Use of ACRP30 Globular Head to Promote Increases in Muscle Mass and Muscle Differentiation

Abstract

The present invention relates to the field of muscle research, in particular to the discovery of a compound effective for increasing muscle mass, muscle cell differentiation, and oxidation of free fatty acids in muscle, useful in methods of treating muscle-related diseases and disorders as well as for augmenting muscle mass in general. The muscle-related diseases or disorders envisaged to be treated by the methods of the invention include, but are not limited to, muscular dystrophy, and other conditions resulting in muscle atrophy or muscle wasting.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 10/296,865, filed Jul. 31, 2003, which is the national stage of international application No. PCT/IB01/01126, filed May 22, 2001, which claims the benefit of U.S. Provisional Application Ser. No. 60/208,251, filed May 31, 2000 and Ser. No. 60/239,735, filed Oct. 11, 2000, the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of muscle research, in particular to the discovery of a compound effective for increasing muscle mass, muscle cell differentiation, and oxidation of free fatty acids in muscle, that should be useful in methods of treating muscle-related diseases and disorders as well as for augmenting muscle mass. The muscle-related diseases or disorders envisaged to be treated by the methods of the invention include, but are not limited to, muscle-related eye diseases and disorders, muscle-related recovery after injuries, muscle-related recovery after surgery, muscle-related disorders of aging, muscular dystrophy, and other conditions resulting in muscle atrophy.

BACKGROUND OF THE INVENTION

[0003] Acrp30 (also known as AdipoQ) is the murine homolog of the human Apm1 protein (this class of proteins is generically referred to as OBG3 herein). The predicted protein structure of Apm1 indicates the presence of: 1) a secreted protein signal peptide, 2) a region containing collagen repeats, and 3) a globular region (FIG. 2), which is highly conserved (FIG. 1).

[0004] Acrp30 is a circulating protein that is produced by the adipose tissue (WO 96/39429 which is hereby incorporated herein in its entirety including any figures, tables, or drawings). Acrp30 mRNA levels are significantly reduced in adipose tissue from obese ob/ob mice. Acrp30 expression is greatly increased during adipocyte differentiation and appears to be regulated by insulin. In addition, Acrp30 has been shown to reduce postprandial blood lipid levels, stimulate weight loss, and reduce plasma triglycerides in normal mice, and to promote the reduction of food intake in genetically obese mice (PCT Application No. 99/07736 which is hereby incorporated herein in its entirety including any figures, tables, or drawings).

[0005] Studies performed using just the globular region of Acrp30, termed globular Acrp30 (gAcrp30) showed that it effectively reduced body weight in mice on a high-fat cafeteria diet, as well as decreased fatty acid levels, decreased triglyceride levels, and improved glucose turnover in post-prandial tests in mice (See US Provisional Application No's 60/176,228 and 60/198,087, both of which are hereby incorporated herein by reference including any drawings, figures, or tables).

SUMMARY OF THE INVENTION

[0006] Globular OBG3 (gOBG3) has previously been linked with obesity, both in a murine model where treatment with gAcrp30 was shown to decrease body weight in mice fed a high fat diet, and in human subjects where an association between some Apm1 single nucleotide polymorphisms (SNPs) and obesity was documented. The instant invention is drawn inter alia to the unexpected effects of gOBG3 on muscle cells, including increasing the oxidation of free fatty acid in muscle cells as well as accelerating muscle re-orientation/re-organization and differentiation.

[0007] In a first aspect, the invention features methods of accelerating muscle cell differentiation, comprising contacting muscle cells with gOBG3 thereby accelerating differentiation of the muscle cells. Preferably the contacting is performed under conditions such that gOBG3 binds to the muscle cells. Preferably the muscle cells are present in an individual. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

[0008] In a second aspect, the invention features methods of accelerating muscle cell reorganization, comprising contacting muscle cells with gOBG3 thereby accelerating reorganization of the muscle cells. Preferably the muscle cells are present in an individual. Preferably the contacting is under conditions such that gOBG3 binds to the muscle cells. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

[0009] In a third aspect, the invention features methods of accelerating muscle repair, comprising contacting muscle cells with gOBG3 thereby accelerating reorganization and differentiation of the muscle cells. Preferably the contacting is under conditions such that gOBG3 binds to the muscle cells. Preferably the muscle cells are present in an individual. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

[0010] In a fourth aspect, the invention features methods of increasing muscle mass in an individual, comprising contacting muscle cells in the individual with gOBG3 thereby accelerating the reorganization and differentiation of the muscle cells and increasing the muscle mass of the individual. Preferably said contacting is under conditions wherein gOBG3 binds to muscle cells. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

[0011] In a fifth aspect, the invention features methods of treating muscle cell disorders in an individual, comprising contacting muscle cells in the individual with gOBG3 thereby accelerating the reorganization and differentiation of the muscle cells, and thereby treating the muscle cell disorders. Preferably the contacting is under conditions wherein gOBG3 binds to muscle cells. In preferred embodiments, the muscle cell disorders are selected from the group consisting of muscle-related eye diseases and disorders, muscle-related recovery after injuries, muscle-related recovery after surgery, muscle-related disorders of aging, muscle atrophy and muscular dystrophy. Preferably, gOBG3 is present in a pharmaceutical composition. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable diluent. gOBG3 can be provided as a polypeptide or as a polynucleotide encoding gOBG3. Preferably, gOBG3 is gApm1.

DEFINITIONS

[0012] Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.

[0013] As used herein, the term "OBG3" refers to any member of the family of homologous proteins that includes Apm1, the human homologue, as well as Acrp30 or AdipoQ, the mouse homologue. These proteins and the polynucleotides encoding them are described in detail in WO 96/39429 and U.S. Patent applications 60/176,228 and 60/198,087, all of which are hereby incorporated by reference herein in their entirety including any drawings, figures, or tables.

[0014] The term "polypeptide" refers to a polymer of amino acids without regard to the length of the polymer. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides. For example, polypeptides which include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide. Also included within the definition are polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0015] As used herein, the term "non-human animal" refers to any non-human vertebrate, birds and more usually mammals, preferably primates, farm animals such as swine, goats, sheep, donkeys, and horses, rabbits or rodents, more preferably rats or mice. As used herein, the term "animal" is used to refer to any vertebrate, preferably a mammal. Both the terms "animal" and "mammal" expressly embrace human subjects unless preceded with the term "non-human".

[0016] The term "individual" as used herein refers to a mammal, including animals, preferably mice, rats, dogs, cattle, sheep, or primates, most preferably humans that perceives a need (or for whom a need is perceived) to accelerate muscle cell differentiation, to accelerate muscle cell reorganization, or to increase muscle mass. "Perceives a need" does not necessarily refer to a clinical need, but may simply be the result of an aesthetic desire to increase muscle mass, or to facilitate athletic training. An individual can also be a "patient".

[0017] The term "patient" as used herein refers to a mammal, including animals, preferably mice, rats, dogs, cattle, sheep, or primates, most preferably humans that are in need of treatment. The term "in need of treatment" as used herein refers to a judgment made by a medical care-provider such as a physician, nurse practitioner, nurse or the like, that a patient could benefit from or requires treatment. This judgment is made based on a variety of factors that are in the realm of the medical care-provider's expertise, but that include the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.

[0018] The term "accelerating muscle cell differentiation" as used herein refers to the ability of compounds of the invention to decrease the number of hours muscle cell differentiation requires in the absence of the compound (or alternatively stated, to increase the process of differentiation). This decrease in hours is at least 5 hours, preferably at least 10 hours, more preferably at least 20 hours. Alternatively, accelerating muscle cell differentiation can refer to the ability of compounds of the invention to increase the numbers of muscle cells undergoing differentiation at a given time, compared to the number in the absence of the compound, or to decrease the amount of apoptosis of muscle cells compared with the amount of apoptosis at a given time in the absence of the compound. Muscle cell differentiation can be determined by visual inspection (Example 1) or through markers of muscle cell differentiation known in the art, some of which are described in Shimokawa et al (1998) (Biochem Biophys Res Commun 246:287-292; hereby incorporated herein by reference in its entirety including drawings, figures, and tables; Example 6).

[0019] The term "accelerating muscle cell reorganization" as used herein refers to the ability of compounds of the invention to increase muscle cell reorganization in the presence of the compound as compared to in its absence. Alternatively, accelerating muscle cell reorganization can refer to the ability of compounds of the invention to increase the numbers of muscle cells undergoing reorganization at a given time, compared to the number in the absence of the compound. Muscle cell reorganization can be determined by visual inspection (Example 1).

[0020] The term "increasing muscle mass" as used herein refers to the ability of compounds of the invention to increase the number of differentiated muscle cells as compared to the numbers in the absence of the compound. Muscle cell differentiation can be determined by visual inspection (Example 1) or through markers of muscle cell differentiation known in the art, some of which are described in Shimokawa et al (1998). Increases in muscle mass can also be determined by measurements of the overall size of a muscle or the strength of a muscle using techniques well-known in the art.

[0021] The term "muscle cell disorders" as used herein refers to disorders where there is a loss of muscle mass, or muscle strength. These would include muscle atrophy or muscle wasting as the result of disease or trauma, or malnutrition, for example. In addition, disorders such as muscle-related eye diseases and disorders, muscle-related recovery after injuries, muscle-related recovery after surgery, muscle-related disorders of aging, and muscular dystrophy are specifically envisioned. Whereas some of these disorders would require a medical care provider's diagnosis (muscular dystrophy for example), others would simply require an individual to detect the change and desire the treatment (some forms of muscle wasting or muscle atrophy).

[0022] The terms "percentage of sequence identity" and "percentage homology" are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue 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 and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al., 1990; Altschul et al., 1993). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool ("BLAST") which is well known in the art (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993, 1997). In particular, five specific BLAST programs are used to perform the following task:

[0023] (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

[0024] (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

[0025] (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

[0026] (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

[0027] (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

[0028] The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992; Henikoff and Henikoff, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990).

[0029] By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65.degree. C. in buffer composed of 6.times.SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 .mu.g/mL denatured salmon sperm DNA. Filters are hybridized for 48 h at 65.degree. C., the preferred hybridization temperature, in prehybridization mixture containing 100 .mu.g/mL denatured salmon sperm DNA and 5-20.times.10.sup.6 cpm of .sup.32P-labeled probe. Alternatively, the hybridization step can be performed at 65.degree. C. in the presence of SSC buffer, 1.times.SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37.degree. C. for 1 h in a solution containing 2.times.SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1.times.SSC at 50.degree. C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2.times.SSC and 0.1% SDS, or 0.5.times.SSC and 0.1% SDS, or 0.1.times.SSC and 0.1% SDS at 68.degree. C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency may also be used and are well known in the art (Sambrook et al., 1989 and Ausubel et al., 1989, both of which are hereby incorporated herein in their entirety including any drawings, figures, or tables). These hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. The hybridization conditions described can be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art. The suitable hybridization conditions may, for example, be adapted according to Hames and Higgins (1985) or Sambrook et al., (1989).

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIG. 1 shows an alignment of the sequences of the human (APM1), and mouse (adipoQ and acrp30) OBG3 proteins.

[0031] FIG. 2 shows a schematic drawing of the protein structure of APM1. The putative signal sequence at the N-terminus (AA 1-15), the unique region (AA 16-42), the collagen region (AA 43-107), and the globular region (AA 108-244) at the carboxy terminus are shown. Two protease cleavage sites at AA 100 and AA 131 are also shown.

[0032] FIG. 3 shows a graphical representation of the effect of the addition of gACRP30 on the oleate oxidation of differentiated C2C12 cells.

[0033] FIG. 4 shows a graphical representation of the effect of gAcrp30 treatment on fatty acid metabolism in muscle isolated from mice. EDL and Soleus muscles were isolated from both legs of each mouse (n=18). One muscle of each pair was incubated in medium with 2.5 .mu.g/mL gAcrp30 (dark gray) and one in medium without gAcrp30 (control-light gray).

[0034] FIGS. 5A and 5B show graphical representations of the effect of gAcrp30 treatment on triglyceride content of muscle and liver isolated from mice. Mice that had received 25 .mu.g of gAcrp30 twice daily showed significantly higher (p=0.002) muscle triglyceride content (FIG. 5A) than those receiving saline (control: light gray; gAcrp30: dark gray). This contrasted with a lack of increase in liver triglycerides (FIG. 5B).

[0035] FIGS. 6A and 6B show SDS-PAGE separations of the purification of Acrp30 and gAcrp30 (6A) and a cleavage product of apm1 (6B). FIG. 6A, Lane II shows the complete form of Acrp30 purified by FPLC. Lane I shows the proteolytic cleavage product gAcrp30. FIG. 6B shows a cleavage product of apm-1 after immunoprecipitation followed by Western blotting. The apparent molecular weight of this truncated form is 27 kDa, corresponding to about 70% of the complete form of apm-1 (lane IV). This truncated form was not detectable when a second anti-serum, specific for the human non-homologous region (HDQETTTQGPGVLLPLPKGA) of the protein was used for immunoprecipitation (Lane V) and the same anti-globular head antiserum for detection. A preimmune serum of the same animal did not detect any protein; a dimer of apm-1 was seen with both specific antibodies (apparent MW 74 kDa).

DETAILED DESCRIPTION OF THE INVENTION

[0036] Globular OBG3 has previously been linked with obesity, both in a murine model where treatment with globular Acrp30 (gAcrp30) was shown to decrease body weight in mice fed a high fat diet, and in human subjects where an association between some Apm1 single nucleotide polymorphisms (SNPs) and obesity was documented. In the instant application, the inventors have shown inter alia that at least some of the effects of gOBG3 are directed toward muscle cells. These effects include increasing the oxidation of free fatty acid in muscle cells as well as accelerating muscle re-organization and differentiation. Although the oxidation of free fatty acid in muscle cells likely is linked to the weight loss previously observed, it also seems to be linked to the acceleration of muscle cell reorganization and differentiation.

[0037] The effect of gAcrp30 on muscle cells was assessed using the murine, skeletal muscle cell line C2C12, and in ex vivo experiments on muscle cells excised from mice. The C2C12 cells were originally isolated from normal C3H mouse thigh muscle 72 hours after the muscle was crushed to increase the yield of mononucleated myogenic cells and designated C2 cells (Yaffe & Saxel (1977) Nature 270:725-727). The C2C12 cell line is a sub-clone of the C2 cell line selected for its ability to differentiate rapidly and to produce extensive contracting myotubes expressing characteristic muscle proteins (Blau et al (1985) Science 230:758-766). Thus, it appears that the C2C12 line is probably a clonal derivative of the satellite muscle cells present in muscle tissue that can replicate and differentiate to form additional muscle fibers.

[0038] Differentiation of C2C12 myocytes is induced when cultures are shifted to medium containing low concentrations of mitogens (Wang & Walsh (1996) Science 273:359-361). During this process myoblasts withdraw permanently from the cell cycle, express muscle specific structural proteins, and fuse into multinucleated myotubes (Davis et al (1987). Extensive cell death is also observed in cultures of C2C12 cells exposed to differentiation medium containing 2% horse serum beginning at 24 hours and reaching a maximum of 20-30% of cells at 48 hours (Wang & Walsh (1996)). After 72 to 96 hours myotubes become abundant and cell death is diminished.

