MXPA00001848A - Adipocyte-specific protein homologs - Google Patents

Abstract

The present invention relates to polynucleotide and polypeptide molecules for zsig39, a novel 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 zsig39 polypeptides.

Description

PROTEIN HOMOLOGOUS SPECIFIC FOR ADIPOCYTES Background of the Invention The energy balance (which involves the metabolism of energy, the nutritional status, the storage of lipids and the like) is an important criterion for health. This homeostasis of energy involves the admission of food and the metabolism of carbohydrates and lipids to generate the necessary energy for voluntary and involuntary functions. The metabolism of proteins can lead to the generation of energy, but preferably leads to the formation or repair of muscles. Among other consequences, an energy homeostasis leads to the over and under formation of adipose tissue. The formation and storage of fat is modulated by insulin. For example, insulin stimulates the transport of glucose to cells, where it is metabolized in a-glycerophosphate which is used in the esterification of fatty acids to allow the storage of them as triglycerides. In addition, adipocytes (ref.032811 fat cells) express a specific transport protein that improves the transfer of free fatty acids to adipocytes. Adipocytes also secrete several proteins that are thought to modulate the homeostatic control of glucose and lipid metabolism. These proteins that secreted additional adipocytes include adipsin, complement factors C3 and B, factor a of tumor necrosis, the product of the ob gene and Acrp30. There is also evidence suggesting the existence of a secretory pathway regulated 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 associated or indirectly with obesity or anorexia. Acrp30 is a polypeptide of 247 amino acids that is expressed exclusively by adipocytes. The Acrp30 polypeptide is composed of a sequence of the amino-terminal signal, a stretch of 27 amino acids of unknown homology, 22 repeats of collagen of perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa and a carboxy terminal globular domain. See, Scherer et al., As described above and International Patent Application No. W096 / 39429. Acrp30, an abundant human serum protein regulated by insulin, shares a structural similarity, particularly in the carboxy-terminal globular domain, to complement the Clq factor and with a summer serum protein of Siberian squirrels in hibernation (Hib27) . The expression of Acrp30 is induced up to 100 times during the differentiation of adipocytes. Acrp30 is suggested for use in the modulation of energy balance and in the identification of adipocytes in test samples. Another secreted protein that seems to be produced exclusively in adipocytes is apMl, described, for example, in Maeda et al., Biochem. Biophys. Res. Co m. 221: 286-9, 1996. A 4517 bp clone had an open reading frame of 244 amino acids and a 3 'untranslated long region. The protein included a signal sequence, a different amino-terminal collagen sequence, 22 repeats of collagen (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminal region with homology to collagen X, Collagen VIII and complement protein Clq. The complement Clq factor consists of six copies of three related polypeptides (A, B and C chains), with each polypeptide being approximately 225 amino acids in length with a domain of nearby amino-terminal collagen and a carboxy-terminal globular region. The regions of six triplex helices are formed by the collagen domains of the six A chains, six B chains and six C chains, which form 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, a B chain and a C chain. The Clq is therefore composed of six globular heads linked by means of six stems similar to collagen to a central fibril region. Sellar et al., Biochem. J. 274: 481-90, 1991. This configuration is often referred to as a flower scent. Acrp30 has a similar aroma structure formed from a single type of polypeptide chain. The molecules capable of modulating the homeostasis of energy are desired for the study of this phenomenon and for the prevention of the treatment of imbalances. Also, molecules capable of modulating the secretory pathways of adipocytes are desired as modulators of indirect energy homeostasis and as research reagents. The present invention provides such 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 of the invention there is provided an isolated polypeptide comprising an amino acid residue sequence that is at least 80% identical to SEQ ID NO: 2, wherein the sequence