MXPA00000611A - Adipocyte-specific protein homologs - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig37, a member of the family of proteins bearing a collagen-like domain and a globular domain. The polypeptides, and polynucleotides encoding them, are involved in dimerization or oligomerization and may be used in the study thereof. The present invention also includes antibodies to the zsig37 polypeptides.

Description

PROTEIN HOMOLOGUES SPECIFIC TO ADIPOCYTES BACKGROUND OF THE INVENTION The energy balance (comprising energy metabolism, nutritional status, lipid storage and the like) is an important criterion for health. This energy homeostasis includes the admission of element and metabolism of carbohydrates and lipids to generate the energy necessary for voluntary and involuntary functions. The metabolism of proteins can lead to the generation of energy, then preferentially leads to the formation or repair of muscle. Among other consequences, a lack of. energy homeostasis leads to an envelope or sub-formation of adipose tissue. The formation and storage of fat is modulated by insulin, for example, insulin stimulates the transport of glucose to the cells, where it is metabolized into a-glycerophosphate or used in the esterification of fatty acids to allow the storage of glucose. same as triglycerides. In addition, adipocytes (fat cells) express a transport protein REF. : 32526 specific that intensifies the transfer of free fatty acids in adipocytes. Adipocytes also secrete several proteins that are thought to modulate the hydrostatic control of glucose and lipid metabolism. These . Adipose-secreted proteins, additional, include adipsin, complement factors C3 and B, a tumor necrosis factor OI, and the gene product ob and Acrp30. There is also evidence that suggests the existence of a secretory route arranged by insulin in adipocytes. Scherer et al., J. Biol. Chem. 270 (45): 26746-9, 1995. The over- or under-secretion of these portions, impacted in part by the over- or under-formation of adipose tissue, can lead to pathological conditions directly or indirectly associated with obesity or anorexia. Acrp30 is a 247 amino acid polypeptide that is expressed exclusively by adipocytes. The Acrp30 polypeptide is composed of an amino-terminal signal sequence, a stretch of 27 amino acids of unknown homology; 22 repeats of perfect Gly-Xaa-Pro collagen or imperfect Gly-Xaa-Xaa collagen and a carboxy-terminal globular domain. See, Scherer et al., As described previously in International Patent Application No. W096 / 39429. Acrp30, an abundant human serum protein regulated by insulin, shares structural similarity, particularly in the carboxy-terminal globular domain, complement factor Clq and a summer serum protein of hibernating Siberian squirrels (Hib27). The expression of Acrp30 is induced by more than 100 times during the differentiation of adipocytes. Acrp30 is suggested for use in the modulation of energy balance and in the identification of adipocytes from test samples. Another secreted protein that appears to be produced exclusively in adipocytes is the apM1, described, for example, in Maeda et al., Biochem, Blophys, Res. Comm. 22: 286-.9, 1996. A 4517 bp clone has an open reading frame of 244 amino acids and a long 3 'untranslated region. The protein includes a signal sequence, a non-collagenous sequence, amino-tminal, 22 repeats of collagen (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminal region with homology to collagen X, collagen X , collagen VIII and complement protein Clq.

The complement factor Clq consists of six copies of three related polypeptides (A, B and C chains), with each polypeptide being approximately 225 amino acids long with a domain of nearby amino-terminal collagen and a carboxy-terminal globular region. If there are triple helical instructions are formed by the collagen domains of the six chains A, six B and six C, forming a central region and six stems. A globular head portion is formed by the association of the globular carboxy-terminal domain of an A chain, B and C. Therefore the Clq is composed of six globular heads linked via six stems type collagen to a central, febrile region. Sellar et al., Biochem. J. 274: 481-90, 1991. This configuration is often referred to as a bouquet of flowers. The Acrp30 has a similar corsage structure formed from an individual type of the polypeptide chain. The molecules capable of modulating energy homeostasis are sought for the study of this phenomenon and for the prevention or treatment of imbalances. Also, molecules capable of modulating the secretory pathways of adipocytes are also sought as indirect modulators of the energy homeostasis and as research reagents. The present invention provides these polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE INVENTION Within one aspect, the invention provides a desired polypeptide containing an amino acid residue sequence that is at least 75% identical in amino acid sequence to residues 26-281 and SEQ ID No. 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro that form a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion. Within one embodiment, the polypeptide is at least 90% identical in the amino acid sequences at residues 22-281 of SEQ ID No. 2. Within a related aspect, the polypeptide is at least 90% identical in sequence. amino acids at residues 22-281 of SEQ ID No. 2. Within another embodiment, the polypeptide comprises residues 1-281 of the SEQ ID No. 2 or SEQ ID No. 44. Within this embodiment, the polypeptide is covalently linked in an amino-terminal or carboxy-terminal manner to a portion selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores. Within a related embodiment, a proteolytic cleavage site is additionally provided between the amino acid residue sequence and the affinity tag. Within another aspect there is provided an isolated polypeptide selected from the group consisting of: a) a polypeptide having an amino acid residue sequence that is 75% identical in amino acid sequence to 99 amino acid residue to amino acid residue 140 of SEQ ID No. 2; b) a polypeptide having an amino acid residue sequence that is 75% identical from an amino acid sequence to amino acid residue 140 and 141 to amino acid residue 281 in SEQ ID No. 2; and c) a polypeptide having an amino acid residue sequence that is 75% identical in sequence from the amino acid to amino acid residue 99 to 281 of SEQ ID No. 2.

Within another aspect, a fusion protein consisting essentially of a first portion and a second portion of a unit is provided by a peptide bond, the first portion comprising polypeptides originated from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in sequence from amino acids to amino acid residue 26 to amino acid residue 281 of SEQ ID No. 2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID No. 2 from amino acid residue 1, 22 or 26 to amino acid residue 281; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID No. 44 from amino acid residue 1, 22 or 26 to amino acid residue 281; d) a portion of zsig37 polypeptide as shown in SEQ ID No. 2 or SEQ ID No. 44, which contains the collagen type domain or a portion of the collagen type domain capable of dimerization or oligomerization; e) a portion of zsig37 polypeptide as shown in SEQ ID No. 2 or SEQ ID No. 44 which contains the globular type domain or an active portion of the globular type domain; or e) a Zsig37 polypeptide portion as shown in SEQ ID No. 2 or SEQ ID No. 44 which includes the collagen type domain and the globular domain; and the second portion comprising another polypeptide. Within one embodiment, the first portion is selected from the group consisting of: a) a polypeptide having the sequence from amino acid residue 99 to amino acid residue 140 of SEQ ID No. 2 or SEQ ID No. 44; b) a polypeptide having the sequence of amino acid residue 140 and 141 to amino acid residue 181 of SEQ ID No. 2 or SEQ ID No. 44; c) a polypeptide having the sequence and amino acid residue 99 to 281 of SEQ ID No. 2 or SEQ ID No. 44. Within another aspect there is provided a fusion protein comprising a secretory signal sequence having the amino acid sequence and amino acid residues 1-21 or 1-25 of SEQ ID No. 2 or SEQ ID No. 44, wherein the secretory signal sequence is operably linked to an additional polypeptide. Within a further aspect, there is provided an expression vector comprising the following operably linked elements: a transcription promoter, a DNA segment that encodes a polypeptide comprising a sequence of amino acid residues that is at least 75% in the amino acid sequence at residues 26-281 of SEQ ID No. 2, wherein the sequence comprises: Gly-Xaa-Xaa repeats or Gly-Xaa-Pro which forms a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and a terminator in transcription. Within one embodiment, the DNA segment codes for a polypeptide that is at least 90% identical in the amino acid sequence to residues 26-281 of SEQ ID No. 2. Within another embodiment, the DNA segment encodes for a polypeptide that is at least 90% identical from the amino acid sequence to residues 22-281 of SEQ ID No. 2. Within another embodiment, the DNA segment encodes a polypeptide comprising residues 1-281 of SEQ. ID No. 2 or SEQ ID No. 44. Within yet another embodiment, the DNA segment codes for a polypeptide covalently linked in an amino-terminal or carboxy-terminal manner to an affinity tag. Within a further embodiment, the DNA segment additionally encodes a secretory signal sequence operably linked to the polypeptide. Within yet another modality, the The secretory signal sequence comprises residues 1-21 or 1-25 of SEQ ID No. 2 or SEQ ID No. 4 4. Within another aspect, there is provided a cultured cell into which an expression vector has been introduced which comprises the following operably linked elements: a transcription promoter, or a segment of DNA encoding a polypeptide comprising a sequence of residues of amino acid that is at least 75% identical to the amino acid sequence at residues 26-281 of SEQ ID No. 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro that form a domain of collagen, wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and a transcription terminator, wherein the cell expresses the polypeptide encoded by the DNA segment. Within yet another aspect there is provided a method for producing a polypeptide comprising: culturing a cell in which an expression vector comprising the following operably linked elements has been introduced. A transcription promoter; a segment of DNA encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in the amino acid sequence to residues 26-281 of SEQ ID No. 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro that form a collagen domain, wherein Xaa is any amino acid, and a carboxy-terminal globular portion; and a transcription terminator; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide. Within another aspect, there is provided a pharmaceutical composition comprising a polypeptide, the polypeptide comprising a sequence of amino acid residues that is at least 75% identical in the amino acid sequence to residues 26-281 of SEQ ID No. 2 , wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro that form a collagen domain, wherein Xaa is any amino acid; a globular, carboxy-terminal portion; in combination with a pharmaceutically acceptable vehicle. Within yet another aspect there is provided an antibody that specifically binds to an epitope of a polypeptide comprising an amino acid residue sequence that is at least 75% identical in amino acid sequence to the residues 26-281 of SEQ ID No. 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro which forms a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion. Within another aspect there is provided a binding protein that specifically binds to an epitope of a polypeptide comprising a sequence of amino acid residues that is at least 75% in the amino acid sequence to residues 26-281 of SEQ ID No .2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro that form collagen domains, wherein Xaa is any amino acid; and a carboxy-terminal globular portion. Within another aspect there is provided an isolated polynucleotide encoding a polypeptide comprising an amino acid residue sequence that is at least 75% identical in the amino acid sequence at residues 26-281 of SEQ ID No. 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro which forms a collagen domain, wherein Xaa is any amino acid, and a carboxy-terminal globular portion. Within one embodiment, the polypeptide is at least 90% identical in the amino acid sequence to residues 26-281 of SEQ ID No. 2. Within another embodiment, the polypeptide is at least 90% in the amino acid sequence at residues 22-281 of SEQ ID No. 2. Within another embodiment, the polypeptide comprises residues 1-281 of SEQ ID No. 2 or SEQ ID No. 44. Within another embodiment, the polynucleotide is DNA. Within another aspect, there is provided an isolated polynucleotide selected from the group consisting of, a) a nucleotide sequence from nucleotide 465 to nucleotide 688 of SEQ ID No. 1; b) a nucleotide sequence from nucleotide 688 to nucleotide 1016 of SEQ ID No. 1; c) a nucleotide sequence from nucleotide 691 to nucleotide 1016 of SEQ ID No. 1; d) a nucleotide sequence from nucleotide 465 to nucleotides 1061 of SEQ ID No. 1; e) a nucleotide sequence from nucleotide 364 to nucleotide 490 of SEQ ID No. 43; f) a nucleotide sequence from nucleotide 490 to nucleotide 912 of SEQ ID No. 43; g) a nucleotide sequence from nucleotide 364 to nucleotide 912 of SEQ ID No. 43; h) a sequence of nucleotides from nucleotide 364 to nucleotide 490 of SEQ ID No. 43; i) a polypeptide encoding a polypeptide having an amino acid residue sequence that is at least 75% identical in sequence from amino acids to amino acid residue 99, 140 or 141 to amino acid residue 281 of SEQ ID No. 2; j) a polynucleotide that encodes a polypeptide having an amino acid residue sequence that is at least 75% identical in amino acid sequence to amino acid residue 99 to amino acid residue 140 of SEQ ID No. 2; k) sequence of complementary nucleotides aa), b), c), d), e), f), g), h), i) oj) and 1) degenerate nucleotide sequences of 'a), b), c) ), d), e), f), g), h), i), j) ok). Within another aspect there is provided an isolated polynucleotide encoding a fusion protein consisting essentially of a first portion and a second portion bound by a binding peptide, the first portion being selected from the group consisting of: a) a polypeptide comprising an amino acid residue sequence that is at least 75% identical in amino acid sequence to amino acid residue 26 to amino acid residue 281 of SEQ ID No. 2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID No. 2 from amino acid residue 1, 22 or 26 to amino acid residue 281; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID No. 44 from amino acid residue 1, 22 or 26 to amino acid residue 281; d) a portion of the zsig37 polypeptide as shown in SEQ ID No. 2 or SEQ ID No. 44, which contains the collagen type domain or a portion of the collagen type domain capable of dimerization or oligomerization; e) a portion of zsig37 polypeptide as shown in SEQ ID No. 2 or SEQ ID No. 44 containing the globular type domain or an active portion of the globular type domain; or f) a portion of the zsig37 polypeptide as shown in SEQ ID No. 2 or SEQ ID No. 44 which includes a collagen type domain and the globular domain; the second portion comprising another polypeptide. Within yet another aspect is provided an isolated polypeptide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-21 or 1-25 of SEQ ID No. 2 or SEQ ID No. 44, where the The secretory signal sequence is operably linked to an additional polypeptide. Within yet another aspect there is provided an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID No. 23 or a sequence complementary to SEQ ID No. 23.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the multiple alignment of the zsig37 polypeptide of the present invention and HUMUPST2 1 (Maeda et al., Biochem. Biophys. Res. Comm. 221 (2): 286-9, 1996); CIQA_HUMAN (Sellar et al., Biochem. J. 274: 481-90, 1991, Reid, Biochem. J. 179: 367-71, 1979, and Reid et al., Biochem. J. 203: 559-69, 1982 ); HP25_TAMAS (Takamatsu et al., Mol Cell. Biol. 13: 1516-21, 1993 and Kondo &Kondo, J. Biol. Chem. 267: 473-8, 1992); HP27_TAMAS (Takamatsu et al., And Kondo &Kondo, referenced above); and CERL_RAT (ada &Ohtani, Brain Res. Mol. Brain Res. 9: 71-7, 1991).

Figure 2 is a matrix illustrating the percent identity of amino acids in a comparison of the six proteins shown in the multiple alignment of Figure 1.

DETAILED DESCRIPTION OF THE INVENTION Before exposing the invention in detail, it will be useful for the understanding of it to define the following terms. The term "affinity tag" is used herein to denote a peptide segment that can bind to a polypeptide to provide for purification or detection of the polypeptide or to provide sites for binding of the polypeptide to a substrate. In principle, any peptide or protein for which an antibody or other specific agent is available can be used as an affinity tag. Affinity tags include a poly-styrein, protein A (Hilsson et al., EMBO J. 4_: 1075, 1985, Nilsson et al., Me th ods Enzymol 198: 3, 1991), glutathione-S- treatment. transferase (Smith and Johnson, Gen e 67_: 31, 1988), substance P, FlagMR peptide (Hopp et al., Biotech ch ol olgy 6_: 1204-1210, 1988; available from Eastman Kodak Co., Nes Haven, CT. ), streptavidin-bound peptide, or other epitope or antigen binding domain. To see in General Ford et al., Protein Expressi on and Purifi cation 2_: 95-104, 1991. DNAs encoding affinity tags are available from potential suppliers (eg, Pharmacia Biotech, Piscataway, NJ). The term "allelic variant" denotes any of two or more alternative forms of a gene occupying the same chromosomal site. Allelic variation arises naturally through mutation, and can result in phenotypic polymorphism within populations. Gene mutations may be absent (no change in the encoded polypeptide) or may encode polypeptides having an altered amino acid sequence. The term "allelic variant" is also used herein to denote a protein encoded by an allelic variant of a gene. The terms "amino-terminal" and "carboxy-terminal" are used herein to denote positions within polypeptides and proteins. Where the context permits, this text is used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence placed in a carboxy-terminal form to a reference sequence within a protein is located close to the carboxyl terminus in the reference sequence, but not necessarily at the carboxyl terminus of the entire protein. The term "complement pair / anticomplement" denotes non-identical portions that form a stable pair, associated non-covalently under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical members of a complement / anti-complement pair. Other exemplary complement / anticomplement pairs include pairs of receptor / ligand, pairs of antibody / antigen (or hapten or epitope) pairs, of homosense / antisense polynucleotides, and the like. Where subsequent dissociation of the complement / anti-complement pair is desirable, the complement / anti-complement pair preferably has a binding affinity < 109 M "1. The term" complement of a polynucleotide molecule "is a polynucleotide molecule having a complementary base sequence and an inverted orientation as compared to a reference sequence, eg, the sequence 5'-ATGCACGGG-3 ' is complementary to 5 'CCCGTGCAT- 3'.

The term "contig" denotes a polynucleotide having a contiguous stretch of an identical sequence complementary to another polynucleotide. The contiguous sequences are said to "overlap" a given stretch of the polynucleotide sequence either in its entirety or along a partial stretch of the polynucleotide. For example, contigs representative of the polynucleotide sequence 5 '-ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3 'and 3' -gt cgacTACCGA-5 '. The term "degenerate nucleotide sequence" denotes a nucleotide sequence that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different user triplets, but they code for the same amino acid residue (ie, GAU triplets and GAC encode each for Asp). The term "expression vector" denotes a DNA molecule, linear or circular, comprising a segment encoding a polypeptide of interest operably linked to the additional segments that provide its transcription. These additional segments can include sequences promoters and terminators, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide is removed from its natural genetic environment and thus is free from other foreign or unwanted coding sequences, and is in a form suitable for use within Protein production systems, genetically engineered. Isolated molecules are those that are separated from their natural environment and include genomic and cDNA clones. The DNA molecules isolated in the present invention are free of other genes with which they are associated in an ordinary manner, but may include naturally occurring 5 'and 3' untranslated clones such as promoters and terminators. The identification of the associated regions will be apparent to one skilled in the art (see, for example, Dynan and Tijan, Nature 316: 774-78, 1985).