[0039] The process of differentiation of the C2C12 cell line is a good model for studies for treatments of damaged muscle tissue following muscle injuries associated with strains and sprains, and tears normally encountered in daily life, as well as during intense athletic training, or as the result of accidental injury, or surgery. In all these instances, the muscle tissue is damaged, needs to re-orient/reorganize, and to grow new muscle fibers. Thus, treatments that enhance the reorientation/reorganization of the cultured muscle cells and their differentiation into muscle fibers should also be useful for accelerating the healing of muscle tissue following injury, as well as for accelerating the augmentation or strengthening of muscle cells during physical therapy, or athletic training.

[0040] Globular Acrp30 was found to induce pronounced re-orientation/re-organization of undifferentiated C2C12 muscle cells, as well as to accelerate the process of differentiation into muscle fibers. Further, there were indications of a decrease in apoptosis of muscle cells during the differentiation process, since the numbers of cells compared to the cells not treated with gOBG3 increased. Treatment of differentiated C2C12 cells with gAcrp30 also caused an increase in fatty acid metabolism, since oleate oxidation was increased approximately 40% (FIG. 3). A significant increase in oleate oxidation was also seen in ex vivo experiments with isolated mouse EDL and soleus muscles (FIG. 4), indicating the strength of the C2C12 cell line as a model system. A concurrent significant increase in triglyceride concentration was also observed in the ex vivo muscle.

[0041] Although not wishing to be limited to one hypothesis, the inventors believe that the increase in metabolism of free fatty acids that results from the addition of gAcrp30 may be involved in the acceleration of muscle cell reorientation/reorganization and differentiation. It is possible that the increase in fatty acid oxidation provides nutrients or energy that are involved in the process, or simply sends a signal. Whatever the exact mechanism involved, it is clear that treatment with gOBG3 has dramatic effects on muscle cells both in vitro and in vivo, for re-organization, differentiation and preventing apoptosis. Thus, it should be useful for treatment of muscle disorders where additional muscle tissue is desired and potentially where prevention of muscle cell death is needed. Examples of such disorders include, but are not limited to, muscle wasting, muscle atrophy, or muscular dystrophy. Augmenting muscle differentiation and growth should be ameliorative to the symptoms, if not curative of the disease. Recruitment of more muscle cells, alignment, and differentiation of more muscle fibers should have a positive effect, and might at least prolong the useful life of the muscles of patients afflicted with muscular dystrophy.

PREFERRED EMBODIMENTS OF THE INVENTION

[0042] I. Muscle-Related Uses of gOBG3

[0043] Methods of Accelerating Muscle Repair:

[0044] The inventors have shown that treatment of muscle cells with gOBG3 leads to their re-organization and differentiation, as well as increased free fatty acid oxidation, processes believed to be important in muscle repair. The muscle cell line used is one created as the result of trauma to skeletal muscle cells and is therefore thought to be a good model for studying muscle repair. Treatments that accelerate muscle cell re-organization, muscle cell differentiation, and/or muscle cell repair would be useful following any kind of muscle injury, including, but not limited to trauma, either accidental, or the result of surgery, or over-exercising. Trauma to muscles can result from blows, tears, cuts, strains, etc. gOBG3 variants and fragments, as well as agonists and antagonists of gOBG3, can be tested for their activity and thus their potential for use as treatments for the acceleration of muscle repair using the assays described in the Examples (particularly Examples 1, 2, 4, 6, 7) or in other assays known to those in the art.

[0045] Methods of Increasing Muscle Mass:

[0046] In addition to muscle repair, the results of treatment of muscle cells with gOBG3 suggest that gOBG3 treatment could also be useful for increasing muscle mass and/or increasing muscle strength and/or muscle endurance. Increasing body mass (for aesthetic or sports-related reasons, for example) also involves the recruitment and development of new muscle cells, which gAcrp30 has been shown to promote in the experiments with C2C12 cells. Further, increased free fatty acid oxidation should also be useful in any kind of endurance or other activities leading to muscle fatigue, since free fatty acids are a better source of energy and generally less easily utilized than glucose stores. To some extent, gOBG3 would be expected to function similarly to the anabolic steroid-type drugs currently used by athletes. gOBG3 variants and fragments, as well as agonists and antagonists of gOBG3, can be tested for their activity and thus their potential for use for increasing muscle mass, strength and/or endurance using the assays described in the Examples (particularly Examples 1, 2, 4, 6, 7) or assays known to those in the art.

[0047] Methods of Treatment of Muscle Disorders:

[0048] For similar reasons as those that suggest gOBG3 would be useful for accelerating muscle repair and increasing muscle mass, and additionally because it appears that gOBG3 may be useful in preventing apoptosis, gOBG3 should also be useful for treating muscle cell disorders. The muscle cell disorders contemplated are those that would improve, or whose symptoms would be ameliorated by treatment with gOBG3. These would include disorders in which the cells need to be strengthened (improve utilisation of free fatty acid), or the amount of muscle fibers increased (increased differentiation of muscle cells). For example, gOBG3 would be expected to be useful for treating the muscle cell disorders muscle atrophy, muscle wasting, and muscular dystrophy. Treatment with gOBG3 is expected to ameliorate some symptoms of these diseases by increasing the strength of the existing muscle cells by increasing their use of free fatty acids, and by increasing the differentiation of additional muscle cells, as well as by preventing the apoptosis of existing muscle cells. For instance, even though in muscular dystrophy the existing muscles are abnormal and their use is gradually lost, it is thought that gOBG3 should be able to increase the useful life of the muscles. gOBG3 variants and fragments, as well as agonists and antagonists of gOBG3, can be tested for their activity and thus their potential for use as treatments for muscle disorders using the assays described in the Examples (particularly Examples 1, 2, 4-8) or in other assays known to those in the art.

II. Globular OBG3 Polypeptides

[0049] Globular OBG3 polypeptides are used in the methods of treating muscle cells of the instant invention. As used herein, the term "gOBG3" refers to the globular portion of any member of the family of homologous proteins that includes Apm1, the human homologue, as well as Acrp30 or AdipoQ, the mouse homologue. Globular OBG3 polypeptides have previously been described in detail in U.S. Provisional patent application Nos. 60/176,228 and 60/198,087, hereby incorporated by reference herein in their entirety including figures, drawings, or tables. As used herein, unless specifically limited, the term is meant to include modified gOBG3 polypeptide sequences, including variants, fragments, analogs and derivatives of the gOBG3 polypeptides as described previously.

[0050] For the purposes of this invention, however, useful gOBG3 polypeptides are those that retain any one or more of the desired activities described herein, including but not limited to the effects on muscle cells that are the subject of the instant invention. These include accelerating re-orientation and differentiation of muscle cells as well as increasing free fatty acid oxidation, and preventing apoptosis. Variants, fragments, analogs and derivatives of these polypeptide sequences can be assayed for their retention of the desired activities using any of the methods/tests described in Examples 1, 2, and 4-8 or any comparable assays.

[0051] Globular OBG3 is the portion of intact OBG3 that does not include the collagen-like tail, or that contains few enough of the collagen residues such that the peptides do not assemble, or if they assemble this does not inhibit their activity. Preferably, this is fewer than 6 collagen residues, fewer than 4, fewer than 2, or more preferably no collagen residues. By "collagen residues" as used herein is meant the amino acids glycine, X, Y, where X and Y can be any amino acid. The collagen-like region of OBG3 is shown in FIG. 2 for APM1.

[0052] The term "activity" as used herein refers to a measurable result of the interaction of molecules. For example, a preferred gOBG3 activity is to accelerate re-orientation of muscle cells, accelerate differentiation of muscle cells, and/or increase free fatty acid oxidation of muscle cells. Representative assays to test for these functions are provided in Examples 1, 2, and 4-9. However, these examples are provided for explanation, not limitation. Those with skill in the art would be able to design other experiments to test for the same retained activity.

[0053] The term the "same retained activity" as used herein refers to the ability of a variant, fragment, analog or derivative of gOBG3 to have the same activity as is demonstrated in Examples 1, 2, and 4-8 and claimed in the instant invention. The variant, fragment, analog or derivative of gOBG3 does not necessarily have to retain all of the activities described herein for gOBG3's action on muscle cells, unless specified, but preferably retains at least one of the activities.

[0054] The "same activity" also relates to the amount of a given activity observed. In the instant application this refers to the amount of oleate oxidation, or amount of acceleration of differentiation, or amount of prevention of apoptosis, for example. Preferably, the "same" activity as it relates to amount, means within 10% of the previously observed amount, but it can include a difference of 20% or 30% or even 50%. However, this is not meant to limit the use of more effective gOBG3 polypeptides.

[0055] "More effective" gOBG3 polypeptides include those with an increased activity compared with the gAcrp30 polypeptide used in experiments described herein. The term "increased" as used herein refers to the ability of gOBG3 polypeptides to increase an activity in some measurable way as compared to an appropriate control. As a result of the presence of a gOBG3 variant, the levels of fatty acid oxidation, or muscle cell differentiation might increase, or the amount of apoptosis might decrease, for example, as compared to appropriate controls, typically the presence of gAcrp30 used in the experiments described herein. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100%.

[0056] "More effective" gOBG3 polypeptides may also include those that lack or have a decreased amount of one activity compared with the gAcrp30 polypeptide used in experiments described herein, and an increased amount of another activity compared with the gAcrp30 polypeptide used in experiments described herein. The term "decreased" as used herein refers to the ability of gOBG3 polypeptides to decrease an activity in some measurable way as compared to an appropriate control, such as the gAcrp30 polypeptide used in experiments described herein. As a result of the presence of a gOBG3 variant, fatty acid oxidation might decrease, for example, as compared to controls in the presence of the gAcrp30 polypeptide used in experiments described herein. Preferably, a decrease in activity is at least 25%, more preferably at least 50%, most preferably at least 100%. The term "lack" as used herein refers to an inability to detect an activity using the methods described herein, or similar methods. A gOBG3 variant could be thought to "lack" activity even though an increase of 5 or 10 or 15% of an effect is observed compared with an assay performed in its absence.

[0057] Finally, "more effective" gOBG3 polypeptides may also include those that lack or have a decreased amount of one activity or all activities compared with the gAcrp30 polypeptide used in experiments described herein, but an increased amount of these activities in vivo as compared with the gAcrp30 polypeptide used in experiments described herein.

[0058] Preferred embodiments of the invention feature gOBG3 polypeptide that consists of the sequence of the globular region shown in FIG. 1, or variants, fragments, analogs, or derivatives thereof. Preferable embodiments include amino acids 108-244 of SEQ ID NO:6 or 111-247 of SEQ ID Nos 2 and 4. Alternative preferable embodiments include amino acids 104 to 247 of the OBG3 proteins described in FIG. 1.

[0059] In other preferred embodiments, the invention features a gOBG3 polypeptide comprising at least 115, but not more than 175 contiguous amino acids of any one of the gOBG3 polypeptide sequences set forth in FIG. 1, wherein no more than 12 of said at least 115 and no more than 175 contiguous amino acids are present in the collagen-like region of OBG3. Preferably, the gOBG3 polypeptide comprises at least 125, but not more than 165, or at least 135, but not more than 155, and no more than 9 are in the collagen-like region; more preferably at least 125 but not more than 165, or 135 but not more than 155, and no more than 6 are in the collagen-like region; or at least 140 and not more than 150, and no more than 3 are present in the collagen-like region. Preferably the gOBG3 polypeptide is human or mouse, but most preferably human.

[0060] Variant gOBG3 polypeptides of the invention may be 1) ones in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) ones in which one or more of the amino acid residues includes a substituent group, or 3) ones in which a modified gOBG3 polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) ones in which the additional amino acids are fused to modify a gOBG3 polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the modified gOBG3 polypeptide or a pre-protein sequence. Such variants are deemed to be within the scope of those skilled in the art. The retention of the desired activity (and thus desired gOBG3 polypeptides) can be determined using the assays described in Examples 1, 2, 4-8 or other assays that achieve the same result.

[0061] Amino acid changes present in a variant polypeptide may be non-conservative amino acid changes but more preferably are conservative amino acid changes. In cases where there are one or more amino acid changes, preferred gOBG3 polypeptides include those that retain the same activities and activity levels as the reference gOBG3 polypeptide sequence, as well as those where the level of one or more activities is increased. Assays for determining gOBG3 polypeptide activities of the invention are described herein in the Examples (1, 2, 4-8) in more detail, but include accelerating muscle differentiation, muscle oleate oxidation, and decreasing muscle apoptosis, both in vitro and in vivo.

[0062] In preferred embodiments, the invention features a variant of a gOBG3 polypeptide that is at least 75% identical to gOBG3 polypeptide sequences selected from the group consisting of 101-244, 108-244, and 132-244 of SEQ ID NO:6, or 104-247, 111-247, and 135-247 of SEQ ID Nos 2 or 4. Preferably, the amino acid sequence is at least 85% identical, more preferably 90% identical, most preferably 95% identical and optionally 100% identical. Preferably the sequence is human or mouse, and most preferably human.

[0063] In yet other preferred embodiments, the invention features a variant of a gOBG3 polypeptide that comprises (or consists of) a 143 contiguous amino acid sequence, wherein at least 100 of the 143 amino acids are identical to amino acids 101-244 of SEQ ID NO:6 or 104-247 of SEQ ID Nos 2 or 4. Preferably, at least 113 of the 143 amino acids are identical, more preferably 127 of the 143 are identical, even more preferably 134 of the 143 are identical, and most preferably all of the amino acids are identical. Preferably the sequence is human or mouse, and most preferably human.

[0064] In yet other preferred embodiments, the invention features a variant of a gOBG3 polypeptide that comprises (or consists of) a 137 contiguous amino acid sequence, wherein at least 100 of the 137 amino acids are identical to amino acids 108-244 of SEQ ID NO:6 or 111-247 of SEQ ID Nos 2 or 4. Preferably, at least 113 of the 137 amino acids are identical, more preferably 127 of the 137 are identical, even more preferably 134 of the 137 are identical, and most preferably all of the amino acids are identical. Preferably the sequence is human or mouse, and most preferably human.

[0065] In yet other preferred embodiments, the invention features a variant of a gOBG3 polypeptide that comprises (or consists of) a 113 contiguous amino acid sequence, wherein at least 80 of the 113 amino acids are identical to amino acids 132-244 of SEQ ID NO:6 or 135-247 of SEQ ID Nos 2 or 4. Preferably, at least 90 of the 113 amino acids are identical, more preferably 100 of the 113 are identical, even more preferably 110 of the 113 are identical, and most preferably all of the amino acids are identical. Preferably the sequence is human or mouse, and most preferably human.

[0066] A polypeptide fragment is a polypeptide having a sequence that is entirely the same as part, but not all, of a given polypeptide sequence, preferably a gOBG3 polypeptide and variants thereof. Such fragments may be "free-standing", i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger non-OBG3 polypeptide of which they form a part or region. However, several fragments may be comprised within a single larger polypeptide. As representative examples of gOBG3 polypeptide fragments of the invention, there may be mentioned those which have from about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, 30 to 55, 40 to 70, 60 to 95, 80 to 130, or 90 to 144 amino acids long. Preferred are those fragments containing at least one amino acid substitution or deletion compared to a gOBG3 polypeptide.