comprises: beta strands corresponding to the amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO: 2 '. Within one embodiment the polypeptide is at least 90% identical to SEQ ID NO: 2. Within another embodiment the polypeptide comprises a collagen-like domain having at least 22 repeats of collagen. Within another embodiment the polypeptide comprises residues 19-243 of SEQ ID NO: 2. Within yet another embodiment the polypeptide is covalently, amino terminally or carboxyterminally linked to a portion selected from the group consisting of affinity tags, toxins , radionucleotides, enzymes and fluorophores. Within another aspect there is provided an isolated epitope polypeptide selected from the group consisting of: a) a polypeptide having an amino acid residue sequence from amino acid residue 30 to amino acid residue 95 of SEQ ID NO: 2; b) a polypeptide having an amino acid residue sequence from amino acid residue 30 to amino acid residue 96 of SEQ ID NO: 2; and c) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to 97 of SEQ ID NO: 2; d) a polypeptide having an amino acid residue sequence from amino acid residue 30 to amino acid residue 98 of SEQ ID NO: 2; e) a polypeptide having a sequence of amino acid residues from the amino acid residue 98 to amino acid residue 243 of SEQ ID NO: 2; f) a polypeptide having a sequence of amino acid residues from amino acid residue 99 to amino acid residue 243 of SEQ ID NO: 2; g) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to amino acid residue 243 of SEQ ID NO: 2; and h) a polypeptide having an amino acid residue sequence that is 90% identical in the amino acid sequence a), b), c), d), e), f), g) or h). Within another aspect there is provided 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 the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 80% identical to SEQ ID NO: 2, wherein the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-17'4 of SEQ ID NO: 2; a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 16 to amino acid residue 243; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2 containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, which contains the globular domain or the receptor binding portion of the globular domain; or f) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, including the collagen-like 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 of amino acid residues up to amino acid residue 95 of SEQ ID NO: 2; b) a polypeptide having the sequence of amino acid residues up to amino acid residue 96 of SEQ ID NO: 2; c) a polypeptide having the sequence of amino acid residues 30 up to the residue 'of amino acids 97 of SEQ ID NO: 2; d) a polypeptide having the sequence from amino acid residue 30 to amino acid residue 98 of SEQ ID NO: 2; e) a polypeptide having the sequence from amino acid residue 30 to amino acid residue 243 of SEQ ID NO: 2; f) a polypeptide having the sequence of amino acid residue 98 to amino acid residue 243 of SEQ ID NO: 2; and g) a polypeptide having the sequence of amino acid residue 99 to amino acid residue 243 of SEQ ID NO: 2. Within another aspect, a fusion protein comprising a sequence of the secretory signal having the sequence of amino acids of amino acid residues 1-15 or 1-18 of SEQ ID NO: 2, wherein the sequence of the secretory signal is operably linked to an additional polypeptide. Within yet another aspect is a pharmaceutical composition comprising a polypeptide as described above, in combination with a pharmaceutically acceptable carrier. An antibody that binds specifically to an epitope of a polypeptide as described above is also provided. There is further provided an isolated polynucleotide encoding a polypeptide comprising an amino acid residue sequence that is at least 80% identical to SEQ ID NO: 2, wherein the sequence comprises: beta strands corresponding to amino acid residues 105- 109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by minus two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO: 2. Within one embodiment the polypeptide is at least 90% identical to SEQ ID NO: 2. In another embodiment, the polypeptide comprises a collagen-like domain having at least 22 repeats of collagen. Within another embodiment, the polynucleotide is DNA. Within yet another aspect there is provided an isolated polynucleotide selected from the group consisting of: a) a nucleotide sequence from nucleotide 243 to nucleotide 962 of SEQ ID NO: 1; b) a nucleotide sequence from nucleotide 252 to nucleotide 962 of SEQ ID NO: 1; c) a nucleotide sequence from nucleotide 285 to