gly Gly Asn Glu
Asn Lys Phe Gly 3140 3145
3150Ser Gln Lys Glu Cys Glu Lys Val Cys Ala Pro Val Leu Ala Lys Pro
3155 3160 3165Gly Val Ile Ser Val Met Gly
Thr 3170 3175163151PRTHomo sapiens 16Gln Gln Gln Gln
Ala Asp Val Lys Asn Gly Ala Ala Ala Asp Ile Ile 1 5
10 15Phe Leu Val Asp Ser Ser Trp Thr Ile Gly
Glu Glu His Phe Gln Leu 20 25
30Val Arg Glu Phe Leu Tyr Asp Val Val Lys Ser Leu Ala Val Gly Glu
35 40 45Asn Asp Phe His Phe Ala Leu Val
Gln Phe Asn Gly Asn Pro His Thr 50 55
60Glu Phe Leu Leu Asn Thr Tyr Arg Thr Lys Gln Glu Val Leu Ser His65
70 75 80Ile Ser Asn Met Ser
Tyr Ile Gly Gly Thr Asn Gln Thr Gly Lys Gly 85
90 95Leu Glu Tyr Ile Met Gln Ser His Leu Thr Lys
Ala Ala Gly Ser Arg 100 105
110Ala Gly Asp Gly Val Pro Gln Val Ile Val Val Leu Thr Asp Gly His
115 120 125Ser Lys Asp Gly Leu Ala Leu
Pro Ser Ala Glu Leu Lys Ser Ala Asp 130 135
140Val Asn Val Phe Ala Ile Gly Val Glu Asp Ala Asp Glu Gly Ala
Leu145 150 155 160Lys Glu
Ile Ala Ser Glu Pro Leu Asn Met His Met Phe Asn Leu Glu
165 170 175Asn Phe Thr Ser Leu His Asp
Ile Val Gly Asn Leu Val Ser Cys Val 180 185
190His Ser Ser Val Ser Pro Glu Arg Ala Gly Asp Thr Glu Thr
Leu Lys 195 200 205Asp Ile Thr Ala
Gln Asp Ser Ala Asp Ile Ile Phe Leu Ile Asp Gly 210
215 220Ser Asn Asn Thr Gly Ser Val Asn Phe Ala Val Ile
Leu Asp Phe Leu225 230 235
240Val Asn Leu Leu Glu Lys Leu Pro Ile Gly Thr Gln Gln Ile Arg Val
245 250 255Gly Val Val Gln Phe
Ser Asp Glu Pro Arg Thr Met Phe Ser Leu Asp 260
265 270Thr Tyr Ser Thr Lys Ala Gln Val Leu Gly Ala Val
Lys Ala Leu Gly 275 280 285Phe Ala
Gly Gly Glu Leu Ala Asn Ile Gly Leu Ala Leu Asp Phe Val 290
295 300Val Glu Asn His Phe Thr Arg Ala Gly Gly Ser
Arg Val Glu Glu Gly305 310 315
320Val Pro Gln Val Leu Val Leu Ile Ser Ala Gly Pro Ser Ser Asp Glu
325 330 335Ile Arg Tyr Gly
Val Val Ala Leu Lys Gln Ala Ser Val Phe Ser Phe 340
345 350Gly Leu Gly Ala Gln Ala Ala Ser Arg Ala Glu
Leu Gln His Ile Ala 355 360 365Thr
Asp Asp Asn Leu Val Phe Thr Val Pro Glu Phe Arg Ser Phe Gly 370
375 380Asp Leu Gln Glu Lys Leu Leu Pro Tyr Ile
Val Gly Val Ala Gln Arg385 390 395
400His Ile Val Leu Lys Pro Pro Thr Ile Val Thr Gln Val Ile Glu
Val 405 410 415Asn Lys Arg
Asp Ile Val Phe Leu Val Asp Gly Ser Ser Ala Leu Gly 420
425 430Leu Ala Asn Phe Asn Ala Ile Arg Asp Phe
Ile Ala Lys Val Ile Gln 435 440
445Arg Leu Glu Ile Gly Gln Asp Leu Ile Gln Val Ala Val Ala Gln Tyr 450
455 460Ala Asp Thr Val Arg Pro Glu Phe
Tyr Phe Asn Thr His Pro Thr Lys465 470
475 480Arg Glu Val Ile Thr Ala Val Arg Lys Met Lys Pro
Leu Asp Gly Ser 485 490
495Ala Leu Tyr Thr Gly Ser Ala Leu Asp Phe Val Arg Asn Asn Leu Phe
500 505 510Thr Ser Ser Ala Gly Tyr
Arg Ala Ala Glu Gly Ile Pro Lys Leu Leu 515 520
525Val Leu Ile Thr Gly Gly Lys Ser Leu Asp Glu Ile Ser Gln
Pro Ala 530 535 540Gln Glu Leu Lys Arg
Ser Ser Ile Met Ala Phe Ala Ile Gly Asn Lys545 550
555 560Gly Ala Asp Gln Ala Glu Leu Glu Glu Ile
Ala Phe Asp Ser Ser Leu 565 570
575Val Phe Ile Pro Ala Glu Phe Arg Ala Ala Pro Leu Gln Gly Met Leu
580 585 590Pro Gly Leu Leu Ala
Pro Leu Arg Thr Leu Ser Gly Thr Pro Glu Val 595
600 605His Ser Asn Lys Arg Asp Ile Ile Phe Leu Leu Asp
Gly Ser Ala Asn 610 615 620Val Gly Lys
Thr Asn Phe Pro Tyr Val Arg Asp Phe Val Met Asn Leu625
630 635 640Val Asn Ser Leu Asp Ile Gly
Asn Asp Asn Ile Arg Val Gly Leu Val 645
650 655Gln Phe Ser Asp Thr Pro Val Thr Glu Phe Ser Leu
Asn Thr Tyr Gln 660 665 670Thr
Lys Ser Asp Ile Leu Gly His Leu Arg Gln Leu Gln Leu Gln Gly 675
680 685Gly Ser Gly Leu Asn Thr Gly Ser Ala
Leu Ser Tyr Val Tyr Ala Asn 690 695
700His Phe Thr Glu Ala Gly Gly Ser Arg Ile Arg Glu His Val Pro Gln705
710 715 720Leu Leu Leu Leu
Leu Thr Ala Gly Gln Ser Glu Asp Ser Tyr Leu Gln 725
730 735Ala Ala Asn Ala Leu Thr Arg Ala Gly Ile
Leu Thr Phe Cys Val Gly 740 745
750Ala Ser Gln Ala Asn Lys Ala Glu Leu Glu Gln Ile Ala Phe Asn Pro
755 760 765Ser Leu Val Tyr Leu Met Asp
Asp Phe Ser Ser Leu Pro Ala Leu Pro 770 775
780Gln Gln Leu Ile Gln Pro Leu Thr Thr Tyr Val Ser Gly Gly Val
Glu785 790 795 800Glu Val
Pro Leu Ala Gln Pro Glu Ser Lys Arg Asp Ile Leu Phe Leu
805 810 815Phe Asp Gly Ser Ala Asn Leu
Val Gly Gln Phe Pro Val Val Arg Asp 820 825
830Phe Leu Tyr Lys Ile Ile Asp Glu Leu Asn Val Lys Pro Glu
Gly Thr 835 840 845Arg Ile Ala Val
Ala Gln Tyr Ser Asp Asp Val Lys Val Glu Ser Arg 850
855 860Phe Asp Glu His Gln Ser Lys Pro Glu Ile Leu Asn
Leu Val Lys Arg865 870 875
880Met Lys Ile Lys Thr Gly Lys Ala Leu Asn Leu Gly Tyr Ala Leu Asp
885 890 895Tyr Ala Gln Arg Tyr
Ile Phe Val Lys Ser Ala Gly Ser Arg Ile Glu 900
905 910Asp Gly Val Leu Gln Phe Leu Val Leu Leu Val Ala
Gly Arg Ser Ser 915 920 925Asp Arg