III. Pharmaceutical Compositions

[0067] The gOBG3 polypeptides of the invention can be administered to a mammal, including a human patient, alone or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s). The pharmaceutical composition is then provided at a therapeutically or aesthetically effective dose. A therapeutically or aesthetically effective dose refers to that amount of gOBG3 sufficient to result in amelioration of symptoms of muscle-related disorders as determined by the methods described herein. A therapeutically or aesthetically effective dose can also refer to the amount of gOBG3 necessary for an increase in muscle mass or an increase in muscle strength or endurance in persons desiring this affect for aesthetic or athletic reasons alone. A therapeutically effective dosage of a gOBG3 polypeptide of the invention is that dosage that is adequate to promote muscle differentiation and/or increased free fatty acid oxidation, or decreased muscle cell apoptosis with continued or periodic use or administration. Techniques for formulation and administration of gOBG3 may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0068] Other diseases or disorders that gOBG3 could be used to treat or prevent include, but are not limited to, muscle atrophy, muscle wasting and muscular dystrophy.

[0069] Routes of Administration

[0070] Suitable routes of administration include oral, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections. A particularly useful method of administering compounds for promoting weight loss involves surgical implantation, for example into the abdominal cavity of the recipient, of a device for delivering gOBG3 over an extended period of time. Sustained release formulations of the invented medicaments particularly are contemplated.

[0071] Composition/Formulation

[0072] Pharmaceutical compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.

[0073] Certain of the medicaments described herein will include a pharmaceutically acceptable carrier and at least one polypeptide that is a gOBG3 polypeptide of the invention. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0074] Pharmaceutical preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

[0075] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0076] 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 nebulizer, with the use of a suitable gaseous propellant, e.g., carbon dioxide. 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.

[0077] 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 aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0078] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0079] Alternatively, the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0080] 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.

[0081] Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.

[0082] Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

[0083] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[0084] Effective Dosage

[0085] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0086] For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system. Such information can be used to more accurately determine useful doses in humans.

[0087] A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds 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 test 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 between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred.

[0088] The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. 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. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g. Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1).

[0089] Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain the weight loss or prevention of weight gain effects. Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.

[0090] Dosage intervals can also be determined using the value for the minimum effective concentration. Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0091] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

[0092] A preferred dosage range for the amount of a gOBG3 polypeptide of the invention, that can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma triglyceride-rich lipoproteins, range from 0.01-50 mg/kg body mass. A more preferred dosage range is from 0.02-25 mg/kg. A still more preferred dosage range is from 0.1-20 mg/kg, while the most preferred range is from 0.2-10 mg/kg. Of course, these daily dosages can be delivered or administered in small amounts periodically during the course of a day.

EXAMPLES

[0093] Other characteristics and advantages of the invention are described in the Examples. The following Examples are provided for illustrative purposes and not as a means of limitation. One of ordinary skill in the art would be able to design equivalent assays and methods based on the disclosure herein, all of which form part of the instant invention.

[0094] Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these publications, patents and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure.

Example 1

Effect of gACRP30 Treatment of Muscle cells on Muscle Differentiation

[0095] C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville, Md.) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine, pen/strep, etc)+10% FCS. Two days later they become 80-90% confluent. At this time, the media is changed to DMEM+2% horse serum to allow differentiation. The media is changed daily. Abundant myotube formation occurs after 3-4 days of being in 2% horse serum.

[0096] To test the effect of the presence of gACRP30 on muscle differentiation, gACRP30 (1 to 2 .mu.g/mL) was added the day after seeding when the cells are still in DMEM w/10% FCS. Two days after plating the cells (one day after gACRP30 was first added), at about 80-90% confluency, the media was changed to DMEM+2% horse serum plus gACRP30.

[0097] The results show that the addition of gACRP30 causes the cells to begin organizing within one day after its addition. In contrast to the random orientation of the cells not treated with gACRP30, those treated with gARCP30 aligned themselves in relation to each other. In addition, differentiation progressed more rapidly in the presence o gAcrp30; after only 2 days of gACRP30 treatment maximal or near maximal differentiation was observed in contrast to the 3 to 4 days needed in its absence.

Example 2

Effect of gACRP30 on Muscle Cell Fatty Acid Oxidation

[0098] C2C12 cells were differentiated in the presence or absence of 2 .mu.g/mL gACRP30 for 4 days. On day 4, oleate oxidation rates were determined by measuring conversion of 1-.sup.14C-oleate (0.2 mM) to .sup.14CO.sub.2 for 90 min. C2C12 cells differentiated in the presence of gACRP30 undergo 40% more oleate oxidation than controls differentiated in the absence of oleate (FIG. 3).

[0099] The effect of gAcrp30 on the rate of oleate oxidation was compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, Va. CRL-1772) and in a hepatocyte cell line (Hepa1-6; ATCC, Manassas, Va. CRL-1830). Cultured cells were maintained according to manufacturer's instructions.

[0100] The oleate oxidation assay was performed as previously described (Muoio et al (1999) Biochem J 338:783-791). Briefly, nearly confluent myocytes were kept in low serum differentiation media (DMEM, 2.5% Horse serum) for 4 days, at which time formation of myotubes became maximal. Hepatocytes were kept in the same DMEM medium supplemented with 10% FCS for 2 days. One hour prior to the experiment the media was removed and 1 mL of preincubation media (MEM, 2.5% Horse serum, 3 mM glucose, 4 mM Glutamine, 25 mM Hepes, 1% FFA free BSA, 0.25 mM Oleate, 5 .mu.g/mL gentamycin) was added. At the start of the oxidation experiment, .sup.14C-Oleic acid (1 .mu.Ci/mL, American Radiolabeled Chemical Inc., St. Louis, Mo.) was added and cells were incubated for 90 min at 37.degree. C. in the absence/presence of 2.5 .mu.g/mL gAcrp30. After the incubation period, 0.75 mL of the media was removed and was assayed for .sup.14C-oxidation products as described for the muscle FFA oxidation experiment.

[0101] Oleate oxidation in C2C12 cells determined over 90 min increased significantly (39%; p=0.036, two-tailed t-Test) in cells treated with gAcrp30. In contrast, no detectable increase in the rate of FFA oxidation was seen in hepatocytes incubated with gAcrp30.

[0102] Triglyceride and Protein Analysis Following Oleate Oxidation in Cultured Cells

[0103] Following the transfer of media for the oleate oxidation assay, cells were placed on ice. To determine triglyceride and protein content, cells were washed with 1 mL of 1.times.PBS to remove residual media. To each well, 300 .mu.L of cell dissociation solution (Sigma) was added. Cells were incubated at 37.degree. C. for 10 min. Plates were tapped to loosen cells, and 0.5 mL of 1.times.PBS was added. The cell suspension was transferred to an eppendorf tube, each well was rinsed with an additional 0.5 mL of 1.times.PBS, and the rinse was transferred to appropriate eppendorf tube. Samples were centrifuged at 1000 rpm for 10 minutes at room temperature. The supernatant was discarded, and 750 .mu.L of 1.times.PBS/2% chaps was added to each cell pellet. The cell suspension was vortexed and was placed on ice for 1 hour. Samples were then centrifuged at 13000 rpm for 20 min at 4.degree. C. Supernatants were transferred to new tubes and frozen at -20.degree. C. until analyzed.

[0104] The amount of triglycerides in each sample was determined using Sigma Diagnostics GPO-TRINDER enzymatic kit. The procedure outlined in the manual was adhered to with the following exceptions: the assay was performed in a 48 well plate, 350 .mu.L of sample volume was assayed, the control blank consisted of 350 .mu.L PBS/2% chaps, and the standard contained 10 .mu.L of the standard provided in the kit plus 690 .mu.L PBS/2% chaps. Analysis of the samples was performed on a Packard Spectra Count at a wavelength of 550 nm.

[0105] Protein analysis was carried out on 25 .mu.L of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions. Analysis of the samples was performed on a Packard Spectra Count at a wavelength of 550 nm.

[0106] Triglyceride production in both C2C12 and Hepa 1-6 cells did not change significantly in the absence/presence of Acrp30 and gAcrp30. The protein content of all cells analyzed was equivalent in the absence/presence of Acrp30 and gAcrp30.

Example 3

Production of Recombinant Acrp30

[0107] An illustrative method for producing recombinant OBG3 is given below. Although the method describes the production of the mouse analog, Acrp30, a person of ordinary skill in the art would be able to use the guidance provided to produce other OBG3 analogs, including but not limited to, the human analog, Apm1.

[0108] Recombinant Acrp30 is cloned in pTRC His B (Invitrogen) between BamH1 and Xho1 and maintained in E. coli DH5-.alpha.. The sequence of the Acrp30 insert corresponds to Acrp 30 genbank U37222 bases 88 to 791, except in position 382 where in #3 G replaces A found in ACRP 30 (V instead of M). The corresponding nucleotide in AdipoQ U49915 is G as in clone #3. The amino acid V is also conserved in the human sequence Apm-1 D45371.

Culture:

[0109] Plate out bacteria in LB agar media containing 100 .mu.g/mL ampicillin. Inoculate 1 colony into 5 mL media (no agar) at 37.degree. C. overnight. Add 2 mL of this initial culture into 500 mL Erlenmeyer flasks containing 200 mL LB media and 100 .mu.g/mL ampicillin. Incubate at 37.degree. C. in an orbital shaker until the OD.sub.600=0.2. Add IPTG to a final concentration of 1 mM (stock solution=1 M). Incubate at 37.degree. C. overnight.

Lysis:

[0110] Pellet the bacteria by centrifugation (Sorvall, 3500 rpm, 15 min, 4.degree. C.) in a pre-weighed tube.

[0111] At 4.degree. C. resuspend the pellet in 3 mL/g of lysis buffer

[0112] Add 40 .mu.L/g PMSF 10 mM

[0113] Add 80 .mu.L/g of lysozyme 10 mg/mL

[0114] Incubate 20 min on ice, shaking intermittently

[0115] Add 30 .mu.L/g 10% sodium deoxycholate

[0116] Incubate at 37.degree. C. until the lysate is viscous

[0117] Freeze in liquid Nitrogen and thaw at 37.degree. C. three times

[0118] Sonicate 2.times., 30 sec, 25% cycle, 2.5 power level

[0119] Centrifuge 30 min, 15000 rpm, 4.degree. C.

[0120] Recover the supernatant

[0121] Note: The lysate can be stored frozen before or after the sonication step.

Batch Purification:

[0122] 1. Pack 1 mL of Probond resin (Invitrogen; 1 mL=2 mL suspended gel) into a 5 mL column. Wash with 5 mL PBS.

[0123] 2. Apply 5 mL bacterial supernatant to the 1 mL of gel. (If volume is very high, use several small columns.)

[0124] 3. Wash with 24 mL phosphate buffer, pH 7.8, followed by a wash with 24 mL phosphate buffer, pH 6.

[0125] 4. Elute with imidazole buffer and collect fractions of 1 mL.

[0126] 5. Analyze fractions by OD at 280 nm or by SDS-PAGE (12.5%; dilution 1/2 in 2.times. sample buffer) under reducing conditions (100.degree. C., 5 min)

[0127] 6. Pool the fractions containing protein (usually fraction numbers 2-4 for concentrations of 0.8-1 mg/mL and fractions 1, 5 and 6 for concentrations of 0.2-0.4 mg/mL).

[0128] 7. Dialyze thoroughly against 1.times.PBS, 24 mM ammonium bicarbonate or 50 mM Tris, pH 7.4 containing 250 nM NaCl. Concentrate by Speed-Vac if needed.

[0129] 8. Analyze protein by the Lowry method.

[0130] 9. Aliquot and store at -20.degree. C.

Purification On Liquid Chromatography System

[0131] 1. Pack 5 mL of Probond resin into a 5 mL column.

[0132] 2. Wash with 4 bed volumes of phosphate buffer pH 7.8, 1 mL/min.

[0133] 3. Inject 25 mL lysate (filtered on 0.45.mu. or centrifuged at 3000 rpm, 30 min, 4.degree. C., Beckman Allegra 6R) at 0.5 mL/min.

[0134] 4. Wash with 4 bed volumes of phosphate buffer, pH 7.8 at 1 mL/min.

[0135] 5. Wash with 12 bed volumes of phosphate buffer pH 5.5 at 1 mL/min.

[0136] 6. Elute bound fraction with phosphate buffer, pH 5.5, containing 1 M imidazole at 1 mL/min.

[0137] 7. Collect fractions, dialyze and analyze protein as described for batch purification, steps 7-9.

Example 4

Effect of ACRP30 on Oleate Oxidation in Isolated Muscle

[0138] Intact muscles were isolated from C57BL6/J mice and the oleate oxidation of the isolated muscle was measured (Clee et al (2000) J Lipid Res 41:521-531; Muoio et al (1999) Am J Physiol 276:E913-921).--Oleate oxidation in isolated muscle was measured as previously described (Cuendet et al (1976) J Clin Invest 58:1078-1088; Le Marchand-Brustel (1978) Am J Physiol 234:E348-E358, hereby incorporated by reference herein in its entirety including any figures, drawings, or tables).

[0139] Two groups of C57BL6/J mice, age 5 and 33 weeks, were used. Mice were kept on a regular diet with free access to food and water. The day/night light cycle was kept at 12 hr ON/12 hr OFF. The older animals were injected twice daily with either saline or 25 .mu.g of gACRP30 for 3-4 days. The younger group of animals was given a high fat meal (6 g butter, 6 g sunflower oil, 10 g nonfat dry milk, 10 g sucrose, 12 ml, distilled water prepared fresh) by gavage (vol.=1% of body weight) at time 0 and was injected with saline or gACRP30 only at time 0 and again at 45 minutes. They were sacrificed at 180 minutes. The gACRP30 used was prepared as described in Example 3.

[0140] After cervical dislocation, soleus and EDL muscles were rapidly isolated from the hind limbs of the mice. The distal tendon of each muscle was tied to a piece of suture to facilitate transfer among different media. All incubations were carried out at 30.degree. C. in 1.5 mL of Krebs-Ringer bicarbonate buffer (118.6 mM NaCl, 4.76 mM KCl, 1.19 mM KH.sub.2PO.sub.4, 1.19 mM MgSO.sub.4, 2.54 mM CaCl.sub.2, 10 mM Hepes, pH7.4) supplemented with 4% bovine serum albumin, FFA free (fraction V, RIA grade, Sigma, Inc., St. Louis, Mo.) and 5 mM glucose (Sigma, Inc., St. Louis, Mo.). The concentration of oleate (Sigma, Inc., St. Louis, Mo.) throughout the experiment was 0.25 mM. All media were oxygenated (95% O.sub.2; 5% CO.sub.2) prior to incubation. The gas mixture was hydrated throughout the experiment by bubbling through a gas washer (Kontes Inc., Vineland, N.J.).