nucleotide 482 of SEQ ID NO: 1; d) a nucleotide sequence from nucleotide '285 to nucleotide 485 of SEQ ID NO: 1, e) a nucleotide sequence from nucleotide 285 to nucleotide 488 of SEQ ID NO: 1; f) a nucleotide sequence from nucleotide 285 to nucleotide 491 of SEQ ID NO: 1; g) a nucleotide sequence from nucleotide 285 to nucleotide 926 of SEQ ID NO: 1; h) a nucleotide sequence from nucleotide 491 to nucleotide 926 of SEQ ID NO: 1; i) a polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical in nucleotide sequence a), b), c), d), e), f), g), and h); j) the nucleotide sequences complementary to a), b), c), d), e), f), g), h) or i); and) degenerate nucleotide sequences of a), b), c), d), e), f), g), h), i), or j). Within another aspect an isolated polynucleotide encoding a fusion protein consisting essentially of a first portion and a second portion bound by a peptide bond is provided., the first portion is selected from the group consisting of: a) a polypeptide comprising an amino acid residue sequence that is at least 80% identical to SEQ ID NO: 2, wherein the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 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 16 to amino acid residue 243; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2 containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, which contains the globular-like domain or an active portion of the globular-like domain; or f) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, including the collagen-like domain and the globular domain; and the second portion comprising another polypeptide. Within another aspect there is provided an isolated polynucleotide encoding a fusion protein comprising a secretory signal sequence having an amino acid sequence of amino acid residues 1-15 or 1-18 of SEQ ID NO: 2, wherein the sequence of the secretory signal is operably linked to an additional polypeptide. Within yet another aspect is an isolated polynucleotide comprising the sequence of nucleotides 1 to nucleotide 729 of SEQ ID NO: 10. An expression vector is also provided comprising the following operably linked elements: a transcription promoter; a segment of DNA encoding a polypeptide as described above; and a transcription terminator. Within one embodiment, the DNA segment encodes a polypeptide that is at least 90% identical to SEQ ID NO: 2. Within another embodiment, the DNA segment encodes a polypeptide that further comprises a collagen-like domain that is at least 22 collagen repeats. Within yet another embodiment the DNA segment encodes a polypeptide covalently linked, amino terminally or carboxy terminally to an affinity tag. Within yet another embodiment the DNA segment further encodes a secretory signal sequence operably linked to the polypeptide. Within yet another embodiment the sequence of the secretory signal comprises residues 1-15 or 1-18 of SEQ ID NO: 2. A cultured cell is also provided within which an expression vector has been introduced comprising the following Operably linked elements: a transcription promoter; a segment of DNA encoding a polypeptide as described above; and a transcription terminator; wherein the cell expresses the polypeptide encoded by the DNA segment.

Within yet another aspect there is provided a method of 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 as described above; and a transcription terminator; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide. Within another aspect is an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 10 or a sequence complementary to SEQ ID NO: 10. Within yet another aspect is a method for modulating the metabolism of free fatty acids by administering a pharmaceutically effective dose of a polypeptide as described above.

Brief Description of the Drawings Figure 1 illustrates a multiple alignment of the zsig39 polypeptide of the present invention and HUMUPST2_1 (SEQ ID NO: 3) (Maeda et al., Biochem. Biophys., Res. Comm. 221 (2): 286-9, 1996); C1QA HUMAN (SEQ ID NO: 4) (Sellar et al., Biochem J. 274: 481-90, 1991, Reid, Biochem. J. 179: 367-71, 1979, and Reid et al., Biochem. 203: 559-69, 1982); HP25_TAMAS (SEQ ID NO: 5) (Takamatsu et al., Mol Cell. Biol. 13: 1516-21, 1993 and Kondo and Kondo, J. Biol. Chem. 267: 473-8, 1992); HP27 TAMAS (SEQ ID NO: 6) (Takamatsu et al., And Kondo and Kondo referred to above); and CERL_RAT (SEQ ID NO: 7) (Wada and Ohtani, Brain Res. Mol. Brain Res. 9: 71-7, 1991). Figure 2 is a matrix showing the percent identity of the amino acid in a comparison of the six proteins shown in the multiple alignment of Figure 1.