An "isolated" polypeptide or protein is a protein polypeptide that is in a condition different from its natural environment, such as separated from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, ie, greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms. The term "operably linked", when referring to DNA segments, denotes that the segments are arranged so that they function in accordance with their intended purposes, for example, start the transcription in the promoter and proceed through the coding segment to the terminator The term "ortholog" denotes a polypeptide or protein obtained from a species that is the functional counterpart of a polypeptide or protein of a different species. The differences of the sequences between the orthologs are the result of the evolution of the species. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are thought to arise between duplication of genes. For example, α-globin, β-globin and myoglobins are paralogs with each other. The term "polynucleotide" denotes a single or double stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'to the 3' end. The polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The sizes of the polynucleotides are expressed as base p (abbreviated "pb"), nucleotides ("nt"), or kilobases ("kb"). When the context permits, the latter two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied the double-stranded molecule is used to denote the full length and it will be understood that it is equivalent to the term "base p". HE it will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; in this way all nucleotides within a double-stranded polynucleotide molecule can not be pd. These unpd ends will generally not exceed 20 nt in length. A "gone polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptide". The "probes and / or primers" as used herein may be RNA or DNA. The DNA can be either cDNA or genomic DNA. The polynucleotide probes and primers are single or double stranded DNA or RNA, generally synthetic oligonucleotides, but can be generated from cloned cDNA or genomic sequence or its complements. Gravitational probes will generally be at least 20 nucleotides in length, although some shorter probes (14-17 nucleotides) may be used. The PCR primers are at least 5 nucleotides length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is searched for analysis. For the normal analysis of the genes, a polynucleotide probe can comprise a complete exon or more. The probe can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescent, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene , OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art. The term "promoter" denotes a portion of a gene that contains DNA sequences that provide for the binding of the RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'regions of non-coding genes. The term "receptor" denotes an associated protein in the cell that binds to a bioactive molecule (i.e., a ligand) and measures the effect of the ligand on the cell. The receptors attached to the Membranes are characterized by a multi-domain structure comprising a ligand, extracellular binding domain and an intracellular effector domain that is typically comprised in signal transduction. The binding of ligand to the receptor results in a conformational change in the receptor that causes an interaction between the effector domain and another (s) molecule (s) in the cells. This interaction in turn leads to an alteration in the metabolism of the cells. Metabolic segments that bind to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increase in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. Most nuclear receptors also exhibit a multi-domain structure, including an amino-terminal transactivation domain, a DNA binding domain and a ligand-binding domain. In general, the results can be linked to the membrane, be cytotoxic or nuclear; monomeric (eg, thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor, and IL-6 receptor). The term "secretory signal sequence" denotes a DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, of larger polypeptide origin via a secretory pathway of a cell in a which is synthesized. The major peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. A "soluble receptor" is a receptor polypeptide that does not bind to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides, which lack the transmembrane and cytoplasmic domains. Soluble receptors may comprise additional amino acid residues, such as affinity tags that provide for the purification of the polypeptide or provide sites for the attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell surface receptors have soluble counterparts that are naturally occurring that are produced by proteolysis or produced from the alternatively spliced mRNAs the receptor polypeptides are to be substantially of the transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide the membrane anchor or signal transduction, respectively. The term "splice variant" is used herein to denote alternative forms of the transcribed RNA of a gene. Splice variations arise naturally through the use of alternative splice sites within a transcribed RNA molecule, or less commonly between RNA molecules transcribed separately, which can result in several RNAs transcribed from the same gene. The splice variants can encode polypeptides having an altered amino acid sequence. The term "splice variant" is also used herein to denote the protein encoded by a splicing variant of a mRNA transcribed from a gene. The molecular weights and lengths of the polymers determined by the analytical methods imprecise (for example, gel electrophoresis) will be understood to be approximate values. When this value is expressed as "approximately" X or "approximately" X, the designated value of X will be understood to be accurate to ± 10%. All references cited herein are incorporated herein by reference in their entirety. The present invention is based in part on the discovery of a new DNA sequence encoding a polypeptide having homology to a related adipocyte complement protein (Acrp30). See, for example, Scherer et al., J. Biol. Chem. 270 (45): 26746-9, 1995. Acrp30 polypeptide is shown in SEQ ID No. 3. Acrp30 appears to be highly related to apMl (HUMUPST2_1 in Figures 1 and 2), with the most significant differences observed in the secretory sequence. The new DNA sequence encodes a polypeptide having an amino-terminal signal sequence, an adjacent n-terminal region of non-homology, a truncated collagen domain composed of repeats of Gly-Xaa-Xaa or Gly-Xaa-Pro and a carboxy-terminal globular portion The new The polynucleotide sequence also contains a 3 'untranslated, long region. The structure of the general polypeptide discussed above is shared by Acrp30 and HUMUPST2_1, except that the collagen-like domain of each of its proteins is longer than that of the zsig37 polypeptides. Also, the DNA sequence of HUMUPST2_1 is characterized by a long 3 'untranslated region. In addition, Acrp30 and all sequences aligned in Figure 1, with the exception of CERL.-RAT, share a cysteine residue conserved at position 187 of the zsig37 polypeptide as shown in Figure 1 and SEQ ID No. 2 Other regions of homology, found in the carboxy-terminal globular portion in the aligned proteins, are identified herein as 'useful primers for the search of other family members. The Acrp30, for example, will be identified in a search using the primers. Also, the zsig37 polypeptides of the present invention include an N-linked, putative glycosylation site of amino acid 93 (Asn) of SEQ ID No. 2. Analysis of the tissue distribution of the mRNA corresponding to this new DNA using a The 30-base pathway (SEQ ID No. 4) showed that the expression was higher in the heart of the placenta, with relatively less intense signals, in the kidney, ovary, adrenal gland and skeletal muscle in minor signals in a wide variety of other tissues present in the Northern blot. The polypeptide has been designed zsig37 polypeptide. The new zsig37 proteins of the present invention were initially identified by querying an EST database for secretory signal sequences, characterized by a methionine start site in the .5 'direction, a hydrophobic region of approximately 13 amino acids and a incision, in an effort to select the secreted proteins. The polypeptides corresponding to the ESTs satisfying these search criteria were compared to the known sequences to identify the secreted proteins having homology to the known ligands. An individual EST sequence was discovered and predicted to be a segregated protein. The new polypeptide encoded by the full-length cDNA allows the identification of a homologous relationship with the related Acrp30 protein, adipocyte complement (SEQ ID No. 3) and the segregated adipocyte protein apMl (HUMPST2_1 in Figures 1 and 2). Some more distant homology was also identified to the C1Q A component, complement chain, two factors observed in the active state of the Siberian marmots in hibernation (HP25_TAMAS and HP27_TAMAS) and a rat brain protein (CERL: RAT), as shows in Figures 1 and. The complete sequence of the zsig37 polypeptide was obtained from a single clone believed to contain it, wherein the clone was obtained from a library of tumor tissue in 'brain. Other libraries that may also be searched for these clones include brain tissue, placenta, kidney, ovary, adrenal gland, skeletal muscle, and adipose tissue and the like. The nucleotide sequence of the N-terminal EST is described in SEQ ID No. 1, and its reduced amino acid sequence is described in SEQ ID No. 2. As generally described above, the zsig37 polypeptide includes a signal sequence, which varies from amino acid 1 (Met) to amino acid residue 21 (Gly). An alternative signal sequence varies from amino acid 1 (Met) to amino acid 25 (Ser). The mature polypeptide therefore varies from amino acid 22 (Leu) or 26 (Arg) to amino acid 281 (Pro). Within the mature polypeptide, an N-terminal region of unknown homology is found, varying between amino acid residue 22 (Leu) and 98 (Lys). In addition, a truncated collagen domain is found between amino acid 99 (Gly) and 140 (Arg). In the domain of truncated collagen, a perfect repetition of Gly-Xaa-Pro and 13 imperfect of Gly-Xaa-Xaa are observed. In contrast, the Acrp30 contains 22 perfect or imperfect repeats. The zsig37 polypeptide also includes a carboxy-terminal globular domain, ranging from about amino acid 151 (Cys) to 281 (Pro). The polypeptide Zsig37, HUMUPST2_1 and Acrp30 appear to be homologues within the collagen domain and in the globular domain, but not in the N-terminal portion of the mature polypeptide. Another aspect of the present invention includes zsig37 polypeptide fragments. Preferred fragments include the collagen type domain of zsig37 polypeptides, ranging from amino acid 99 (Gly) to amino acid 140 (Arg) of SEQ ID No. 2, a portion of zsig37 polypeptide which contains the collagen type domain or a portion of the collagen type domain capable of dimerization or oligomerization. These fragments are particularly useful in the study of the dimerization or oligomerization of collagen or in the formation of fusion proteins as described more fully below. Polynucleotides encoding this fragment are also encompassed by the present invention, including the group consisting of (a) polynucleotide molecules comprising a sequence of the nucleotides as shown in SEQ ID No. 1 from nucleotide 1, 171, 234, 246 or 465 to nucleotide 589; (b) polynucleotide molecules that encode a zsig37 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID No. 2 from amino acid residue 99 (Gly) to amino acid residue 140 (Arg); (c) molecules complementary to (a) or (b); and (d) degenerate nucleotide sequences encoding a fragment of the collagen type domain of the z s Ig37 polypeptide. Other preferred fragments include the globular domain of zsig37 polypeptides, ranging from amino acid 140 (Arg) or 141 (Cys) to 281 (Pro) of SEQ ID No. 2, a portion of the zsig37 polypeptide that contains the globular type domain or an active portion of the globular type domain. Its fragments are particularly useful in the study or modulation of energy balance or neurotransmission, particularly neurotransmission related to diet or effort. Anti-microbial activity may also be present in these fragments. Polynucleotides encoding these fragments are also encompassed by the present invention, including the group consisting of (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID No. 1 from a nucleotide 587 or 590 to the nucleotide 1016; (b) polynucleotide molecules that encode a zsig37 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID No. 2 from amino acid residue 141 (Gly) to amino acid residue 281 (Pro); (c) molecules complementary to (a) or (b); and (d) degenerate nucleotide sequences encoding a fragment) the globular domain of the zsig37 polypeptide.

Another fragment of the zsig37 polypeptide of the present invention includes both the collagen type domain and the globular domain ranging from amino acid residue 99 (Gly) to 281 (Pro) of SEQ ID No. 2. The polynucleotides encoding these fragments they are also encompassed by the present invention, including the group consisting of (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID No. 1 from nucleotide 465 to nucleotide 1016; (b) polynucleotide molecules encoding a zsig37 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID No. 2 from amino acid residue 99 (Gly) to amino acid residue 281 (Pro); (c) molecules complementary to (a) or (b); and (f) degenerate nucleotide sequences encoding a fragment of the collagen-globular domain domain of the zsig37 polypeptide. The present invention also contemplates degenerate probes based on the polynucleotides described above. The probes corresponding to the complements of the polynucleotides discussed above are also encompassed.

The present invention also provides polynucleotide molecules, including DNA or RNA molecules, that encode the zsig37 polypeptides described herein. Those skilled in the art will readily recognize that, in view of degeneracy in the genetic code, considerable sequence variation between these polynucleotide molecules is possible. SEQ ID No. 23 is a degenerate DNA sequence encompassing all of the DNAs encoding the zsig37 polypeptide of SEQ ID No. 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID No. 23 also provides all the RNA sequences coding for SEQ ID No. 2 by substituting U (uracil) for T (thymine). Thus, polynucleotides encoding the zsig37 polypeptide comprising nucleotide 1 to nucleotide 842 of SEQ ID No. 23 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth one-letter codes used within SEQ ID No. 23 to denote the positions of the degenerate nucleotides. "Resolutions" are the nucleotides denoted by a code letter. "Complement" indicates the code for the complementary nucleotide (s). For example, the code Y denotes either C (cytosine) or T, and its complement R denotes A (adenine) or G (Guanine), A which is complementary to T, and G which is complementary to C.

Table 1 Nucleotide Resolution Complement Resolution A A T T C C G G G G C C T T A A RA | GY CT Y c | t R AG MA | CK GT KG | TM AC SC | GS CG WA | TW AT HA | C | TD AGT BC | G | TV ACG VA | C | GB CGT DA | G | TH ACT NA | C | G | TN ACGT The degenerate codons used in SEQ ID NO: 23 envelop all possible codons for a given amino acid, are set forth in Table 2.

Table 2 Amino Acid Codon Code Codon a Degenerate letter Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT SN Thr T ACÁ ACC ACG ACT CAN Pro P CCA CCC CCG CCT CCN Wing A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG He I ATA OATC ATT ATH Leu L CTA ATC ATG ATT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr and TAC TAT TAY Trp W TGG TGG Ter • TAA TAG TGA TRR Asn 1 sp B RAY GlulGln Z SAR Any X NNN One skilled in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of all possible codons coding for each amino acid. For example, the degenerate codon for serine (SN) can encode, in some circumstances, for arginine (AGR), the degenerate arginine pair codon (MGN) can, in some circumstances, code for serine (AGY). A similar relationship exists between the codons that code for phenylalanine and leucine. In this way, some polynucleotides encompassed by the degenerate sequence can code for variant amino acid sequences, but one skilled in the art can easily identify these variant sequences by reference to the amino acid sequence of SEQ ID No. 2. The variant sequences can easily be tested for the functionality as described herein. One skilled in the art will appreciate that different species may exhibit "preferential use of the codon". In general, see, Grantham, et al., Nuc. Acids Res. 8_: 1893-912, 1980; Hass, et al. Curr. Biol. 6_: 315-24, 1996; Ain-Hobson, et al., Gene 13: 355-64; 1981; Grosjean and Fiers, Gene 18: 199- 209, 1982; Hol, Nuc. Acids Res. U.-3075-87, 1986; Ike ura, J. Mol. Biol. 158: 573-97, 1982. As used herein, the term "preferential codon usage" or "preferential codons" is a term of the art that refers to the most commonly used protein transmission codons. often in cells of a certain species, thus favoring one or a few representatives of the possible codons coding for each amino acid (see Table 2). For example, the amino acid threonine (Thr) can be encoded by AC, ACC, ACG, or ACT, but in mammalian cells, ACC is the most commonly used codon; in other species, for example, insect cells, and yeast, virus or bacteria, different Thr codons may be preferential. Preferential codons for particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of the sequences of the preferential codons in the recombinant DNA for example can enhance the production of the protein by making the translation of the protein more efficient within a particular type or species of cell. Therefore, the The sequence of the degenerate codon described in SEQ ID No. 23 serves as a template to optimize the expression of the polynucleotides in various cell types and species commonly used in the art and described herein. Sequences containing preferential codons can be tested and optimized for expression in several species, and tested for functionality as described herein. The fragments of Zsig37 can be evaluated with respect to their anti-microbial properties according to the procedures known in the art. See, for example, Barsum et al., Eur. Breathe J. 8 (5): 709-14, 1995; Sandovs ky-Losica et al., J. Med. See. Mycol (England) 28 (4): 279-87, 1990; Mehentee et al., J. Gen. Microbiol (England) 135 (PT 8): 2181-8, 1989; Segal and Savage, Journal of Medical and Veterinary Mycology 24: 477-479, 1986 and the like. If desired, the performance of the zsig37 polypeptide fragment in this regard can be compared to known proteins that are functional in this regard, such as proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like. In addition, fragments of the zsig37 polypeptide can be evaluated in combination with one or more antimicrobial agents to identify the synergistic effects. One skilled in the art will recognize that the anti-microbial properties of the zsig37 peptides, fusion proteins, agonists, antagonists and antibodies can be assessed in a similar manner. As neurotransmitters or modulators of neurotransmission, the zsig37 polypeptide fragments as well as the zsig37 polypeptides, fusion proteins, agonists, antagonists or antibodies of the present invention can also modulate calcium ion concentration, muscle concentration, hormone secretion, DNA synthesis or cell growth, production of inositol-phosphate, release of arachidonate, activation of phospholipase-C, gastric emptying, activation of human neutrophils or ADCC capacity, production of superoxide anions and the like. The evaluation of these properties can be carried out by known methods, such as those skilled in the present. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist or intracellular calcium level can be assessed by methods known in the art, such as that described by Regulatory Peptides 45: 341-52, 1993, and the like. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist on muscle concentration can be assessed by methods known in the art, such as those described by Smits & Lebebvre, J. Auton. Pharmacol. 1: 383-92, 1994, Belloli et al., J. Vet. Pharmacol. Therap. 17: 379-83, 1994, Maggi et al., Regulatory Peptides 53: 259-74, 1994, and the like. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist or secretion of hormones can be assessed by methods known in the art, such as those for the release of prolactin described by Henriksen et al., J. Of Receptor &; Signal Transduction Research 15 (1-): 529-41, 1995, and the like. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist on DNA synthesis or cell growth can be assessed by methods known in the art, such as those described by Dobrzanskí et al., Regulatory Peptides 45: 341-52, 1993 , and similar. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist in the production of inositol-phosphate can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52, 193, and similar.

Also, the impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist or arachidonate release can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and Similar. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist on phospholipase-C activation can be assessed by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and similar. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist in gastric emptying can be assessed, by methods known in the art, such as those described by Varga et al., Eur. J. Pharmacol. 286: 109-112, 1995, and the like. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist on the activation of human neutrophils and ADCC capacity can be assessed by methods known in the art, such as those described by Wozniak et al., Immunology 78: 629-34. , 1993, and the like. The impact of the zsig37 polypeptide, fragment, fusion, agonist or antagonist in the production of superoxide anion can be assessed in methods known in the art, such as those described by Wozniak et al., Immunology 78: 629-34, 1993, and the like. The present invention also provides zsig37 fusion proteins. For example, the fusion proteins of the present invention encompass (1) a polypeptide selected from the group comprising: (a) polypeptide molecules comprising a sequence of amino acid residues as shown in-SEQ ID No. 2 from of amino acid residue 1 (Met), 22 (Leu) or 26 (Arg) of amino acid residue 281 (Pro); (b) polypeptide molecules ranging from amino acid 99 (Gly) to amino acid 140 (Arg) of SEQ ID No. 2, a portion of zsig37 polypeptide also containing the collagen type or a portion of the collagen type domain capable of dimerization or oligomerization; (c) polypeptide molecules ranging from amino acid 140 (Arg) or 141 (Cys) to 281 (Pro) of SEQ ID No. 2, a portion of the zsig37 polypeptide that contains the globular type domain or an active portion of the domain globular type; or (d) polypeptide molecules ranging from amino acid 99 (Gly) to 281 (Pro), a portion of zsig37 polypeptide that includes the collagen type domain and the globular domain; and (2) another polypeptide. The other polypeptide can be an alternative or additional globular domain, a collagen-like domain alternative or additional, a signal peptide to facilitate the acceptance of the fusion protein and the like. The globular domain of the complement binding IgG, thus, the globular domain of the zsig37 polypeptide, fragment or fusion may have a similar role. The zsig37 polypeptides, which vary from amino acids (Met) to amino acid 281 ° (Pro); the mature, alternative zsig37 polypeptides ranging from amino acid 22 (Leu) or amino acid 26 (Arg) to amino acid 281 (Pro); or alternative secretory guide fragments thereof, fragments ranging from amino acid 1 (Met) to amino acid 21 (gly) or amino acid 25 (Ser) can be used in the study of protein secretion from cells. In preferred embodiments of this aspect of the present invention, mature polypeptides are formed as fusion proteins with secretory, putative signal sequences; plasmids having regulatory regions capable of directing the expression of the fusion protein are introduced into the test cells; and the secretion of the mature protein is monitored. In other preferred embodiments of this aspect of the present invention, alternative secretion guide fragments are formed as fusion proteins with the alternative proteins selected for secretion; the plasmids that have regulatory regions capable of directing the expression of the fusion protein are introduced into the test cells; and the secretion of the protein is monitored. Monitoring can be done by techniques known in the art, such as HPLC and the like. Highly conserved acids, particularly those in the carboxy-terminal globular domain of the zsig37 polypeptide, can be used as a tool to identify both members of the family. For example, the inverted poly-eraser-reverse transcript (RT-PCR) chain reaction can be used to amplify the sequences encoding the conserved functions of the RNA obtained from a variety of tissue exponents. In particular, highly degenerate primers designed from conserved sequences are useful for this purpose.

In particular, the following primers are useful for this purpose. 1) Amino acids 269-274 of SEQ ID No. 2 (corresponding to nucleotides 975-992 of SEQ ID No. 1); 2) Amino acids 191-196 of SEQ ID No. 2 (corresponding to nucleotides 741-758 of SEQ ID No. 1); 3) Amino Acids 163-168 of SEQ ID No. 2 (corresponding to nucleotides 657-674 of SEQ ID No. 1); 4) Amino acids 173-178 of SEQ ID No. 2 (corresponding to nucleotides 687-704 of SEQ ID No. 1); and 5) Amino Acids 243-248 of SEQ ID No. 2 (corresponding to nucleotides 897-914 of SEQ ID No. 1). The present invention also contemplates degenerate probes based on the polynucleotides described above. The probes corresponding to the complements of the polynucleotides discussed above are also contemplated. Within the preferred embodiments of the invention, the isolated polynucleotides will hybridize to cell size regions of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20 or a sequence complementary thereto, under severe conditions. In general, severe conditions are selected to be at about 5 ° C below the thermal melting point (Tm) for the specific sequence at a defined ionic concentration and pH. The Tm is the temperature (under defined ionic concentration and pH) at which 50% of the objective sequence hybridizes to a probe that perfectly matches. Typical conditions of severity are those in which the salt concentration of up to about 0.03 M at pH 7 and the temperature is at least about 60 ° C. Within another aspect of the present invention, there is provided a pharmaceutical composition comprising the purified zsgi37 polypeptide in combination with a pharmaceutically acceptable carrier. This pharmaceutical composition will be used to modulate the energy balance in mammals and to protect endothelial cells from damage. With respect to the modulation of the energy balance, the zsig37 polypeptides modulate cellular metabolic reactions. These metabolic reactions include adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose admission, protein synthesis, thermogenesis, oxygen utilization and the like. The expression pattern of zsig37 polypeptide indicates the expression in tissues of endothelial cells. With respect to the production of endothelial cells, the zsig37 polypeptide can be used in the concentration of organs, for cryopreservation, for surgical pre-treatment to prevent injury due to ischemia and / or inflammation or similar procedures. Expression of the Zsig37 polypeptide in the heart suggests that the protein can modulate the release of acetylcholine and / or norepinephrine. The polypeptides may also find use as neurotransmitters and as modulators of neurotransmission, as indicated by the expression of the polypeptide in tissues associated with the sympathetic or para-sympathetic nervous system. In this regard, zsig37 polypeptides can find utility in the modulation of nutrient admission, as demonstrated, for example, by the admission of 2-deoxy glucose in the brain and the like. Among other methods known in the art or described herein, the energy balance of mammals can be assessed by monitoring one or more of the following metabolic functions: adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, use of oxygen or similar. These functions Metabolics are monitored by techniques (tests or animal models) known to a person skilled in the art, as will be more fully explained later. For example, the glucurregulatory effects of insulin are predominantly exerted in the liver, skeletal muscle and adipose tissue. Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that result, for example, in the inhibition of glucose production and the stimulation of glucose utilization. In the liver, insulin stimulates the admission of glucose and inhibits glycogenesis and glycogenolysis. In the skeletal muscle adipose tissue, insulin acts to stimulate the admission, storage and utilization of glucose. The methods recognized in the art exist to monitor all the metabolic functions cited above. In this way, one skilled in the art is able to evaluate the zsig37 polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists for the functions of metabolic modulation. The example modulation techniques are discussed below.

Adipogenesis, gluconeogenesis and glycogenolysis are interrelated components of the energy balance of mammals, which can be evaluated by known techniques using, for example, ob / ob mice or db / db mice. Ob / ob mice are crossed mice that are homozygous for a mutation in ob-site inactivation (obese). These ob / ob mice are hyperphagic and hypometabolic, and are believed to be efficient in the production of the ob protein in circulation. The db / db mice are crossed mice that are homozygous for a mutation in inactivation in the db site (diabetes). The db / db mice exhibit a phenotype similar to that of the db / db mice, except that the ob / ob mice, except that the deb / db mice and also exhibit a diabetic phenotype. These db / db mice are believed to be resistant to the effects of the ob protein in circulation. Also, several in vitro methods of evaluating these parameters are known in the art. Insulin-stimulated lipogenesis, for example, can be monitored by measuring the incorporation of 14C-acetate into triglyceride (Mackall et al., J. Biol.