[0141] Muscles were rinsed for 30 min in incubation media with oxygenation. The muscles were then transferred to fresh media (1.5 mL) and incubated at 30.degree. C. in the presence of 1 .mu.Ci/mL [1-.sup.14C] oleic acid (ARC) and 1 .mu.Ci/mL [5-.sup.3H]-glucose (Amersham). The incubation vials containing this media were sealed with a rubber septum from which a center well carrying a piece of Whatman paper (1.5 cm.times.11.5 cm) was suspended.

[0142] After an initial incubation period of 10 min with constant oxygenation, gas circulation was removed to close the system to the outside environment. The muscles were incubated for an additional 90 min at 30.degree. C. At the end of this period, 0.45 mL of Solvable (Packard Instruments, Meriden, Conn.) was injected onto the Whatman paper in the center well. The oxidation of oleate by the muscle was stopped at this time point by transferring the vial onto ice.

[0143] After 5 min, the muscle was removed from the medium, was cleaned of connective tissue, was weighed and was frozen for further analysis. An aliquot of 0.5 mL medium was removed, the vials were closed again and 1 mL of 35% perchloric acid was injected with a syringe into the media by piercing through the rubber septum with a needle. The CO.sub.2 released from the acidified media was collected by the Solvable in the center well.

[0144] After a 90 min collection period at 30.degree. C., the Whatman paper was removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, Conn.). The amount of .sup.14C radioactivity was quantitated by liquid scintillation counting. The rate of oleate oxidation was expressed as nmol oleate produced in 90 min/g muscle.

[0145] To test the effect of gACRP30 or ACRP30 on oleate oxidation, these proteins were added to the media at a final concentration of 2.5 .mu.g/mL and maintained in the media throughout the procedure. Two muscles of different oxidative capacity (soleus and extensor digitorum longus (EDL)) were tested (FIG. 4). EDL and Soleus muscles were isolated from both legs of normal C57BL/6J mice (n=18). One muscle of each pair was incubated in medium with 2.5 .mu.g/ml, gAcrp30 (dark gray) and one in medium without gAcrp30 (control--light gray). This experimental design allowed us to compare oleate oxidation in pairs of muscles isolated from the same animal. .sup.14C-Oleate oxidation was determined over 90 minutes. Incubation of EDL and soleus muscles for 90 minutes in medium containing 2.5 .mu.g/ml gAcrp30 leads to a statistically significant increase in oleate oxidation (p<0.05, paired, one-tailed, t-Test) or (p=0.0041, Repeated Measures Analysis of Variance, Univariate Tests of Hypotheses for Within Subject Effects) in both muscle types.

[0146] Both muscle types showed a significant response to gAcrp30. The relative increase in FFA oxidation was 17% (p=0.03) and 10% (p=0.04) for EDL and soleus, respectively. In humans, muscles represent approximately 25% of body weight. Therefore, even a moderate increase in free fatty acid oxidation can have quantitatively important consequences on overall energy utilization.

[0147] In summary, these experiments show that gAcrp30 acts on muscle cells in vivo and ex vivo. The invention is drawn, inter alia, to the effects of gAcrp30 on muscle resulting in increased lean muscle mass and exercise capability. The increase in oleate oxidation seen in these whole muscle experiments, suggests that experiments done using the simpler C2C12 cell line model are predictive for effects in vivo.

[0148] The hindlimb muscle and liver triglyceride content was measured after gAcrp30 treatment of mice. Hind limb muscles as well as liver samples were removed from treated and untreated animals and the triglyceride and free fatty acid concentration was determined following a standard lipid extraction method (Shimabukuro et al (1997) Proc Natl Acad Sci USA 94:4637-4641, hereby incorporated herein by reference in its entirety including any figures, drawings, or tables) followed by TG and FFA analysis using standard test kits.

[0149] Short-term treatment of animals with gAcrp30 (2 injections of 25 .mu.g each given within 3 hours before sacrifice) did not change the triglyceride content either of hind limb muscle or liver tissue. However, after 3 days of treatment, during which period normal C57BL/6J mice consumed a regular rodent diet, mice that had received 25 .mu.g of gAcrp30 twice daily showed significantly higher (p=0.002) muscle triglyceride content (FIG. 5A) than those receiving saline (control: light gray; gAcrp30: dark gray). This contrasted with a lack of increase in liver triglycerides (FIG. 5B). Furthermore, no detectable increase in muscle TG was observed after the 16-day treatment shown independently by directly measuring the muscle TG content and by oil red O staining of frozen microscope sections. In summary, the data indicate that the increase in TG content was transient.

[0150] Ketone bodies (KB) are produced in the liver as a result of free fatty acid oxidation, but KB formation does not occur significantly in muscle. In mice receiving the high fat test meal and saline injection, the level of plasma KB increased significantly over the next 3 hours (183.+-.12%, n=6). Animals treated with gAcrp30, on the other hand, showed no increase in plasma KB concentrations. Although not wishing to be limited by any particular theory, this suggests that gAcrp30 inhibits either directly KB formation or can decrease KB production by inhibiting liver FFA oxidation.

Example 5

Methods of Testing the Effect of gOBG3 on Apoptosis

[0151] Prior experiments indicated that gAcrp30 either increased cell division or decreased apoptosis is C2C12 cells undergoing differentiation (Example 1). In order to assess the effect C2C12 cells can be transferred into medium containing 0.5% horse serum (rather than 2% horse serum for differentiation), which rapidly induces the onset of apoptosis (Wang & Walsh (1996) hereby incorporated herein by reference in its entirety including any figures, tables, or drawings). Cells would then be incubated for 16 hours either in the presence or absence of gACRP30 and then the level of apoptosis compared by staining for ApopTag as described in Wang & Walsh (1996).

Example 6

Methods of Assessing the Acceleration of Myoblast Differentiation with gOBG3

[0152] Prior experiments have also indicated that gAcrp30 accelerates re-organization/reorientation and differentiation of C2C12 muscle cells in vitro (Example 1). Rapid, highly sensitive methods are available to facilitate screening of the activity of variants of gAcrp30 (and antagonists and agonists) for their effect on differentiation. These assays include, among others, a TaqMan PCR-based method that assesses mRNA levels of muscle-specific markers of differentiation, including, but not limited to, muscle regulatory factor, myogen, alpha-actin, thermoregulatory uncoupling protein (UCP2), glucose transporter isotype glut4, myf5, beta-actin, UCP1, UCP3, and glut1 (Shimokawa et al (1998) Biochem Biophys Res Commun 246:287-292, hereby incorporated by reference herein in its entirety including any figures, drawings, or tables). Levels of mRNA would be compared between the gAcrp30 treated and the untreated cultures of differentiating C2C12 cells.

Example 7

Effect of gOBG3 on Muscle Cells In Vivo

[0153] Experiments can be performed on normal mice as well as mice fed a high fat diet, to assess increases in the differentiation of muscle cells over a time course of treatment using the TaqMan PCR-based method described in Example 6, for example. Other muscle cell parameters discussed herein can also be assessed over time. The animal experiments can be performed as follows, for example:

[0154] Experiment 1: 10-week-old male C57BL/6J mice were put on a very high fat/sucrose purified diet for 19 days to promote weight gain (see Example 4); the average body weight at this time was 30 g. The mice were then surgically implanted with an osmotic pump (Alzet, Newark, Del.) delivering either 2.5 .mu.g/day of gAcrp30, 5 .mu.g/day of Acrp30, or physiological saline. The mice were continued on the high fat diet and their body weight was recorded over the following 10-day period. Muscle parameters can be assessed over this same time period.

[0155] Experiment 2: mature 9 month old, male obese C57BL/6J mice that had been on the same high fat/sucrose diet for 6 months; the average body weight when the study began was 52.5.+-.0.8 g. Three groups of 8 mice were treated with saline, Acrp30 or gAcrp30 for 16 days. Animals in the treated group received twice daily 25 .mu.g of protein subcutaneously. Body weights were recorded at appropriate time points (daily or weekly). Muscle parameters can also be assessed over the same time period.

Example 8

Mouse Models of Muscular Dystrophy

[0156] In order to further assess the efficacy of gAcrp30 and analogs, variants, and fragments thereof (as well as antagonists or agonists as needed) mouse models of muscular dystrophy can be used including, but not limited to, 129P1/ReJ-Lama2.sup.dy, C57BL/10ScSn-Dmd.sup.mdx/J, C57BL/6J-Lama2.sup.dy, C57BL/6J-Lama2.sup.dy-2J, C57BL/6Ros-Dmd.sup.mdx-2Cv, C57BL/6Ros-Dmd.sup.mdx-3Cv, C57BL/6Ros-Dmd.sup.mdx-4Cv, C57BL/6Ros-Dmd.sup.mdx52Cv, and D.B/20Ei-Lama2.sup.dy-6J/+, all of which are available from Jackson Laboratories. The experiments would include beginning to treat mice as young as possible (and then starting at increasingly later time points) with gAcrp30 as described in Example 7 and comparing the onset and severity of disease symptoms with untreated mice.

Example 9

Cellular Binding and Uptake of gOBG-3 as Detected by Fluorescence Microscopy

[0157] Fluorecein isothiocyanate (FITC) conjugation of gOBG3: Purified gOBG3 at 1 mg/mL concentration was labeled with FITC using Sigma's FluoroTag FITC conjugation kit (Stock No. FITC-1). Protocol outlined in the Sigma Handbook for small scale conjugation was followed for gOBG3 labeling.

[0158] Cell Culture: C2C12 mouse skeletal muscle cells (ATCC, Manassas, Va. CRL-1772) and Hepa-1-6 mouse hepatocytes (ATCC, Manassas, Va. CRL-1830) were seeded into 6 well plates at a cell density of 2.times.10.sup.5 cells per well. C2C12 and Hepa-1-6 cells were cultured according to repository's instructions for 24-48 hours prior to analysis. Assay was performed when cells were 80% confluent.

[0159] FITC labeled gOBG3 cellular binding and uptake using microscopy in C2C12 and Hepa 1-6 cells. FITC-gOBG3 (50 nM/mL) was added to each cell culture well. Cells were incubated for 1 hour at 37.degree. C., 5% CO.sub.2. Cells were washed 2.times. with PBS, cells were scraped from well into 1 mL of PBS. Cell suspension was transferred to an eppendorf tube and centrifuged at 1000 rpm for 2 minutes. Supernatant was removed and cells resuspended in 200 .mu.L of PBS. Binding and uptake of FITC-gOBG3 was analyzed by fluorescence microscopy under 40.times. magnification.

[0160] Analysis of C2C12 and Hepa 1-6 cells reveals identical phenotypes with respect to FITC-gOBG3 binding and uptake profiles. FITC-gOBG3 appears to be localized within vesicles in the cytoplasm of both mouse hepatocytes and mouse myoblasts, suggesting that binding and uptake of FITC-gOBG3 is occurring. Thus, this assay may be useful for identifying agents that facilitate or prevent the uptake and/or binding of OBG3 or gOBG3 polypeptides to cells by adding the substance to be tested with, before, or after the FITC-gOBG3.

Example 10

Detection of Apm-1 Fragment in Human Plasma after Immunoprecipitation

[0161] The recombinant form of Acrp30 protein used has an apparent molecular weight of 37 kDa and forms a dimer of 74 kDa (FIG. 6 A, Lane II). A proteolytic fragment that contains the entire globular head region (gAcrp30) and that migrates with an apparent molecular weight of 18 kDa was generated using acetylated trypsin (FIG. 6A, lane I). Both protein preparations (Acrp30 and gAcrp30) were essentially endotoxin free; "ActiClean Etox" affinity columns were used to remove potential endotoxin contaminations (Sterogene Bioseparations Inc., Carlsbad, Calif.) following the manufacturer's protocol. Endotoxin levels were determined by Endosafe, Charleston, S.C. As determined by N-terminal sequencing of purified gAcrp30, the site of cleavage was just before amino acid 104.

[0162] Immunoprecipitation of human plasma Apm followed by Western blotting was used to detect a cleavage product of Apm-1, the human homolog of Acrp30, using a globular head specific anti-serum for the immunoprecipitation step as well as for the detection step. Preimmune serum or serum raised against the globular head domain or human non-homologous region (HDQETTTQGPGVLLPLPKGA) were cross-linked to protein A (Sigma Chemical CO, Saint Louis, Mo.) using dimethyl-pimelimidate-dihydrochloride (Sigma Chemical Co, Saint Louis, Mo.). After washing (0.2 M salt), proteins were eluted from protein A, were separated by SDS-PAGE, and were transferred to Protran.RTM. pure nitrocellulose membrane (Schleicher and Schuell, Keene, N.H.) using standard procedures. Apm-1 products were visualized using globular head domain antibodies labeled with biotin; horseradish peroxidase conjugated to Streptavidin and CN/DAB substrate kit (Pierce, Rockford, Ill.) according to manufacturer's instructions.

[0163] The apparent molecular weight of this truncated form was 27 kDa, corresponding to about 70% of the complete form of Apm-1 (FIG. 6B, Lane IV). This truncated form was not detectable when immunoprecipitation was performed using a different antibody directed against the human non-homologous region (HDQETTTQGPGVLLPLPKGA) of Apm-1; this domain is located toward the NH.sub.2 terminal end of the protein outside of the globular domain (FIG. 6, Lane V). Both anti-Apm-1 antibodies directed against either the globular or the non-globular domain identified the full-length form of the protein, as well as a low abundance dimer of apparent MW 74 kDa.

Example 11

Tissue Distribution of gACRP30

[0164] Intra venous Injection of .sup.125I (g)Acrp30

[0165] The disappearance from plasma and subsequent tissue distribution of Acrp30 and the globular head (gAcrp30) was investigated by injecting .sup.125I (g)Acrp30 into CD-1 mice.

[0166] CD-1 mice were kept on regular diet with free access to food and water. The day/night light cycle was kept at 12 hr ON/12 hr OFF. Acrp30 and gACRP30 were prepared as previously described and labeled with .sup.125I to a specific activity of approximately 100 cpm/ng using Iodobeads (Pierce). The labeled protein was prepared immediately before injection and 0.1-0.5 .mu.Ci were injected through the tail vein. Clearance of the labeled protein from plasma was followed over 3 hrs after which the animals were perfused systemically with PBS+EDTA to remove all blood. Tissues were isolated (liver, adipose, kidney, skeletal muscle, brain), weighed, and the specific activity in plasma and tissue was determined.

[0167] Acrp30 showed a plasma half-life of about 7 hours. The plasma turnover of gAcrp30 was significantly faster showing a half-life close to 1 hour. The majority of the labeled Acrp30 protein was found in kidney and in skeletal muscle tissue, indicating that it represents a functional target tissue. A much smaller part of the injected protein was found in adipose and liver tissue and only a very small pool was seen in brain.

[0168] Immunohistochemistry Studies

[0169] Frozen sections of skeletal muscle from mice were stained using an antibody to the globular head of Acrp30 coupled with calorimetric detection. Briefly, C57 mice were sacrificed and various skeletal muscles were isolated. Muscles were frozen in an isopentane/liquid nitrogen bath. Tissue was then sectioned at 8 microns using a Leica 3050 cryostat and sections were picked up on microscope slides. Slides were dried overnight and fixed with 4.degree. C. acetone after which sections were pretreated for endogenous peroxidase and biotin prior to IHC. An antibody to gAcrp30 was used on the slides at a concentration of 1:20,000. The primary antibody was detected using a biotin-labeled streptavidin system. The chromagen was DAB with hematoxylin counterstain.