DETAILED DESCRIPTION OF THE INVENTION Prior to describing the invention in detail, it may be useful for the understanding thereof to define the following terms. The term "affinity tag" is used herein to denote a segment of polypeptide that can be attached to a second polypeptide to provide purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate. In the beginning, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag. Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. _4: 1075, 1985, Nilsson et al., Methods Enzymol 198: 3, 1991), glutathione S transferase ( Smith and Johnson, Gene 67_: 31, 1988), substance P, Flag ™ peptide (Hopp et al., Biotechnology, 6: 1204-1210, 1988, available from Eastman Kodak Co., New Haven, CT), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Nati, Acad. Sci. USA 82: 7925-4, 1995), the streptavidin binding peptide, or other antigenic epitope or binding domain. See generally Ford et al., Protein Expression and Purification 2: 95-107, 1991. The DNAs encoding the affinity tags are available from commercial 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 lead to polymorphism within populations. Mutations of the gene can be silent (without change in the encoded polypeptide) or can 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. Wherever the context permits, these terms are used with reference to a particular sequence or a portion of a polypeptide or protein to denote a proximity or relative position. For example, a certain sequence placed carboxyl-terminal with respect to a reference sequence within a protein is located close to the carboxyl terminal of the reference sequence, but is not necessarily in the carboxyl terminal of the complete protein. The term "collagen" or collagen-like domain "refers to a series of repeating triplet amino acid sequences," repeats "or" collagen repeats ", Gly-Xaa-Pro or Gly-Xaa-Xaa, wherein Xaa It is any amino acid residue. Such domains can contain as many as 22 repeats of collagen or more. Fragments or proteins containing such collagen-like domains can form homomeric constructs (dimers or oligomers of the same fragment or protein). In addition, such fragments or protein containing such collagen-like domains can form heteromeric constructs (dimers or oligomers of different fragments or proteins). The term "complement / anti-complement pair" denotes non-identical portions that form a stable pair, associated non-covalently, under appropriate conditions. For example, biotin and avidin (or streptavidin) are prototypical elements of a complement / anti-complement pair. Other exemplary complement / anti-complement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. Where the subsequent dissociation of the complement / anti-complement pair is' desirable, the complement / anti-complement pair preferably has a binding affinity of <109 M "1. The term" polynucleotide molecule complements "denotes polynucleotide molecules having a complementary base sequence and the reverse orientation when compared to a reference sequence, eg, the 5 'ATGCACGGG 3' sequence. it is complementary with 5 'CCCGTGCAT 3'.

The term "contiguous" denotes a polynucleotide having a contiguous stretch of a sequence identical or complementary to another polynucleotide. The contiguous sequences are said to "overlap" on a given stretch of the polynucleotide sequence either in its entirety or along a partial stretch of the polynucleotide. For example, the contiguous representatives for the sequence of polynucleotides 5'-ATGGCTTAGCTT-3 'are 5'-TAGCTTgagtct-3' and 3'-gtcgacTACCGA-5 '. The term "degenerate nucleotide sequence" denotes the nucleotide sequence that includes one or more degenerate codons (when compared to a reference polynucleotide molecule that encodes a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (ie, the triplets of GAU and GAC each encode the 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 are provided for transcription. Such additional segments may include the promoter and terminator sequences, 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 the plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic environment and is therefore free of other foreign or unwanted coding sequences, and is in a form suitable for use within of genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include the cDNA and the genomic clones. The isolated DNA molecules are free of other genes with which they are ordinarily associated, but may include the 5 'and 3' untranslated regions that are naturally present such as the promoters and terminators. The identification of the associated regions will be apparent to a person with ordinary skill in the art (see for example, Dynan and Tijan, Nature 316: 774-78, 1985). A polypeptide or protein is a polypeptide or protein that is found in a condition other than its natural environment, such as apart 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 dimer or alternatively derived or glycosylated forms. The term "operatively linked", when referring to the DNA segments, denotes that the segments are arranged so that they function in concert with their intended purposes, for example the transcription is initiated at the promoter and proceeds through the segment of coding 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 sequence differences between orthologs are the result of speciation. The "paralogs" are different but structurally related proteins, made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, ß-globin, and myoglobin are paralogs with each other. The term "polynucleotide" denotes a single-stranded or double-stranded polymer of the deoxyribonucleotide or ribonucleotide bases read from the 5 'end to the 3' end. 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 the base pairs (abbreviated "pb"), nucleotides ("nt"), or kilobases ("kb"). Wherever the context permits, these last two terms may describe polynucleotides that are single-stranded or double-stranded. When the term is applied to double-stranded molecules, it is used to denote the full length and it will be understood that they will be equivalent to the term "base pairs". 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.; therefore, all nucleotides within a double-stranded polynucleotide molecule can not be damaged.