Chem. 251: 6462, 1976) or accumulation of triglycerides (Kletzien et al., Mol.Pharmacol. 41: 393-398, 1992).

The admission of glucose can be evaluated, for example, in a test for the transport of glucose stimulated by insulin. Differentiated or transfected L6 myotubes (maintained in the absence of G418) were placed in DMEM containing 1 g / 1 glucose, 0.5 to 1.0% BSA, 20 mM Hepes, and 2 mM glutamine. After two to five hours of culture, the medium is replaced with glucose- DMEM, h containing 0.5 to 1.0% BSA, 20 mM Hepes, 1 mM pyruvate and 2 mM glutamine. Appropriate concentrations of insulin or IGF-1, or a series of dilutions of the test substance are added, and the cells are incubated for 20-30 minutes. Deoxyglucose 3H or 14C is added to a final concentration of approximately 50 1M, and the cells are incubated for approximately 10-30 minutes. The cells are then rapidly rinsed with cold buffer (e.g., PBS), then lysed with a suitable lysis agent (e.g., 1% SDS or 1N NaOH). The cell lysate is then evaluated by counting in a desyntilation counter. The reactivity - associated with the cells is taken as a measure of glucose transport after subtracting the specific binding as determined by incubating cells in the presence of cytokinesin b, a glucose transport inhibitor.

Other methods include those described for example, by Manchester et al., Am. J. Physiol. 266 (Endocrinol Metab 29): E326E333, 1994 (glucose transport stimulated by insulin). Protein synthesis can be evaluated, for example, by comparing the precipitation of methionine-labeled 35S proteins after incubation of the test cells with 35S-methionine and 35S-methionine of a putative modulator of protein synthesis. Thermogenesis can be evaluated as described by B. Stanley in the Biology of Neuropeptide and and Related Peptides. W. Colmers and C. Ahlestedt (eds.), Humana Press, Ottawa, 1993, p. 457-509; C. Billington et al., Am. J. Physiol. 260.-R321, 1991; N. Zarjevski et al., Endocrinology 133: 1753, 1993; C. Billington et al., Am. J. Physiol. 266.-R1765, 1994; Heller et al., A. J. Physiol. 252 (4 Pt 2): R661-7, 1987; and Heller et al., A. J. Physiol. 245 (3): R321-8, 1983. Also, the metabolic rate, which can be measured by a variety of techniques, is an indirect measure of thermogenesis. Oxygen utilization can be evaluated as described by Hller et al., Pflugers Arch 369 (1): 55-9, 1977. This method also comprises an analysis of the hypothalmic temperature and the production of heat metabolic. The use and thermoregulation of oxygen has been evaluated in humans as described by Has eil et al., J. Appl. Physiol. 51 (4): 948-54, 1981. Among other methods known in the art or described herein, tissue protection of mammalian endothelial cells can be evaluated by monitoring the function of endothelial tissue. For example, heart function (aorta) can be assessed by monitoring the release of acetylcholine, norepinephrine release, or similar parameters. These parameters are monitored by techniques (tests in animal models) known to a person skilled in the art, as more fully discussed later. The release of acetylcholine and norepinephrine can be monitored by HPLC. Levy, Electrophysiology of the Synoa trial and Atrrioven tricular Nodes, Alan R. Liss, Inc., 1987-197, 1998, describes the measurement of norepinephrine in the coronary sinus effluent. In addition, the animals can be moved electrically, with the results monitored as described by Elsner, European Heart Journal 16 (Supplement N) 52-8, 195, and Reiffel and Kuenhnert, PACE 17 (Part 1): 349-65, 1994 Among other methods known in the art or described herein, the functions of neurotransmission can be assessed by monitoring the admission of 2-deoxy-glucose in the brain. This parameter is monitored by techniques (tests or animal models) known to a person skilled in the art, for example, autoradiography. Useful monitoring techniques are described, for example, by Kilduff et al., J. Neurosci. 10 2463-75, 1990, with related techniques used to evaluate the "heart in hibernation" as described in Gerber et al. Circulation 94 (4) C: 651-8, 1996, and Fallavllita et al., Circulation 95 (7): 1900-1909, 1997. In addition, the zsig37 polypeptides, fragments, fusions, agonists or antagonists thereof can be therapeutically useful for applications modulated by neurotransmitters or antimicrobials. For example, the Clq complement component plays a role in the defense of the host against infectious agents, such as bacteria and viruses. Clq is known to inhibit several specialized functions. For example, Clq activates the complement cascade via interaction with bound antibody or C-reactive protein (CRP). Also, Clq interacts directly with certain bacteria, RNA viruses, microplasma, uric acid crystals, the lipid A component of bacterial endotoxin and membranes in certain organelles intracellular The Clq that binds to the Clq receptor is thought to promote phagocytosis. Clq also appears to enhance the antibody formation aspect of the host defense system. See, for example, Johnston, Pediatr. Infect. Dis. J. 12 (11): 933-41, 1993. Thus, soluble Cql-like molecules may be useful, antimicrobial agents, which promote the lysis or phagocytosis of infectious agents. The zsgi37 polypeptides of the present invention also exhibit homology to portions that are believed to modulate neutrotransmission. As shown in Figure 1, zsig37 polypeptides are homologous to the following proteins: HP25_TAMAS (Takamatsu et al., Mol. Cell. Biol. 13: 1516-21, 1993 and Kondo &Kondoreferido above) and CERL_RAT (Wada & Ohtani, Brain Res. Mol. Brain Res. 9_: 11-1, 1991). The HP25 and HP27 are polypeptides found in active serum (estibal) of Siberian marmots in hibernation. He CERL (present in the cerebellum of rats.) Thus, zsig37 polypeptides, fragments, fusions, agonists or antagonists may be useful in the modulation of neurotransmission, for example, by binding to neurotransmitters or receptors therefor.

The correlation of radiation hybrids is a somatic cell genetic technique developed to construct contiguous high-resolution maps of mammalian chromosomes (Cox et al., Science 250: 245-250, 1990). The partial or complete knowledge of the gene sequence allows the design of PCR primers suitable for use with chromosomal radiation hybrid correlation panels. Commercially available hybrid, radiation correlation panels covering the entire human genome such as the Stanford G3 RH panel and the GeneBridge 4 RH panel (Research Genetics, Inc., Huntsville, AL), are available. These panels allow for chromosomal, PCR-based, rapid locations and gene sorting, sequence-tagged sites (STS), and other non-polymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously correlated markers. Accurate knowledge of a gene position can be useful in a number of ways including the content 1) determination if a sequence is part of an existing contig and obtaining the surrounding, additional genetic sequences in various forms such as YAC-, BAC- or CDNA, 2) provide a possible candidate gene for a hereditable disease that shows a link to the same chromosomal region, and 3) for cross-referenced model organisms such as mice that may be helpful in determining what function a particular gene may have. The results showed that the gene coding for the zsig37 polypeptide correlated to human chromosome 17, region 17q25.2, by PCR using the Hybrid Correlation Panel Number 2, the Somatic, Human / Rodent Cells, NIGMS (National Institute of General Medical Sciences, Coriell Institute of Medical Research). The present invention also provides reagents that will find use in diagnostic applications. For example, the zsig37 gene, a probe comprising the DNA or RNA of zsig37, or a sub-sequence thereof can be used to determine whether the zsig37 gene is present on chromosome 17 or whether a mutation has occurred. Chromosomal aberrations detectable at the zsig37 gene site include, but are not limited to, aneuploid, changes in gene copy number, insertions, deletions, changes in the restriction and array site. These aberrations can occur within the coding sequence, within introns, or within the flanking sequences including the regulatory and promoter regions in the 5 'direction, and may be manifested as physical alterations within a coding sequence or changes in the level of expression gene In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as described above, under conditions wherein the polynucleotide will hybridize to the complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the reaction product of indicative controls and a genetic abnormality in the patient. Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA. The polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO: 1, and complement of SEQ ID NO: 1, or one equivalent of RNA of the same. Suitable assay methods in this regard include molecular genetic techniques known to those skilled in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, Ligation chain reaction (Barany, PCR Methoods and Applicatioons 1_: 5-16, 1991), ribonuclease protection assays, and other genetic linkage techniques known in the art (Sambrook et al., Ibid.; Ausubel et al., Ibid .; Marian, Chest 108: 255-65, 1995). The ribonuclease protection assays (see, for example, Ausubel et al., Ibid. , ch. 4) comprise the hybridization of an RNA probe to a patient's RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to Nrasa. The hybridized regions of RNA are protected from digestion. Within the PCR assays, a genetic sample of the patient is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in the size and quantity of the recovered product is indicative of mutations in the patient. Other PCR-based techniques that can be used are the analysis of single-strand conformational polymorphism (SSCP) (Hayashi, PCR Methods and Applications 1: 34-8, 1991). A condition associated with 17q25.2 is glycogen storage disease II. In this way, the zsig37 polypeptide can be useful in the study, prevention or treatment, for example, gene therapy, of this condition. If a mammal has a mutated zsig37 gene or lacks it, the zsig37 gene can be introduced into mammalian cells. In one embodiment, a gene encoding a zsig37 polypeptide in vi is introduced into the viral vector. These vectors include an attenuated or defective DNA virus, such as, but limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which completely or almost completely lack the viral genes, are preferred. A defective virus is not infective after introduction into the cell. The use of defective viral vectors allows administration to cells in a specific localized area, without interest that the vector can infect other cells. The examples of particular vectors include, but are not limited to, the defective herpesvirus 1 vector (HSV1) (Kaplitt et al., Moldee, Cell. Neurosci., 2 ^: 320-330 (1991)), an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-630 (1992), and a defective adeno-associated virus vector (Samulski et al., J. Virol., _61_: 3096-3101 (1987); Samulski et al., J. Virol., _63_: 3822 -3828 (1989) In one embodiment, the gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Patent No. 5, 399, 346; Mann et al., Cell, 33: 153 (1983); Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Marko itz et al., J. Virol. _62_: 1120 (1988); Temin et al., U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al .; and Blood, 82: 845 (1993). Alternatively, the vector can be introduced by lipofection in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for transfection in vi of a gene coding for a marker (Felgner et al., Proc. Nati, Acad. Sci. USA, 84: 7413-7417 (1987); see, Mackey et al., Proc. Nati. Acad. Sci. USA, 8_5: 8027-8031 (1988) The use of lipofection to introduce exogenous genes into specific organs in vi vi has certain practical advantages.The molecular recognition of liposomes to specific cells represents an area of benefit. Transfection to particular cells represents a beneficial area It is clear that the direction of transfection to a particular type of cell would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney and brain.The lipids can be chemically coupled to other molecules for the purpose of recognition Peptides searched for, eg, hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules can be chemically coupled to liposomes. It is possible to remove the cells from the body and introduce the vector as a plasmid of naked DNA and then re-implant the transformed cells in the body. The naked DNA vector for gene therapy can be introduced in the desired host cells by methods known in the art, for example, transfection, Electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transponder. See, for example, u et al., J. Biol. Chem 267: 963-967 (1992); Wu et al., J. Biol. Chem. 263: 14621-14624 (1988); and Johnston and Tang, Methods in Cell Biology 4J_: 353-65 (1994). Another aspect of the present invention comprises antisense polynucleotide compositions that are complementary to a segment of the polynucleotides set forth in SEQ ID NO: 1. These synthetic antisense oligonucleotides are designed to bind to the mRNA encoding the zsig37 polypeptides and inhibit a translation of this to mRNA. These antisense oligonucleotides are useful for inhibiting the expression of genes encoding zsig37 polypeptides in cell culture or in a subject. Zsig37 polypeptides can be used in the energy efficiency analysis of a mammal. The zsig37 polypeptides found in serum or tissue samples may be indicative of a ability of mammals to store food, with more highly efficient mammals that tend towards obesity. More specifically, the present invention contemplates methods for detecting the zsig37 polypeptide comprising: displaying a sample possibly containing the zsig37 polypeptide to an antibody bound to a solid support, wherein the antibody binds to an epitope of a zsig37 polypeptide; wash the immobilized antibody-polypeptide to remove unbound contaminants; exposing the immobilized antibody-polypeptide to a second antibody directed to a second epitope of a zsig37 polypeptide, wherein the second antibody is associated with a detectable label; and detect the detectable mark. The concentration of the zsig37 polypeptide in the test sample appears to be indicative of the energy efficiency of a mammal. This transformation can help in the nutritional analysis of a mammal. Potentially, this information can be useful in the identification and / or search of the deficient tissue of energy.

Within further aspects of the invention, antibodies are provided that specifically bind to the zsig37 polypeptides described above. These antibodies are useful for, among other uses as described herein, the preparation of anti-idiotypic antibody. A further aspect of the present invention provides methods for identifying agonists or antagonists of the zsig37 polypeptides described above, agonists or antagonists that may have vble properties as discussed further herein. Within a modality, a method is provided for identifying the zsig37 polypeptide agonists which comprises providing cells responsive thereto, culturing the cells in the presence of a test compound and comparing the cell response with the cell grown in the presence of the zsig37 polypeptide, and selecting the test compounds for which the cellular response is of the same type. Within another embodiment, there is provided a method for identifying antagonists of the zsig37 polypeptide, which comprises providing cells responsive to a zsig37 polypeptide, culturing a first portion of the cells in the presence of the zsig37 polypeptide, culturing a second portion of the cells in the presence of the zsig37 polypeptide and a test compound, and detecting a decrease in a cellular response of the second portion of the cells compared to the first portion of the cells. In addition to those assays described herein, samples for the inhibition of zsig37 activity can be tested within a variety of assays designed to measure receptor binding or stimulation / inhibition of zsig37-dependent cellular responses. For example, zsig37-sensitive cell lines can be transfected with an indicator gene construct that is sensitive to a cell path stimulated by zsig37. Indicator gene constructs of this type are known in the art, and will generally comprise a zsig37-DNA response element operably linked to a gene encoding a mountable protein, such as luciferase. DNA response elements may include, but not be limited to, AMP response elements, cyclic (CRE), hormone response elements (HRE), insulin response element (IRE) (Nasrin et al., Prot. Nati, Acad. Sci. USA 87: 5273-7, 1990) and the serum response elements (SER) (Shaw et al., Cell 56: 563-72, 1989). The cyclic AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6, 1988 and Haneber, Molec. Endocrinol 4 (8): 1087-94, 1990. Hormone response elements are reviewed in Beato, Cell 56: 335-44; 1989. Candidate compounds, solutions, mixtures, substrates are tested for the ability to inhibit zsig37 activity of target cells as evidenced by a decrease in Zsig37 stimulation of reporter gene expression. Assays of this type will detect compounds that directly block the binding of zsig37 to cell surface receptors, as well as compounds that block processes in the cell pathway subsequent to receptor-ligand binding. In the alternative, compounds or other samples can be tested for direct blocking of zsig37 binding to the receptor using zsig37 labeled with a detectable label (eg, 125 I, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zsig37 to the receptor is indicative of the inhibitory activity, which can be confirmed through the secondary trials. The receptors used within the binding assays can be cellular receptors or immobilized receptors, isolated. A further aspect of the invention provides a method for studying insulin. These methods of the present invention comprise concealing adipocytes in a culture medium comprising the zsig37 polypeptide, monoclonal antibody, agonist or antagonist of the same insulin, and observing the changes in the secretion of adipocyte proteins or their differentiation. Anti-microbial protective agents can be activated directly or activated indirectly. These agents that operate via the membrane association or the mechanism of formation of forums of action are directly linked to the offensive microbe. The antimicrobial agents can also act via an enzymatic mechanism, breaking the microbial protective substances or the cell wall / membrane thereof. Antimicrobial agents capable of inhibiting the proliferation of microorganisms in the action of the integrity of the switch microorganism either by the mechanism discussed above are useful in methods to prevent contamination in cell culture by microbes susceptible to that anti-microbial activity. These techniques comprise culturing cells in the presence of an effective amount of this zsig37 polypeptide or an agonist or antagonist thereof. Also, • zsig37 polypeptides or agonists thereof can be used as cell culture reagents in in vitro studies of the infection of microorganisms, exogenous, such as bacterial, viral or fungal infection. These portions can also be used in in vivo animal models of infection. The present invention also provides methods for studying similar metabolism of mammals. These methods of the present invention comprise incubating cells to be studied, for example, vascular, human endothelial cells, zsig37 polypeptide, monoclonal antibody, agonist or antagonist thereof and observing changes in adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis. , glucose intake, or the like. A further aspect of the invention provides in method for studying dimerization or oligomerization. These methods of the present invention cise incubating zsig37 polypeptide or fusion fragments or proteins thereof containing a collagen-like domain alone or in combination with other polypeptides carrying collagen-like domains and observing the associations formed between the collagen-like domains. These associations are indicated by HPLC, circular dichroism or the like. - As noted previously, isolated polynucleotides of the present invention include RNA and DNA. Methods for isolating RNA and DNA are well known in the art. It is generally preferred to isolate the RNA from the tumor of the brain, heart, placenta, similar adipose tissue, although DNA can also be prepared using the RNA of other species or isolated as genomic DNA. Total RNA can be prepared using guanidine HCl extraction followed by isolation. by centrifugation in a CsCl gradient (Chirg in et al., Biochemistry 18: 52-94, 1979). Poly (A) + RNA is prepared from the total RNA using the method of Aviv and Leder (Proc. Nati, Acad. Sci. USA 69: 1408-1412, 1972). Cementary DNA (cDNA) is prepared from poly (A) + RNA using known methods.

The polynucleotides encoding the zsig37 polypeptides are then identified and isolated for example by hybridization or PCR. The present invention further provides polypeptides and counterpart polynucleotides from other species (orthologs or paralogs). These species include, dogs are not limited to species of mammals, poultry, amphibians, reptiles, fish, insects and other vertebrates and invertebrates. Of particular interest are the zsig37 polypeptides of other mammalian species, including murine, rat, porcine, ovine, bovine, canine, feline, equine and other primate proteins. Orthologs of human proteins can be cloned using the information and csitions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences described herein. A library of the mRNA from a positive cell or cell line is then prepared. A DNA that codes for the polypeptide zsig37 can then be isolated by a variety of methods, such as when probing with a partial cete human cDNA or with one or more sets of degenerate probes based on the described sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using the designed primers of the sequences described herein. Within a further method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the zsig37 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Orthologs can also be identified by detecting species-specific EST species databases using the sequences provided herein. An EST that codes for an ortholog of murine zsig37 was found by detecting a mouse EST database as described in detail below. The mouse ortholog (SEQ ID NO: 43) has 77% identity with the human sequence at the nucleotide level and 77% identity at the amino acid level.

Those skilled in the art that the sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 represent an individual allele of the DNA of human SEQ ID NO: 2 Zsig37 and protein and that allelic variation and alternative splicing is expected to be present. Allelic variants of these sequences can be cloned by probing the cDNA or genomic libraries of different individuals according to normal procedures. Allelic variants of the DNA sequence shown in SEQ ID NO: l, include those that contain missing mutations and those in which the mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins that are allelic variants of SEQ ID NO: 2. CDNAs generated from the alternatively spliced mRNAs, which retain the properties of the zsig37 polypeptide are included within the scope of the present invention, as are the polypeptides encoded by these cDNA and mRNA. The allelic variants and splice variants of these sequences can be cloned by probing the cDNA or genomic libraries of different individuals or tissues according to standard procedures known in the art.