[0170] Cross-sectional views of skeletal muscle show distinct outlining of muscle fibers with Acrp30 staining. A positive signal is also seen in any blood vessels in the tissue. This is not surprising since the animals were not perfused before tissues were isolated and Acrp30 should thus be present in the plasma that would be in these vessels. This staining appears to be specific given that tissues stained with preimmune at the same concentration were clean. Also it should be noted that tissues without much Acrp30, such as liver, do not show much Acrp30 staining.

[0171] The substantial presence of Acrp30 in skeletal muscle supports the inventor's belief that muscle is in fact a target tissue of this molecule.

Example 12

Generation of Globular ACRP3 of by Enzymatic Cleavage

[0172] Incubate purified Acrp30 (obtained as described above or through equivalent method) with acetylated Trypsin-Type V-S from Bovine Pancreas (Sigma E.C.=3.4.21.4) at 400 u/mg protein at 25.degree. C. for 10 min.

[0173] Stop reaction by running the sample over a Poly-Prep Column (Biorad 731-1550) at +4.degree. C. containing immobilized Trypsin inhibitor.

[0174] Collect 1.0 mL fractions. Determine protein concentration.

[0175] Pool the protein containing fractions and dialyze extensively against PBS using dialysis tubing with M.W. cutoff=10,000 da.

[0176] Concentrate on Amicon YM-10 Centricon Filter (Millipore, M.W. cutoff=10,000 da). Sterile filter.

[0177] Determine final protein concentration using Markwell's modified Lowry procedure (1981) or BCA protein assay (Pierce Chemical Co, Rockford, Ill.) and BSA as standard.

[0178] Check purity and efficiency of cleavage by SDS-PAGE analysis using a 4-20% gradient gel. The intact Acrp30 migrates as a single band at approx. 37 kda due to co-transcribed vector sequences attached to the histidine tag at the N-terminus of Acrp30. The cleaved Acrp30 forms a band at approx. 18 kda (gAcrp30). Additional degradation products, all smaller than 10 kda are also generated from the N-terminal region. These are separated from the desired 18 kda band by dialysis with semipermeable membranes with a MW cutoff of 10,000. The actual cleavage site using this method has been identified as the one after amino acid 103.

REFERENCES

[0179] Dehouck et al. J Neurochem 54:1798-801, 1990 [0180] Shapiro, and Scherer, Curr. Biol. 8:335-338, 1998 [0181] Uysal, et al. Nature 389:610-614, 1997

Sequence CWU 1

1

711152DNAmus musculus 1gaattcggca cgaggg atg cta ctg ttg caa gct ctc ctg ttc ctc tta atc 52Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile1 5 10ctg ccc agt cat gcc gaa gat gac gtt act aca act gaa gag cta gct 100Leu Pro Ser His Ala Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala 15 20 25cct gct ttg gtc cct cca ccc aag gga act tgt gca ggt tgg atg gca 148Pro Ala Leu Val Pro Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala 30 35 40ggc atc cca gga cat tct ggc cac aat ggc aca cca ggc cgt gat ggc 196Gly Ile Pro Gly His Ser Gly His Asn Gly Thr Pro Gly Arg Asp Gly45 50 55 60aga gat ggc act cct gga gag aag gga gag aaa gga gat tca ggt ctt 244Arg Asp Gly Thr Pro Gly Glu Lys Gly Glu Lys Gly Asp Ser Gly Leu 65 70 75ctt ggt cct aag ggt gag aca gga gat gtt gga atg aca gga gct gaa 292Leu Gly Pro Lys Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu 80 85 90ggg cct cgg ggc ttc ccc gga acc cct ggc agg aaa gga gag cct gga 340Gly Pro Arg Gly Phe Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro Gly 95 100 105gaa gcc gct tat gtg tat cgc tca ggc ttc agt gtg ggg ctg gag acc 388Glu Ala Ala Tyr Val Tyr Arg Ser Gly Phe Ser Val Gly Leu Glu Thr 110 115 120cgc gtc act gtt ccc aat gta ccc att cgc ttt act aag atc ttc tac 436Arg Val Thr Val Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr125 130 135 140aac caa cag aat cat tat gac aac agc act ggc aag ttc tac tgc aac 484Asn Gln Gln Asn His Tyr Asp Asn Ser Thr Gly Lys Phe Tyr Cys Asn 145 150 155att ccg gga ctc tac tac ttc tct tac cac atc acg gtg tac atg aaa 532Ile Pro Gly Leu Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys 160 165 170gat gtg aag gtg agc ctc ttc aag aag gac aag gcc gtt ctc ttc acc 580Asp Val Lys Val Ser Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr 175 180 185tac gac cag tat cag gaa aag aat gtg gac cag gcc tct ggc tct gtg 628Tyr Asp Gln Tyr Gln Glu Lys Asn Val Asp Gln Ala Ser Gly Ser Val 190 195 200ctc ctc cat ctg gag gtg gga gac caa gtc tgg ctc cag gtg tat ggg 676Leu Leu His Leu Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly205 210 215 220gat ggg gac cac aat gga ctc tat gca gat aac gtc aac gac tct aca 724Asp Gly Asp His Asn Gly Leu Tyr Ala Asp Asn Val Asn Asp Ser Thr 225 230 235ttt act ggc ttt ctt ctc ttc cat gat acc aac tga ctgcaactac 770Phe Thr Gly Phe Leu Leu Phe His Asp Thr Asn 240 245tcatagccca tacaccagga gaatcatgga acgtcgacac actttcagct tagtttgaga 830gattgatttt attgcttagt ttgagagtcc tgagtattat ccacacgtgt actcacttgt 890tcattaaacg actttataaa aaataatttg tgttcctagt ccagaaaaaa aggcactccc 950tggtctccac gactcttaca tggtagcaat aacagaatga aaatcacatt tggtatgggg 1010gcttcacaat attcgcatga ctgtctggaa gtagaccatg ctatttttct gctcactgta 1070cacaaatatt gttcacataa accctataat gtaaatatga aatacagtga ttatcttctc 1130aaaaaaaact cgtgccgaat tc 11522247PRTmus musculus 2Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His1 5 10 15Ala Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 20 25 30Pro Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 40 45His Ser Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 50 55 60Pro Gly Glu Lys Gly Glu Lys Gly Asp Ser Gly Leu Leu Gly Pro Lys65 70 75 80Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 90 95Phe Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr 100 105 110Val Tyr Arg Ser Gly Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val 115 120 125Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn 130 135 140His Tyr Asp Asn Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu145 150 155 160Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val 165 170 175Ser Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr 180 185 190Gln Glu Lys Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu 195 200 205Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His 210 215 220Asn Gly Leu Tyr Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe225 230 235 240Leu Leu Phe His Asp Thr Asn 24531276DNAmus musculus 3ctctaaagat tgtcagtgga tctgacgaca ccaaaagggc tcagg atg cta ctg ttg 57Met Leu Leu Leu1caa gct ctc ctg ttc ctc tta atc ctg ccc agt cat gcc gaa gat gac 105Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His Ala Glu Asp Asp5 10 15 20gtt act aca act gaa gag cta gct cct gct ttg gtc cct cca ccc aag 153Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val Pro Pro Pro Lys 25 30 35gga act tgt gca ggt tgg atg gca ggc atc cca gga cat cct ggc cac 201Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly His 40 45 50aat ggc aca cca ggc cgt gat ggc aga gat ggc act cct gga gag aag 249Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu Lys 55 60 65gga gag aaa gga gat gca ggt ctt ctt ggt cct aag ggt gag aca gga 297Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys Gly Glu Thr Gly 70 75 80gat gtt gga atg aca gga gct gaa ggg cca cgg ggc ttc ccc gga acc 345Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly Thr85 90 95 100cct ggc agg aaa gga gag cct gga gaa gcc gct tat atg tat cgc tca 393Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr Met Tyr Arg Ser 105 110 115gcg ttc agt gtg ggg ctg gag acc cgc gtc act gtt ccc aat gta ccc 441Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val Pro Asn Val Pro 120 125 130att cgc ttt act aag atc ttc tac aac caa cag aat cat tat gac ggc 489Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp Gly 135 140 145agc act ggc aag ttc tac tgc aac att ccg gga ctc tac tac ttc tct 537Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu Tyr Tyr Phe Ser 150 155 160tac cac atc acg gtg tac atg aaa gat gtg aag gtg agc ctc ttc aag 585Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe Lys165 170 175 180aag gac aag gcc gtt ctc ttc acc tac gac cag tat cag gaa aag aat 633Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Lys Asn 185 190 195gtg gac cag gcc tct ggc tct gtg ctc ctc cat ctg gag gtg gga gac 681Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly Asp 200 205 210caa gtc tgg ctc cag gtg tat ggg gat ggg gac cac aat gga ctc tat 729Gln Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His Asn Gly Leu Tyr 215 220 225gca gat aac gtc aac gac tct aca ttt act ggc ttt ctt ctc tac cat 777Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His 230 235 240gat acc aac tga ctgcaactac ccatagccca tacaccagga gaatcatgga 829Asp Thr Asn245acagtcgaca cactttcagc ttagtttgag agattgattt tattgcttag tttgagagtc 889ctgagtatta tccacacgtg tactcacttg ttcattaaac gactttataa aaaataattt 949gtgttcctag tccagaaaaa aaggcactcc ctggtctcca cgactcttac atggtagcaa 1009taacagaatg aaaatcacat ttggtatggg ggcttcacaa tattcgcatg actgtctgga 1069agtagaccat gctatttttc tgctcactgt acacaaatat tgttcacata aaccctataa 1129tgtaaatatg aaatacagtg attactcttc tcacaggctg agtgtatgaa tgtctaaaga 1189cccataagta ttaaagtggt agggataaat tggaaaaaaa aaaaaaaaaa aagaaaaact 1249ttagagcaca ctggcggccg ttactag 12764247PRTmus musculus 4Met Leu Leu Leu Gln Ala Leu Leu Phe Leu Leu Ile Leu Pro Ser His1 5 10 15Ala Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Ala Leu Val 20 25 30Pro Pro Pro Lys Gly Thr Cys Ala Gly Trp Met Ala Gly Ile Pro Gly 35 40 45His Pro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 50 55 60Pro Gly Glu Lys Gly Glu Lys Gly Asp Ala Gly Leu Leu Gly Pro Lys65 70 75 80Gly Glu Thr Gly Asp Val Gly Met Thr Gly Ala Glu Gly Pro Arg Gly 85 90 95Phe Pro Gly Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Ala Ala Tyr 100 105 110Met Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val 115 120 125Pro Asn Val Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn 130 135 140His Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn Ile Pro Gly Leu145 150 155 160Tyr Tyr Phe Ser Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val 165 170 175Ser Leu Phe Lys Lys Asp Lys Ala Val Leu Phe Thr Tyr Asp Gln Tyr 180 185 190Gln Glu Lys Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu 195 200 205Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Asp Gly Asp His 210 215 220Asn Gly Leu Tyr Ala Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe225 230 235 240Leu Leu Tyr His Asp Thr Asn 24554517DNAHomo sapiens 5ctgattccat accagagggg ctcagg atg ctg ttg ctg gga gct gtt cta ctg 53Met Leu Leu Leu Gly Ala Val Leu Leu1 5cta tta gct ctg ccc ggg cat gac cag gaa acc acg act caa ggg ccc 101Leu Leu Ala Leu Pro Gly His Asp Gln Glu Thr Thr Thr Gln Gly Pro10 15 20 25gga gtc ctg ctt ccc ctg ccc aag ggg gcc tgc aca ggt tgg atg gcg 149Gly Val Leu Leu Pro Leu Pro Lys Gly Ala Cys Thr Gly Trp Met Ala 30 35 40ggc atc cca ggg cat ccg ggc cat aat ggg gcc cca ggc cgt gat ggc 197Gly Ile Pro Gly His Pro Gly His Asn Gly Ala Pro Gly Arg Asp Gly 45 50 55aga gat ggc acc cct ggt gag aag ggt gag aaa gga gat cca ggt ctt 245Arg Asp Gly Thr Pro Gly Glu Lys Gly Glu Lys Gly Asp Pro Gly Leu 60 65 70att ggt cct aag gga gac atc ggt gaa acc gga gta ccc ggg gct gaa 293Ile Gly Pro Lys Gly Asp Ile Gly Glu Thr Gly Val Pro Gly Ala Glu 75 80 85ggt ccc cga ggc ttt ccg gga atc caa ggc agg aaa gga gaa cct gga 341Gly Pro Arg Gly Phe Pro Gly Ile Gln Gly Arg Lys Gly Glu Pro Gly90 95 100 105gaa ggt gcc tat gta tac cgc tca gca ttc agt gtg gga ttg gag act 389Glu Gly Ala Tyr Val Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr 110 115 120tac gtt act atc ccc aac atg ccc att cgc ttt acc aag atc ttc tac 437Tyr Val Thr Ile Pro Asn Met Pro Ile Arg Phe Thr Lys Ile Phe Tyr 125 130 135aat cag caa aac cac tat gat ggc tcc act ggt aaa ttc cac tgc aac 485Asn Gln Gln Asn His Tyr Asp Gly Ser Thr Gly Lys Phe His Cys Asn 140 145 150att cct ggg ctg tac tac ttt gcc tac cac atc aca gtc tat atg aag 533Ile Pro Gly Leu Tyr Tyr Phe Ala Tyr His Ile Thr Val Tyr Met Lys 155 160 165gat gtg aag gtc agc ctc ttc aag aag gac aag gct atg ctc ttc acc 581Asp Val Lys Val Ser Leu Phe Lys Lys Asp Lys Ala Met Leu Phe Thr170 175 180 185tat gat cag tac cag gaa aat aat gtg gac cag gcc tcc ggc tct gtg 629Tyr Asp Gln Tyr Gln Glu Asn Asn Val Asp Gln Ala Ser Gly Ser Val 190 195 200ctc ctg cat ctg gag gtg ggc gac caa gtc tgg ctc cag gtg tat ggg 677Leu Leu His Leu Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly 205 210 215gaa gga gag cgt aat gga ctc tat gct gat aat gac aat gac tcc acc 725Glu Gly Glu Arg Asn Gly Leu Tyr Ala Asp Asn Asp Asn Asp Ser Thr 220 225 230ttc aca ggc ttt ctt ctc tac cat gac acc aac tga tcaccactaa 771Phe Thr Gly Phe Leu Leu Tyr His Asp Thr Asn 235 240ctcagagcct cctccaggcc aaacagcccc aaagtcaatt aaaggctttc agtacggtta 831ggaagttgat tattatttag ttggaggcct ttagatatta ttcattcatt tactcattca 891tttattcatt cattcatcaa gtaactttaa aaaaatcata tgctatgttc ccagtcctgg 951ggagcttcac aaacatgacc agataactga ctagaaagaa gtagttgaca gtgctatttt 1011gtgcccactg tctctcctga tgctcatatc aatcctataa ggcacaggga acaagcattc 1071tcctgttttt acagattgta tcctgaggct gagagagtta agtgaatgtc taaggtcaca 1131cagtattaag tgacagtgct agaaatcaaa cccagagctg tggactttgt tcactagact 1191gtgccctttt atagaggtac atgttctctt tggagtgttg gtaggtgtct gtttcccacc 1251tcacctgaga gccattgaat ttgccttcct catgaattaa aacctccccc aagcagagct 1311tcctcagaga aagtggttct atgatgaagt cctgtcttgg aaggactact actcaatggc 1371ccctgcacta ctctacttcc tcttacctat gtcccttctc atgcctttcc ctccaacggg 1431gaaagccaac tccatctcta agtgctgaac tcatccctgt tcctcaaggc cacctggcca 1491ggagcttctc tgatgtgata tccacttttt tttttttttg agatggagtc tcactctgtc 1551acccaggctg gagtacagtg acacgacctc ggctcactgc agcctccttc tcctgggtcc 1611aagcaattat tgtgcctcag cctcccgagt agctgagact tcaggtgcat tccaccacac 1671atggctaatt tttgtatttt tagtagaaat ggggtttcgt catgttggcc aggctggtct 1731cgaactcctg gcctaggtga tccacccgcc tcgacctccc aaagtgctgg gattacaggc 1791atgagccacc atgcccagtc gatatctcac tttttatttt gccatggatg agagtcctgg 1851gtgtgaggaa cacctcccac caggctagag gcaactgccc aggaaggact gtgcttccgt 1911cacctctaaa tcccttgcag atccttgata aatgcctcat gaagaccaat ctcttgaatc 1971ccatatctac ccagaattaa ctccattcca gtctctgcat gtaatcagtt ttatccacag 2031aaacattttc attttaggaa atccctggtt taagtatcaa tccttgttca gctggacaat 2091atgaatcttt tccactgaag ttagggatga ctgtgatttt cagaacacgt ccagaatttt 2151tcatcaagaa ggtagcttga gcctgaaatg caaaacccat ggaggaattc tgaagccatt 2211gtctccttga gtaccaacag ggtcagggaa gactgggcct cctgaattta ttattgttct 2271ttaagaatta caggttgagg tagttgatgg tggtaaacat tctctcagga gacaataact 2331ccagtgatgt ttttcaaaga ttttagcaaa aacagagtaa atagcattct ctatcaatat 2391ataaatttaa aaaactatct ttttgcttac agttttaaat tctgaacaat ttctcttata 2451tgtgtattgc taatcattaa ggtattattt tttccacata taaagctttg tctttttgtt 2511gttgttgttg tttttaagat ggagtttccc tctgttgcca ggctagagtg cagtggcatg 2571atctcggctt actgcaacct ttgcctccca ggtttaagcg attcttctgc ctcagcctcc 2631cgagtagctg ggaccacagg tgcctaccac catgccaggc taatttttgt atttttagta 2691aagacagggt ttcaccatat tggccaggct ggtctcgaac tcctgacctt gtgatctgcc 2751cgcctccatt gtgttgttat ttgtgagaaa gatagatatg aggtttagag agggatgaag 2811aggtgagagt aagccttgtg ttagtcagaa ctctgtgttg tgaatgtcat tcacaacaga 2871aaacccaaaa tattatgcaa actactgtaa gcaagaaaaa taaaggaaaa atggaaacat 2931ttattccttt gcataataga aattaccaga gttgttctgt ctttagataa ggtttgaacc 2991aaagctcaaa acaatcaaga cccttttctg tatgtccttc tgttctgcct tccgcagtgt 3051aggctttacc ctcaggtgct acacagtata gttctagggt ttccctcccg atatcaaaaa 3111gactgtggcc tgcccagctc tcgtatcccc aagccacacc atctggctaa atggacatca 3171tgttttctgg tgatgcccaa agaggagaga ggaagctctc tttcccagat gccccagcaa 3231gtgtaacctt gcatctcatt gctctggctg agttgtgtgc ctgtttctga ccaatcactg 3291agtcaggagg atgaaatatt catattgact taattgcagc ttaagttagg ggtatgtaga 3351ggtattttcc ctaaagcaaa attgggacac tgttatcaga aataggagag tggatgatag 3411atgcaaaata atacctgtcc acaacaaact cttaatgctg tgtttgagct ttcatgagtt 3471tcccagagag acatagctgg aaaattccta ttgattttct ctaaaatttc aacaagtagc 3531taaagtctgg ctatgctcac agtctcacat ctggtggggg tgggctcctt acagaacacg 3591ctttcacagt taccctaaac tctctggggc agggttattc ctttgtggaa ccagaggcac 3651agagacagtc aactgaggcc caacagaggc ctgagagaaa ctgaggtcaa gatttcagga 3711ttaatggtcc tgtgatgctt tgaagtacaa ttgtggattt gtccaattct ctttagttct 3771gtcagctttt gcttcatata ttttagcgct ctattattag atatatacat gtttagtatt 3831atgtcttatt ggtgcattta ctctcttatc attatgtaat gtccttcttt atctgtgata 3891attttctgtg ttctgaagtc tactttgtct aaaaataaca tacgcactca acttcctttt 3951ctttcttcct tcctttcttt cttccttcct ttctttctct ctctctcttt ccttccttcc 4011ttcctccttt tctctctctc tctctctctc tctcttttct tgacagactc tcgttctgtg 4071gccctggctg gagttcagtg gtgtgatctt ggctcactgc tacctctacc atgagcaatt 4131ctcctgcctc agcctcccaa gtagctggaa ctacaggctc atgccactgc gcccagctaa 4191tttttgtatt tttcgtagag acggggtttc accacattcg tcaggttggt ttcaaactcc 4251tgactttgtg atccacccgc ctcggcctcc caaagtgctg ggattacagg catgagccat 4311cacacctggt caactttctt ttgattagtg tttttgtggt atatcttttt ccatcatgtt 4371actttaaata tatctatatt attgtattta aaatgtgttt cttacagact gcatgtagtt 4431gggtataatt tttatccagt ctaaaaatat ctgtctttta