Val Asp Gly Pro Ala Ser Asn Leu Lys Gln Ser Gly Val Val 930
935 940Pro Phe Ile Phe Gln Ala Lys Asn Ala Asp Pro
Ala Glu Leu Glu Gln945 950 955
960Ile Val Leu Ser Pro Ala Phe Ile Leu Ala Ala Glu Ser Leu Pro Lys
965 970 975Ile Gly Asp Leu
His Pro Gln Ile Val Asn Leu Leu Lys Ser Val His 980
985 990Asn Gly Ala Pro Ala Pro Val Ser Gly Glu Lys
Asp Val Val Phe Leu 995 1000
1005Leu Asp Gly Ser Glu Gly Val Arg Ser Gly Phe Pro Leu Leu Lys Glu
1010 1015 1020Phe Val Gln Arg Val Val Glu
Ser Leu Asp Val Gly Gln Asp Arg Val1025 1030
1035 1040Arg Val Ala Val Val Gln Tyr Ser Asp Arg Thr Arg
Pro Glu Phe Tyr 1045 1050
1055Leu Asn Ser Tyr Met Asn Lys Gln Asp Val Val Asn Ala Val Arg Gln
1060 1065 1070Leu Thr Leu Leu Gly Gly
Pro Thr Pro Asn Thr Gly Ala Ala Leu Glu 1075 1080
1085Phe Val Leu Arg Asn Ile Leu Val Ser Ser Ala Gly Ser Arg
Ile Thr 1090 1095 1100Glu Gly Val Pro
Gln Leu Leu Ile Val Leu Thr Ala Asp Arg Ser Gly1105 1110
1115 1120Asp Asp Val Arg Asn Pro Ser Val Val
Val Lys Arg Gly Gly Ala Val 1125 1130
1135Pro Ile Gly Ile Gly Ile Gly Asn Ala Asp Ile Thr Glu Met Gln
Thr 1140 1145 1150Ile Ser Phe
Ile Pro Asp Phe Ala Val Ala Ile Pro Thr Phe Arg Gln 1155
1160 1165Leu Gly Thr Val Gln Gln Val Ile Ser Glu Arg
Val Thr Gln Leu Thr 1170 1175 1180Arg
Glu Glu Leu Ser Arg Leu Gln Pro Val Leu Gln Pro Leu Pro Ser1185
1190 1195 1200Pro Gly Val Gly Gly Lys
Arg Asp Val Val Phe Leu Ile Asp Gly Ser 1205
1210 1215Gln Ser Ala Gly Pro Glu Phe Gln Tyr Val Arg Thr
Leu Ile Glu Arg 1220 1225
1230Leu Val Asp Tyr Leu Asp Val Gly Phe Asp Thr Thr Arg Val Ala Val
1235 1240 1245Ile Gln Phe Ser Asp Asp Pro
Lys Ala Glu Phe Leu Leu Asn Ala His 1250 1255
1260Ser Ser Lys Asp Glu Val Gln Asn Ala Val Gln Arg Leu Arg Pro
Lys1265 1270 1275 1280Gly
Gly Arg Gln Ile Asn Val Gly Asn Ala Leu Glu Tyr Val Ser Arg
1285 1290 1295Asn Ile Phe Lys Arg Pro Leu
Gly Ser Arg Ile Glu Glu Gly Val Pro 1300 1305
1310Gln Phe Leu Val Leu Ile Ser Ser Gly Lys Ser Asp Asp Glu
Val Val 1315 1320 1325Val Pro Ala
Val Glu Leu Lys Gln Phe Gly Val Ala Pro Phe Thr Ile 1330
1335 1340Ala Arg Asn Ala Asp Gln Glu Glu Leu Val Lys Ile
Ser Leu Ser Pro1345 1350 1355
1360Glu Tyr Val Phe Ser Val Ser Thr Phe Arg Glu Leu Pro Ser Leu Glu
1365 1370 1375Gln Lys Leu Leu Thr
Pro Ile Thr Thr Leu Thr Ser Glu Gln Ile Gln 1380
1385 1390Lys Leu Leu Ala Ser Thr Arg Tyr Pro Pro Pro Ala
Val Glu Ser Asp 1395 1400 1405Ala
Ala Asp Ile Val Phe Leu Ile Asp Ser Ser Glu Gly Val Arg Pro 1410
1415 1420Asp Gly Phe Ala His Ile Arg Asp Phe Val
Ser Arg Ile Val Arg Arg1425 1430 1435
1440Leu Asn Ile Gly Pro Ser Lys Val Arg Val Gly Val Val Gln Phe
Ser 1445 1450 1455Asn Asp
Val Phe Pro Glu Phe Tyr Leu Lys Thr Tyr Arg Ser Gln Ala 1460
1465 1470Pro Val Leu Asp Ala Ile Arg Arg Leu
Arg Leu Arg Gly Gly Ser Pro 1475 1480
1485Leu Asn Thr Gly Lys Ala Leu Glu Phe Val Ala Arg Asn Leu Phe Val
1490 1495 1500Lys Ser Ala Gly Ser Arg Ile
Glu Asp Gly Val Pro Gln His Leu Val1505 1510
1515 1520Leu Val Leu Gly Gly Lys Ser Gln Asp Asp Val Ser
Arg Phe Ala Gln 1525 1530
1535Val Ile Arg Ser Ser Gly Ile Val Ser Leu Gly Val Gly Asp Arg Asn
1540 1545 1550Ile Asp Arg Thr Glu Leu
Gln Thr Ile Thr Asn Asp Pro Arg Leu Val 1555 1560
1565Phe Thr Val Arg Glu Phe Arg Glu Leu Pro Asn Ile Glu Glu
Arg Ile 1570 1575 1580Met Asn Ser Phe
Gly Pro Ser Ala Ala Thr Pro Ala Pro Pro Gly Val1585 1590
1595 1600Asp Thr Pro Pro Pro Ser Arg Pro Glu
Lys Lys Lys Ala Asp Ile Val 1605 1610
1615Phe Leu Leu Asp Gly Ser Ile Asn Phe Arg Arg Asp Ser Phe Gln
Glu 1620 1625 1630Val Leu Arg
Phe Val Ser Glu Ile Val Asp Thr Val Tyr Glu Asp Gly 1635
1640 1645Asp Ser Ile Gln Val Gly Leu Val Gln Tyr Asn
Ser Asp Pro Thr Asp 1650 1655 1660Glu
Phe Phe Leu Lys Asp Phe Ser Thr Lys Arg Gln Ile Ile Asp Ala1665
1670 1675 1680Ile Asn Lys Val Val Tyr
Lys Gly Gly Arg His Ala Asn Thr Lys Val 1685
1690 1695Gly Leu Glu His Leu Arg Val Asn His Phe Val Pro
Glu Ala Gly Ser 1700 1705
1710Arg Leu Asp Gln Arg Val Pro Gln Ile Ala Phe Val Ile Thr Gly Gly
1715 1720 1725Lys Ser Val Glu Asp Ala Gln
Asp Val Ser Leu Ala Leu Thr Gln Arg 1730 1735
1740Gly Val Lys Val Phe Ala Val Gly Val Arg Asn Ile Asp Ser Glu
Glu1745 1750 1755 1760Val
Gly Lys Ile Ala Ser Asn Ser Ala Thr Ala Phe Arg Val Gly Asn
1765 1770 1775Val Gln Glu Leu Ser Glu Leu
Ser Glu Gln Val Leu Glu Thr Leu His 1780 1785
1790Asp Ala Met His Glu Thr Leu Cys Pro Gly Val Thr Asp Ala
Ala Lys 1795 1800 1805Ala Cys Asn
Leu Asp Val Ile Leu Gly Phe Asp Gly Ser Arg Asp Gln 1810
1815 1820Asn Val Phe Val Ala Gln Lys Gly Phe Glu Ser Lys
Val Asp Ala Ile1825 1830 1835
1840Leu Asn Arg Ile Ser Gln Met His Arg Val Ser Cys Ser Gly Gly Arg
1845 1850 1855Ser Pro Thr Val Arg
Val Ser Val Val Ala Asn Thr Pro Ser Gly Pro 1860