The present invention also provides isolated zsig37 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO: 2 and their species counterparts / orthologs. The term "substantially homologous" is used herein to excite polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NO: 2 or their orthologs or paralogs. These polypeptides will more preferably be at least 90% identical, and more preferably 95% or more identical to SEQ ID NO: 2 or their orthologs or paralogs. The percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Nati Acad. Sci. USA 89: 10915-10919, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap-opening penalty of 10, an extension and separation purpose of 1, and the scoring matrix " blosum 62"by Henikoff and Henikoff (ibid) as shown in table 3 (amino acids are indicated by codes normal of a letter). The identity percent is then calculated as: Total number of identical matches x? Oo [length of the longest sequence plus the number of separations entered in the longest sequence in order to align the two sequences] Table 3 ARNDCQEGHILMFP s TWYVA 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 10 E '-1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 SL - 1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 15 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 - 1 0 -2 -3 -2 1 2 -1 5 P -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 - 1 -1 -2 -2 -3 -3 -1 -2 -4 ns 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 20 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 V * -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 '-1 -2 -1 3, •• 3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 The sequence identity of the polynucleotide molecules is determined by similar methods using a ratio as described above. The substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, which are amino acid substitutions, conservative (see Table 4) and other substitutions that do not significantly affect folding or activity of the protein or polypeptide; small deletions, typically from one to about 30 amino acids; and small amino-or carboxyl-terminal extensions, such as an omino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. Polypeptides comprising affinity tags can additionally comprise a proteolytic cleavage site between the zsig37 polypeptide and the affinity tag. Preferred sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 4 Conservative amino acid substitutions Basic Arginine Usina Histidine Acid Glutamic Acid Aspartic Acid Polar Glutamine Asparagine Hydrophobic Leucine Valine Aromatic Phenylalanine Tryptophan Tyrosine Small: Glycine Alanine Serine Threonine Methionine The proteins of the present invention can also comprise amino acid residues that occur naturally. Amino acids that do not occur naturally include, without limitation, trans-3-methylproline, 2,4-methaneproline, cis-4-hydroxy proline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyltreonin, hydroxyethylcysteine, hydroxy-ethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine-carboxylic acid, dehydroproline, 3- and 4-methylproline, 3, 3-dimethylproline, ter-leucine, normalin, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Various methods are known in the art for incorporating amino acid residues that do not occur naturally in proteins. For example, an in vitro system can be employed where non-homosense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating the tRNA are known in the art. The transcription and translation of the plasmids containing non-homosentide mutations is carried out in a cell-free system comprising an S30 extract of E. coli and commercially available enzymes and others. reagents The proteins are purified by chromatography. See, for example Robertson et al., J. Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzimol. 202: 301, 1991; Chung et al., Proc. Nati Acad. Sci. USA 90: 10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of the mutated mRNA and the chemically aminoacylated suppressor tRNAs (Turcatti et al., J Biol. Chem. 271: 1999-1-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid to be replaced (e.g., phenylalanine) and the presence of amino acids (s) that do not occur naturally, desired ( for example, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The amino acid that does not occur naturally is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted to species that occur unnaturally by in vitro chemical modification. Chemical modification can be combined with sequence-directed mutagenesis to extend additionally the range of substitutions (Wynn and Richards, Protein Sci. 2: 395-403, 1993). A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, amino acids that do not occur naturally, and unnatural amino acids can be substituted for the amino acid residues of zsig37. The essential amino acids in the polypeptides of the present invention can be identified according to methods known in the art, such as sequence directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Nati, Acad. Sci. USA 88: 4498-502, 1991). In this latter technique, individual mutations of alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for biological activity (eg, ability to modulate energy balance) as described below to identify residues of amino acids that are critical to the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-708, 1996. The sites of the ligand- The receptor or other biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photon affinity, in conjunction with the amino acid mutation of the putative contact site. See, for example, from Vos et al., Science 255: 306-12, 1992; Smith et al., FEBS Olett. 309: 59-64, 1992. The identities of the essential amino acids can also be deferred from the analysis of the homologies with the related polypeptides. Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and detection, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-7, 1988) or Boie and Sauer (Proc. Nati. Acad. Sci. USA 86: 2152-6, 1989). Briefly, these authors describe methods for simultaneously screening two or more positions in a polypeptide, selecting the functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem 30: 10832-7, 1991; Ladner et al.

United States No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988). The zsig37 DNA variants described and the polypeptide sequences can be generated through the DNA change as described by Stem er, Nature 370: 389-91, 1994, Stemmer, Proc. Nati Acad. Sci USA 91: 10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, the variant DNAs are generated by an in vitro homologous recommendation by random fragmentation of an origin DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of source DNA, such as allelic variants or DNA from different species, to introduce additional variability into the process. The selection or detection for the desired activity followed by the additional mutagenesis and assay interactions provides a rapid "evolution" of the sequences when selecting the desirable mutations while simultaneously selecting against the deleterious changes.

Mutagenesis methods as described above can be combined with automated, high throughput detection methods to detect the activity of mutagenized, thunked polypeptides in host cells. The mutagenized DNA molecules that code for the active polypeptides (e.g., ability to modulate the energy balance) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods discussed above, one skilled in the art can identify and / or prepare a variety of polypeptides that are substantially homologous at residues 22 to 281 of SEQ ID No. 2 or allelic variants thereof and retain the properties of modulation or other energy balance of the wild type protein. These polypeptides may include additional amino acids, such as additional collagen repeats of the Gly Xaa-Pro or Gly-Xaa-Xaa type. These polypeptides also they may include additional polypeptide segments as generally described above. The polypeptides of the present invention, which include full length proteins, fragments thereof and fusion proteins, can produce in genetically driven host cells, according to conventional techniques. Suitable host cells are those types of cells that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and eukaryotic, cultured cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating the cloned DNA molecules and for introducing the exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al. (eds.), Current Porotocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a zsig37 polypeptide of the present invention is operably linked to other elements genes required for their expression, which generally include a transcription promoter and a terminator within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers can be provided in separate vectors, and replication of the exogenous DNA can be provided by the integration of the host cell genome. The selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many of these elements are described in the literature and are available through commercial providers. To direct a zsig37 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as the leader sequence, signal sequence, pre-pro-sequence or pre-sequence) is provided in the expression vector. The secretory signal sequence can be that of the zsig37 polypeptide, it can be derived from another secreted protein (for example, t-PA) or is synthesized from n ovo. The secretory signal sequence binds to the zsig37 polypeptide DNA sequence in the correct reading frame. The secretory signal sequences are commonly placed 5 'to the DNA sequence encoding the polypeptide of interest, although certain signal sequences can be placed anywhere in the DNA sequence of interest (see, eg, Welch et al, United States Patent No. 5, 037, 743; Holland et al., U.S. Patent No. 5,143,830). In contrast, the ratio of the zsig37 polypeptide signal sequence (amino acids 1-21 or 1-25 of SEQ ID NO: 2) can be used to direct the secretion of an alternative protein by analogous methods. The secretory signal sequence contained in the polypeptides of the present invention can be used to direct other polypeptides in the secretory pathway. The present invention provides these fusion polypeptides. A signal fusion polypeptide can be made where a secretory signal sequence derived from the amino acid residues 1-22 or 1-25 of SEQ ID N: 2 is operably linked to another polypeptide using methods known in the art and described herein. The contained secretory signal sequence of the fusion polypeptides of the present invention are preferably fused in an amino-terminal manner to an additional polypeptide to direct the additional polypeptide in the secretory pathway. These constructions have numerous applications known in the art. For example, these novel secretory signal fusion fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor. These fusions can be used in vi ve or in vi tro to direct the polypeptides through the secretory route. Cultured mammalian cells are also suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981: Graham and Van der Eb , virology 5_2: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), transfection mediated by DEAE-dextran (Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), liposome-mediated transfection (Ha ley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 1_5: 80, 1993), and viral vectors (A Miller and G. Rosman, BioTechniques _7'980-90 '1989; Q. Wang and M. Finer, Nature Med. 2: 714-16, 1996). The production of recombinant polypeptides in cultured mammalian cells is described, for example, by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134. Preferred cultured mammalian cells include COS-1 cell lines (ATCC Noo, CRL 165CJ), COS-7 (ATCC No. CRL 1651), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen Virol 36: 59-72, 1977) and those of Chinese hamster ovary (e.g., CHO-K1; ATCC No. CCL 61). Additional suitable cell lines are known in the art and are available from public constants such as the American Species Crop collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as the promoters of SV-40, or cytomegalovirus. See, for example, U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter. Drug selection is generally used to select cultured mammalian cells in which foreign DNA has been inserted. These cells are commonly referred to as "transfectant". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants". A selectable, preferred marker is a gene that codes for resistance to the antibiotic neomycin. The selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems can also be used to increase the level of expression of the gene of interest, a process referred to as "amplification". Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of the selective agent to select the cells that produce high levels of the products in the introduced genes. A selectable, amplifiable, preferred marker is dihydrofolate reductase, which confers methotrexate resistance. Other drug resistance genes (eg, hygromycin resistance, multidrug resistance, pyromycin acetyltrasferase) may also be used. Alternative markers that introduce an altered phenotype, such as the green fluorescent protein, or proteins on the cell surface such as CD4, CD8, MHC Class I, placental alkaline phosphatase can be used to classify the transfected cells from non-transfected cells by a means such as a Separation of magnetic beads or classification by FACS. Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and poultry cells. The use of Agroba cterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci.

(Bangalor) 11: 47-58, 1987. Transformation into insect cells and the production of polypeptides strangers in the present is described by Guarino et al., U.S. Patent No. 5,162.22 and WIPO publication WO 94/06463. The insect cells can be infected with recombinant baculovirus, which is derived from the nuclear polyhedrosis virus of Au tomatica ca l i forni ca (AcNPV). See, King and Possee, The Baculovirus Expression System: A Laboratory Guide, London Chapman & Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; and Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Toto, NJ, Humana Press, 1995. A second method for making recombinant zsig37 baculovirus uses a transposon-based system described by Luckow (Luckow et al., J. Virol. 67_: 4566-79, 1993). This system, which uses transfer vectors, is sold in the Bac-to-BacMR equipment (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl ™ (Life Technologies) containing a Tn7 transposon to move the DNA encoding the zsig37 polypeptide into a baculovirus genome maintained at E. Coli as a large plasmid called a "bacmid". See, Hill-Perkins and Possee, J. Gen. Virol. l_l: 91 1-6, 1990; Bonning et al., J. Gen. Virol. 75: 1551-6, 1994; and Chazenbalk and Rapoport, J.Biol. Chem. 270: 1543-9, 1995. In addition, the transfer vectors can include a function in the structure with the DNA encoding the epitope tag on the C- or N-terminus of the expressed zsig37 polypeptide, eg, a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nati, Acad. Sci. 8_2: 7952-4, 1985). Using a technique known in the art, a transfer vector containing zsig37 is transformed into E. Col i, and is detected by bacmides containing an interrupted lacZ gene indicative of the recombinant baculovirus. The bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect cells of Spodop t was giperda fru, e.g., Sf9 cells. Recombinant viruses expressing zsig37 are produced in a subsequent manner. Recombinant viral concentrations are made by methods commonly used in the art. The recombinant virus is used to infect host cells, typically a cell line derived from the autumnal worm, Spodoptera frugiperda. See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994. Another suitable cell line is the High FiveOMR cell line (Invitrogen) derived from Tri chopl usi a n i (U.S. Patent No. 5,300,435). The commercially available serum free media is used to grow and maintain the cells. Suitable media are Sf IIMR (Life Technologies) or ESF 921MR (Expression Systems) for Sf9 cells; and Ex cell0405MR (JRH Biosciences, Lenexa, KS) or Express FiveOMR (Life Technologies) for T cells. neither . The cells are cultured from an inoculation density of about 2-5 x 10 5 cells at a density of 1-2 x 10 6 cells at which time a recombinant viral concentration is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically wax of 3. The procedures used are generally described in available laboratory manuals (King and Possee, ibid., O'Reilly et al., ibid; Richardson, ibid.). Subsequent purification of the zsig37 polypeptide from the supernatant can be achieved using methods described herein. Fungal cells, including yeast cells, can also be used within the present invention. Yeast species of particular interest in this regard include Sa ccha romyces cerevisia e, Pi chi a pa s tori s, and Pi chia me than ol i ca. The methods to transform S cells. Cerebu tion with exogenous DNA and producing recombinant polypeptides thereof are described for example in Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murria et al., U.S. Patent No. 4,845,075. The transformed cells are selected by the phenotype determined by the selectable marker, commonly the drug resistance, the ability to grow in the presence of a particular nutrient (e.g., leucine). A preferred vector system for use in Sa ccha romyces cerevisia e is the POT1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected for growth in the medium containing glucose. Promoters and terminators suitable for use in yeast include those from enzyme genes glycolytics (see, for example, Kawasaki U.S. Patent No. 4,599,311; Kingsam et al., U.S. Patent No. 4,977,092) and the alcohol-dehydrogenase gene. See also, U.S. Patent No. 4,990,446; '5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula plymorph, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose are known in the art, See, for example, Glessoon et al. ., J.Gen. Microbiol 132: 3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells can be used according to the methods of Mchnight et al., U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are described by Su ino et al., U.S. Patent No. 5,162,228. Methods for transforming Neurspora are described by Lambowitz, U.S. Patent No. 4, 486, 533. The use of Pichia methanolica as a host for the production of recombinant proteins is described in WIPO publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methalonica are commonly prepared as double-stranded circular plasmids, which are preferably linearized before transformation. For the production of polypeptides in P. Methalonica it is preferred that the promoter and the terminator in the plasmid be those of a P. Methalonica gene, such as an alcohol utilization gene of P. Methalonica (AUG1 or AUG2). Other useful promoters include those of dihydroxyacetoone synthase (DHAS), formate dehydrogenase (FMD), and catalase genes (CAT). To facilitate the integration of DNA in the host chromosome, it is preferred to make the complete expression segment of the plasmid flanked at both ends by the host DNA sequences. A selectable marker, preferred for use in Pichia methanolica is an ADE2 gene from P. Methalonica, which codes for phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine. For large-scale industrial processes, where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For the production of secreted proteins, the host cells deficient in the vacuolar protease genes (PEP4 and PRB1) swe prefer. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest in the P cells. Me tha l on i ca. The cells of P. It is evaluated by Electroporation using an exponentially falling electric impulse field, which has a field strength of 2.5 to 4.5 kV / cm, preferably about 3.75 kV / c, and a time constant (t) of 1 to 40 milliseconds, more preferably about 20 milliseconds. Prokaryotic host cells, including strains of the bacteria Escheri chia coli, Ba cillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA frequencies cloned in the present are well known in the art (see, for example, Sambrook et al., Ibid). When a zsig37 polypeptide of the bacteria such as E is expressed. Col i, the polypeptide can be retained in the cytoplasm, typically as insoluble granules, or it can be directed to the periplasmic space by a sequence of bacterial secretion. Edn the previous case, the cells are smoothed, and the granulate is not recovered and denatured using, for example, guanidine isothiosanate or urea. The denatured polypeptide can then be refolded and dimerized by dilution of the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic space in a soluble and functional form by interrupting the cells (for example, by sonication or osmic shock) to release the contents of the periplasmic space and recover the protein, avoiding this way the need for denaturalization and refolding. The transformed or transfected host cells are cultured according to conventional procedures and a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable means, including defined means and means complexes are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, as required. The growth medium will generally be selected for cells containing the exogenously added DNA, for example, drug selection or deficiency in an essential nutrient that is complemented by the selectable marker carried in the expression vector or co-transfected into a cell Guest. The expressed recombinant zsig37 polypeptides (or chimeric zsig37 polypeptides) can be purified using conventional purification methods and / or fractionation and media. Precipitation with ammonium sulfate and extraction with acid or chaotrope can be used for the fractionation of the samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and inverted phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyethylamide, specialty silicas, and the like.

Preferred are PEI, DEAE, QAE and Q derivatives. Exemplary chromatographic media include those media derivatized with phenyl, butyl or octyl groups, such as phenyl sepharose FF (Pharmacia), Toyopearl-butyl-650 (Toso Haas, Montgomeryville , PA), Octyl Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica based resins, cellulosic resins, agarose beads, crosslinked agarose beads, polystyrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they are to be used. These reports can be modified with reactive groups that allow the binding of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of chemistry of coupling chemicals include activation by cyanogen bromide, activation by N-hydroxysuccinamide, activation by epoxide, activation by sulfhydryl, activation by hydra, and carboxyl and amino derivatives for the carbodiimide coupling chemicals. These and other solid media are well known and widely used in the art and are available from commercial suppliers. Methods for attaching the receptor polypeptides to the support medium are well known in the art. The selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principies & Methods, Pharmacia LLKB Biotechnology, Uppsala, Sweden, 1988. The polypeptides of the present invention can be isolated by exploiting their structural or binding properties. For example, immobilized metal ion adsorption chromatography (IMAC) can be used to purify histidine rich proteins or proteins having a His tag. Briefly, a gene is first charged with divalent metal ions to form a chelate (E. Sulkowski, Trends in Biochem. 3_: 1-1, 1985). The proteins rich in histidine will be absorbed into this matrix with different affinities, depending on the metal ion used and will be eluted by competitive elution, lowering the pH, or the use of strong chelating agents. These purification methods include protein purification glycolysates by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529 -39). Within a further preferred embodiment of the invention, a function of the polypeptide of interest and an affinity tag (eg, maltose binding protein), FLAG, Glu-Glu, an immunoglobulin domain) can be constructed to facilitate purification as discussed in greater detail in the sections of later examples. Protein retraction procedures (and optionally reoxidation) can be used advantageously. It is preferred to purify the protein at a purity of greater than 80%, more preferably greater than 90% purity, most preferably greater than 95%, and particularly preferably is a pharmaceutically pure state that is greater than 99.9% pure with with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin. Zsig37 polypeptides or fragments thereof can also be prepared through chemical synthesis. These zsig37 polypeptides can be monomers or multimers; be glycosylated and non-glycosylated; be pegylated, not pegylated; and may or may not include an initial methionine amino acid residue. A ligand-binding polypeptide, such as a polypeptide binding to the zsig37 polypeptide, can also be used for the purification of the ligand. The polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polyestene, cross-linked polyacrylamide, or similar materials that are stable under the conditions of use. Methods for packaging solid support polypeptides are known in the art, and include amine chemistry, activation with cyanogen bromide, activation with N-hydroxysuccinamide, activation with epoxide, activation with sulfhydryl, and activation with hydrazide. The resulting medium will generally be configured in the form of a column, and the fluids containing ligands are passed through the column one or more times to allow the ligand to bind to the ligand-binding polypeptide. The ligand is then diluted using changes in saline concentration, chaotropic agents (guanidine HCl), or a pH to break the ligand-receptor binding. A test system using a ligand binding receptor (or an antibody, a member and a complement / anti-complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway , NJ) can be used advantageously. This receptor, antibody, member and a complement / anti-complement pair or fragment is immobilized on the surface of a piece of the receptor. The use of this instrument is described by Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. A receptor, antibody, member or fragment is covalently linked using amine or sulfhydryl chemistry, to dextran fibers that bind to gold film of the flow cell. A test sample is passed through the cell. If a ligand, epitope or opposite member of the complement / anti-complement pair is present in the sample, will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in the surface plasmon resonance of the gold film. This system will allow determination of on and off speeds, from which the binding affinity can be calculated, and the assessment of the binding stoichiometry. Ligand-binding polypeptides can also be used within another assay system known in the art. These systems include Scatchard analysis for binding affinity determination (see, Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric assays (Cunningham et al., Science 253: 545-48, 1991; Cunningham et al., Science 245: 821-25, 1991). The zsig37 polypeptides can also be used to prepare antibodies that specifically bind to the zsig37 polypeptide epitopes, peptides or polypeptides. Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, JGR, Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982), As will be apparent to one skilled in the art, polyclonal antibodies can be generated from inoculation of a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, horses and rats as well as transgenic animals such as sheep, cows, goats or transgenic pigs. The antibodies can also be expressed in yeast and fungi in modified form as well as insect and mammalian cells. The zsig37 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal or produce an immunoresponse. Suitable antigens will include the zsig37 polypeptide encoded by SEQ ID NO: 2 of amino acid residue 22-281 of SEQ ID NO: 2, from amino acid residue 26-281 of SEQ ID NO: 2, or a fragment of residue from amino acid 9-21, contiguous thereof. The immunogenicity of a zsig37 polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freud's adjuvant. The polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsig37 or a portion thereof with an immunoglobulin polypeptide or with an affinity tag. The polypeptide immunogen can be a full-length molecule or a portion thereof. If the polypeptide portion is "aftenoid type", this portion can be advantageously attached or articulated to a macromolecular carrier (such as limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. As used herein, the term "antibodies" includes polyclonal antibodies, affinity purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F (ab ') 2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as antigen-binding peptides, synthetics and polypeptides, are also included. Non-human antibodies can be humanized by grafting only non-human CDRs onto the human structure and constant regions, or incorporate the complete non-human variable domains (optionally "mask" it with a human-type surface by replacing exposed residues, where the result is a "capped" antibody). In some cases, humanized antibodies can retain non-human residues from the domains of the human variable region structure to enhance the appropriate binding characteristics. Through the humanization of antibodies, the biological half-life can be increased, and the potential for adverse immune reactions in administration to humans is reduced. Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zsig37 peptide protein, which is display in libraries of antibody selection in phage or similar vectors (e.g., through the use of protein or zsig37 peptide immobilized or labeled). Antibodies are defined to bind specifically if: 1) they exhibit a threshold level of binding activity and / or 2) they do not significantly cross-react with the related polypeptide molecules. First, the antibodies where they bind specifically if they bind to a zsig37 polypeptide, peptide or epitope with a binding affinity (Ka) of 106 mol-1 or greater, preferably 107 mol-1 major, most preferably 108 mol-1 greater, more preferably 109 mol-1 major. The binding affinity of the antibody can easily be terminated with respect to the art, for example, by Scatchard analysis (Scatchard, Ann, NY Acad. Sci. 51: 660-672, 1949). Second, the antibodies bind specifically if they do not cross-react significantly with the related polypeptides. The antibodies do not cross-react significantly with the related polypeptide molecules, for example, if they detect the zsig37 polypeptide but not the known related polypeptides using a normal Western blot analysis (Ausubel et al., Ibid.). Examples of related polypeptides include other members of a family of proteins such as Acrp30 (SEQ ID NO: 3), in the polypeptides shown in the alignment of Figure 1 and the like. They may also include, if desired, orthologs and human zsig37 polypeptides, mutants.