Such unpaired ends will generally not exceed 20 nt in length. A "polypeptide" is a polymer of amino acid residues joined by peptide linkages, produced either naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides". The "probes and / or primers" as used herein may be RNA or DNA. The DNA can be either the cDNA or the genomic DNA. The polynucleotide probes and primers are single-stranded or double-stranded DNA or RNA, the oligonucleotides generally synthetic, but can be generated from the cloned cDNA or the genomic sequences or their complements. The analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes (14-17 nucleotides) may be used. The PCR primers are at least 5 nucleotides in length, preferably 15 or more nt, more preferably 20-30 nt. Short polynucleotides can be used when a small region of the gene is targeted for analysis. For an approximate analysis of the genes, a polynucleotide probe can comprise a complete exon or more. The probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescent agent, 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 the DNA sequences that are provided for the binding of the RNA polymerase and the initiation of transcription. Promoter sequences are commonly, but not always, found in the 5 'non-coding regions of the genes. A "protein" is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other nonpeptide substituents can be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined here in terms of their skeleton structures of the amino acids; substituents such as carbohydrate groups are generally not specified, but may nevertheless be present. The term "receptor" denotes a protein associated with the cell that binds to a bioactive molecule (i.e., a ligand) and has a mediating effect of the ligand on the cell. The membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand binding domain and an intracellular effector domain that is typically involved in signal transduction. The binding of the ligand to the receptor leads to a conformational change in the receptor, which causes an interaction between the effector domain and another (s) molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids , adhesion of cells, hydrolysis of inositol lipids and hydrolysis of phospholipids. Most nuclear receptors also exhibit a structure of multiple domains, including a transactivation, amino-terminal domain, a DNA binding domain and a ligand binding domain. In general, the receptors can be cytosolic or nuclear, bound to the membrane; monomeric (for example, the thyroid stimulating hormone receptor, the beta-adrenergic receptor) or multimeric (for example, the PDGF receptor, the growth hormone receptor, the IL-3 receptor, the GM-CSF receptor, the G-CSF receptor, the erythropoietin receptor and the IL-6 receptor). The term "secretory signal sequence" denotes a DNA sequence encoding a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The largest peptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway. A "soluble receptor" is a polypeptide of the receptor that is not bound to a cell membrane. Soluble receptors are very commonly receptor polypeptides that bind to the ligand, which lack 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 binding the polypeptide to a substrate, or immunoglobulin constant region sequences. Many receptors on the surface of the cell have soluble counterparts, which are naturally present, which are produced by proteolysis or translationally from the mRNAs divided alternatively. The receptor polypeptides are said to be substantially free of segments of transmembrane or intracellular polypeptides when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively. The term "splice variant" is used herein to denote alternative forms of RNA transcribed from a gene. Splicing variations arise naturally through the use of alternative splicing sites within a transcribed RNA molecule, or less commonly between the separately transcribed RNA molecules, and can lead to several mRNAs 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 