attggtgttt agacaattta 4491tatttaataa aatggtggaa tttaaa 45176244PRTHomo sapiens 6Met Leu Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His1 5 10 15Asp Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro 20 25 30Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly 35 40 45His Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 50 55 60Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile65 70 75 80Gly Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 90 95Ile Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg 100 105 110Ser Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met 115 120 125Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp 130 135 140Gly Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe145 150 155 160Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe 165 170 175Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn 180 185 190Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly 195 200 205Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu 210 215 220Tyr Ala Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr225 230 235 240His Asp Thr Asn720966DNAHomo sapiensmisc_feature1..48115' regulatory region 7gctgatctgc tgcctcagcc ttcccaaagt gctgtaattt attaggcata agccactgtg 60cctgcctagt gttgtacatt ctgtgggttt tgacaattgt atgcatctac atgtatgtac 120catttatagt attcctgttt ttaattttag ccattctagt aggcatgtag tgatatctca 180tggtgatttt aatttgcgtt tccgtaatgg ttaataatgc tgaacatctt tgcatgtgct 240tgtttgtcat ttgtgtttcc tacttggtga aataattgtt catgtccttt gtccattttc 300taattgaatt tttttttacc atttagtttt gagatttctt tatacaatct agatccaaat 360ctcttgtctc aaatatggtt tgcaaataca ttcctctaat tcatatattg ccttttcctc 420ctcttaacag gatgtttcac agagcaaaag ttttagtttt gttgaaatct cacttttcat 480ttttttcttt agtggattgt gcttttgttg tcatatgtaa gaactcttca ctggccctag 540atccttgtat tggtttccta agattgccat agcaaatcac catgaactta gtgacaaaaa 600gacagaaatt tattttcact tcctactgtg ggcagactag acgttaatta ttttcatgta 660tgctcattcc tatgacatct ttctgatata ataattatag ttattcttaa gcttcaccct 720tttttctatt agctttgtta ccttgggtgt cactttttct tttttgacat tgtgacctat 780gccagatcat gtctgttagt acttagccct ccattcacct ctccataatc ccttttgtat 840tcctggagct tgatgcctga aatgacacat cctacattcc tttgccagat gggtaccagt 900tagcttgtgc acatgggaga caaccgtgaa aagactgaag tggggaagaa gggaggagct 960gttgtgtttc agtgagcgcc cttggcagtg gcggtgacag tggctcctgt tcagtggcaa 1020tggtggagca gctagcaaga catgcagtaa gcgcaggctc ataggctatg gtccaggagc 1080agtcaccgat tcctggtctt taggcaatat catctccctt tgcttctcca gcctttctaa 1140aattattgta ccttgactag tacaattttt tagtattggg ggtagtccaa ggacacaggc 1200tttaaaaagt atgaattcag ggttgcctac ctgcattgac tgcgcttgaa tcatgatggc 1260cttctggtcg gtggcaggag gtgacagtcc aaatcatgca gtagcaaacc agatacttaa 1320attatcatct gagatacttc agaagtacag ccgtagccat accttcagaa gagataaaga 1380aatgttctcc tggccaggcg cggtggctca cgcctgtcat tccagcactt tgggaggccg 1440agggggtgga tcacctgagg tcgggagttc gagaccagcc tgaccaacat ggggaaaccc 1500tgtctctact aaaaatacaa aattagcggg gcgtggtggc acatgcccat aatcccagct 1560actcgggagg ctaaggcagg ataatcgctt gaacctgaga ggcagaggtt gcggtgaact 1620gagatcatgc catagtactc cagcctgggc aacaagagtg aaactccatc tcaaaaaaaa 1680aaaaaagaaa aaaagataaa gaaatgttct cctttcttgc catttctagg ggtttgggga 1740tggcgtacat tgctgcaggg cgtgctcact ctaccatctt gctccaatct ttatttttca 1800aaatacagtg cttatgcttg gttacttcag ttaagattat ttttaaaaat cataattaag 1860caaaaatata tggccatgct taaacatatt taagataaat taagtgattt ggcctgtttc 1920agtatcccaa ctcacatgct aacaggggct tgacctgtag ctacggtacc ctggaggaaa 1980tgatcgcatt tatttggtta tttcggtcta agtagtaata gttctgtcct gggaaaaaga 2040ctagcctcaa ggcatttctg attgaatgtt tttcaattac agtctttaaa ccagtatgcc 2100acagaactgg ctctttccac atgacggcct ttgtggtggg tggcagattg ccctgaggcc 2160tcgcaaaatg ctaggctttc acaatgtcac tgactgacag ccaggcccag cacagtcttg 2220gtgtgattgt ggggctaaag ttattccacc ttgtgcaata gctacagcct tctctaacca 2280gctgcattct tataaagtta gaagaaaata cttttttttt tttgagatgg attctcgctc 2340tgttgcccag gctggagtgc aatggtgcga tctcggctcg ctgcaacctc cgcctcctgg 2400gttcaaacga ttctcctccc tcagaccccc gagtagctgg gattgcaggt gcctgccacc 2460acgcccggct aacttttttg tatttttagt ggagacgggg tttcaccatc ttcgtcaggc 2520tggtctcaga ctcctgacct caagtgatct gcccgcctca gcctcccaaa atgctgggat 2580tacaggcatg agctactgtg cccggccaaa gaaaatactt tttatgccag ccctgaaact 2640accctgaagc acatacatca accttgaggc ctcacactcc atcaagaggg gtgaagggca 2700tgaggaatta gaaagcatag ggatttttag ttagacagat ctggttcaaa tcctagactt 2760gtgccttgaa caaattattt accctcattg aactctagat tcattatttg taaaatgaaa 2820gacaataata gttatctcca aaggaaagtt gaatatgatc attcatttat tcattaattc 2880aacatttatt attgcctact ttgtgccagg ttctattcta ggaactaagg gatacaactt 2940tgaataggca aaatctctgc tctcctgaag tttacttttt tttttttttt ttgagacaga 3000gtttcactct tgtcacccag gctggagcgc aatggtgctc ttggctcact gcaacctcca 3060cctcctgggt tcaagtgatt ctcttgtctc agcctcccaa gtagctggga ctacaggtat 3120gtgccaccac gcccggctat ttctgcattt ttagtagaga tggggtttca ccatgttggc 3180cagactggtc tcaaactcct gatctcaggt gatatgcctg tcttggcctt ccaaagtact 3240gggattacag gcctgagcca ctgcacctga cctgaagttt atgttctatt aaatagcaac 3300agacagtaac ataaaccaaa aataaatagg aaaacaccat aacaaaaatc aaacagtgat 3360ataattgaga gttgcttcta tttctttttg ttgtcttctt ggttcaatca gcctgctaaa 3420ctatatggaa cctcattttc atgggccact tatttaagcc gggggacctt ggaaagtctc 3480tcatgtctct catctcaacg gcctaatgtg acttctcttg aaatatttgg acattagcag 3540gaagctgagg ctttacatca gatctttact ttaatggtgg acttgacttt actggtagat 3600ttttaggctc tgtgtggact gtggagatga tatctggggg gcaggcagac acttgccctg 3660cctctgtctg agaaaattct gttttggatg tcttgttgaa gttggtgctg gcatcctaag 3720cccttgctgg ggtcgtaatt taattcatca gaatgtgtgg cttgcaagaa ccggctcaga 3780tcctgcsctt caaaaacaaa acatgagcgt gccaagaaag tccaaggtgt tgaatgttgc 3840cacttcaagc ctaaactttc taggaacacc taagtgggtg gcagcttcca gttctccagg 3900ctgcttctag gccagagctg ggttccacaa gagacagaat aggcatatat atgcttaagg 3960aactggaaaa acaggctctc tctctctcac aaacacacac acacacatac caaggtagct 4020gtcaaaatgt tatccgaaat tttggaacca aaaaatcttg aaagatggta ttccaatatc 4080acattttatg taagttttct attatattag attcaaatta cgattcgagg ccacaagctt 4140taagaattca gggccttttt aacttgccaa gccccacacc actccaggaa cttccccaca 4200ccccagttct cagaattcat gtgcaaggtc tttcctaaat ccagggtcca ggtcagagag 4260tggaggatgt gctctatttc ttacctgatt gcagacccct ctgacagtgc tcccttctga 4320agcactcact gtctgaacgt acacagtctc agacttaatc atgcacagtg agcaagactg 4380tggtgtgata attggcgtcc ctgacttatt agggcaaatc tatgggaggg ggagacctcc 4440tggaccactg agcaattaat tcatttacat taggaagttt ctccgtcaga tgcaggaaaa 4500aaatcttgtt ttcctgctgt ggttttgact tttgccccat cttctgttgc tgttgtagga 4560ggcaaaataa gggtcaaggc ctggaaacac aagtgctttg actgaagctc cacttggctt 4620ccgaagccca agctgggttg taccaggttc cctagggtgc aggctgtggg caactgccag 4680ggacatgtgc ctgcccaccg gcctctggcc ctcactgagt tggccaatgg gaaatgacaa 4740ttgtgaggtg gggactgcct gcccccgtga gtaccaggct gttgaggctg ggccatctcc 4800tcctcacttc cattctgact gcagtctgtg gttctgattc cataccagag ggtaagagca 4860attctgtgaa gttccaggct gggtggggga tgcatgcata gcctctggct gggatcaccc 4920aggctctccc gtccgtagta gtgtgggagt ggatacaggt ggatactctg gtcagagcag 4980cactggtgga ggcagatatg cactgggctt cttcctccgt tctcccacag ccccaagaga 5040gaaagggtta tttcagacat tccttctaag atgcatggaa ccattctgaa ttttacccag 5100ttcgctctgt agcaggatac ctattgagaa aaagttaggg tcagtaaggt ggaagggtct 5160gtccacagat gaagtccaat tcgattaagg gggataaggg aatacattgt ctcttagctt 5220gaccaggtag ggcaaaggaa gaagcatata tgaaggcagc ttcagaaaag tcaagctgag 5280cactgacttc agactggaat taggaatcca gctctgccac tttattctac tcagcaaata 5340tttactgagc aaattctatg ggctagacag tggattgggt tcacaagata caatgagtgt 5400gacatggttg ttgtctatgg atttggggat atatgtaggt atagggatat cttacaaggt 5460aatcaagagg ttctaatgag gccagccatg gtggctcaca cctgtaatcc cagcaatttg 5520ggagaccgag gcgggtggat cacctgaggt caggagttcc agactagcct gaccaacatg 5580gtgaaacccc gcctctacca aaaatacaaa aattagttgg gcgtgatggc aggtgcctgt 5640aatcccagct tctcgggagg ctgaggcagg agaattgtct gaacctggga ggcagaggtt 5700gcagtgagcc gagattgttg ccactgcatt ccagcctggg tgacagagcg agactttgtg 5760tcaaaaaaaa aaaaaaaaag aaagaaaaga aaaagaggct ctaatgagat aaaatgagaa 5820aagcctggca tgtagtggca acttatgaaa aattgtaatt aaaaaaaaac attttctgac 5880agaagaaact ggatctacct ggtttttctg aagcctaatc ctgctcgccc cagtgagtgc 5940tgtttctgag gcatcctggt tgttttgagc tgtggatgct gaaggttaga gtgggaggga 6000ttttagaggt taggtctgcc cctcttgtgt tagaggacat ggatccctgg tctggagagg 6060ttctggtttt tggatcaagc ctcacaaggg gtggcaccaa ctcactccta ggaactccgc 6120tagaaggaag gccagctctg cctaattcgg ttggggagat gggggtccct ttatgctagc 6180agaatatgtc cgaaggagca tgatggtgtc agctttgttc atgaaggcca gtggtacaca 6240gggagcccgg cagcttcctc agcagtccct gctgccactc ttccttaagt cttgaggagt 6300ctttttttgg cacaatctca gctcactgca acctccgcct cccaggttca agcgattctc 6360ctgcctcagt ctcccaagta gctgagacta caggcatgcg ccaccacgcc cagctaattt 6420ttatattttt agtagagatg gggttcacca tattggccag gatggtctcg atctcttgac 6480ctcatattcc acctgcctcg gcctcccaaa gtgctggtat tacaggtgtg agccactgcg 6540cctggccgag gagtcttaag ctgagatcac agcattgcac tccagcctgg gcaaaaagag 6600caaaactcca tctcaaaaaa aaaaaaaaaa tagacacaag actggctcct tgtctttttt 6660ggggacaggg tctcactcta tcacccaggc tggagtgcag tggtgcaatc acagctcact 6720gcagcctcga tttcccaggc tcaagtgacc ctcccatctt agcctcctga gtagctggga 6780ctacaggtgt gtgcaaccat gcctggctaa tttttaaaaa ttttttgtag agatgaggtc 6840tcactatatt ggctgggggg cctcaaactc ctgggctcag cagtcctccc acctcagcct 6900cccaaaaggc tgggattata tgcttgctct ttttaaggtg gctgtaggga caaactttcc 6960acctactcct tgtcaagcca gtggaccggt ggtcccagac atacggctaa agtcaagagg 7020tgatgtcttt tggagagata ctttcaatca ggaatttcaa tcagaaattc aatcatgtgg 7080agagagactt atcctaaaaa tgtggtggtg cgtgggatgc tctgttttat tagttccttg 7140acagtatgta tgtgtgtgag tgtgtgtgtg tgcgcgcgca cactcatttg gatgggtgtg 7200tatgtgtgtg ggggggtggt gcgtacgtat gtggatgtgt ggatgtggtg tgtgggtgtg 7260cgcgtgcata ggtggaggtg tgtgtatggg tgcgggtatg tgtgtgtgtt gggcatggag 7320atattgacag ctctcccagg gctgagtgaa ggctttcggg caaagctcct gggagctagg 7380caaagctgag ttgattcctg gttatgccat ttattattgg gttgcaccgt gtgaaactgc 7440caatattcta cactttgact tttatttatt tttattttta ttttttttga gacagagttt 7500cacacttgtc acctaggctg gagtgcagtg gcgcgatctc agctcactgc aacctctgcc 7560tcatggattc aagtgattct cctgcctcag cctcccaagt agctggaatt acaggtgccc 7620gctaccacgc ctgactaatt tttgtatttt tggtagagac gggatttcac catgttgtcc 7680aggctggtct gaaactcctg acatcaggta atccacccac ctcagcctcc caaagtgctg 7740ggattacagg catgagccac tgcgcccggc ccattttgac ttttaaaaat gggagtttga 7800tataattcaa tccagtggtt gaattagcta gcatcgttcc ctctccaagt ctcaggttct 7860cctacacgtt agagtcaaaa gcagggctat gggaagatta agtaaaataa attttgaaaa 7920tgccttatga aaattacact ccaaagaact cgcgccagtg tcagtgttct catgttcctc 7980atctcacatg atcacatttc gcggattagg aagctgagtc tgagaagctc cgtgtagtgc 8040tttttcggag gcaccgtgat gtgatggaag gctcactcgt taggaagtca gaacagagtc 8100tctgagggat catttcctta atctgtcagt ttcctcatct ctgaagttgg gctcatttcc 8160ttccttcatg gagttattgt aaagatgaag ataaataacg tgtaaaatct agcatgggaa 8220ctggcttcta taaggttcta ataagtgcat tcctactcct tcccctcagc cttcccattt 8280gtaaaagcaa ggcaggggtg aggtgatttc tggggctcct tttggctctg acatttgagg 8340attttgtatc cttttttttt tcagagtctt gctctgtcac ccaggttgga gtgcagctca 8400atgcaaattc cgcctcccag gctcaagcaa ttcttatgtc tcagcctcct gagtacctgg 8460gattacaggc aggcaccacc acccccagct aattttttgt attttcagta gagacggggt 8520tttgccatat tggccaggct ggtcttgaac tcctgacttc atgtgaccca cccatctcag 8580cctcccaaag tgctgagatg acaggtgtga gctaccgtgc ctggccaatt ttgtgtgctt 8640taatgccctt ttctgctgga agagttggca ccaggtttgg tgatctcttt cccccacacg 8700gctctgcctc ctgccagtcc cagaggggac cctgtccttg catttcacag gattctgctg 8760ttgcaactga aattccagta ggtcaaagtg aaatttctca tacactttaa catgaagata 8820aatgatcaca gtatggccct ttaggatcct gagaacatca cggtcatccc ctggtataat 8880tttaaaagca gatgaatcca tgcctgtgcg aggtttgcca ggaaagccag tgctgggatt 8940acagtggaag tctttttatg ctactttttt cttgtatccc tcaccccatg gggtggcata 9000ttgaaaggca ggatgtgtga ccacgatact tttctcctcc tggactatgt ctaagagtct 9060gttattgggt tctgaagatc agagtttaat ttccgactcc tctctgtgta gctctgggat 9120cttggaaagc cacttaacct ttctgaagtc ccctttcctc atctctaaaa tgcatacact 9180catcactaac atttactgag cactgacatg tgccagacac cattctaagc attttacaca 9240gactacacca tttgatcttc caacaaacag aacactgaaa cgcattacag gtcagaacaa 9300atgatttgtg cctaagcacc aagaccgtag agcccgtgct ccctattcta ccctatcctg 9360tctctcaaaa tgattgtgag aatcgaatga gacactaggt gagaaaaggg ttttataaat 9420agcattttaa aaatttttta aagtccacaa aatttttaat tttaatacag ataaaataga 9480tccctttgtt ttataaaaag taacaaaatt tgttatacaa caactatgtt atttattaat 9540tttgcctttt tgtatgctgc caggaaagaa acattaagaa atcttaaatt gattatggtg 9600aatcagaagg tctgcctgga ctttttattg ctctaactgt acagctgatc atactacctc 9660attttttttt atgacacttc aagggtgcgc ttagcttcat cactccttcg ttgccaaaag 9720ctttgtgacc aaaaacaatt aagcagattc ctgagtcact aaatgacaca taaccagagt 9780tgagacttag gaacttttag tgccatgcta agcccacagg gacacaacaa atagcatttt 9840acaaaggcaa agaattgtga cacttgagat ttagcttgtt gatccttgta aaagttttct 9900ttttaggcat aattgagttt tagatcatag tactcactat tacttagtaa taattttttt 9960ctgatagaaa tacagtgtaa caggccgggc gcagtggctc atgcctgtaa tcccagcact 10020ttgggaggcc gaggcgggcg gatcacttga ggtcaggagt ttgagaccag cccggccaac 10080atggtgaaat cccatctcta ctaaaaatac aaaaaattag ccaggtgtgg tcgtggattc 10140ctgtgatccc agctacttgg gagggtgagg caggagcatc agttgaaccc aggaggcgga 10200ggttgcagtg agccaagatg gtgccattgc actccagcct gggccacaaa gcgagactcc 10260acttcagaaa caaaaaaaaa aagagagaga gagaaaagaa ggaaggaagg aaggaaggaa 10320ggaaagaagg aaggaaggaa ggaaagaagg aaggaaggaa ggaaggaaag aaggaaggaa 10380ggaaagaagg aaggaaggaa agaaggaagg aaggaaggaa ggaagggtaa caagcaaagt 10440gtaacaatgg caatatctaa aaaaataggt atttttatat gtttgtcgtt ttatatatat 10500gacccccact ttagagatga ggaaactgag agattaagga aacgatccct gagagactct 10560gttctgactt ccaaatcggt gagctttcca tcgcatcacg gtgcctccga aagcatgaca 10620cggagcttct cagacttagc ttcctaatcc gctaaacggg attatgtgag atgaggaaca 10680tgagaacgct gacatgggtg agggttcctt ggagtatcat tttcatgtgg cattttcaaa 10740acttatttta cctaatcttc ccaaagccct gcttttgact ctaatgtgtc tcctgagact 10800tggagagcgc aagatgctag cgacagagca agactccatc tccagataaa taaataagta 10860aaataaaaaa gaacacaaat aattttgaaa atttttttga aaattaggca cgtttgcact 10920gaccttcaat tgttattaat tgctggtttc ccacccagaa ttaagttgga atgcaacttt 10980cttttacaat cagagtccgt tcttggtctt ggaaacttct gaggctcctg tgctaatccc 11040actcttgtat ttttggcacc tctaccccgt gccactgtca tggaacccag gctgatcgca 11100cctattagtg gagaaatmtg tccataatac tgaagtttgg ggacaaacag tgttccctta 11160gggtaggaga aagagatctt tatttttaac aaagggggag gagccagaaa actccagaga 11220cccctgagtt tgccctctct ccaaggtttg gggtaagccc cccgtcaccc tttatctctg 11280gggctttcac atattctgga ttctctcctc ctgtttccca gcagaaaagg atggagcctc 11340acagattctt cccatttctg gagaaaaaca tgcatggagc tcaaagttct tctcaggagt 11400tttattgcca aagccataat aagaaagggt ggaggtgaca agcagtgagg aagtttaaag 11460atgcatgaaa tctgtaaagt ctcagaacaa gaattctcct aaaatgcaaa aggggctttg 11520ctggtctccc cttggcttct catgtagctc acctcttttt tcttatcttg agactagtca 11580aacctaagct gtttctcatt ttatttccag aagctattga gaacactctc ctgaattctt 11640caaattcagt agagggcgac aaatgtacat ataaatgatg gtagtgggtc ttaaataaag 11700actcatgaca cctaaagggg cagcacctga gtctgattgc acctgtttct gttgctgttt 11760ctgtctctct tctctctgtc tgccatttca ttatcaatgg ttactttact tataagatca 11820tattagaacc tgatatttga taaatgatgc atcagatcta tagtgagaga aaaaattaat 11880gcaattaaag gtgttgtaac agctagtctt caagtgggga gaaatcattt gagtacctta 11940ggtcacagct tacatcaaaa caaaaaatca gagctacatt aaaaagtgaa attttaacta 12000tatcaaacaa tagaaaaaaa cagaagaaaa ttgaatactt actaaatctt agcatgaata 12060agaactgttt aacacttaga ggcaaggact gggcgtggtg gctcatgctt ttaatcccag 12120gactttggga gcccaaggcg ggcggatcac ctgaggtcag gagtttgaga ctagcctggc 12180caacatggtg aaaccccgtc tctactaaaa aatgcaaaaa ttagctgcgt gtggtggtgc 12240atgcctgtaa tctcagctac ttgggaggct aaggcatgag aatcgcttga acctgggagg 12300tggaggctgt agtgagccga gattgtgcca ctgcactaca gcctgggtga cagtgtgaaa 12360tcctctctct caaaaaaaaa aaaaaaaaaa agcaaactag agcagtgagg taccattatt 12420tcctttgctc actaaactga caacacacaa atgtttttta taatacccaa agctgatgag 12480ggtagttaag gtatgccctt ttatacacac actaatgatg tactactggt tggcagtata 12540acatatgctg ccatgtgggg atatgtatca ggagacttaa aaatgtgcat accttttggt 12600ccagtaattt acttctggga atctgtcata acagaataat aatcttgggg aaagctacat 12660gcctaaggat atttaaaata ttatttaaaa atcaaagtat aatttcttac agaatataaa 12720ataatatttt aaaatgaaaa tatgctaaaa gtttgatgaa atataaatgg tcaaatatat 12780attgattata tccacttact agactagcac tcactctgag acgttaaaaa tagtcattat 12840aaaaactaga aaatgccaaa gacaaaataa aggaataaag ttttacataa agtatgattc 12900cactatgttt aaaaataaac agagacattc ttggagttga gtattgtttt cttttctgtc 12960atgtccaaag aactatataa ctattatttt taatgaacta tatatgtaat atatacatat 13020agtttatatg tatatacaaa atttatctca tatatatgat aaagatgaaa gatgagttgg 13080atgtgccacg tgaagtgggt agtatagaaa cccaggtaat ggggcatagg agtgggattc 13140cagataccag gcccatgttt ttggggtgag attgccaatc acggtctttc ttccatccct 13200cacagaggag taggtttgtc ttcaacaaac cttcagttgt cctgaagaca aacctaattc 13260tggagacttc atataatcta gaagagacaa gcaaactgat gaaaaatagt gaatttttaa 13320ggtaaaataa agtacatgga ctacactttg tttagaatca gattcttggg attaaccaca 13380ttaacccaca gagggtctta gtgatgcctc taatccagga tcctaggacc tatttctctc