1865 1870Val Glu Ala Phe Asp Phe Asp Glu Tyr Gln Pro Glu
Met Leu Glu Lys 1875 1880 1885Phe
Arg Asn Met Arg Ser Gln His Pro Tyr Val Leu Thr Glu Asp Thr 1890
1895 1900Leu Lys Val Tyr Leu Asn Lys Phe Arg Gln
Ser Ser Pro Asp Ser Val1905 1910 1915
1920Lys Val Val Ile His Phe Thr Asp Gly Ala Asp Gly Asp Leu Ala
Asp 1925 1930 1935Leu His
Arg Ala Ser Glu Asn Leu Arg Gln Glu Gly Val Arg Ala Leu 1940
1945 1950Ile Leu Val Gly Leu Glu Arg Val Val
Asn Leu Glu Arg Leu Met His 1955 1960
1965Leu Glu Phe Gly Arg Gly Phe Met Tyr Asp Arg Pro Leu Arg Leu Asn
1970 1975 1980Leu Leu Asp Leu Asp Tyr Glu
Leu Ala Glu Gln Leu Asp Asn Ile Ala1985 1990
1995 2000Glu Lys Ala Cys Cys Gly Val Pro Cys Lys Cys Ser
Gly Gln Arg Gly 2005 2010
2015Asp Arg Gly Pro Ile Gly Ser Ile Gly Pro Lys Gly Ile Pro Gly Glu
2020 2025 2030Asp Gly Tyr Arg Gly Tyr
Pro Gly Asp Glu Gly Gly Pro Gly Glu Arg 2035 2040
2045Gly Pro Pro Gly Val Asn Gly Thr Gln Gly Phe Gln Gly Cys
Pro Gly 2050 2055 2060Gln Arg Gly Val
Lys Gly Ser Arg Gly Phe Pro Gly Glu Lys Gly Glu2065 2070
2075 2080Val Gly Glu Ile Gly Leu Asp Gly Leu
Asp Gly Glu Asp Gly Asp Lys 2085 2090
2095Gly Leu Pro Gly Ser Ser Gly Glu Lys Gly Asn Pro Gly Arg Arg
Gly 2100 2105 2110Asp Lys Gly
Pro Arg Gly Glu Lys Gly Glu Arg Gly Asp Val Gly Ile 2115
2120 2125Arg Gly Asp Pro Gly Asn Pro Gly Gln Asp Ser
Gln Glu Arg Gly Pro 2130 2135 2140Lys
Gly Glu Thr Gly Asp Leu Gly Pro Met Gly Val Pro Gly Arg Asp2145
2150 2155 2160Gly Val Pro Gly Gly Pro
Gly Glu Thr Gly Lys Asn Gly Gly Phe Gly 2165
2170 2175Arg Arg Gly Pro Pro Gly Ala Lys Gly Asn Lys Gly
Gly Pro Gly Gln 2180 2185
2190Pro Gly Phe Glu Gly Glu Gln Gly Thr Arg Gly Ala Gln Gly Pro Ala
2195 2200 2205Gly Pro Ala Gly Pro Pro Gly
Leu Ile Gly Glu Gln Gly Ile Ser Gly 2210 2215
2220Pro Arg Gly Ser Gly Gly Ala Arg Gly Ala Pro Gly Glu Arg Gly
Arg2225 2230 2235 2240Thr
Gly Pro Leu Gly Arg Lys Gly Glu Pro Gly Glu Pro Gly Pro Lys
2245 2250 2255Gly Gly Ile Gly Asn Pro Gly
Pro Arg Gly Glu Thr Gly Asp Asp Gly 2260 2265
2270Arg Asp Gly Val Gly Ser Glu Gly Arg Arg Gly Lys Lys Gly
Glu Arg 2275 2280 2285Gly Phe Pro
Gly Tyr Pro Gly Pro Lys Gly Asn Pro Gly Glu Pro Gly 2290
2295 2300Leu Asn Gly Thr Thr Gly Pro Lys Gly Ile Arg Gly
Arg Arg Gly Asn2305 2310 2315
2320Ser Gly Pro Pro Gly Ile Val Gly Gln Lys Gly Arg Pro Gly Tyr Pro
2325 2330 2335Gly Pro Ala Gly Pro
Arg Gly Asn Arg Gly Asp Ser Ile Asp Gln Cys 2340
2345 2350Ala Leu Ile Gln Ser Ile Lys Asp Lys Cys Pro Cys
Cys Tyr Gly Pro 2355 2360 2365Leu
Glu Cys Pro Val Phe Pro Thr Glu Leu Ala Phe Ala Leu Asp Thr 2370
2375 2380Ser Glu Gly Val Asn Gln Asp Thr Phe Gly
Arg Met Arg Asp Val Val2385 2390 2395
2400Leu Ser Ile Val Asn Val Leu Thr Ile Ala Glu Ser Asn Cys Pro
Thr 2405 2410 2415Gly Ala
Arg Val Ala Val Val Thr Tyr Asn Asn Glu Val Thr Thr Glu 2420
2425 2430Ile Arg Phe Ala Asp Ser Lys Arg Lys
Ser Val Leu Leu Asp Lys Ile 2435 2440
2445Lys Asn Leu Gln Val Ala Leu Thr Ser Lys Gln Gln Ser Leu Glu Thr
2450 2455 2460Ala Met Ser Phe Val Ala Arg
Asn Thr Phe Lys Arg Val Arg Asn Gly2465 2470
2475 2480Phe Leu Met Arg Lys Val Ala Val Phe Phe Ser Asn
Thr Pro Thr Arg 2485 2490
2495Ala Ser Pro Gln Leu Arg Glu Ala Val Leu Lys Leu Ser Asp Ala Gly
2500 2505 2510Ile Thr Pro Leu Phe Leu
Thr Arg Gln Glu Asp Arg Gln Leu Ile Asn 2515 2520
2525Ala Leu Gln Ile Asn Asn Thr Ala Val Gly His Ala Leu Val
Leu Pro 2530 2535 2540Ala Gly Arg Asp
Leu Thr Asp Phe Leu Glu Asn Val Leu Thr Cys His2545 2550
2555 2560Val Cys Leu Asp Ile Cys Asn Ile Asp
Pro Ser Cys Gly Phe Gly Ser 2565 2570
2575Trp Arg Pro Ser Phe Arg Asp Arg Arg Ala Ala Gly Ser Asp Val
Asp 2580 2585 2590Ile Asp Met
Ala Phe Ile Leu Asp Ser Ala Glu Thr Thr Thr Leu Phe 2595
2600 2605Gln Phe Asn Glu Met Lys Lys Tyr Ile Ala Tyr
Leu Val Arg Gln Leu 2610 2615 2620Asp
Met Ser Pro Asp Pro Lys Ala Ser Gln His Phe Ala Arg Val Ala2625
2630 2635 2640Val Val Gln His Ala Pro
Ser Glu Ser Val Asp Asn Ala Ser Met Pro 2645
2650 2655Pro Val Lys Val Glu Phe Ser Leu Thr Asp Tyr Gly
Ser Lys Glu Lys 2660 2665
2670Leu Val Asp Phe Leu Ser Arg Gly Met Thr Gln Leu Gln Gly Thr Arg
2675 2680 2685Ala Leu Gly Ser Ala Ile Glu
Tyr Thr Ile Glu Asn Val Phe Glu Ser 2690 2695
2700Ala Pro Asn Pro Arg Asp Leu Lys Ile Val Val Leu Met Leu Thr
Gly2705 2710 2715 2720Glu
Val Pro Glu Gln Gln Leu Glu Glu Ala Gln Arg Val Ile Leu Gln
2725 2730 2735Ala Lys Cys Lys Gly Tyr Phe
Phe Val Val Leu Gly Ile Gly Arg Lys 2740 2745
2750Val Asn Ile Lys Glu Val Tyr Thr Phe Ala Ser Glu Pro Asn
Asp Val 2755 2760 2765Phe Phe Lys
Leu Val Asp Lys Ser Thr Glu Leu Asn Glu Glu Pro Leu 2770
2775 2780Met Arg Phe Gly Arg Leu Leu Pro Ser Phe Val Ser
Ser Glu Asn Ala2785 2790 2795