In addition, the antibodies can be "detected against" known related polypeptides to avoid a population that binds specifically to the inventive polypeptides. For example, antibodies formulated to human zsig37 polypeptides are absorbed into related polypeptides adhered to the insoluble matrix, antibodies specific to human zsig37 polypeptides will flow through the matrix under appropriate buffering conditions. This detection allows the isolation of polyclonal and monoclonal antibodies that do not cross-react to the closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Coligan, et al. (Eds.), National Institutes of Health, John Wiley and Sons, Inc. , nineteen ninety five) . The detection and isolation of specific antibodies is well known in the art (see, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. In Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principies and Practice, Goding, J.W. (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Revf. Immunol. 2_: 67-101, 1984). The examples Representative of these assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), blot analysis or Western blot, inhibition or competition assays, and intercalation assay. The genes encoding the polypeptides having the potential zsig37 polypeptide binding domains, "binding proteins", can be obtained by detecting random or direct peptide libraries displayed in phage (phage display) or in bacteria such as E.

Col i. The nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through mutated random genesis and random synthesis of polynucleotides. Alternatively, the restricted phage display library can also be produced. These peptide display libraries can be used to detect peptides that interact with a known target which can be a protein or polypeptide, such as a receptor ligand, a synthetic biological macromolecule, or organic or inorganic substances. The techniques to create and detect these peptide display libraries are known in the art (Ladner et al., U.S. Patent No. 5,223,409; Ladner et al., U.S. Patent No. 5,403,484 and Ladner et al., U.S. Patent No. 5, 571, 698) and peptide display libraries and kits for detecting these libraries are commercially available, for example, from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Polypeptide display libraries can be detected using the zsig37 sequences described herein to identify proteins that bind to zsig37. These "binding proteins" interact with the zsig37 polypeptides can be used essentially the same as an antibody, to label cells, to isolate homologous polypeptides by affinity purification; conjugated directly or indirectly to drugs, toxins, radionuclides, and the like. These binding proteins can also be used in analytical methods such as to detect libraries of expression and neutralizing activity. The binding proteins can also be used for diagnostic assays to determine polypeptide circulation levels; to detect or quantify soluble polypeptides as a marker of the underlying disease or disease. To increase the half-life of these binding proteins, they can be conjugated. The biological properties can be modified by dimerizing or multimerizing for use, agonists or antagonists. Binding peptides can be detected against related, known polypeptides, as described above. Antibodies and zsig37 binding proteins can be used to label cells expressing zsig37; to isolate zsig37 by affinity purification; for diagnostic assay to determine circulating levels of the zsig37 polypeptides; to detect or quantify soluble zsig37 as a marker of the underlying disease or disease; the analytical methods that use FACS; to detect expression libraries; to generate anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block the modulating activity of the energy balance of zsíg37 polypeptides or similar activity in vi tro and in vi vo. Suitable direct labels or tags include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect brands or labels exhibit their use of biotin-avidin or other complement / anti-complement pairs as intermediates. In addition, zsig37 antibodies or fragments thereof can be used in vi tro to detect denatured zsig37 or fragments thereof in assays, for example Western blots or other assays known in the art. The antibodies or binding proteins herein can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like, and these conjugates used for in vitro diagnostic or therapeutic applications. For example, the polypeptides or antibodies of the present invention can be used to identify or treat diseases or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for example). Specifically, zsig37 polypeptides or anti-zsig37 antibodies, or fragments or bioactive portions thereof, can be coupled to detectable or cytotoxic molecules and distributed to a mammal that has cells, tissue or organs that express the anti-complementary molecule. Suitable detectable molecules can be linked directly or indirectly to the polypeptide or antibody and include radionuclides, enzymes, substrate, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can be linked directly or indirectly to the polypeptide or antibody, in include bacterial or plant toxins (eg, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly linked through a medium of a chelating moiety, for example). The polypeptides or antibodies can also be conjugated to cytotoxic drugs, such as adriamycin. For indirect binding of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary / anticomplementary pair, where the other member binds to the polypeptide or antibody portion. For this For purposes, biotin / s treptavidin is an example of the complementary / anti-complementary pair. In another embodiment, the polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for the inhibition of targeted cells or tissue or ablation (e.g., to treat cancer or tissue cells). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a search domain), a fusion protein that includes only the search domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a molecule complementary to a cell or tissue type of interest. In cases where the domain is unique, the fusion protein includes a complementary molecule, the anti-complementary molecule can be conjugated to a detectable or cytotoxic molecule. These complement-domain molecule fusion proteins thus represent a generic search vehicle for the cell / tissue-specific distribution of the generic conjugates of the cytotoxic / detectable-anti-complementary, generic molecule. Polypeptide conjugates or antibody, bioactive agents described herein may be distributed intravenously, intra-erially, intraductally with DMSO, intramuscular, subcutaneous, int raperitoneal, also by transdermal methods, by electro-transfer, orally or by inhalant route. The polynucleotides encoding the zsig37 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit the activity of zsig37. If a mammal has a zsig37 gene mutated or absent, the zsig37 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zsig37 polypeptide is introduced in vi ve into a viral vector. These vectors include an attenuated or defective DNA virus, such as, but not limited to, simple herpes virus (HSV), papilloma virus, Epstein Barr virus (EBV), adeno virus, adeno-associated virus (AAV), and the like. Defective viruses, which are either completely or almost completely free of viral genes are preferred. A defective virus is not infective after introduction into the cell. The use of defective viral vectors allows administration to cells in a localized area,specific, without the interest that the vector can infect other cells. Examples of particular vectors include, but are not limited to, vector of defective herpes simplex virus 1 (HSV1) (Kaplitt et al., Molec.Cell.Neurosci.2_: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Strat ford-Pperricaudet et al., J. Clin. Invest. _9_0: 626-30, 1992; and a defective adeno-associated virus vector (Samuski et al., J. Virol. 61_: 3096-101, 1987; Samulski et al., J. Virol. 63_: 3822-8, 1989). In another embodiment, a zsig37 gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Patent No. 5, 399, 3 6; 'Mann et al. Cell 33: 153, 1983; Temin et al., U.S. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289; Markowitz et al., J. Virol. 6_2: 1120, 1988; Temin et al., U.S. Patent No. 5,124,263; a WIPO Publication WO 95/07358; and Kuo et al., Blood _d_2: 845, 1993. Alternatively, the vector can be introduced by lipofection in vi ve using liposomes. Synthetic cationic lipids can be used to prepare liposomes for transfection in vi vo a gene coding for a marker (Felgner et al., Proc. Nati, Acad. Sci USA _84_: 7413-7, 1987; Mackey et al., Proc. Nati. Acad. Sci. USA 85: 0827-31, 1988 ). The use of lipofection to introduce exogenous genes into specific organs has certain practical advantages. The molecular recognition of liposomes to specific cells represents an area of benefit. More particularly, the direction of transfection to particular cells represents an area of benefit. For example, the direction of transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney and brain. The lipids can be chemically coupled to other molecules for the purpose of recognition. Peptides sought (eg, hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be chemically coupled to liposomes. It is possible to remove the target cells from the body; to introduce vector as a naked DNA plasmid; and then re-implant the cells formed in the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, for example, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, for example, Wu et al, J. Biol. Chem. 267: 963-7, 1992; Wu et al., J. Biol. Chem. 263: 14621-4, 1988. The antisense methodology can be used to inhibit the transcription of the zsig37 gene, such as to inhibit cell proliferation in vi vo. Polynucleotides that are complementary to a segment of a polynucleotide encoding zsig37 (eg, a polynucleotide as set forth in SEQ ID NO: 1) are designed to bind to mRNA encoding zsig37 and to inhibit translation of this to mRNA. . These antisense polynucleotides are used to inhibit the expression of the genes encoding the zsig37 polypeptide in the cell culture or in a subject. Transgenic mice, engineered to express the zsig37 gene, and mice that exhibit a complete absence of zsig37 gene function, are referred to as "excellent mice" (Snouwaert et al., Science 257: 1083, 1992), can also be generate (Lowell et al., Nature 366: 740-42, 1993). These mice can be used to study the zsig37 gene and the protein modified in this way in an in vi vo system. For pharmaceutical use, the proteins of the present invention are formulated for parenteral distribution, particularly intravenously or subcutaneously according to conventional methods. The intravenous administration will be by injection or bolus infusion during a typical period of one to several hours. In general, the pharmaceutical formulations will include a zsig37 protein in combination with a pharmaceutically acceptable carrier, such as saline, 5% dextrose buffered saline in water or the like. The formulations will additionally include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent the loss of protein on the surfaces of the flasks, etc. Formulation methods are well known in the art and are described, for example, in Remington: The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co., Easton PA, 19th ed., 1995. Therapeutic bases are defined by the doctor according to accepted standards taking into account the nature and severity of the condition to be treated, patient traits, etc. The determination of the dose is within the level of ordinary skill in the art. The invention is further illustrated by the following non-limiting examples.

Example 1 Extension of the EST Sequence The novel polynucleotides encoding the zsig37 polypeptide of the present invention are initially identified by selecting an EST from an EST database, producing a protein sequence based on it, and searching databases of known sequences for the secreted protein that is more homologous to the predicted protein based on the EST. ESTs that potentially encode proteins that have biologically interesting homology to known, secreted proteins were identified for further study. An individual EST sequence was discovered and predicted to be homologous to the adipocyte-specific protein. See, for example, Scherer et al., J ^ Biol. Chem. 270 (45): 26746-9, 1995. For identify the corresponding cDNA, a clone considered likely to contain the entire coding sequence was used for sequencing. Using an Invitrogen S.N.A.P. Miniprep kit (Invitrogen, Corp., San Diego CA) according to the manufacturer's instructions 5 ml of an overnight culture in LB + 50 μg / ml ampicillin was prepared. The template was sequenced in a DNA sequencer model 377 ABIPRISM MR (Perkin-Elmer Cetus, Norwalk, Ct using the ready-to-sequence sequencing reaction equipment in the ABI PRISMMR dye terminator cycle (Perkin-Elmer Corp.) according to the manufacturer's instructions ZC695 oligonucleotides (SEQ ID NO: 5) ZC694 (SEQ ID NO: 6) to the SP6 and T7 promoters in the vector containing the clone were used as sequencing monitors: Oligonucleotides ZC13210 (SEQ ID NO: 7), ZC13588 (SEQ ID NO: 8), ZC13532 (SEQ ID NO: 9), ZC13641 (SEQ ID NO: 10), ZC13586 (SEQ ID NO: 11), ZC13651 (SEQ ID NO: 12), ZC13622 (SEQ ID NO 13), ZC13625 (SEQ ID NO: 14), ZC13650 (SEQ ID NO 15), ZC13589 (SEQ ID NO: 16), ZC13624 (SEQ ID NO 17), ZC13531 (SEQ ID NO: 18) , ZC13587 (SEQ ID NO: 19), and ZC323 (SEQ ID NO: 20) were used to terminate the clone sequence. The Sequencing reactions were carried out in a temperature Cycling System and Hybaid OmniGene (Natinal Labnet Co., Woodbridge, NY). The SEQUENCHERMR sequence analysis computation program (Gene Codes Corporation, Ann Arbor, MI) was used for data analysis. The resulting 2769 bp sequence is described in SEQ ID NO: 1. The comparison of the EST sequence originally derived with the sequence depicted in SEQ ID NO: 1 showed that there was an ambiguity of base pairs (an unknown "N" residue). ) and no insertion of base pairs that results in the identification of leucine in the resolution of ambiguity and zero structure changes between the reduced amino acid sequences.

Example 2 Fabric Distribution Northern were made using multiple human tissue transfers from Clontech (Palo Alto, CA). A DNA probe at 30 bases (ZC12447; SEQ ID NO: 4) at the 5 'end of the nucleotide sequence of the mature protein shown in SEQ ID NO: 1 was radioactively labeled with 32P using T4-polynucleide-kinase and forward reaction buffer (GIBCO BRL, Githersurg, MD) according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA). The EXPRESSHYB solution (Clontech, Palo Alto CA) was used for pre-hybridization and as a hybridization solution for Northern blots. Hybridization took place overnight at 50 ° C, and the blots were then washed in 2X SSC and 0.1% SDS at RT, followed by a wash in IX SSC and 0.1% SDS at 68 ° C (approximately 5 ° C). less than the melting point). A transcript size was observed at approximately 2.8 kb. The intensity of the signal was higher for the heart and placenta, with the relatively less intense signals in the kidney, ovary, adrenal gland and skeletal muscle and minor signals in a wide variety of other tissues present in the Northern blot. The additional Northern blot analysis was done using a Northern tissue transfer, Gut gut. The transfer was prepared using mRNA, from the human colorectal adenocarcinoma cell line SW480 (Clontech, Palo Alto, CA), human small intestine tissue (Clontech), human stomach tissue (Clontech), human intestinal smooth muscle cell line (His; ATCC No. CRL-1692; American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD), normal human colon cell line (FHC; ATCC No. CRL-1831; American Type Culture Collection) and fetal, normal, human small intestine cell line (FHs74 Int, ATCC No. CCL241, American Type Culture Collection). Total RNAs were isolated from Hism, FHC and FHs74 Int. By guanidino acid method (Cheomczyns ki et al., Anal. Biochem. 162: 156-9, 1987). The poly A + RNA was selected by diluting the total RNA through a column that maintained the poly A + RNA (Aviv et al., Proc. Nat. Acad. Sci. 69: 1408-12, 1972). The μg of the poly A + RNA of each sample was separated on a 1.5% agarose gel in 2.2 M formaldehyde and phosphate buffer. The RNAs were transferred onto a Nytran membrane (Schleicher and Schuell, Keene, NH) in 20X SSC overnight. The transfer was treated in a UV Stratalinker 2400 (Stratagene, La Jolla, CA) at 0.12 Joules. The transfer was cooked at 80 ° C for one hour.

The full length DNA (Shown in SEQ ID NO: 1) was amplified by PCR and radiolabelled with 32P using a Radiprime agglomeration kit (Amersham, Arlington Heigts, IL) according to the manufacturer's specifications. The blot was hybridized in EXPRESSHYB (Clontech) at 56 ° overnight. The transfer was washed at room temperature in 2X SSC and SDS, at 01%, then in 2X SSC and 0.1% SDS at 65 ° C, and finally at 65 ° at 0. IX SSC and 0.1% SDS. The results showed that zsig37 hybridized to all tissues except the HISM line of smooth, human, non-human muscle cells.

Example 3 Co-chromosomal relationship of Zsig37 gene The Zsig37 gene was co-related to human chromosome 17, region 17q25.2, by PCR using a panela of co-relation number 2 of human / rodent somatic cell hybrids NIGMS (National Institute of General Medical Sciences, Coriell Institute of Medical Research). The panel consists of DNA isolated from 24 hybrids of human / rodent somatic cells each retaining a chromosome specific human and the DNA of origin. For the correlation of the Zsig37 gene, 20 μl reactions were established in a 96-well microtiter plate (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler.

(Stratagene). Each of the 27 PCR reactions consisted of 2 μl of the 10X KlenTaq buffer (Clontech Laboratories, Inc., Palo Alto, CA), 1.6 μl of dNTPs mixture (2.5 M each, PERKIN-ELMER, Foster City, CA) 1 μl of homosentide primer (SEQ ID No .: 21), 1 μl of antisense primer (SEQ ID No .: 22), 2 μl RediLoad (Research Genetics, Inc.), 0.4 μl of 50X Advantage ClenTaq polymerase mix (Clontech Laboratories, Inc.), 25 ng of DNA from a single hybrid clone or control and ddH20 for a total volume of 20 μl. The reactions were overlaid with an equal amount of mineral oil and sealed. The conditions of the PCR cycler were as follows: an initial cycle of 5 minutes of denaturation at 95 ° C, 35 cycles of 1 minute of denaturation at 95 ° C, 1 minute of fixation at 60 ° C and 1.5 minutes of extension at 72 ° C, followed by a final extension cycle of 7 minutes at 72 ° C. The reactions were separated by electrophoresis on a GTG NuSieve 3% agarose gel (FMC Bioproducts, Rockland, ME.

Example 4 Creation of mammalian expression vectors Zsig37NEE / pZP9 and zsig37CEE / pZP9 Two expression vectors for the zsig37, zSIG37NEE / pZ P9 and zSIG37CEE / pZP9 polypeptide were prepared, where the constructs were designed to express a zsig37 polypeptide having a Glu-Glu or C- or N-terminal tag.

Zsig37NEE / pZP9 An AND fragment of zsig-37 generated by 800 bp PCR was created using ZC15040 (SEQ ID NO: 24) and ZC15033 (SEQ ID NO: 25) as PCR primers and the template described in example one above. The PCR reaction was incubated at 94 ° C, for 3 minutes, and then ran for 5 cycles of 94 ° C for 30 seconds, 30 ° C for 20 seconds and 72 ° C for 1 minute, followed by 25 cycles at 94 ° C. ° C for 30 seconds, 64 ° C for 20 seconds and 72 ° C for 1 minute. An extension of 5 minutes at 72 ° C followed. The resulting PCR product was run on a 0.9% TBE agarose gel with IX TBE buffer. A band of predicted size was excised and the DNA was purified from the gel with a Qiaex II resin (Qiagen) according to the manufacturer's instructions. The DNA was digested with the restriction enzymes of Bam HO and Xba I followed by extraction and precipitation. The cleaved, restriction-digested zsig37 DNA fragment was subcloned into the NEE / pZP9 plasmid that has been cut with the restriction enzymes Bam Hl and Xba I. The zsig37NEE / pZ P9 expression vector incorporates the TPA guide and binds to the Glu-Glu tag (SEQ ID NO: 26) to the n-terminus of the polynucleotide sequence encoding the zsig37 polypeptide. The NEE / pZ09 plasmid (deposited in the American Species Culture Collection, 12301 Parklawn Drive, Rockville, MD, ATCC No. 98668) is a mammalian expression vector that contains an expression cartridge that has a metallothionein-1 promoter. mouse, a TPA guide peptide followed by the sequence coding for the Glu-Glu tag (SEQ ID NO: 26), multiple reaction sites for the insertion of the coding sequences and a human growth hormone terminator. The plasmid also contains a origin of E col i for replication, a mammalian selectable marker expression unit having an SV40 promoter, enhancer and origin of replication, a DHRF gene and an SV40 terminator. zsig37CEE / pZP9 A DNA fragment of zsig37 generated by PCR, of 866 bp was created according to the procedure set forth above using ZC15721 (SEQ ID NO: 27) and ZC15035 (SEQ ID NO: 28) as the PCR primers. The purified PCR fragment was digested with the restriction enzymes Eco Ra and Bam Hl, gel purified using the Qiaex II resin as described above. The zsig37 DNA digested by restriction and cleaved was subcloned into the CEE / pZP9 plasmid which is cut with Eco Ri and Ban Hl. The zsig37CEE / pZ P9 expression vector uses a native zsig37 signal peptide, and the Glu epitope (SEQ ID NO.26) binds to the C-terminus as a purification aid plasmid CEE / pZP9 (deposited in the American collection Species Culture, 12301 Parkawn Drive, Rockville, MD, ATCC No. 98668) is a mammalian expression vector that contains an expression cartridge that has the expression promoter of mouse ionein-1 metalot, multiple restriction sites for the insertion of the coding sequence, a sequence coding for the Glu-Glu tag (SEQ ID NO.26), a terminator codon and a terminator of human growth hormone . The plasmid also has an origin of replication E. col i, a mammalian selectable marker, expression unit having an SV4.0 promoter, enhancer and origin of replication, a DHFR gene and an SV40 terminator. For the N- and C-labeled constructs, approximately 30 ng of the restriction-digested inserts and 50 ng of the corresponding vectors were ligated at room temperature for 4 hours. A microtiter of each ligation reaction was electrophoresed independently in competent DH10B cells (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions and placed on LB plates containing 50 mg / ml of ampicillin, and they incubated during the night. The colonies were detected by PCR as described above. For the detections of zsig37NEE / pZP9 and z s ig37CEE / pZ P9 the primers were ZC13006 (SEQ ID NO: 29) and ZC13007 (SEQ ID NO: 20) The PCR reaction was incubated at 94 ° C for 2.5 minutes, and then run for 25 cycles of 94 ° C for 10 seconds, 58 ° C for 2 0 seconds and 72 ° C for 1 minute. An extension was followed for 5 minutes at 72 ° C. The insert sequence of the positive clones, 1013 bp for zsig37NEE and a fragment of 950 bp for zsig37CEE / pZP9 were verified by sequence analysis. A large-scale plasmid preparation was made using a QUIAGEN Maxi prep. (Qiagen) according to the manufacturer's instructions.