13440tgtgagatgc tttctcccaa ctccttggtg agagtgggaa gactaagacc tcagcaatct 13500gaggtggagg cctaagatcc ccctaagatc ggaggcagaa tctgagaggg gataaaagtc 13560cctatacctg tattgggccc ttttctggga gggggatatc aaagaatgat tttgagacag 13620ggaggctttt gactacctgt gccacttgag ctctttgcta gggctccaga atacatattt 13680caaatacatt ccccctccct ccttccttcc ctcttccact cttccttttt atcttccttt 13740cttcttttcc ttcctccttc ccttcctttc tctggctctc tcatgatttc ttttcctcat 13800tataaaagtg cttatttagt ccctactctg ctattagtgt gttagtcttt gtcccctggt 13860acttgctgtt taatggagaa atgggtgagc aaaacagaaa ttacagcaga gtgcaataat 13920agagctaagc caggtgtata aatccattct cacactgctg taaaaaacta ctgggtaatt 13980tataaagaaa agaggtttaa ttgactcaca gttccacagg ctgtacagga agcatggctg 14040gggaggcctc agaaaactta caatcatggt ggaagaaaga gcgaagggga agcaagcaca 14100tcacacagca gcaggagaga gagagagaaa gagagagaga gagaatatag gggaagtgct 14160acacactttc aaccagatct tgtgagaatt cacctactat catgagaaca gcaagggata 14220agtctgcctc catgattcag tcacctccta ccaggcccct tctccaacac atgtcgacgt 14280gctatttggg tggggacaca gacccaaacc atattaccag ggcactggag aaacacagag 14340gggaaagaac cagccaagga gtgagatgga gaacaaggag gacttcttga aacagatgac 14400atccaaactg ggtcctgaaa gctgaataga gattagacag gggaggaggg gcagctaaag 14460atggctcagg caaacaaagg gccaggggat atgttcatgg gatgatgtgt ctctcgttgt 14520ctgcttaaca caaggtgagt ctctccctcc ctctctctct ctttttctct gtgtgtgttt 14580gtgtgtgtgc atgtgtgcaa atgtaatata cccaatagtc aaacatgtgc cccaggagag 14640gggtagagga agaaagagaa tgagagagta agaaggagga atagacacag aaaatgagag 14700agaagggggg aaagaaaaag aagaaaggag ccagaggaga gaagctggtt agcattgaat 14760ggagcaatct gtgtcatcgt acttgggaaa cccaaggatg gattcttggc aagtcgactc 14820ttggagcttt ccctgtgctt ggtcctgtgc tcagacatgg gaaaattaga ggagtgtcat 14880ctgtgcaatc actgaattca taatcttggt gaggaaagga gactacacac agggaataat 14940gctaagtatt acagatttca gggcagaaag agatcaaggt gggctgcaat attcagaaaa 15000gtcttcctgg aaaagttgaa tacttagaaa gcagctccta gaagtagact ctgctgagat 15060ggacggagtc ctttgtaggt cccaactggg tgtgtgtgtg gggtctgtct ctccatggcy 15120gacagtgcac atgtggattc cagggctcag gatgctgttg ctgggagctg ttctactgct 15180attagctctg cccggkcatg accaggaaac cacgactcaa gggcccggag tcctgcttcc 15240cctgcccaag ggggcctgca caggttggat ggcgggcatc ccagggcatc cgggccataa 15300tggggcccca ggccgtgatg gcagagatgg cacccctggt gagaagggtg agaaaggaga 15360tccaggtaag aatgtttctg gcctctttca tcacagacct cctacactga tataaactat 15420atgaagkcat tcattattaa ctaaggccta gacacaggga gaaagcaaag cttttttatg 15480ttaaccataa gcaacctgar gtgatttggg gttggtcttc caaggatgag tgtagatggt 15540gcctctataa ccaagacttt ggctttgctg catctgcagc tccttttcca tcccctttcc 15600catcttcacc ctcatcccta ttcccagtac attcatattc tgattcctct ttctgtctgc 15660ttaacttcca tttcacccag tggcattcaa ccacatttac tgcacacccc ctgaaaggct 15720cagtcctgcc tttggggaac tcttgatcta ggtaagatgt ctaatgtgca aggctctgtt 15780ggtggttacc acaagaaagt ctactctaaa aatgtcaaac tgaatgtgaa caagtattca 15840aagtatggag catagagaaa atrtactcac cgtggacctg atgaagaatg aaggcttcaa 15900ggaggaggca gagcttcagc taggccttga atgatgggta ggcagaatag aggaggagag 15960acatcctaga tggagggggt agaattgcaa aaccagggtt gatggtgcca gcacataaag 16020ggctggcagg gtggagggtc tatgatagag acctatagga gataaagata gagttgaaat 16080tatgggagcc tccatgtctg tgggagatat agaaggagga ggtaacacct ctctcctttt 16140gggagctctt attggtttct tgatctataa gtcaagaagg ttgtgagtgg gagccacagg 16200gatggtaatt taggctgtaa ccaacctagg caggagttct gttctttgta gtcactgagg 16260tcttctcatt ccttaggtct tattggtcct aagggagaca tcggtgaaac cggagtaccc 16320ggggctgaag gtccccgagg ctttccggga atccaaggca ggaaaggaga acctggagaa 16380ggtgcctatg tataccgctc agcattcagt gtgggattgg agacttacgt tactatcccc 16440aacatgccca ttcgctttac caagatcttc tacaatcagc aaaaccacta tgatggctcc 16500actggtaaat tccactgcaa cattcctggg ctgtactact ttgcctacca catcacagtc 16560tatatgaagg atgtgaaggt cagcctcttc aagaaggaca aggctatgct cttcacctat 16620gatcagtacc aggaaaataa tgtggaccag gcctccggct ctgtgctcct gcatctggag 16680gtgggcgacc aagtctggct ccaggtgtat ggggaaggag agcgtaatgg actctatgct 16740gataatgaca atgactccac cttcacaggc tttcttctct accatgacac caactgatca 16800ccactaactc agagcctcct ccaggccaaa cagccccaaa gtcaattaaa ggctttcagt 16860acggttagga agttgattat tatttagttg gaggccttta gatattattc attcatttac 16920tcattcattt attcattcat tcatcaagta actttaaaaa aatcatatgc tatgttccca 16980gtcctgggga gcttcacaaa catgaccaga taactgacta gaaagaagta gttgacagtg 17040ctattttgtg cccactgtct ctcctgatgc tcatatcaat cctataaggc acagggaaca 17100agcattctcc tgtttttaca gattgtatcc tgaggctgag agagttaagt gaatgtctaa 17160ggtcacacaa gtattaagtg acagtgctag aaatcaaacc cagagctgtg gactttgttc 17220actagactgt gcccttttat agaggtacat gttctctttg gagtgttggt aggtgtctgt 17280ttcccacctc acctgagagc cattgaattt gccttcctca tgaattaaaa cctcccccaa 17340gcagagcttc ctcagagaaa gtggttctat gatgaagtcc tgtcttggaa ggactactac 17400tcaatggccc ctgcactact ctacttcctc ttacctatgt cccttctcat gcctttccct 17460ccaacgggga aagccaactc catctctaag tgctgaactc atccctgttc ctcaaggcca 17520cctggccagg agcttctctg atgtgatatc cacttttttt tttttttgag atggagtctc 17580actctgtcac ccaggctgga gtacagtgac acgacctcgg ctcactgcag cctccttctc 17640ctgggtccaa gcaattattg tgcctcagcc tcccgagtag ctgagacttc aggtgcattc 17700caccacacat ggctaatttt tgtattttta gtagaaatgg ggtttcgtca tgttggccag 17760gctggtctcg aactcctggc ctaggtgatc cacccgcctc gacctcccaa agtgctggga 17820ttacaggcat gagccaccat gcccagtcga tatctcactt tttattttgc catggatgag 17880agtcctgggt gtgaggaaca cctcccacca ggctagaggc aactgcccag gaaggactgt 17940gcttccgtca cctctaaatc ccttgcagat ccttgataaa tgcctcatga agaccaatct 18000cttgaatccc gtatctaccc agaattaact ccattccagt ctctgcatgt aatcagtttt 18060atccacagaa acattttcat tttaggaaat ccctggtttt aagtatcaat ccttgttcag 18120ctggacaata tgaatctttt ccactgaagt tagggatgac tgtgattttc agaacacgtc 18180cagaattttt catcaagaag gtagcttgag cctgaaatgc aaaacccatg gaggaattct 18240gaagccattg tctccttgag taccaacagg gtcagggaag actgggcctc ctgaatttat 18300tattgttctt taagaattac aggttgaggt agttgatggt ggtaaacatt ctctcaggag 18360acaataactc cagtgatgtt cttcaaagat tttagcaaaa acagagtaaa tagcattctc 18420tatcaatata taaatttaaa aaactatctt tttgcttaca gttttaaatc ctgaacaatt 18480ctctcttaca tgtgtattgc taatcattaa ggtattattt tttccacata taaagctttg 18540tctttttgtt gttgttgttg tttttaagat ggagtttccc tctgttgcca ggctagagtg 18600cagtggcatg atctcggctt actgcaacct ttgcctccca ggttcaagcg attcttctgc 18660ctcagcctcc cgagtagctg ggaccacagg tgcctaccac catgccaggc taatttttgt 18720atttttagta aagacagggt ttcaccatat tggccaggct ggtctcgaac tcctgacctt 18780gtgatctgcc cacctccatt tttgttgtta ttttttgaga aagatagata tgaggtttag 18840agagggatga agaggtgaga gtaagccttg tgttagtcag aactctgtgt tgtgaatgtc 18900attcacaaca gaaaacccaa aatattatgc aaactactgt aagcaagaaa aataaaggaa 18960aaatggaaac atttattcct ttgcataata gaaattacca gagttgttct gtctttagat 19020aaggtttgaa ccaaagctca aaacaatcaa gacccttttc tgtatgtcct tctgttctgc 19080cttccgcagt gtaggcttta ccctcaggtg ctacacagta tagttctagg gtttccctcc 19140cgatatcaaa aagactgtgg cctgcccagc tctcgtatcc ccaagccaca ccatctggct 19200aaatggacat catgttttct ggtgatgccc aaagaggaga gaggaagctc tctttcccag 19260atgccccagc aagtgtaacc ttgcatctca ttgctctggc tgagttgtgt gcctgtttct 19320gaccaatcac tgagtcagga ggatgaaata ttcatattga cttaattgca gcttaagtta 19380ggggtatgta gaggtatttt ccctaaagca aaattgggac actgttatca gaaataggag 19440agtggatgat agatgcaaaa taatacctgt ccacaacaaa ctcttaatgc tgtgtttgag 19500ctttcatgag tttcccagag agacatagct ggaaaattcc tattgatttt ctctaaaatt 19560tcaacaagta gctaaagtct ggctatgctc acagtctcac atctggttgg ggtgggctcc 19620ttacagaaca cgctttcaca gttaccctaa actctctggg gcagggttat tcctttgtgg 19680aaccagaggc acagagagag tcaactgagg ccaaaagagg cctgagagaa actgaggtca 19740agatttcagg attaatggtc ctgtgatgct ttgaagtaca attgtggatt tgtccaattc 19800tctttagttc tgtcagcttt tgcttcatat attttagcgc tctattatta gatatataca 19860tgtttagtat tatgtcttat tggtgcattt actctcttat cattatgtaa tgtccttctt 19920tatctgtgat aattttctgt gttctgaagt ctactttgtc taaaaataac atacgcactc 19980aacttccttt tctttcttcc ttcctttctt tcttccttcc tttctttctc tctctctctc 20040tttccttcct tccttcctcc ttttctttct ctctctctct ctctctcttt ttttgacaga 20100ctctcgttct gtggccctgg ctggagttca gtggtgtgat cttggctcac tgctacctct 20160accatgagca attctcctgc ctcagcctcc caagtagctg gaactacagg ctcatgccac 20220tgcgcccagc taatttttgt atttttcgta gagacggggt ttcaccacat tcgtcaggtt 20280ggtttcaaac tcctgacttt gtgatccacc cgcctcggcc tcccaaagtg ctgggattac 20340aggcatgagc catcacacct ggtcaacttt cttttgatta gtgtttttgt ggtatatctt 20400tttccatcat gttactttaa atatatctat attattgtat ttaaaatgtg tttcttacag 20460actgcatgta gttgggtata atttttatcc agtctaaaaa tatctgtctt ttaattggtg 20520tttagacaat ttatatttaa taaaattgtt gaatttaaga tggatgactg ttttatttgt 20580ttgctgttca ccacttctgt tttattctct ttccagaatt cttttggatt gtttaaatat 20640ttcataatat tttatcttaa tttatttatt gggtatttgc ctatatctct ttgtggtatt 20700ttttagtggt tgcttgaggg attacaatgt acttaacttt tcacagtgtg cataaagtta 20760atattttgcc acttgcagta aaccgtagaa ggcttataat catattagta cctctatcca 20820ctttctttta tgttgtagtt gtcatatata ttacatctat atacactgaa acattatagg 20880caatgttatg atttttgcat tcgtcagtca tatatatatt ttaaagaatt taagaggaga 20940aaaatacata ttcagatatt catcat 20966