2800Phe Tyr Leu Ser Pro Asp Ile Arg Lys Gln Cys Asp Trp Phe Gln Gly
2805 2810 2815Asp Gln Pro Thr Lys
Asn Leu Val Lys Phe Gly His Lys Gln Val Asn 2820
2825 2830Val Pro Asn Asn Val Thr Ser Ser Pro Thr Ser Asn
Pro Val Thr Thr 2835 2840 2845Thr
Lys Pro Val Thr Thr Thr Lys Pro Val Thr Thr Thr Thr Lys Pro 2850
2855 2860Val Thr Thr Thr Thr Lys Pro Val Thr Ile
Ile Asn Gln Pro Ser Val2865 2870 2875
2880Lys Pro Ala Ala Ala Lys Pro Ala Pro Ala Lys Pro Val Ala Ala
Lys 2885 2890 2895Pro Val
Ala Thr Lys Thr Ala Thr Val Arg Pro Pro Val Ala Val Lys 2900
2905 2910Pro Ala Thr Ala Ala Lys Pro Val Ala
Ala Lys Pro Ala Ala Val Arg 2915 2920
2925Pro Pro Ala Ala Ala Ala Lys Pro Val Ala Thr Lys Pro Glu Val Pro
2930 2935 2940Arg Pro Gln Ala Ala Lys Pro
Ala Ala Thr Lys Pro Ala Thr Thr Lys2945 2950
2955 2960Pro Val Val Lys Met Leu Arg Glu Val Gln Val Phe
Glu Ile Thr Glu 2965 2970
2975Asn Ser Ala Lys Leu His Trp Glu Arg Pro Glu Pro Pro Gly Pro Tyr
2980 2985 2990Phe Tyr Asp Leu Thr Val
Thr Ser Ala His Asp Gln Ser Leu Val Leu 2995 3000
3005Lys Gln Asn Leu Thr Val Thr Asp Arg Val Ile Gly Gly Leu
Leu Ala 3010 3015 3020Gly Gln Thr Tyr
His Val Ala Val Val Cys Tyr Leu Arg Ser Gln Val3025 3030
3035 3040Arg Ala Thr Tyr His Gly Ser Phe Ser
Thr Lys Lys Ser Gln Pro Pro 3045 3050
3055Pro Pro Gln Pro Ala Arg Ser Ala Ser Ser Ser Thr Ile Asn Leu
Met 3060 3065 3070Val Ser Thr
Glu Pro Leu Ala Leu Thr Glu Thr Asp Ile Cys Lys Leu 3075
3080 3085Pro Lys Asp Glu Gly Thr Cys Arg Asp Phe Ile
Leu Lys Trp Tyr Tyr 3090 3095 3100Asp
Pro Asn Thr Lys Ser Cys Ala Arg Phe Trp Tyr Gly Gly Cys Gly3105
3110 3115 3120Gly Asn Glu Asn Lys Phe
Gly Ser Gln Lys Glu Cys Glu Lys Val Cys 3125
3130 3135Ala Pro Val Leu Ala Lys Pro Gly Val Ile Ser Val
Met Gly Thr 3140 3145
3150174164DNAHomo sapiens 17cactctggct gggagcagaa ggcagcctcg gtctctgggc
ggcggcggcg gccctctctg 60ccctggccgc gctgtgtggt gaccgcaggc ccgagacatg
agggcggccc gtgctctgct 120gcccctgctg ctgcaggcct gctggacagc cgcgcaggat
gagccggaga ccccgagggc 180cgtggccttc caggactgcc ccgtggacct gttctttgtg
ctggacacct ctgagagcgt 240ggccctgagg ctgaagccct acggggccct cgtggacaaa
gtcaagtcct tcaccaagcg 300cttcatcgac aacctgaggg acaggtacta ccgctgtgac
cgaaacctgg tgtggaacgc 360aggcgcgctg cactacagtg acgaggtgga gatcatccaa
ggcctcacgc gcatgcctgg 420cggccgcgac gcactcaaaa gcagcgtgga cgcggtcaag
tactttggga agggcaccta 480caccgactgc gctatcaaga aggggctgga gcagctcctc
gtggggggct cccacctgaa 540ggagaataag tacctgattg tggtgaccga cgggcacccc
ctggagggct acaaggaacc 600ctgtgggggg ctggaggatg ctgtgaacga ggccaagcac
ctgggcgtca aagtcttctc 660ggtggccatc acacccgacc acctggagcc gcgtctgagc
atcatcgcca cggaccacac 720gtaccggcgc aacttcacgg cggctgactg gggccagagc
cgcgacgcag aggaggccat 780cagccagacc atcgacacca tcgtggacat gatcaaaaat
aacgttgagc aagtgtgctg 840ctccttcgaa tgccagcctg caagaggacc tccgggcctc
cggggcgacc ccggctttga 900gggagaacga ggcaagccgg ggctcccagg agagaaggga
gaagccggag atcctggaag 960acccggggac ctcggacctg ttgggtacca gggaatgaag
ggagaaaaag ggagccgtgg 1020ggagaagggc tccaggggac caaagggcta caagggagag
aagggcaagc gtggcatcga 1080cggggtggac ggcgtgaagg gggagatggg gtacccaggc
ctgccaggct gcaagggctc 1140gccgggtttt gacggcattc aaggaccccc tggccccaag
ggagaccccg gcgcctttgg 1200actgaaagga gaaaagggcg agcctggagc tgacggggag
gccgggagac caggagctcg 1260gggaccatct ggagacgagg ggccagccgg agagcctggg
ccccccggag agaaaggaga 1320ggcgggcgac gaggggaacc caggacctga cggtgccccc
ggggagcggg gtggccctgg 1380agagagagga ccacggggga ccccaggccc gcggggacca
agaggagacc ctggtgaagc 1440tggcccgcag ggtgatcagg gaagagaagg gcccgttggt
gtccctggag acccgggcga 1500ggctggccct atcggaccta aaggctaccg aggcgatgag
ggtcccccag ggtccgaggg 1560tgccagagga gccccaggac ctgccggacc ccctggagac
ccggggctga tgggagaaag 1620gggagaagac ggccccgctg gaaatggcac cgagggcttc
cccggcttcc ccgggtatcc 1680cgggaacagg ggcgctcccg ggataaacgg cacgaagggc
taccccggcc tcaaggggga 1740cgagggagaa gccggggacc ccggagacga taacaacgac
attgcacccc gaggagtcaa 1800aggagcaaag gggtaccggg gtcccgaggg cccccaggga
cccccaggac accaaggacc 1860gcctgggccg gacgaatgcg agattttgga catcatcatg
aaaatgtgct cttgctgtga 1920atgcaagtgc ggccccatcg acctcctgtt cgtgctggac
agctcagaga gcattggcct 1980gcagaacttc gagattgcca aggacttcgt cgtcaaggtc
atcgaccggc tgagccggga 2040cgagctggtc aagttcgagc cagggcagtc gtacgcgggt
gtggtgcagt acagccacag 2100ccagatgcag gagcacgtga gcctgcgcag ccccagcatc
cggaacgtgc aggagctcaa 2160ggaagccatc aagagcctgc agtggatggc gggcggcacc
ttcacggggg aggccctgca 2220gtacacgcgg gaccagctgc tgccgcccag cccgaacaac
cgcatcgccc tggtcatcac 2280tgacgggcgc tcagacactc agagggacac cacaccgctc
aacgtgctct gcagccccgg 2340catccaggtg gtctccgtgg gcatcaaaga cgtgtttgac
ttcatcccag gctcagacca 2400gctcaatgtc atttcttgcc aaggcctggc accatcccag