a protein encoded by a splicing variant of a mRNA transcribed from a gene. The weights and molecular lengths of the polymers determined by imprecise analytical methods (for example, gel electrophoresis) will be understood to be approximate values. When such a value is expressed as "close to" X or "approximately" X, the established value of X will be understood to be accurate to + 10%. All references cited here are incorporated for reference in their entirety. The present invention is based in part on the discovery that a novel DNA sequence encoding a polypeptide has homology to a protein related to the adipocyte complement (Acrp30). See, for example, Scherer et al., J. Biol. Chem. 270 (45): 26746-9, 1995. The Acrp30 polypeptide is shown in SEQ ID NO: 8. The Acrp30 appears to be highly related to the human apMl (HUMUPST2_1 in Figures 1 and 2, SEQ ID NO: 3), with the most significant differences observed in the secretory sequence. The novel 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 the Gly-Xaa-Xaa or Gly-Xaa-Pro repeats and a carboxy-terminal globular portion. The novel polynucleotide sequence also contains a 3 'untranslated long region. The structure of the general polypeptide described above is shared by Acrp30 (SEQ ID NO: 8) and HUMUPST2_1 (SEQ ID NO: 3). Also the DNA sequence of HUMUPST2_1 (SEQ ID NO: 9) is characterized by a 3 'untranslated long region. However, Acrp30 and all of the sequences aligned in Figure 1, with the exception of CERL_RAT (SEQ ID NO: 7), share a cysteine residue conserved at position 144 of the zsig39 polypeptide as shown in Figure 1 and SEQ ID NO: 2. Other regions of homology, found in the The carboxy-terminal globular portion in the aligned proteins are identified here as useful primers for the search for other elements of the family. The Acrp30, for example, could be identified in a search using the primers. Also, the zsig39 polypeptides of the present invention include a 'putative cell binding site, the RGD motif or radical at amino acid residues 77-79 of SEQ ID NO: 2. See, for example, Rouslahti and Pierschbacher , Cell 44: 517-7, 1986, and d'Souza et al., Trends Biochem. Sci. 16: 246-50, 1991, for descriptions of the motif or radical of the RGD peptide and its role in adhesion. The analysis of the tissue distribution of the mRNA corresponding to this novel DNA was carried out as described in Example 2 here. A transcription size was observed at approximately 1.2 kb. The intensity of the signal was higher for the small intestine and the heart, with relatively less intense signals in the pancreas, the musculoskeletal system, the kidney and the thyroid, and with signals of lower intensity in the placenta, the lung, the liver, the spleen, the prostate, the ovaries, the colon, the stomach, the spinal cord, the lymph nodes, the trachea, the adrenal gland and the bone marrow. The polypeptide has been designated the zsig39 polypeptide. A spot spot indicated the expression of the zsig39 polypeptide in the subthalamic nucleus, the hippocampus, the medulla oblongata and the thalamus. A spot of human intestines showed expression in the SW480 cell line of human colorectal adenocarcinoma, small intestine tissue, stomach tissue, normal human colon cell line, FHC; and the FHs74 Int cell line of the normal fetal small intestine. The novel zsig39 polypeptides of the present invention were initially identified by questioning an EST database to verify the secretory signal sequences, characterized by an upstream methionine starting site, a hydrophobic region of approximately 13 amino acids and a targeting site , in an effort to select secreted proteins. The polypeptides corresponding to the ESTs that satisfy these search criteria were compared with the known sequences to identify the secreted proteins having a homology with respect to the known ligands. A single EST sequence was discovered and predicted to be a secreted protein. The novel polypeptide encoded by the full length cDNA makes it possible to identify a homologous relationship with