Example 5 Transfection and Expression of zsig37NRR and CEE polypeptides BHF 570 cells (ATCC No. CRL-10314) were plated in 10 cm tissue culture dishes and grown to approximately 50 to 70% confluency overnight at 37 ° C, 5% CO2, in the DMEM / FBS medium (DMEM, Gibco / BRL) High Glucose, (Gibco BRL, Gaithersburg, MD), 5% fetal bovine serum (Hyclone, Logan, ET), 2 μM L-glutamine (JRH Biosciences, Lenexa , KS) 1 μM sodium pyruvate (Gibco BRL)). The cells were then transfected with the zsig37NEE / pZP9 plasmid (brand N-terminal Glu-Glu) or zsig37CEE / pZP9 (C-terminal Glu-Glu label) using lipofectamine MR (Gibco BRL), in serum free medium (SF) formulation (DMEM, Gibco / BRL, higher glucose, (Gibco BRL Gaithersburg, MD), L-glutamine, 2 mM 2 mM sodium pyruvate, 10 ug / ml transferrin, 5 μg / ml insulin, 10 ug / ml fetuin and 2 mg / ml selenium). Sixteen micrograms of zsig37NEE / pZP9 and 16 μg of zsig37CEE / pZP9 were separately diluted in 15 ml tubes to a total final volume of 640 μl of SF medium. In separate tubes, 35 μl of lipofectamineRM (Gibco BRL) was mixed with 605 μl of the SF medium. The lipofectamineRM mixture was added to the DNA mixture and allowed to incubate for approximately 30 minutes at room temperature. Five milliliters of the SF medium were added to the DNA mixture: lipofectamine MR The cells were rinsed once with 5 ml of the SF medium, aspirated and the DNA mixture: lipofectamine MR was added. The cells were incubated at 37 ° C for 5 hours, then 6 were added. 4 ml of DMEM / 10% FBS, 1% PSN medium to the plate. The plate was incubated at 37 ° C overnight and the DNA: lipofectamine ™ mixture was replaced with fresh FBS / DMEM medium, the next day. On day 2 after of the transfection the cells were divided in the selection medium (ESTEP # 1 with 1 μM MTX) in plates of 150 mm at 1:50, 1: 100 and 1: 200. The plates were burst on day 5 after transfection with fresh selection medium.

Detection colonies Approximately 10-12 days after transfection, a culture box of 150 mM of methotrexate-resistant colonies was chosen through each transfection, the media was aspirated, the plates were washed with 10 ml of serum-free ESTEP 2 medium ( 668.7g / 50L DMEM (Gibco), 5.5 g / 50 L of pyruvic acid, 96% sodium salt (Mallinckrodt), 185.0 g / 50L NaHC03, (Mallinkrodt), . 0 mg / ml, 25 ml / 50 L of insulin, 10.0 mg / ml and 25 ml / 50 L of trans ferrine). The washing medium was aspirated and replaced with 5 ml of serum-free ESTEP 2. A Sterile Teflon mesh (Spectrum Medical Industries, Los Angeles, CA) pre-moistened in serum free ESTEP 2 was then placed on the cells. A sterile nitrocellulose filter pre-moistened in serum free ESTEP 2 was then placed on the mesh. The orientation marks in The nitrocellulose was then transferred to the culture box. The plates were then incubated for 5-6 hours in a 5% C02 incubator at 37 ° C. After incubation, the filter was removed, and the medium was aspirated and replaced with the DMEM / 5% FBS medium, IX PSN (Gibco BRL). The filter was then placed in a sealable bag containing 50 ml of buffer (25 mM Tris, 25 mM glycine, 5 mM β-mercaptotanol) and incubated in a 65 ° C water bath for 10 minutes. The filters were blocked in 10% non-fat dry milk / Western A buffer (Western A: 50 mM Tris, pH 7.4, 5mM EDTA, 0.05% NP-40, 150mM NaCl and 0.25% gelatine) for 15 minutes at room temperature on a 'rotary shaker. The filter was then incubated with an anti-Glu-Glu HRP antibody conjugate at a dilution of 1: 1000 in 2.5% fat-free dry milk / Western A buffer (Western A: 50 mM Tris, pH 74., 5 Mm EDTA , NP-40 at 0.05%, 150 mM NaCl and 0.25% gelatin) overnight at 4 ° C on a rotary shaker. The filter was then washed three times at room temperature in PBS plus Tween 20, at 0.1%, 5-15 minutes per wash. The filter was developed with ECL reagent (Amersham Copr., Arington Heights, IL) according to the manufacturer's instructions. exposed to the movie (Hyperfilm ECL, Amersham) for about 5 minutes. The film was aligned with the plates containing the colonies. Using the film as a guide, suitable colonies were selected. Sterile 3 mm colonization discs (PGC Scientific Coprp., Frederick, MD) were soaked in trypsin, and placed in the colonies. Twelve colonies for each construct were transferred in 200 μl of the selection medium in a 96-well plate. A series of dilutions of seven ten-fold dilutions were carried out for each colony. Cells were cultured for a week at 37 ° C at which time the wells that had received the lowest dilution of cells that are now at the optimum density were screened, trypsinized and transferred to a plate of twelve wells containing medium of selection. The 150 mm culture box was also tripded and the rest of the cells mixed and subjected to Western analysis and a microplasma test. The mixture was frozen for storage. The clones were extended directly from the 12-well plate in two T-75 flasks. A flask was maintained to continue growth cell, the second flask was cultured in serum free ESTEP 2 that was harvested from the Western blot analysis. The clones of each of the expression constructs, based on the Western blot analysis, were selected, mixed and transferred to a large scale culture.

EXAMPLE 7 Large scale mammal expression of zsig37CEE A T-162 flask, containing confluent cells expressing zsig37CEE and one containing zsig37NEE obtained from the expression procedure described above, were expanded into four T-162 flasks each. One of the four resulting flasks was used to freeze four cryo-flasks, and the other three flasks were used to generate a Nunc cell factory. The cells of the three T-162 flasks of zsig37CEE and zsig37NEE were used to independently seed two Nunc cell factories (10 layers, commercially available from VWR). Briefly, the cells of the T-162 flasks described above were separated using trypsin, were mixed and added to the medium ESTEP 1 of 1.5 liters (66.8.7g / 50L DMEM (Gibco), . 5 / 50L of pyruvic acid, 96% sodium salt (Mallinckrodt), 185.0 g / 50L of NaHCO3 (Mallinkrodt), 5.0mg / ml and 25mL / 50L of insulin (JRH Biosciences), 10.0 mg / ml and 25 ml / 50L of transferrin (JRH Biosciences) 2.5L / 50L fetal bovine serum (characterized) (Hyclone), 1 μM MTX, with pH adjusted to 7.05 +/- 0.05) preheated to 37 ° C. The media containing the cells were then mixed in the Nunc cell factories via a funnel. The cell factories were placed in a 37 ° C / 5.0% C02 incubator plate. At a confluence of 80-100%, a visual contamination test (phenol red color change) was performed on the contents of the Nunc cell factories). Since no contamination was observed, the supernatant from the confluent factories was mixed in a small collection vessel, sampled and discarded. The adherent cells were then washed once with 400 ml of PBS. To separate the cells from the factories, 100 ml of trypsin was added to each and they were removed and the cells were then incubated for 5 to 10 minutes in residual trypsin. The cells are they were collected following two 200 ml washes with the ESTEP 1 medium. To each of the ten bottles containing the ESTEP 1 medium (1.5 liters each, at 37 ° C) were added 40 ml of the collected cells. A 1.5-liter bottle was then used to refill a Nunc factory. Each cell factory was collocated in an incubator of 37 ° C / 5.0% Co2- At a confluence of 80-90%, a visual contamination test (phenol red color change) was performed in the Nunc cell factories . Since no contamination was observed, the supernatant from the confluent factories was poured into a small collection container, it was sampled and discarded. The cells were then washed once with 4,000 mg of PBS. 1.5 liters of ESTEP 2 medium (668.7g / 50L DMEM (Gibco), 5.5 g / 50L of pyruvic acid, 96% sodium salt (Mallinckrodt), 185.0 g / 50L of NaHCO3 (Mallinkrodt), 5.0 mg / ml, were added. ml / 50 L of insulin, 10.0 mg / ml and 25 ml / 50 L of transferrin) to each Nunc cell factory. The cell factories were incubated at 37 ° C / 5% C02. At approximately 48 hours, a visual contamination test (phenol red color change) was performed in the Nunc cell factories.

Supernatant from each factory was poured into the small collection containers. Fresh serum-free media (1.5 liters) were poured into each Nunc cell factory, and the factories were incubated at 37 ° C / 5.0% C02. One ml of the supernatant collection for each construct was transferred to a microscope slide, and subjected for microscopic analysis for contamination. The contents of the small collection containers for each construction were mixed and filtered immediately. A second harvest was then performed, substantially as described earlier at 48 hours and the cell factories were subsequently discarded. An aseptically assembled filter series apparatus was used for the aseptic filtration of the harvest supernatant (conditioning medium). The assembly was as follows: the pipe was tied with wire from an Opti-Cap filter (Millipore Corp., Bedford, MA) and a Gelman Supercap 50 filter (Gelman Sciences, Ann Arbor, MI). The Supercap 50 filter was also attached to a capped, sterile container located in a hood; the tubing located upstream of the Millipore Opti-cap filter was inserted into a pump peristaltic and the free end of the pipe was placed in the large collection vessel. The peristaltic pump was run between 200 and 300 rpm, until all the conditioned medium passed through the final 0.22 μm filter in a sterile collection vessel. The filtrate was placed in a cold room pending purification at 4 ° C. The 10X concentrated medium with 5 kDa cut-off concentration (Millipore Corp., Bedford, MA) according to the manufacturer's instructions was subjected to the ransfer analysis. Western using an anti-FLAG antibody (Kodak).

Zsig37CEE 5 flasks T-162 = 0.12 mg / 1, 38 kDa: 1 Factory, FBS = 0.12 mg / L, 38 kDa; 10 Factories, FBS = 0.12 mg / L, 38 kDa; 10 Factories (# 1), SF = 1.2 mg / L, 38 kDa; and 10 Factories (# 2), SF = 3.56 mg / L, 38 kDa Zsig37NEE 5-flasks T-162 = 0.137 mg / L, 35 kDa; 1 Factory, FBS = 0.137 mg / L, 35 kDa; 10 Factories, FBS = 0.137 mg / L, 35 kDa; 10 Factories (# 1), SF = 1.37 mg / L, 35 kDa; Y Factories (# 2), SF = 4.11 mg / L, 35 kDa Example 7 Purification of zsig37 NEE and zsig37 CEE Unless otherwise noted, all operations were carried out at 41 C. The following procedure was used to purify zsig37 containing N-terminal or C-terminal Glu-Glu (EE) tags. A total of 25 liters of conditioned medium from unborn hamster kidney cells (BHK) was sequentially sterile filtered through a 0.2 mM Millipore capsule filter (Bedford MA). Opticap and a 0.2 mM Supercap 50. (Ann Arbor, MI) The material was then concentrated to approximately 1.3 liters using a Míllipore ProFlux A30 tangential flow concentrator equipped with a membrane S10Y3 A cut icon of 3,000 kDa (Bedford, Ma) The concentrated material was again sterile filtered with the Gelman filter as described above. A mixture of protease inhibitors was added to conditioned media, final concentrates of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co., St. Louis, MO), 0.001 mM leupeptin.

(Boehringer-Mannheim, Indianapolis, IN), 0.001 M pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). A sample of 25.0 ml of anti-SE Sepharose, prepared as described below, was added to the sample for batch absorption and the mixture was gently stirred on a Wheaton roller culture apparatus (Millville, NJ) for 18.0 hours at 4 ° C. The mixture was then poured into an Econo 5.0 x 20.9 column (Bio-Rad, Laboratories, Hercules, CA) and the gel was washed with 30 column volumes of phosphate buffered saline (PBS). The non-retained flow fraction was discarded. Once the absorbance other than 280 nM is less than 0.05, the flow through the column was reduced to zero and the anti-SE sepharose gel was washed, as batches with 2.0 column volumes of PBS containing 0.4 mg / ml peptide EE (AnaSpec, San José, CA). The peptide used had the sequence Glu-Tyr-met-Pro-Val-Asp, SEQ ID NO: 31). After 1.0 hours at 4 ° C, the flow was resumed and the eluded protein was collected. This fraction was referred to as the peptide elution. The anti-EE Sepharose gel was then washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and the glycine wash was collected separately. The pH of the fraction eluted with glycine was adjusted to 7.0 by the addition of a small volume of 10X PBS and stored at 4 ° C for further analysis, if necessary. The peptide elution was concentrated to 5.0 ml using a 15,000 molecular weight cut-off membrane concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions. The concentrated peptide elution was separated from the free peptide by chromatography on a 1.50 x 50 cm sephadex G-50 column (Pharmacia, Piscataway, NJ) in PSB at a flow rate of 1.5 / min using BioCad Sprint HPLC (PerSeptive BioSystems , Framingham, MA). Two-ml fractions were collected in the absorbance 280 nM was monitored. The first peak of the absorbing material at 280 nM and elution near the hollow volume of the column was collected. This fraction was NEE zsig37 pure or CEE zsig37. Pure material was concentrated as described above, analyzed by SDS-PAGE and Western blotting with anti-EE antibodies, and samples were taken for amino acid analysis and N-terminal sequencing. The rest of the sample was formed in aliquot, and stored at minus 80 ° C according to normal procedures. SECT electrophoresis of zsig37 on SDS-PAGE gels in the absence of reducing agents showed a major band stained with Cooie blue of apparent molecular weight of 39,000 and several minor components of molecular weights between 60,000 and 116,000. All bands showed cross-reactivity with anti-EE antibodies in Western blots. In the presence of the reducing agent, the only band observed was the 39,000 kDa protein and its intensity of tension with Coomassie blue increased. This band also showed cross-reactivity with the anti-EE antibody in Western blots. For CEE zsig37, gel electrophoresis of SDS-PAGE in the absence of reducing agents showed a major band stained with Coomassie blue of apparent molecular weight of 39,000 and several minor molecular weight components between 60,000 and 116,000. In the Western blots, only bands of apparent molecular weights of 150,000, 116,000 and 60,000 showed cross-activity with the anti-EE antibody. In the presence of reducing agents, only the band stained with blue Coomassie at 39,000 kDa was observed and this material showed cross-activity with the anti-EE antibody in Western blots. Under these conditions, a small amount of cross-reactivity material at 150,000 kDa was also seen.

Preparation of Safarosa anti-EE A 100 ml bed volume of the G-safarose protein (Pharmacia, Poiscataway, NJ) was washed three times with 100 ml of PBS containing 0.2% sodium azide using a 0.45 micron filter unit of 500 ml Nalgene. The gel was washed with 6.0 volumes of 200 mM triethanolamine. (TEA, Signa, St. Louis, MO), and an equal volume of the antibody EE containing 900 mg of antibody was added. After an overnight incubation at 4 ° C, the unbound antibody was removed by washing the resin with 5 volumes of 200 mM TEA as described above. The resin was redispersed in 2 volumes of TEA, transferred to a suitable vessel, and dimethyl-pillimidate-2HCl (Pierce, Rockford, IL), dissolved in TEA, was added to a final concentration of 36 mg / ml gel. The gel was rotated at room temperature for 45 minutes and the liquid was removed using the filter unit as described above. The nonspecific sites in the gel were then blocked by incubating for 10 minutes at room temperature with 5 volumes of ethanolamine in 20 mM in 200 mM TEA. The gel was washed with 5 volumes of PBS containing 0.2% sodium azide and stored in this solution at 4 ° C.

Example 8 Adhesion and Proliferation Tests The ability of zsig37 to stimulate adhesion and extension of TF-1 cells was assessed as follows. A series of dilutions were prepared from zsig37 labeled with C-terminal Glu-Glu from 10 to 0.625 μg / ml, in either buffer of PSA coating and ELISA (0.1 M NaC03) and each was placed in a 96-well plate (Costar, Pleasanton, CA) at 100 μl / well. Plates were incubated at 37 ° C, 5% C02 for 2 hours. The plates were then washed 3X with RPMI / 10% FBS (RPMI 1640, 2mM L-glutamine, 110μg / ml sodium pyruvate, PSN and 10% heat inactivated fetal bovine serum) and allowed to block for 15 minutes .

TF-1 cells (derived from acute myeloid leukemia cells) were redispersed in 10% RPMI / FBS and plated at 10,000 cells / well in 96-well plates coated with zsig37 to a final volume of 120 μl / water well. The plate was incubated under 5% C02 for 2 hours. The plates were then washed 3X with PBS. 200 μl / well of growth medium (RPMI / 10% FBS 5ng / ml GM-CSF) was added. The cells were inspected microscopically before and after washing. A dye incorporation assay was also used to quantitatively measure the number of adherent cells based on a colorimetric change an increase in the TM fluorescence signal. Alamar blue (AccuMed, Chicago, IL) was added to the 96-well plates and the cells were then incubated at 37 ° C under 5% CO. The plates were then screened using a flurometer with an excitation wavelength of 544 nm and an emission wavelength of 590 nm. There were no adherent cells in the plates coated with zsig37CEE-PBS than in the plates coated with zsig37CEE-0.1 M NaC03. The addition of soluble zsig37 does not block the adhesion of cells to the bound zsig37.

A second trial was made using TF-1, DA-1 (an IL-3-dependent cell line derived from the lymphatic modules of a mouse with a B cell lymphoma by growth in the medium and L-3 (provided by Dr Kenneth Kaushansky, University of Washington, Seattle, WA)), pre-B mouse marrow cells (p53 - / - IL-7 dependent, B220 +, thil low, Sca-1 +), and A7BaF cell lines -3 as described previously to 5.00 cells / well. BHK cells were also plated at 500 cells / well. Zsig37 improved the growth of A7-BaF-3 cells and slightly inhibited the growth of DA-1 cells.

Example 9 Mouse orthologous sequence The polynucleotides encoding the human zsig37 polypeptide of the present invention were used to detect a mouse EST database for the homologous mouse sequences. An individual sequence of ESTs was described and predicted to the human zsig37 sequence. To identify the corresponding cDNA, a clone considered likely to contain the sequences of Complete coding or use for sequencing. TM Using an Invitrogen S.N.A.P. Miniprep kit (Invitrogen Corp.) according to manufacturer's instructions 5ml overnight culture in LB + 50 μg / ml ampicn was prepared. The template was sequenced in a DNA sequencer model 377 TM ABIPRISM (Perkm-Elmer Cetus, Norwalk, CT) using the ABIPRISM ready-to-use large dye determination cycle sequencing reaction set (Per in-Elmer Corp). according to the manufacturer's instructions. The oligonucleotides ZC694 (SEQ ID NO: 6), ZC6768 (SEQ ID NO: 32), ZC18297 (SEQ ID NO: 33), SEQ ID NO: 36), ZC18456 (SEQ ID NO: 37), ZC18457) (SEQ ID NO: 38), ZC18560 (SEQ ID NO: 39), ZC18561 (SEQ ID NO: 40), ZC18687 (SEQ ID NO: 41) and ZC18688 (SEQ ID NO: 42) were used to complete the clone sequence. The sequencing reactions were carried out in a Hybadi OmniGene temperature cyclization system (National Labnet Co., Woodbridge, NY). The sequence analysis computation program SEQUENCHERMR DE 3.1 (Gene Codes Corporation, An Arbor, MI) was used for data analysis. The sequence of the resulting 2559 bp is described in SE ID NO: 43 and the reduced amino acid sequence in sequence SEQ ID NO: 4 4. The Alignment with the nucleotide sequence zsig37 (SEQ ID NO: 1) shows 77% identity at the nucleotide level. The putative amino acid sequences (SE ID NO: 44) have 77% identity to the human polypeptide sequence (SEQ ID NO: 2).

Example 10 Cell-based assays The zsig37 polypeptides were evaluated in a high production in vitro assay to identify substances that selectively activate cell responses in immortalized osteoblast cell lines. A line of mature osteoblast cells derived from p53 - / - (deficient) mice, CCC4, which is transfected with an acid containing inducible serum response element (SRE) that drives the expression of luciferase was used in the assay. These cells also express exogenous PTH, receptors of PDG and bFGF. The stimulation of the BE and in this way the expression of luciferaza in the CCC4 cells indicates that the chemical entity is probably going to stimulate the mitogenesis in the osteobastos.