ggccggcccg gcctctcgct 2460ggtcaaggag aactatgcag agctgctgga ggatgccttc
ctgaagaatg tcaccgccca 2520gatctgcata gacaagaagt gtccagatta cacctgcccc
atcacgttct cctccccggc 2580tgacatcacc atcctgctgg acggctccgc cagcgtgggc
agccacaact ttgacaccac 2640caagcgcttc gccaagcgcc tggccgagcg cttcctcaca
gcgggcagga cggaccccgc 2700ccacgacgtg cgggtggcgg tggtgcagta cagcggcacg
ggccagcagc gcccagagcg 2760ggcgtcgctg cagttcctgc agaactacac ggccctggcc
agtgccgtcg atgccatgga 2820ctttatcaac gacgccaccg acgtcaacga tgccctgggc
tatgtgaccc gcttctaccg 2880cgaggcctcg tccggcgctg ccaagaagag gctgctgctc
ttctcagatg gcaactcgca 2940gggcgccacg cccgctgcca tcgagaaggc cgtgcaggaa
gcccagcggg caggcatcga 3000gatcttcgtg gtggtcgtgg gccgccaggt gaatgagccc
cacatccgcg tcctggtcac 3060cggcaagacg gccgagtacg acgtggccta cggcgagagc
cacctgttcc gtgtccccag 3120ctaccaggcc ctgctccgcg gtgtcttcca ccagacagtc
tccaggaagg tggcgctggg 3180ctagcccacc ctgcacgccg gcaccaaacc ctgtcctccc
acccctcccc actcatcact 3240aaacagagcc caagcttgga aagccaggac acaacgctgc
tgcctgcttt gtgcagggtc 3300ctccggggct cagccctgag ttggcatcac ctgcgcaggg
ccctctgggg ctcagctctg 3360agctagtgtc acctgcacag ggccctctga ggctcagccc
tgagctggcg tcacctgtgc 3420agggccctct ggggctcagc cctgagctgg cctcacctgg
gttccccacc ccgggctctc 3480ctgccctgcc ctcctgcccg ccctccctcc tgcctgcgca
gctccttccc taggcacctc 3540tgtgctgcat cccaccagcc tgagcaagac gcctctcggg
gcctgtgccg cactagcctc 3600cctctcctct gtccccatag ctggtttttc ccaccaatcc
tcacctaaca gttactttac 3660aattaaactc aaagcaagct cttctcctca gcttggggca
gccattggcc tctgtctcgt 3720tttgggaaac caaggtcagg aggccgttgc agacataaat
ctcggcgact cggccccgtc 3780tcctgagggt cctgctggtg accggcctgg accttggccc
tacagccctg gaggccgctg 3840ctgaccagca ctgaccccga cctcagagag tactcgcagg
ggcgctggct gcactcaaga 3900ccctcgagat taacggtgct aaccccgtct gctcctccct
cccgcagaga ctggggcctg 3960gactggacat gagagcccct tggtgccaca gagggctgtg
tcttactaga aacaacgcaa 4020acctctcctt cctcagaata gtgatgtgtt cgacgtttta
tcaaaggccc cctttctatg 4080ttcatgttag ttttgctcct tctgtgtttt tttctgaacc
atatccatgt tgctgacttt 4140tccaaataaa ggttttcact cctc
4164183387DNAHomo sapiens 18agggccacag gtgctgccaa
gatgctccag ggcacctgct ccgtgctcct gctctgggga 60atcctggggg ccatccaggc
ccagcagcag gaggtcatct cgccggacac taccgagaga 120aacaacaact gcccagagaa
gaccgactgc cccatccacg tgtacttcgt gctggacacc 180tcggagagcg tcaccatgca
gtcccccacg gacatcctgc tcttccacat gaagcagttc 240gtgccgcagt tcatcagcca
gctgcagaac gagttctacc tggaccaggt ggcgctgagc 300tggcgctacg gcggcctgca
cttctctgac caggtggagg tgttcagccc accgggcagc 360gaccgggcct ccttcatcaa
gaacctgcag ggcatcagct ccttccgccg cggcaccttc 420accgactgcg cgctggccaa
catgacggag cagatccggc aggaccgcag caagggcacc 480gtccacttcg ccgtggtcat
caccgacggc cacgtcaccg gcagcccctg cgggggcatc 540aagctgcagg ccgagcgggc
ccgcgaggag ggcatccggc tcttcgccgt ggcccccaac 600cagaacctga aggagcaggg
cctgcgggac atcgccagca cgccgcacga gctctaccgc 660aacgactacg ccaccatgct
gcccgactcc accgagatca accaggacac catcaaccgc 720atcatcaagg tcatgaaaca
cgaagcctac ggagagtgct acaaggtgag ctgcctggaa 780atccctgggc cctctgggcc
caagggctac cgtggacaga agggtgccaa gggcaacatg 840ggtgagccgg gagagcctgg
ccagaaggga agacagggag acccgggcat cgaaggcccc 900attggattcc caggacccaa
gggcgttcct ggcttcaaag gagagaaggg tgaatttgga 960gccgacggtc gcaagggggc
ccctggcctg gctggcaaga acgggaccga tggacagaag 1020ggcaagctgg ggcgcatcgg
acctcctggc tgcaagggag accctggaaa ccggggcccc 1080gacggttacc cgggggaagc
agggagtcca ggggagcgag gagaccaagg cggcaagggg 1140gaccctggcc gcccaggacg
cagagggccc ccgggagaaa tcggggccaa gggaagcaag 1200gggtatcaag gcaacaatgg
agccccagga agtcctggtg tgaaaggagc caagggcggg 1260cctgggcccc gcggacccaa
aggcgagccg gggcgcaggg gagaccccgg caccaagggc 1320agcccaggca gcgatggccc
caagggggag aagggggacc ctggccctga gggcccccgc 1380ggcctggctg gagaggttgg
caacaaagga gccaagggag accgaggctt gcctggaccc 1440agaggccccc agggagctct
tggggagccc ggaaagcagg gatctcgggg agaccccggt 1500gatgcaggac cccgtggaga
ctcaggacag ccaggcccca agggagaccc cggcaggcct 1560ggattcagct acccaggacc
ccgaggagca cccggagaaa aaggcgagcc cggcccacgc 1620ggccccgagg gaggccgagg
cgactttggc ttgaaaggag aacctgggag gaaaggagag 1680aaaggagagc ctgcggatcc
tggtccccct ggtgagccag gccctcgggg gccaagagga 1740gtcccaggac ccgagggtga
gcccggcccc cctggagacc ccggtctcac ggagtgtgac 1800gtcatgacct acgtgaggga
gacctgcggg tgctgcgact gtgagaagcg ctgtggcgcc 1860ctggacgtgg tcttcgtcat
cgacagctcc gagagcattg ggtacaccaa cttcacactg 1920gagaagaact tcgtcatcaa
cgtggtcaac aggctgggtg ccatcgctaa ggaccccaag 1980tccgagacag ggacgcgtgt
gggcgtggtg cagtacagcc acgagggcac ctttgaggcc 2040atccagctgg acgacgaaca
tatcgactcc ctgtcgagct tcaaggaggc tgtcaagaac 2100ctcgagtgga ttgcgggcgg
cacctggaca ccctcagccc tcaagtttgc ctacgaccgc 2160ctcatcaagg agagccggcg
ccagaagaca cgtgtgtttg cggtggtcat cacggacggg 2220cgccacgacc ctcgggacga