the Acrp30 protein related to the adipocyte complement (SEQ ID NO: 8) and the apMl protein secreted with adipocytes (HUMUPST2_1 in Figures 1 and 2). , SEQ ID NO: 3). A somewhat more distant homology was also identified with respect to the A chain of the Clq complement component, two factors observed in the active state of Siberian marmots in hibernation (HP25_TAMAS (SEQ ID NO: 5) and HP27_TAMAS (SEQ ID NO: 6) and a rat brain protein (CERL_RAT, SEQ ID NO: 7), as shown in Figures 1 and 2. The complete sequence of the zsig39 polypeptide it was obtained from a single clone believed to contain it, where the clone was obtained from a library of lung tissues. Other libraries that can also be investigated for such clones include the heart, the small intestine, the pancreas, the musculoskeletal system, the kidney, the thyroid, the subthalamic nucleus, the hippocampus, the medulla oblongata, the thalamus and the like. The nucleotide sequence of the N-terminal EST is described in SEQ ID NO: 1, and its deduced amino acid sequence is described in SEQ ID NO: 2. As generally described above, the zsig39 polypeptide includes a signal sequence. , which varies from amino acid 1 (Met) to amino acid residue 15 (Gly). A sequence of the alternative signal varies from amino acid 1 (Met) to amino acid 18 (Pro). The mature polypeptide therefore varies from amino acid 16 (Ser) or 19 (Leu) to amino acid 243 (Ala). Within the mature polypeptide, an N-terminal region of limited homology is found, which varies between amino acid residue 20 (Asp) 'and 29 (Pro), where the cysteine at position 28 can provide a similar structure / function that the cysteine found in position 36 in HUMUPST2_1 and in the N-terminal region of HP25_TAMAS and HP27_TAMAS. In addition, a collagen domain is found between amino acid 30 (Gly) and 95 (Ala), 96 (Gly), 97 (Glu) or 98 (Cys). In the collagen domain, 9 perfect Gly-Xaa-Pro repeats and 13 or 14 imperfect Gly-Xaa-Xaa repeats are observed. The Acrp30 contains 22 perfect or imperfect repeats.

The zsig39 polypeptide also includes a carboxy-terminal globular domain, ranging from about amino acid 98 (Cys) or 99 (Ser) to 243 (Ala). The globular domain of ACRP30 has been determined to have a beta-strand "gelatin roll" topology (Shapiro and Scherer, Curr. Biol. _8: 335-8, 1998) and the zsig39 sequence as represented by SEQ ID. NO: 2 contains all 10 beta strands of this structure (amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226- 231 of SEQ ID NO: 2). These threads have been designated "A", "A" ', "B", "B"', "C", "D", "E", "F", "G" and "H" respectively. Also, the two receptor binding loops, amino acid residues 111-139 and 170-182 of SEQ ID NO: 2, are represented. The receptor region of the core receptor was predicted to include amino acid residues 111-135 and 170-174 of SEQ ID NO: 2. Those skilled in the art will recognize that these limits are approximate, and are based on the alignments with the known proteins and the predictions of protein folds. The residues of amino acids 149 (Glu), 151 (Tyr), 199 (Leu) and 227 (Phe) seem to be conserved through the superfamily that includes CD40, TNFa, ACRP30 and zsig39.

The proteins of the present invention comprise an amino acid residue sequence that is at least 80% identical to SEQ ID NO: 2. Within certain embodiments of the invention, the sequence is at least 90% or 95% identical to the SEC. ID NO: 2. Another aspect of the present invention includes fragments of zsig39 polypeptides. Preferred fragments include the collagen-like domain of zsig39 polypeptides, ranging from amino acid 30 (Gly) to amino acid 95 (Ala), 96 (Gly), 97 (Glu) or 98 (Cys) of SEQ ID NO. : 2, a portion of the zsig39 polypeptide that contains the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization. These fragments are particularly useful in the study of collagen dimerization or oligomerization or in the formation of fusion proteins as described more fully below. The polynucleotides that encode such 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.