The CCC4 lines were trypsinized and adjusted to 5 x 10 4 cells / ral in plaque medium (alpha-MEM, 1% heat-inactivated fetal bovine serum, 1 mM Na pyruvate and 2 mM L-glutamate) and placed in plate (200 μl / well in opaque white microtiter plate Dinatech, Microlite (Dynatech, Chantilly, VA) and incubated overnight at 37 ° C. 5% C02.The growth medium was then aspirated and delayed with 50 μl / well of the assay medium (HAM of F-12, 0.5% bovine serum albumin HEPES 20 mM 1 mM Na pyruvate and 2 mM L-glutamate) Serial dilutions of zsig37 were made in the assay medium ( 0.29-1000 ng / ml final test concentration) and were added to the wells.Zsig37 samples were titrated in triplicate.Ceric (negative) and bFGF (positive) controls were also used.The final concentration of bFGF was 3ng / ml The controls were assessed in quadruplicate.The plates were incubated for 4 hours at 37 ° C, 5% C02. He aspirated and the plates were rinsed once with PBS. 25 μl of the lysis buffer (Luciferase Assay Reagent, E1501, Promega Corp., Madison, Wl) was added to each well. The plates were incubated for 15 minutes at room temperature ambient. Fifty microliters / well of Luciferaza substrate (Luciferase Assay Regent, R1501, Promega Corp. = Was added in the luciferaza activity was detected using a LUMINOSKAN Labsystems at 2 seconds / well following a delay of 7 seconds. not induced) was subtracted from all the readings that are expressed in table 5 as a percentage of the maximum induction produced by 3 ng / ml of bFGF.Zsig37 stimulates the expression of luciferase in this assay indicating that it stimulates osteoblasts. zsig37 stimulates a maximum of 73 to 75% at 1000 ng / ml.An imitation assay of the opposite growth factor was performed to determine if zsig37 is acting as an imitator to the growth factor, particularly the receptor ligands of tyrosine kinase PDGF, bFGF and EGF (negative to insulin R) A clonal cell line derived from Swiss 3T3 mice, which is transfected with a plasmid that contains an element of serum response induc ible (SER) that drives the expression of luciferaza that was used in the trial. These cells also express endogenous receptors of PMA, EGF and bFGF. The stimulation of the BE and of this way the expression of luciferaza in the Swiss 3T3 cells indicates that the chemical entity probably mimics the activity of the growth factor PGDF, bFGF and EGF. The Swiss 3T3 cells were tripded and adjusted to 5 x 10 4 cells / ml in the plaque-placing medium, plated and incubated as described above. The growth medium is then aspirated and replaced as 50 μl / well of assay medium (F-12 HAM, 0.5% bovine serum albumin, HEPES mM). Serial assay dilutions of zsig37 were made in the assay medium (0.29-1000 ng / ml final test concentration) and added to the wells. Samples of zsig37 were evaluated in triplicate. A ceric (negative) and bFGF (positive) control to promote cell proliferation was also used. The final concentration of bFGF was 3 ng / ml. The controls were tested in quadruplicate. The plates were incubated for 5 hours at 37 ° C, 5% Co2. The assay medium was then aspirated and the plates rinsed once with PBS. 25 μl of the lysis buffer (Luciferase Assay Reagent, E1501, Promega Corp., Madison, Wl) was added to each well. The plates are incubated for 15 minutes at room temperature. Forty microliters / well of Luciferaza substrate (Luciferase Assay Reagent, E1501, Promega Corp.) were added and the luciferase activity was detected using a LUMINOSKAN® Labsystems at 2 seconds / well after a delay of one second. The average (uninduced) baseline signal was subtracted from all readings that are expressed as a percentage of the maximum induction produced by 3 ng / ml of bFGF. A 5 hour treatment of this cell line with bFGF, DDGF, EGF or PMA leads to a 25-50 fold induction of SRE-luciferase expression. Zsig37 does not appear to stimulate the expression of luciferaza in this assay. Zsig37 stimulates 0.2 to 0.1% maximum at 1000 ng / ml.

Example 10 In vivo administration of zsig37 via adenoviral distribution Twenty-four mice and 24 female C57B16 / J, approximately 12 week old (Jackson Labs, Bar Harbor, ME) were weighed, body temperature was measured and meal ingestion was monitored daily for 4 days prior to injection (days -4 to -1). On day O, the mice were divided into 3 groups and received 0.1 ml of virus (1.8 x 10 11 particles of AdV-vací virus / .O. Ml or 5 x 1011 particles of AdV-zsig37-CEE virus / 0.1 ml) by intravenous injection into the vein of the tail, or no injection at all. The injection should result in infection of the host's liver and the expression of the slightly distributed gene should begin in the space of 24 hours and continue for 1 to 4 weeks. Three groups of mice were tested. Group 1 untreated, n = 8, each male and female. Group 2, empty of AdV (empty virus) n = 8 each male and female. Group 3, adVzsig37 CEE, n = 8 each male and female. The body temperatures, animal weights and the weight of the ingested food were monitored during the 3 weeks of the study. No difference was found between the groups. On day 21, the female mice were euthanized and sacrificed by cervical dislocation, and on day 22, the males were made. The animals had their blood drawn and the tissues collected for necropsy. The normal serum chemistry panel was made at the time of sacrifice. The parameters metabolic, kidney, and liver were all within normal ranges. However, there was a difference between the treatment group of the zsig37 group of the group treated with the empty virus. The zsig37 animals had a higher average lipometric index than the controls with the empty virus. The difference was not significant, however, additional research was guaranteed. Three total fatty acids were evaluated in the remaining serum of each animal. A statistically significant difference in serum free fatty acid levels was seen between the male mice (p = 0.0379) that receive virus and those that receive the virus coding for zsig37; zsig37 mice have higher levels. A difference, although not statistically significant, was also seen in the females (p = 0.3357). The liver, spleen, kidney, thymus, heart and cells were weighed after the removal. No difference was found between the treatment groups. The histopathological analysis of these tissues and bone marrow revealed no difference between the treatment groups. To confirm the previous results, a second detection was made as before with the following modifications. Three groups; year treated and fasting, b) no AdV and fasting, c) AdV-zsig37-CEE and fasting, containing 20 C57B16 / J, 10 males and females were tested. The mice fasted overnight and 100 μl of serum was collected to establish a basal level for the following parameters: fasting glucose, TO, alkaline phosphatase, cholesterol, triglycerides, free fatty acids and insulin. The body weights were taken three times a week. On day 0, the mice were injected into the lateral vein with 0.1 ml of the appropriate virus solution. Blood was collected on day 17 after fasting overnight. After 3 weeks, the mice were sacrificed and all the blood was collected. A portion of the blood was mixed with EDTA to hold the CBC and the rest was retested and detected as described above. The organs were collected and the bodies were saved for his opathology. From the foregoing, it is considered that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit of the scope of the invention. invention. Accordingly, the invention is not limited except by the appended claims.

LIST OF SEQUENCES < 110 > ZymoGenetics. Inc < 120 > SPECIFIC PROTEIN HOMOLOGOUS ADIPOCYTES < 130 > 97-30PC < 150 > 60 / 053,154 < 151 > 1997-07-18 < 160 > 44 < 170 FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 2769 < 212 > DNA < 213 > Homo sapien < 220 > < 221 > CDS < 222 > (171), (1013) 400 > 1 gaattcgaat tcctttgttt ccactgggac ggaatcggag ctctggaggc tgggctggcc 60 aagcgccccg aaggcccgat gcctgacggc tcatgcggcc tccttgtttg cagggcctgg 120 gcaaaaattt acactgagtc ccactcttcg ctccagggcc cggcaggaag atg ggc 176 Met Gly 1 aka cgt gga cag gga etc ttg ctg gcg tac tgc ctg etc ctt gee ttt 224 Ser Arg Gly Gln Gly Leu Leu Leu Ala Tyr Cys Leu Leu Leu Ala Phe 5 10 * • 15 gee tet ggc ctg gtc ctg agt cgc gtg ecc cat gtc cag ggg gaa cag 272 Wing Being Gly Leu Val Leu Being Arg Val Pro His Val Gln Gly Glu Gln 20 25 30 cag gag tgg gag ggg act gag gag ctg ceg tcc ect ceg gac cat gee 320 Gln Glu Trp Glu Gly Thr Glu Glu Leu Pro Pro Pro Asp His Wing 35 40 45 50 gag agg gct gaa gaa caa cat gaa aaa tac agg ccc agt cag gac cag 368 Glu Arg Wing Glu Glu Gln His Glu Lys Tyr Arg Pro Ser Gln Asp Glp 55 60 65 ggg etc ect gct tcc cgg tgc ttg cgc tgc tgt gac ect ggt acc tcc 416 Gly Leu Pro Wing Being Arg Cys Leu Arg Cys Cys Asp Pro Gly Thr Ser 70 75 80 atg tac cec gcg acc gee gtg ccc cag atc aac atc act atc ttg aaa 464 Met Tyr Pro Ala Thr Ala Val Pro Gn Ilt Asn He Thr He Leu Lys 85 90 95 ggg gag aag ggt gac cgc gga gat cga ggc etc ca ggg aaa tat ggc "512 Gly Glu Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln Gly Lys Tyr Gly 100 105 110 aaa here ggc tea gca ggg gee agg ggc falls act gga ccc aaa ggg cag 560 Lys Thr Gly Be Wing Gly Wing Arg Gly His Thr Gly Pro Lys Gly Gln 115 120 125 130 aag ggc tcc atg ggg gee ect ggg gag cgg tgc aag age falls tac gee 608 Lys Gly Ser Met Gly Wing Pro Gly Glu Arg Cys Lys Ser His Tyr Wing 135 140 145 gee ttt teg gg ggc cgg aag aag ccc atg falls age aac falls tac tac 656 Wing Phe Ser Val Gly Arg Lys Lys Pro Met His Ser Asn His Tyr Tyr 150 155 160 cag acg gtg atc tcc gac acg gag ttc gtg aac etc tac gac falls ttc 704 Gln Thr Val He Phe Asp Thr Glu Phe Val Asn Leu Tyr Asp His Phe 165 170 175 aac atc tcc acc ggc aag tcc tac tcc tac gcc ccc gcc etc tac tcc 752 Asn Met Phe Thr Gly Lys Phe Tyr Tys Tyr Val Pro Gly Leu Tyr Phe 180 185 190 ttc age etc aac gtg falls acc tgg aac cag aag gag acc tac ctg falls 800 Phe Ser Leu Asn Val His Thr Trp Asn Gln Lys Glu Thr Tyr Leu His 195 200 205 210 atc atg aag aac gag gag gag gtg gtg atc ttg ttc gcg cag gtg ggc 848 lie Met Lys Asn Glu Glu Glu Val Val He Leu Phe Ala Gln Val Gly 215 220 225 gac cgc age atg atg cag age cag age ctg atg ctg gag ctg cga gag 896 Asp Arg Ser He Met Gln Ser Gln Ser Leu Met Leu Glu Leu Arg Glu 230 235 240 cag gac cag gtg tgg gta cgc etc tac aag ggc gaa cgt gag aac gee 944 Gln Asp Gln Val Trp Val Arg Leu Tyr Lys Gly Glu Arg Glu Asn Wing 245 250 255 ate ttc age gag gag ctg gac acc tac atc acc ttc agt ggc tac ctg 992 He Phe Ser Glu Glu Leu Asp Thr Tyr He Thr Phe Ser Gly Tyr Leu 260 265 270 gtc aag falls gee acc gag ccc tagctggccg gccacctcct ttcctctcgc 1043 Val Lys His Wing Thr Glu Pro 275 280 caccttccac ccctgcgctg tgctgacccc agggctcagc accaggctga ccccaccgcc 1103 tcttccccga tccctggact ccgactccct ggctttggca ttcagtgaga cgccctgcac 1163 acacagaaag ccaaagcgat cggtgctccc agatcccgca gcctctggag agagctgacg 1223 gcagatgaaa tcaccagggc ggggcacccg cgagaaccct ctgggacctt ccgcggccct 1283 ctctgcacac ateetcaagt gaccccgcac ggcgagacgc gggtggcggc agggcgtccc 1343 agggtgcggc accgcggctc cagtccttgg aaataattag gcaaattcta aaggtctcaa 1403 aaggagcaaa gtaaaccgtg gaggacaaag aaaagggttg ttatttttgt ctttccagcc 1463 agcctgctgg ctcccaagag agaggccttt tcagttgaga ctctgcttaa gagaagatec 1523 aaagttaaag ctctggggtc aggggagggg ccgggggca g gaaactaect ctggcttaat 1583 tcttttaagc cacgtaggaa ctttcttgag ggataggtgg accctgacat ccctgtggcc 1643 ttgcccaagg gctctgctgg tctttctgag tcacagctgc gaggtgatgg gggctggggc 1703 cccaggcgtc agcctcccag agggacagct gagccccctg ccttggctcc aggttggtag 1763 aagcagccga agggctcctg acagtggcca gggaeccctg ggtcccccag gcctgcagat 1823 gtttctatga ggggcagagc tcctggtaca tccatgtgtg gctctgctcc acccctgtgc 1883 caccccagag ccctgggggg tggtctccat gcctgccacc ctggcatcgg ctttctgtgc 1943 cgcctcccac acaaatcage cccagaaggc cccggggctt tggcttctgt tttttataaa 2003 cagcactgca acacctcaag gtctcccatc tcctcgtggg etaageatca ccgcttccac 2063 ttggttggca gtgtgttgtg gcaaggctga tccagacccc ttctgccccc actgccctca 2123 tccaggcctc tgaccagtag cctgagaggg * gctttttcta ggcttcagag caggggagag 2183 ctggaagggg etagaaaget cccgcttgtc tgtttctcag gctcctgtga gcctcagtcc 2243 gtcaagagga tgagaccaga agtacacatc ceaateaccc gtgtcaggat tcactctcag 2303 gagctgggtg gcaggagagg caatagcccc tgtggcaatt gcaggaccag ctggagcagg 2363 gttgcggtgt ctccgcggtg ctctcgccct gcccatggcc accccagact ctgatctcca 2423 ggaaccccat agcccctctc cacctcaccc catgttgatg cccagggtca ctcttgctac 2483 ccgctgggcc cccaaacccc cgctgcctct cttccttccc cccatccccc acctggtttt 2543 gactaatect gcttccctct ctgggcctgg ctgccgggat ctggggtccc taagtccctc 2603 tctttaaaga acttctgcgg gtcagactct gaagccgagt tgctgtgggc gtgcccggaa 2663 gcagagcgcc acactcgctg cttaa gctcc cccapctctt tccagaaaac attaaaetca 2723 gaattgtgtt ttcagcaaaa aaaaaaaaaa aaaaaagggc ggccgc 2769 < 210 > 2 < 211 > 281 < 212 > PRT < 213 > Homo sapien 400 > 2 Met Gly Ser Arg Gly Gln Gly Leu Leu Leu Wing Tyr Cys Leu Leu Leu 1 5 10 15 Ala Phe Ala Ser Gly Leu Val Leu Ser Arg Val Pro His Val Gln Gly 25 30 Glu Gln Gln Glu Trp Glu Gly Thr Glu Glu Glu Leu Pro Ser Pro Pro Asp 40 45 His Wing Glu Arg Wing Glu Glu Gln His Glu Lys Tyr Arg Pro Ser Gn 50 55 60 Asp Gln Gly Leu Pro Wing Being Arg Cys Leu Arg Cys Cys Asp Pro Gly 65 70 75 80 Thr Ser Met Tyr Pro Wing Thr Wing Val Pro Gln He Asn He Thr He 85 90 95 Leu Lys Gly Glu Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln Gly Lys 100 105 110 Tyr Gly Lys Thr Gly Ser Wing Gly Wing Arg Gly His Thr Gly Pro Lys 115 120 125 Gly Gln Lys Gly Ser Met Gly Ala Pro Gly Glu Arg Cys Lys Ser His 130 135 140 Tyr Ala Ala Phe Ser Val Gly Arg Lys Lys Pro Met His Ser Asn His 145 150 155 160 Tyr Tyr Gln Thr Val He Phe Asp Thr Glu Phe Val Asn Leu Tyr Asp 165 170 175 His Phe Asn Met Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro Gly Leu 180 185 190 Tyr Phe Phe Ser Leu Asn Val His Thr Trp Asn Gln Lys Glu Thr Tyr 195 200 205 Leu His He Met Lys Asn Glu Glu Glu Val Val He Leu Phe Ala Gln 210 215 220 Val Gly Asp Arg Ser He Met Gln Ser Gln Ser Leu Met Leu Glu Leu 225 230 235 240 Arg Glu Gln Asp Gln Val Trp Val Arg Leu Tyr Lys Gly Glu Arg Glu 245 250 255 Asn Ala He Phe Ser Glu Glu Leu Asp Thr Tyr He Thr Phe Ser Gly 260 265 270 Tyr Leu Val Lys His Ala Thr Glu Pro 275 280 < 210 > 3 < 211 > 247 < 212 > PRT < 213 > Homo sapien < 400 > 3 Met Leu Leu Leu Gln Leu Leu Leu Phe Leu Leu He Leu Pro Ser His "1 5 10 15 Wing Glu Asp Asp Val Thr Thr Thr Glu Glu Leu Ala Pro Wing Leu Val 25 30 Pro Pro Pro Lys Gly Thr Cys Wing Gly Trp Met Wing Gly He Pro Gly 40 45 His Pro Gly His Asn Gly Thr Pro Gly Arg Asp Gly Arg Asp Gly Thr 50 55 60 Pro Gly Glu Lys Gly Glu Lys Gly Asp Wing Gly Leu Leu Gly Pro Lys 65 70 75 80 Gly Glu Thr Gly Asp Val Gly Met Thr Glv Wing Glu Gly Pro Arg Gly 85 90 95 Phe Pro Gln Thr Pro Gly Arg Lys Gly Glu Pro Gly Glu Wing Ala Tyr 100 105 110 Met Tyr Arg Ser Wing Phe Ser Val Gly Leu Glu Thr Arg Val Thr Val 115 120 125 Pro Asn Val Pro He Arg Phe Thr Lys He Phe Tyr Asn Gln Gln Asn 130 135 140 His Tyr Asp Gly Ser Thr Gly Lys Phe Tyr Cys Asn He Pro Gly Leu 145 150 155 160 Tyr Tyr Phe Ser Tyr His He Thr Val Tyr Met Lys Asp Val Lys Val 165 170 175 Ser Leu Phe Lys Lys Asp Lys Wing Val Leu Phe Thr Tyr Asp Gln Tyr 180 185 190 Gln Glu Lys Asn Val Asp Gln Wing Ser Gly Ser Val Leu Leu His Leu 195 200 205 Glu Val Gly Asp Gln Val Trp Leu Gn V; "1 Tyr Gly Asp Gly Asp His 210 215 220 Asn Gly Leu Tyr Wing Asp Asn Val Asn Asp Ser Thr Phe Thr Gly Phe 225 230 *. 235 240 Leu Leu Tyr His Asp Thr Asn 245 < 210 > 4 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide 2C12447 < 400 > 4 atggggcacg cgactcagga ccaggccaga 30 < 210 > 5 < 211 > 19 < 212 * > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC695 < 400 > 5 gattataggtg acactatag - * 19 < 210 > 6 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Ol igonucleotide ZC694 < 400 > 6 taatacgact cactataggg 20 < 210 > 7 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13210 < 400 > 7 aagcaccggg aagcagggag 20 < 210 > 8 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13588 < 400 8 cgggcacgta gcagtagaac 20 < 210 > 9 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13532 < 400 > 9 gagagggctg aagaacaaca 20 < 210 > 10 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Ol gonucleotide ZC13641 < 400 > 10 aaggtggcga gaggaaagga 20 < 210 > 11 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13586 < 400 > 11 tgttcaccgg caagttctac 20 < 210 > 12 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13651 < 400 > 12 ctttgtcctc cacggtttac 20 < 210 > 13 < 211 > 20 < 212 > DNA < 213 Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13622 < 400 > 13 tttcctctcg ccaccttcca 20 < 210 > 14 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 Oligonucleotide ZC13625 400 > 14 cttcggctgc ttctaaccaa c 21 < 210 > 15 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13650 < 400 > 15 gtaaaccgtg gaggacaaag 20 < 210 > 16 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13859 < 400 > 16 gctgccaacc aacacaacca c 21 < 210 > 17 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13624 < 400 > 17 gcaggattag tcaaaacc 18 < 210 > 18 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonuceotide ZC13531 < 400 > 18 aacatggggt gaggtggaga 20 < 210 > 19 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13587 < 400 > 19 tcctcgtggg ctaagcatca 20 < 210 > 20 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13623 < 400 > 20 atctccagga accccatagc 20 < 210 > 21 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14444 < 400 > 21 tctccaggaa ccccatag 18 < 210 > 22 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > 01igonucleoti eoZC14445 < 400 > 22 gcaggattag tcaaaacc 18 < 210 > 23 < 211 > 843 < 212 > DNA 213 > Artificial Sequence < 220 > < 223 > Nucleotide sequence encoding zsig37 polypeptide < 400 > 23 atgggnwsnm gnggncargg nytnytnytn gcntaytgyy tnytnytngc nttygcnwsn 60 ggnytngtny tnwsnmgngt nccncaygtn carggngarc arcargartg ggarggnacn 120 gargarytnc cnwsnccncc ngaycaygcrr. garmgngcng argarca ca ygaraartay 180 gnccnwsnc argaycargg nytnccngcn wsnmgntgyy tnmgntgytg ygayccnggn 240 acnwsnatgt ayccngcnac ngcngtnccn carathaaya thacnathyt naarggngar 300 aarggngaym gnggngaymg nggnytncar ggnaatayg gnaaracngg nsngcnggn 360 gcnmgnggnc ayacnggncc naarggncar aarggnwsna tgggngcncc nggngarmgn 420 tgyaarwsnc aytaygcngc nttywsngtn ggpmgnaara arccnatgca ywsnaaycay 480 taytacara cngtnathtt ygayacngar ttygtnaayy tntaygayca yttyaayatg 540 ttyacnggna arttytaytg ytaygtnccn ggnytntayt tyttywsnyt naaygtncay 600 acntggaayc araargarac ntayytncay athatgaara aygargarga rgtngtnath 660 ytnttygcnc argtnggnga ymgnwsnath atgcarwsnc arwsnytnat gytngarytn 720 mgngarcarg aycargtntg ggtnmgpytn tayaarggng armgngaraa ygcnathtty 780 wsngargary tngayacnta yathacntty wsnggntayy tngtnaarca ygcnacngar 840 cen 843 < 210 > 24 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15040 < 400 > 24 acteatteta gactagggct cggt 24 < 210 > 25 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15033 < 400 > 25 atgaatggat ccctggtcct gagt 24 < 210 > 26 < 211 > 7 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Glu-Glu 400 peptide affinity tag > 26 Gl u Glu Tyr Met Pro Met Glu 1 5 < 210 > 27 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15721 í. < 400 > 27 ctgtaggaat tcatgggctc ccgt 24 < 210 > 28 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15035 < 400 > 28 attcatggat ccgggctcgg tggc 24 < 210 > 29 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13006 < 400 > 29 ggctgtcctc taagcgtcac 20 < 210 > 30 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13007 < 400 > 30 aggggtcaca gggatgcca 19 < 210 > 31 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > Glu-Glu peptide < 400 > 31 Gly Tyr Met Pro Val Asp 1 5 < 210 > 32 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Ol igonucleotide ZC6768 < 400 > 32 gcaattaacc ctcactaaag ggaac 25 < 210 > 33 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oigonucleotide ZC18297 < 400 > 33 tcctgaaagg cgagaaaggt g 21 < 210 > 34 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18298 < 400 > 34 ttccctgagt ctgagctagg 20 < 210 > 35 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18402 < 400 > 35 tccagagtga ctggggaagt g 21 < 210 > 36 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 < 223 > Oligonucleotide ZC18403 < 400 > 36 agtgacgagt tcgacaccta c 21 < 210 > 37 < 211 > twenty-one . < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18456 < 400 > 37 tgtgttccca ttcctggaca c 21 < 210 > 38 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18457 < 400 > 38 tccttccagc tggctggaaa g 21 < 210 > 39 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18560 < 400 > 39 agaatgcagg gataggtcag 20 < 210 > 40 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18561 < 400 > 40 tcagaggatc ctgacagcag 20 < 210 > 41 < 211 > 21 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18687 < 400 > 41 tggacacgtg agagggactt c 21 < 210 > 42 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC18688 < 400 > 42 agcagtagaa cttcccagtg 20 < 210 > 43 < 211 > 2559 < 212 > DNA < 213 > Mus muscle < 220 > < 221 > CDS < 222 > (70) ... (912) < 223 > orthologous mouse < 400 > 43 gaattcggat cctggaagag atgggattgt tataggcgga aagagagaaa cccagagaag 60 tccaggaag atg ggc tcc tgt gca cag gga ttc atg ctg gga tgc tgc ctg 111 Met Gly Ser Cys Wing Gln Gly Phe Met Leu Gly Cys Cys Leu 1 5 10 ctg ctg gcc atc acc tgg ggc ccc atc ctg age ctt gtg cea cgc gtt 159 Leu Leu Wing He Thr Trp Gly Pro He Leu Ser Leu Val Pro Arg Val 15 20 25 30 cag gag gaa cag gag tgg ga ga ga ga ga ga ga ctg cea tet ect 207 Gln Glu Glu Gln Gln Glu Trp Glu Glu Thr Glu Glu Leu Pro Ser Pro 35 40 45 ctg gat ect gtg here agg ect gaa gaa here cga gag aag tat age ect .255 Leu Asp Pro Val Thr Arg Pro Glu Glu Thr Arg Glu Lys Tyr Ser Pro 50 55 60 cgc cag ggt gag gac etc ccc act tet cgg tgc tac cga tgc tgt gac 303 Arg Gln Gly Glu Asp Leu Pro Thr Ser Arg Cys Tyr Arg Cys Cys Asp 65 70 75 ccc age here ect gta tac cag here att ect cea ccc cag atc aac atc 351 Pro Ser Thr Pro Val Tyr Gln Thr He Pro Pro Pro Gln He Asn He 80 85 90 acc atc ctg aaa ggc gag aaa ggt gac cga ggg gat cga ggc etc cag 399 Thr He Leu Lys Gly Glu Lys Gly Asp Arg Gly Asp Arg Gly Leu Gln 95 100 105 110 ggg aag tac ggc aaa ata ggt tet here ggt ccc agg ggc cat gtt ggc 447 Gly Lys Tyr Gly Lys He Gly Ser Thr Gly Pro Arg Gly His Val Gly 115 120 125 ccc aaa ggg cag aag gga tcc att gga gcc ect ggg aac falls tgc to ag 495 Pro Lys Gly Gln Lys Gly Ser He Gly Wing Pro Gly Asn His Cys Lys 130 135 140 age cag tac gca gcc ttc tcc gtg ggc cgg aag aag gct ttg fall age 543 Ser Gln Tyr Ala Ala Phe Ser Val Gly Arg Lys Lys Ala Leu His Ser 145 150 155 aac gac tac tcc cag ccc gtg gtc tcc gac acg gag ttt gtg aac etc 591 Asn Asp Tyr Phe Gln Pro Val Val Phe Asp Thr Glu Phe Val Asn Leu 160 165 170 tac aaa falls ttc aat atg ttc act ggg aag ttc tac tgc tat gtg ceg 639 Tyr Lys His Phe Asn Met Phe Thr Gly Lys Phe Tyr Cys Tyr Val Pro 175 180 185 190 ggc atc tac ttc ttc age etc aac gtg falls act tgg aac cag aag gag 687 Gly He Tyr Phe Phe Ser Leu Asn Val His Thr Trp Asn Gln Lys Glu 195 200 205 acg tac ctg falls atc atg aag gag gag gag gtg gtg atc ctg tat 735 Thr Tyr Leu His He Met Lys Asn Glu Glu Glu Val Val He Leu Tyr 210 215 220 gcg cag gtg age gac cgc age atc atg cag agt cag age ctg atg atg 783 Wing Gln Val Being Asp Arg Being He Met Gln Being Gln Being Leu Met Met - 225 230 235 gag ctg cgg gag gag gat gag gtc tgg gtg cgt etc ttc aag ggc gag 831 Glu Leu Arg Glu Glu Asp Glu Val Val Val Arg Leu Phe Lys Gly Glu 240 245 250 cgt gag aac gcc att ttc agt gac gag ttc gac acc tac atc acc ttc 879 Arg Glu Asn Wing He Phe Ser Asp Glu Phe Asp Thr Tyr He Thr Phe 255 260 265 270 agt ggc tac ctg gtc aag gcc cea tet gag ccc tagtggacac tcctgtggag 932 Gly Tyr Leu Val Ser Pro Lys Ala Ser Glu Pro 275 280 • cttttgtgga ctgctgacc't ccttgcctgg caccctgacc tatccctgca ttctacagac 992 actggagtcc tgccccgggc tgaccccatt ttctctctgc tccatcctgg cttccttggc 1052 cttggcttcc aaagttttgg cttttgacaa gatgecettg gccactggga atcccaaagg 1112 atggtgcgat cccagatctg gctgctactc taageagaga gctgccggca gatgaaatca 1172 ttgggcggg g agcctgtgag gatattgggg ggcctccagc tccttctgtg tacacagcct 1232 tagacgaccc tgtgctgtgt tgtcccgtgg ccacagggtg ttecagagea cagcccctgt 1292 gtgttcccat tcctggacac aagtaagcaa-. atatcatggg tttcttagga acgaagtcaa 1352 gcagaaaaga gaaagaaagg tggtgttagt tttggctttc cagccagctg gaaggaggga 1412 gagagagaga tggggagaga gagctatttg tattggggaa actgaggcat aggaaaaaca 1472 cagagtaget tgaatggcaa gcagtttgtg ggtttggaaa ccacatctga ettaacteta 1532 gatcacatat gagctttcct ggggacagca ggactgacct ccgagctctg ttgacatgct 1592 atagccttgc ccaggggctg gtcaatcttt ctgagccaca ctagtaaaag ggttggagga 1652 gaacagcaag tgccccctgt ggttggctct gggctggtgg cagcatcctg cttgccccaa 1712 ctcacaggat cctgacagca gctgggaacc tcagggactc ctgcagcttt ctctgtaaga 1772 aataaagctc ctactatgtc ccagtacctc tctgctctgc tccacttccc cagtcactct 1832 ggaccccagg gtgggagggc tctcttgcct gttgggacat cagttcccct tcctccttct 1892 tggtgaatta accatggaag gaccagggct cggatttggg ttcccaaact gcccttcacc 1952 atccctagtg tcctgcttcc ttcccagttc agcatcctgt ctgggaactt gatactttaa 2012 cctgctagag cggatgagtc tgatagacct gcccagccct gacacagccc tagtcagctt 2072 atggacacgt gagagggact tcctttgaga cccagagctg gggtagagct ataaaaatct 2132 acctattccc gggtcaaccc caagtggtag aagaggacac aggctatccc gccctagctc 2192 agactcaggg aaggcctcag gcctgattgt ctgactgcag agagcctgtg ttctttcccc 2252 atctcacccc gtgttgatcc ccagggcctg ggccactgga tatctgcttt gtgccaacta 2312 ggccttgctt gctgcttcct ggtggccctt ggttaggatc cctctctttt ccttctggag 2372 ctcaatgtac gtatatgcca cctccgaagg ggcttctgct ggtcagactc tccaagccac 2432 ttccatgggt gtgcctacag cagaggctgc tgcctcctgt gctctaccct gctctttcca 2492 gaaaacatta aacttgccat ggcgattcac agcaaaaaaa aaaaaaaaaa aaaaaaaagg 2552 gcggccg 2559 < 210 > 44 < 211 > 281 < 212 > PRT < 213 > Mus muscle < 400 > 44 Met Gly Ser Cys Wing Gln Gly Phe Met Leu Gly C Cyyss CCyyss Leu Leu Leu 1 5 10 15 Wing He Thr Trp Gly Pro He Leu Ser Leu Val P Prroo A Arrgg Val Gln Glu 20 25 30 Glu G n Gln Glu Trp Glu Glu Thr Glu Glu Leu P Prroo S Seerr Pro Leu Asp 35 40 4 455 Pro Val Thr Arg Pro Glu Glu Thr Arg Glu Lys T Tyyrr S Seerr Pro Arg Gln 50 55 6 600 Gly Glu Asp Leu Pro Thr Ser Arg Cys Tyr Arg C Cyyss CCyyss Asp Pro Ser 65 70 75 80 Thr Pro Val Tyr Gln Thr Pro Pro Pro Gln H Hee AAssnn He Thr He 85 90 95 Leu Lys Gly Glu Lys Gly Asp Arg Gly Asp Arg G Gllyy LLeeuu Gln Gly Lys 100 105 110 Tyr Gly VVaall Gly Pro Lys Gly CCyyss Lys Ser Gl n Tyr Ala Ala Phe Ser Val Gly Arg Lys Lys Ala L Leeuu HHiiss Ser Asn Asp 145 150 155 160 Tyr Phe Gln Pro Val Val Phe Asp Thr Glu Phe V Vaall A Assnn Leu Tyr Lys 165 170 175 His Phe Asn Met Phe Thr Gly Lys Phe Tyr Cys T Tyyrr V Vaall Pro Gly He 180 185 190 Tyr Phe L Lyyss Glu Thr Tyr Leu His He Met Lys Asn Glu Gl u Glu Val Val H H ee LLeeuu Tyr Ala Gln 210 215 220 Val Ser Asp Arg Ser Met Met Gln Ser Gln Ser Leu Met Met Glu Leu 225 230 235 240 Arg Glu Glu Asp Glu Val Trp Val Arg Leu Rhe Lys Gly Glu Arg Glu 245 250 255 Asn Ala He Phe Ser Asp Glu Phe Asp Thr Tyr He Thr Phe Ser Gly 260 265 270 Tyr Leu Val Lys Pro Wing Ser Gl u Pro 275 280