tgacctcaac ttgcgggcgc tgtgcgatcg cgacgtcaca 2280gtgacggcca tcggcatcgg
ggacatgttc cacgagaagc acgagagtga aaacctctac 2340tccatcgcct gcgacaagcc
acagcaggtg cgcaacatga cgctgttctc cgacctggtc 2400gctgagaagt tcatcgatga
catggaggac gtcctctgcc cggaccctca gatcgtgtgc 2460ccagaccttc cctgccaaac
agagctgtcc gtggcacagt gcacgcagcg gcccgtggac 2520atcgtcttcc tgctggacgg
ctccgagcgg ctgggtgagc agaacttcca caaggcccgg 2580cgcttcgtgg agcaggtggc
gcggcggctg acgctggccc ggagggacga cgaccctctc 2640aacgcacgcg tggcgctgct
gcagtttggt ggccccggcg agcagcaggt ggccttcccg 2700ctgagccaca acctcactgc
catccacgag gcgctggaga ccacacaata cctgaactcc 2760ttctcgcacg tgggcgcagg
cgtggtgcac gccatcaatg ccatcgtgcg cagcccgcgt 2820ggcggggccc ggaggcacgc
agagctgtcc ttcgtgttcc tcacggacgg cgtcacgggc 2880aacgacagtc tgcacgagtc
ggcgcactcc atgcgcaacg agaacgtggt acccaccgtc 2940ctggccttgg gcagcgacgt
ggacatggac gtgctcacca cgctcagcct gggtgaccgc 3000gccgccgtgt tccacgagaa
ggactatgac agcctggcgc aacccggctt cttcgaccgc 3060ttcatccgct ggatctgcta
gcgccgccgc ccgggccccg cagtcgaggg tcgtgagccc 3120accccgtcca tggtgctaag
cgggcccggg tcccacacgg ccagcaccgc tgctcactcg 3180gacgacgccc tgggcctgca
cctctccagc tcctcccacg gggtccccgt agccccggcc 3240cccgcccagc cccaggtctc
cccaggccct ccgcaggctg cccggcctcc ctccccctgc 3300agccatccca aggctcctga
cctacctggc ccctgagctc tggagcaagc cctgacccaa 3360taaaggcttt gaacccaaaa
aaaaaaa 33871910558DNAHomo sapiens
19cagtttggag ctcagtcttc caccaaaggc cgttcagttc tcctgggctc cagcctcctg
60caaggactgc aagagttttc ctccgcagct ctgagtctcc acttttttgg tggagaaagg
120ctgcaaaaag aaaaagagac gcagtgagtg ggaaaagtat gcatcctatt caaacctaat
180tgaatcgagg agcccaggga cacacgcctt caggtttgct caggggttca tatttggtgc
240ttagacaaat tcaaaatgag gaaacatcgg cacttgccct tagtggccgt cttttgcctc
300tttctctcag gctttcctac aactcatgcc cagcagcagc aagcagatgt caaaaatggt
360gcggctgctg atataatatt tctagtggat tcctcttgga ccattggaga ggaacatttc
420caacttgttc gagagtttct atatgatgtt gtaaaatcct tagctgtggg agaaaatgat
480ttccattttg ctctggtcca gttcaacgga aacccacata ccgagttcct gttaaatacg
540tatcgtacta aacaagaagt cctttctcat atttccaaca tgtcttatat tgggggaacc
600aatcagactg gaaaaggatt agaatacata atgcaaagcc acctcaccaa ggctgctgga
660agccgggccg gtgacggagt ccctcaggtt atcgtagtgt taactgatgg acactcgaag
720gatggccttg ctctgccctc agcggaactt aagtctgctg atgttaacgt gtttgcaatt
780ggagttgagg atgcagatga aggagcgtta aaagaaatag caagtgaacc gctcaatatg
840catatgttca acctagagaa ttttacctca cttcatgaca tagtaggaaa cttagtgtcc
900tgtgtgcatt catccgtgag tccagaaagg gctggggaca cggaaaccct taaagacatc
960acagcacaag actctgctga cattattttc cttattgatg gatcaaacaa caccggaagt
1020gtcaatttcg cagtcattct cgacttcctt gtaaatctcc ttgagaaact cccaattgga
1080actcagcaga tccgagtggg ggtggtccag tttagcgatg agcccagaac catgttttcc
1140ttggacacct actccaccaa ggcccaggtt ctgggtgcag tgaaagccct cgggtttgct
1200ggtggggagt tggccaatat cggcctcgcc cttgatttcg tggtggagaa ccacttcacc
1260cgggcagggg gcagccgcgt ggaggaaggg gttccccagg tgctggtcct cataagtgcc
1320gggccttcta gtgacgagat tcgctacggg gtggtagcac tgaagcaggc tagcgtgttc
1380tcattcggcc ttggagccca ggccgcctcc agggcagagc ttcagcacat agctaccgat
1440gacaacttgg tgtttactgt cccggaattc cgtagctttg gggacctcca ggagaaatta
1500ctgccgtaca ttgttggcgt ggcccaaagg cacattgtct tgaaaccgcc aaccattgtc
1560acacaagtca ttgaagtcaa caagagagac atagtcttcc tggtggatgg ctcatctgca
1620ctgggactgg ccaacttcaa tgccatccga gacttcattg ctaaagtcat ccagaggctg
1680gaaatcggac aggatcttat ccaggtggca gtggcccagt atgcagacac tgtgaggcct
1740gaattttatt tcaataccca tccaacaaaa agggaagtca taaccgctgt gcggaaaatg
1800aagcccctgg acggctcggc cctgtacacg ggctctgctc tagactttgt tcgtaacaac
1860ctattcacga gttcagccgg ctaccgggct gccgagggga ttcctaagct tttggtgctg
1920atcacaggtg gtaagtccct agatgaaatc agccagcctg cccaggagct gaagagaagc
1980agcataatgg cctttgccat tgggaacaag ggtgccgatc aggctgagct ggaagagatc
2040gctttcgact cctccctggt gttcatccca gctgagttcc gagccgcccc attgcaaggc
2100atgctgcctg gcttgctggc acctctcagg accctctctg gaacccctga agttcactca
2160aacaaaagag atatcatctt tcttttggat ggatcagcca acgttggaaa aaccaatttc
2220ccttatgtgc gcgactttgt aatgaaccta gttaacagcc ttgatattgg aaatgacaat
2280attcgtgttg gtttagtgca atttagtgac actcctgtaa cggagttctc tttaaacaca
2340taccagacca agtcagatat ccttggtcat ctgaggcagc tgcagctcca gggaggttcg
2400ggcctgaaca caggctcagc cctaagctat gtctatgcca accacttcac ggaagctggc
2460ggcagcagga tccgtgaaca cgtgccgcag ctcctgcttc tgctcacagc tgggcagtct
2520gaggactcct atttgcaagc tgccaacgcc ttgacacgcg cgggcatcct gactttttgt
2580gtgggagcta gccaggcgaa taaggcagag cttgagcaga ttgcttttaa cccaagcctg
2640gtgtatctca tggatgattt cagctccctg ccagctttgc ctcagcagct gattcagccc
2700ctaaccacat atgttagtgg aggtgtggag gaagtaccac tcgctcagcc agagagcaag