Claims

1. A method of accelerating muscle cell differentiation, comprising contacting muscle cells with gOBG3 under conditions wherein said gOBG3 binds to said cells thereby accelerating differentiation of said cells.

2. The method of claim 1, wherein said cells are in an individual.

3. The method of claim 2, wherein said gOBG3 is present in a pharmaceutical composition.

4. The method of claim 3, wherein said gOBG3 is gApm1.

5. A method of accelerating muscle cell reorganization, comprising contacting muscle cells with, gOBG3 under conditions wherein said gOBG3 binds to said cells thereby accelerating reorganization of said cells.

6. The method of claim 5, wherein said cells are in an individual.

7. The method of claim 6, wherein said gOBG3 is present in a pharmaceutical composition.

8. The method of claim 7, wherein said gOBG3 is gApm1.

9. A method of increasing muscle mass in an individual, comprising contacting muscle cells in said individual with gOBG3 under conditions wherein said gOBG3 binds to said cells thereby accelerating the reorganization and differentiation of said cells and increasing said muscle mass.

10. The method of claim 9, wherein said gOBG3 is present in a pharmaceutical composition.

11. The method of claim 10, wherein said gOBG3 is gApm1.

12. A method of treating muscle cell disorders in an individual, comprising contacting muscle cells in said individual with gOBG3 under conditions wherein said gOBG3 binds to said cells thereby accelerating the reorganization and differentiation of said cells, and thereby treating said muscle cell disorders.

13. The method of claim 12, wherein said muscle cell disorders are selected from the group consisting of muscle-related eye disorders, muscle-related recovery after injuries, muscle-related recovery after surgery, muscle-related disorders of aging, muscle atrophy, muscle wasting, and muscular dystrophy.

14. The method of claim 13, wherein said gOBG3 is present in a pharmaceutical composition.

15. The method of claim 14, wherein said gOBG3 is gApm1.