Claims (31)

1. An isolated polypeptide comprising a sequence of amino acid residues that is at least 75% identical in the amino acid sequence at residues 26-281 of SEQ ID NO: 2, wherein the sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro which form a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion.

2. An isolated polypeptide according to claim 2, characterized in that the polypeptide that is at least 90% identical in the amino acid sequence in residues 22-281 of SEQ ID NO: 2

3. An isolated polypeptide according to claim 2, characterized in that the polypeptide comprises residues 1-281 of SEQ ID NO: 2 or residues 1-281 of SEQ ID NO: 44.

4. An isolated polypeptide according to claim 1, covalently linked to an amino-terminal or carboxy-terminal manner to a portion selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores.

5. An isolated polypeptide according to claim 4, further comprising a site of proteolytic cleavage between the sequence of amino acid residues and the affinity tag.

6. An isolated polypeptide selected from a group consisting of: a) A polypeptide having an amino acid residue sequence that is at least 75% identical in amino acid sequence to 99 amino acid residue up to the amino residue acids 140 of SEQ ID NO: 2. b) A polypeptide having an amino acid residue sequence that is 75% identical in amino acid sequence to amino acid residue 140 or 141 to the amino acid residue of the amino acid residue SEQ ID NO: 2; and c) A polypeptide having an amino acid residue sequence that is 75% identical in the amino acid sequence to amino acid residue 99 to 281 of SEQ ID NO: 2.

7. A fusion protein consisting essentially of a first portion and a second portion bound by a peptide bond, the first portion comprising a polypeptide selected from a group consisting of: a) A polypeptide comprising an amino residue sequence acids which is at least 75% identical in the amino acid sequence to the amino acid residue 26 to the amino acid residue 281 of SEQ ID NO: 2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from the amino acid residue 1, 22 or 26 to the amino acid residue 281; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 44 from the amino acid residue 1, 22 or 26 to the amino acid residue 281; d) a portion of the zsig37 polypeptide as / shown in SEQ ID NO: 2 or SEQ ID NO. 44, which contains the collagen type domain or a portion of the collagen type domain capable of dimerization or oligomerization; e) a portion of the zsig37 polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO. 44, which contains the globular type domain or an active portion of the globular type domain; f) a portion of the zsig37 polypeptide as shown in SEQ ID NO: 2 or SEQ ID NO. 44, which includes the collagen type domain of the globular domain; and the second portion comprising another polypeptide.

8. A fusion protein according to claim 7, characterized in that the first portion is selected from the group consisting of: a) a polypeptide having the sequence from the amino acid residue 99 to the amino acid residue 140 of SEQ ID NO: 2 or SEQ ID NO. 44; b) a polypeptide having the amino acid residue sequence 140 or 141 to amino acid residue 281 of SEQ ID NO: 2 or SEQ ID NO. 44; c) a polypeptide having the amino acid residue sequence 99 to 281 of SEQ ID NO: 2 or SEQ ID NO. 44;

9. A fusion protein comprising a further secretory sequence having the amino acid sequence of the amino acid residues 1-21 or 1-25 of SEQ ID NO: 2 or SEQ ID NO: 44, wherein the additional secretory sequence is operably linked to an additional polypeptide.

10. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26,281 of SEQ ID NO: 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly Xaa. Pro forming a collagen domain wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and a transcription terminator.

11. An expression vector according to claim 10, characterized in that the DNA segment codes for a polypeptide that is at least 90% identical to the amino acid sequence at residues 26-281 of SEQ ID NO: 2.

12. An expression vector according to claim 11, characterized in that the DNA segment codes for a polypeptide that is at least 90% identical to the amino acid sequence at residues 26-281 of SEQ ID NO: 2.

13. An expression vector according to claim 11, characterized in that the DNA segment codes for a polypeptide comprising residues 1-281 of SEQ ID NO: 2 or 1- 281 of SEQ ID NO: 44.

14. An expression vector according to claim 11, characterized in that the DNA segment codes for a polypeptide covalently linked in an amino-terminal or carboxy-terminal manner to an affinity tag.

15. An expression vector according to claim 11, characterized in that the DNA segment encodes additionally for a secretory signal sequence operably linked to the polypeptide.

16. An expression vector according to claim 15, characterized in that the secretory additional sequence comprises residues 1-21 or 1-25 of SEQ ID NO: 2 or SEQ ID NO: 44.

17. A cultured cell in which an expression vector has been introduced comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in the amino acid sequence of residues 26-281 of SEQ ID NO: 2, wherein the sequence comprises : Repetitions of Gly-Xaa-Xaa or Gly-Xaa-Pro that form a collagen domain where Xaa is any amino acid; and a carboxy-terminal globular portion; Y a transcription terminator wherein the cells express the polypeptide encoded by the DNA segment.

18. A method for producing a polypeptide comprising: culturing a cell into which a special vector has been introduced comprising the following operably linked elements: A transcription promoter a DNA segment encoding a polypeptide comprising an amino residue sequence acids which is at least 75% identical to the amino acid sequence residues 26-281 of SEQ ID NO: 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa, Pro forming a domain of collagen wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and a transcription terminator; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.

19. A pharmaceutical composition comprising a polypeptide, the polypeptide comprising a sequence of amino acid residues that is at least 75% identical in the amino acid sequence to residues 26-281 of SEQ ID NO: 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa, Pro which form a collagen domain wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and in combination with a pharmaceutically acceptable vehicle.

20. A portion that specifically binds to an epitope of a polypeptide comprising an amino acid residue sequence that is at least 75% identical in the amino acid sequence to residues 26-281 of SEQ ID NO: 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa, Pro which form a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion.

21. A binding protein that specifically binds to an epitope of a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in the amino acid sequence to residues 26-281 of SEQ ID NO: 2; wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa, Pro which form a collagen domain wherein Xaa is any amino acid; and a carboxy-terminal globular portion.

22. An isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in the amino acid sequence at residues 26-281 of SEQ ID NO. 2, wherein the sequence comprises: repeats of Gly-Xaa-Xaa or Gly-Xaa, Pro which form a collagen domain wherein Xaa is any amino acid; and a carboxy-terminal globular portion.

23. An isolated polynucleotide according to claim 22, wherein the polypeptide is at least 90% identical in sequence of amino acids to residues 26-281 of SEQ ID NO. 2.

24. An isolated polynucleotide according to claim 23, characterized in that the polypeptide is at least 90% identical to the amino acid sequence in residues 26-281 of SEQ ID NO. 2.

25. An isolated polynucleotide according to claim 22, characterized in that the polypeptide comprises residues 1-281 of SEQ ID NO. 2 or residues 1-281 of SEQ ID No. 44.

26. An isolated polynucleotide according to claim 22, characterized in that the polynucleotide is DNA.

27. An isolated polynucleotide selected from a group consisting of, a) a nucleotide sequence from nucleotide 465 to nucleotide 688 of SEQ ID NO: 1; b) a nucleotide sequence from the nucleotide 688 to the nucleotide 1016 of SEQ ID NO: 1; c) a nucleotide sequence from the nucleotide 691 to the nucleotide 1016 of SEQ ID NO: 1; d) a nucleotide sequence from nucleotide 465 to nucleotide 1016 of SEQ ID NO: 1; e) a nucleotide sequence from nucleotide 364 to nucleotide 490 of SEQ ID NO: 1; f) a nucleotide sequence from nucleotide 490 to nucleotide 912 of SEQ ID NO: 43; g) a nucleotide sequence from nucleotide 364 to nucleotide 912 of SEQ ID NO: 43; h) a nucleotide sequence from nucleotide 364 to nucleotide 490 of SEQ ID NO: 43; i) a polynucleotide encoding a polypeptide having a sequence of amino acid residues that is at least 75% identical in sequence from amino acids to the amino acid residue 99, 140 or 141 to amino acid residue 281 of SEQ ID NO: 2; j) a polynucleotide encoding a polypeptide having an amino acid residue sequence that is at least 75% identical in sequence from amino acids to amino acid residue 99 to amino acid residue 140 of SEQ ID NO: 2; k) complementary nucleotide sequences aa), b), c), d), e), f), g) h), i), oj) and 1) generation nucleotide sequences of a), b), c) ), d), e), f), g) h), i), j) ok).

28. An isolated polynucleotide encoding a fusion protein consisting essentially of a first portion and a second portion measured by a peptide bond, the first portion being selected from the group consisting of: a) a polypeptide comprising an amino residue sequence acid that is at least 75% identical in the amino acid sequence to the amino acid residue 26 to the amino acid residue of SEQ ID NO: 2; b) a polypeptide comprising a sequence of amino acid residues as shown • in SEQ ID NO: 2 from the amino acid residue 1, 22, 26 to the amino acid residue 281. c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 44 from the residue of amino acid 1, 22 or 26 to the amino acid residue 281; d) a portion of the zsig37 polypeptide, as shown in SEQ ID NO: 2 or SEQ ID NO: 44, which contains the collagen type domain or a portion of the collagen type domain capable of dimerization or oligomeation; e) a portion of the zsig37 polypeptide, as shown in SEQ ID NO: 2 or SEQ ID NO: 44, which contains the globular type domain or an active portion of the globular type domain; or f) a portion of the zsig37 polypeptide, as shown in SEQ ID NO: 2 or SEQ ID NO: 44, which includes the collagen type domain and the globular domain; and the second portion comprising another polypeptide.

29. An isolated polypeptide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of residues 1-21 or 1-25 of SEQ ID NO: 2, wherein the sequence of the secretory signal it is operably linked to an additional polypeptide.

30. An isolated polynucleotide comprising nucleotide 1 to nucleotide sequence 843 of SEQ ID NO: 23.

31. An oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 23 or a sequence complementary to SEQ ID NO: 23.