2760cgagacattc tgttcctctt tgacggctca gccaatcttg tgggccagtt ccctgttgtc
2820cgtgactttc tctacaagat tatcgatgag ctcaatgtga agccagaggg gacccgaatt
2880gcggtggctc agtacagcga tgatgtcaag gtggagtccc gttttgatga gcaccagagt
2940aagcctgaga tcctgaatct tgtgaagaga atgaagatca agacgggcaa agccctcaac
3000ctgggctacg cgctggacta tgcacagagg tacatttttg tgaagtctgc tggcagccgg
3060atcgaggatg gagtgcttca gttcctggtg ctgctggtcg caggaaggtc atctgaccgt
3120gtggatgggc cagcaagtaa cctgaagcag agtggggttg tgcctttcat cttccaagcc
3180aagaacgcag accctgctga gttagagcag atcgtgctgt ctccagcgtt tatcctggct
3240gcagagtcgc ttcccaagat tggagatctt catccacaga tagtgaatct cttaaaatca
3300gtgcacaacg gagcaccagc accagtttca ggtgaaaagg acgtggtgtt tctgcttgat
3360ggctctgagg gcgtcaggag cggcttccct ctgttgaaag agtttgtcca gagagtggtg
3420gaaagcctgg atgtgggcca ggaccgggtc cgcgtggccg tggtgcagta cagcgaccgg
3480accaggcccg agttctacct gaattcatac atgaacaagc aggacgtcgt caacgctgtc
3540cgccagctga ccctgctggg agggccgacc cccaacaccg gggccgccct ggagtttgtc
3600ctgaggaaca tcctggtcag ctctgcggga agcaggataa cagaaggtgt gccccagctg
3660ctgatcgtcc tcacggccga caggtctggg gatgatgtgc ggaacccctc cgtggtcgtg
3720aagaggggtg gggctgtgcc cattggcatt ggcatcggga acgctgacat cacagagatg
3780cagaccatct ccttcatccc ggactttgcc gtggccattc ccacctttcg ccagctgggg
3840accgtccaac aggtcatctc tgagagggtg acccagctca cccgcgagga gctgagcagg
3900ctgcagccgg tgttgcagcc tctaccgagc ccaggtgttg gtggcaagag ggacgtggtc
3960tttctcatcg atgggtccca aagtgccggg cctgagttcc agtacgttcg caccctcata
4020gagaggctgg ttgactacct ggacgtgggc tttgacacca cccgggtggc tgtcatccag
4080ttcagcgatg accccaaggc ggagttcctg ctgaacgccc attccagcaa ggatgaagtg
4140cagaacgcgg tgcagcggct gaggcccaag ggagggcggc agatcaacgt gggcaatgcc
4200ctggagtacg tgtccaggaa catcttcaag aggcccctgg ggagccgcat tgaagagggc
4260gtcccacagt tcctggtcct catctcgtct ggaaagtctg acgatgaggt ggtcgtcccg
4320gcggtggagc tcaagcagtt tggcgtggcc cctttcacga tcgccaggaa cgcagaccag
4380gaggagctgg tgaagatctc gctgagcccc gaatatgtgt tctcggtgag caccttccgg
4440gagctgccca gcctggagca gaaactgctg acgcccatca cgaccctgac ctcagagcag
4500atccagaagc tcttagccag cactcgctat ccacctccag cagttgagag tgatgctgca
4560gacattgtct ttctgatcga cagctctgag ggagttaggc cagatggctt tgcacatatt
4620cgagattttg ttagcaggat tgttcgaaga ctcaacatcg gccccagtaa agtgagagtt
4680ggggtcgtgc agttcagcaa tgatgtcttc ccagaattct atctgaaaac ctacagatcc
4740caggccccgg tgctggacgc catacggcgc ctgaggctca gaggggggtc cccactgaac
4800actggcaagg ctctcgaatt tgtggcaaga aacctctttg ttaagtctgc ggggagtcgc
4860atagaagacg gggtgcccca acacctggtc ctggtcctgg gtggaaaatc ccaggacgat
4920gtgtccaggt tcgcccaggt gatccgttcc tcgggcattg tgagtttagg ggtaggagac
4980cggaacatcg acagaacaga gctgcagacc atcaccaatg accccagact ggtcttcaca
5040gtgcgagagt tcagagagct tcccaacata gaagaaagaa tcatgaactc gtttggaccc
5100tccgcagcca ctcctgcacc tccaggggtg gacacccctc ctccttcacg gccagagaag
5160aagaaagcag acattgtgtt cctgttggat ggttccatca acttcaggag ggacagtttc
5220caggaagtgc ttcgttttgt gtctgaaata gtggacacag tttatgaaga tggcgactcc
5280atccaagtgg ggcttgtcca gtacaactct gaccccactg acgaattctt cctgaaggac
5340ttctctacca agaggcagat tattgacgcc atcaacaaag tggtctacaa agggggaaga
5400cacgccaaca ctaaggtggg ccttgagcac ctgcgggtaa accactttgt gcctgaggca
5460ggcagccgcc tggaccagcg ggtccctcag attgcctttg tgatcacggg aggaaagtcg
5520gtggaagatg cacaggatgt gagcctggcc ctcacccaga ggggggtcaa agtgtttgct
5580gttggagtga ggaatatcga ctcggaggag gttggaaaga tagcgtccaa cagcgccaca
5640gcgttccgcg tgggcaacgt ccaggagctg tccgaactga gcgagcaagt tttggaaact
5700ttgcatgatg cgatgcatga aaccctttgc cctggtgtaa ctgatgctgc caaagcttgt
5760aatctggatg tgattctggg gtttgatggt tctagagacc agaatgtttt tgtggcccag
5820aagggcttcg agtccaaggt ggacgccatc ttgaacagaa tcagccagat gcacagggtc
5880agctgcagcg gtggccgctc gcccaccgtg cgtgtgtcag tggtggccaa cacgccctcg
5940ggcccggtgg aggcctttga ctttgacgag taccagccag agatgctcga gaagttccgg
6000aacatgcgca gccagcaccc ctacgtcctc acggaggaca ccctgaaggt ctacctgaac
6060aagttcagac agtcctcgcc ggacagcgtg aaggtggtca ttcattttac tgatggagca
6120gacggagatc tggctgattt acacagagca tctgagaacc tccgccaaga aggagtccgt
6180gccttgatcc tggtgggcct tgaacgagtg gtcaacttgg agcggctaat gcatctggag
6240tttgggcgag ggtttatgta tgacaggccc ctgaggctta acttgctgga cttggattat
6300gaactagcgg agcagcttga caacattgcc gagaaagctt gctgtggggt tccctgcaag
6360tgctctgggc agaggggaga ccgcgggccc atcggcagca tcgggccaaa gggtattcct
6420ggagaagacg gctaccgagg ctatcctggt gatgagggtg gacccggtga gcgtggtccg
6480cctggtgtga acggcactca aggtttccag ggctgcccgg gccagagagg agtaaagggc
6540tctcggggat tcccaggaga gaagggcgaa gtaggagaaa ttggactgga tggtctggat
6600ggtgaagatg gagacaaagg attgcctggt tcttctggag agaaagggaa tcctggaaga
6660aggggtgata aaggacctcg aggagagaaa ggagaaagag gagatgttgg gattcgaggg
6720gacccgggta acccaggaca agacagccag gagagaggac ccaaaggaga aaccggtgac
6780ctcggcccca tgggtgtccc agggagagat ggagtacctg gaggacctgg agaaactggg
6840aagaatggtg gctttggccg aaggggaccc cccggagcta agggcaacaa gggcggtcct
6900ggccagccgg gctttgaggg agagcagggg accagaggtg cacagggccc agctggtcct
6960gctggtcctc cagggctgat aggagaacaa ggcatttctg gacctagggg aagcggaggt
7020gcccgtggcg ctcctggaga acgaggcaga accggtccac tgggaagaaa gggtgagccc
7080ggagagccag gaccaaaagg aggaatcggg aacccgggcc ctcgtgggga gacgggagat
7140gacgggagag acggagttgg cagtgaagga cgcagaggca aaaaaggaga aagaggattt
7200cctggatacc caggaccaaa gggtaaccca ggtgaacctg ggctaaatgg aacaacagga
7260cccaaaggca tcagaggccg aaggggaaat tcgggacctc cagggatagt tggacagaag
7320gggagacctg gctacccagg accagctggt ccaaggggca acaggggcga ctccatcgat
7380caatgtgccc tcatccaaag catcaaagat aaatgccctt gctgttacgg gcccctggag
7440tgccccgtct tcccaacaga actagccttt gctttagaca cctctgaggg agtcaaccaa
7500gacactttcg gccggatgcg agatgtggtc ttgagtattg tgaatgtcct gaccattgct
7560gagagcaact gcccgacggg ggcccgggtg gctgtggtca cctacaacaa cgaggtgacc
7620acggagatcc ggtttgctga ctccaagagg aagtcggtcc tcctggacaa gattaagaac
7680cttcaggtgg ctctgacatc caaacagcag agtctggaga ctgccatgtc gtttgtggcc
7740aggaacacat ttaagcgtgt gaggaacgga ttcctaatga ggaaagtggc tgttttcttc
7800agcaacacac ccacaagagc atccccacag ctcagagagg ctgtgctcaa actctcagat
7860gcggggatca cccccttgtt ccttacaagg caggaagacc ggcagctcat caacgctttg
7920cagatcaata acacagcagt ggggcatgcg cttgtcctgc ctgcagggag agacctcaca
7980gacttcctgg agaatgtcct cacgtgtcat gtttgcttgg acatctgcaa catcgaccca
8040tcctgtggat ttggcagttg gaggccttcc ttcagggaca ggagagcggc agggagtgat
8100gtggacatcg acatggcttt catcttagac agcgctgaga ccaccaccct gttccagttc
8160aatgagatga agaagtacat agcgtacctg gtcagacaac tggacatgag cccagatccc
8220aaggcctccc agcacttcgc cagagtggca gttgtgcagc acgcgccctc tgagtccgtg
8280gacaatgcca gcatgccacc tgtgaaggtg gaattctccc tgactgacta tggctccaag
8340gagaagctgg tggacttcct cagcagggga atgacacagt tgcagggaac cagggcctta
8400ggcagtgcca ttgaatacac catagagaat gtctttgaaa gtgccccaaa cccacgggac
8460ctgaaaattg tggtcctgat gctgacgggc gaggtgccgg agcagcagct ggaggaggcc
8520cagagagtca tcctgcaggc caaatgcaag ggctacttct tcgtggtcct gggcattggc
8580aggaaggtga acatcaagga ggtatacacc ttcgccagtg agccaaacga cgtcttcttc
8640aaattagtgg acaagtccac cgagctcaac gaggagcctt tgatgcgctt cgggaggctg
8700ttgccgtcct tcgtcagcag tgaaaatgct ttttacttgt ccccagatat caggaaacag
8760tgtgattggt tccaagggga ccaacccaca aagaaccttg tgaagtttgg tcacaaacaa
8820gtaaatgttc cgaataacgt tacttcaagt cctacatcca acccagtgac gacaacgaag
8880ccggtgacta cgacgaagcc ggtgaccacc acaacaaagc ctgtaaccac cacaacaaag
8940cctgtgacta ttataaatca gccatctgtg aagccagccg ctgcaaagcc ggcccctgcg
9000aaacctgtgg ctgccaagcc tgtggccaca aagacggcca ctgttagacc cccagtggcg
9060gtgaagccag caacagcagc gaagcctgta gcagcaaagc cagcagctgt aagacccccc
9120gctgctgctg caaaaccagt ggcgaccaag cctgaggtcc ctaggccaca ggcagccaaa
9180ccagctgcca ccaagccagc caccactaag cccgtggtta agatgctccg tgaagtccag
9240gtgtttgaga taacagagaa cagcgccaaa ctccactggg agaggcctga gccccccggt
9300ccttattttt atgacctcac cgtcacctca gcccatgatc agtccctggt tctgaagcag
9360aacctcacgg tcacggaccg cgtcattgga ggcctgctcg ctgggcagac ataccatgtg
9420gctgtggtct gctacctgag gtctcaggtc agagccacct accacggaag tttcagtaca
9480aagaaatctc agcccccacc tccacagcca gcaaggtcag cttctagttc aaccatcaat
9540ctaatggtga gcacagaacc attggctctc actgaaacag atatatgcaa gttgccgaaa
9600gacgaaggaa cttgcaggga tttcatatta aaatggtact atgatccaaa caccaaaagc
9660tgtgcaagat tctggtatgg aggttgtggt ggaaacgaaa acaaatttgg atcacagaaa
9720gaatgtgaaa aggtttgcgc tcctgtgctc gccaaacccg gagtcatcag tgtgatggga
9780acctaagcgt gggtggccaa catcatatac ctcttgaaga agaaggagtc agccatcgcc
9840aacttgtctc tgtagaagct ccgggtgtag attcccttgc actgtatcat ttcatgcttt
9900gatttacact cgaactcggg agggaacatc ctgctgcatg acctatcagt atggtgctaa
9960tgtgtctgtg gaccctcgct ctctgtctcc agcagttctc tcgaatactt tgaatgttgt
10020gtaacagtta gccactgctg gtgtttatgt gaacattcct atcaatccaa attccctctg
10080gagtttcatg ttatgcctgt tgcaggcaaa tgtaaagtct agaaaataat gcaaatgtca
10140cggctactct atatactttt gcttggttca ttttttttcc cttttagtta agcatgactt
10200tagatgggaa gcctgtgtat cgtggagaaa caagagacca actttttcat tccctgcccc
10260caatttccca gactagattt caagctaatt ttctttttct gaagcctcta acaaatgatc
10320tagttcagaa ggaagcaaaa tcccttaatc tatgtgcacc gttgggacca atgccttaat
10380taaagaattt aaaaaagttg taatagagaa tatttttggc attcctctca atgttgtgtg
10440tttttttttt ttgtgtgctg gagggagggg atttaatttt aattttaaaa tgtttaggaa
10500atttatacaa agaaactttt taataaagta tattgaaagt ttaaaaaaaa aaaaaaaa
10558
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