N0: 1 from nucleotide 1, 198, 242, 251 or 285 to nucleotide 482, 485, 488 or 491; (b) polynucleotide molecules that encode a zsig39 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 30 (Gly) to amino acid residue 96 ( Gly), 97 (Glu), 98 (Cys); (c) complementary molecules for (a) or (b); and (f) degenerate nucleotide sequences encoding a fragment of the collagen-like domain of the zsig39 polypeptide. Such fragments or proteins containing such collagen-like domains can form homomeric constructs (dimers or oligomers of the same fragment or protein). In addition, such fragments or proteins containing such collagen-like domains can form heteromeric constructs. (dimers or oligomers of different fragments or proteins). Other components of the heteromeric constructs may include Acrp30 and other polypeptides characterized by the collagen-like domains that are described herein or are known in the art. These homomeric and heteromeric constructions are contemplated by the present invention. Other preferred fragments include the globular domain of the zsig39 polypeptides, ranging from amino acids 98 (Cys) or 99 (Ser) to 243 (Ala) of SEQ ID NO: 2, particularly from amino acid residue 105 to 231 of SEQ ID NO: 2, a portion of the zsig39 polypeptide that contains the globular-like domain or an active portion of the globular-like domain. These fragments are particularly useful in the study or modulation of energy balance or neurotransmission, particularly neurotransmission related to stress or diet. The antimicrobial activity may also be present in such fragments. The globular domain of Acrp30 proteins has been shown to resemble a multimer of trimers. The trimers can be homo or heteromeric (Shapiro and Scherer, ibid.). Such fragments could also be useful for studying the multimerization and binding of the zsig39 receptor and other related proteins such as Acrp30 and TNFa. Polynucleotides encoding such 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 nucleotide 489 or 492 through nucleotide 926 or 1347; (b) polynucleotide molecules that encode a zsig39 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 98 (Cys) or 99 (Ser) to amino acid residue 243 (Ala); (c) complementary molecules for (a) or (b); and (f) degenerate nucleotide sequences encoding a fragment of the globular domain of the zsig39 polypeptide. Another zsig39 polypeptide fragment of the present invention includes both the collagen-like domain and the globular domain ranging from amino acid residue 30 (Gly) to 243 (Ala) of SEQ ID NO: 2. Polynucleotides encoding such 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 nucleotide 285 to nucleotide 926 or 1347; (b) polynucleotide molecules that encode a zsig39 polypeptide fragment that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 30 (Gly) to amino acid residue 243 (To); (c) complementary molecules with (a) or (b); and (f) degenerate nucleotide sequences encoding a fragment of the globular domain / domain similar to the collagen of the zsig39 polypeptide. Fragments of zsig39 can be evaluated for their antimicrobial properties according to procedures known in the art. See, for example, Barsum et al., Eur. Respir. J. 8 (5): 709-14, 1995; Sandosvsky-Losica et al., J. Med. Vet. Mycol (Enqland) 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 functioning of the zsig39 polypeptide fragment in this respect can be compared with proteins that are known to be functional in this respect, such as proline-rich proteins, lysozyme, histatins, lactoperoxidase or the like. In addition, fragments of zsig39 polypeptides can be evaluated in combination with one or more antimicrobial agents to identify the synergistic effects. A person of ordinary skill in the art will recognize that the antimicrobial properties of zsig39 polypeptides, fusion proteins, agonists, antagonists and antibodies can be evaluated similarly. As the neurotransmitters or modulators of neurotransmission, fragments of the zsig39 polypeptide as well as zsig39 polypeptides, fusion proteins, agonists, antagonists or antibodies of the present invention can also modulate the concentration of the calcium ion, the contraction of the muscles, the secretion of hormones, the synthesis of DNA or cell growth, the rotation or production of inositol phosphate, the release of arachidonate, the activation of phospholipase-C, gastric emptying, the activation of human neutrophils or the capacity of ADCC, the production of superoxide anion and the like. The evaluation of these properties can be carried out by known methods, such as those described herein. The impact of the zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on the intracellular calcium level can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52 , 1993, and the like. The impact of the zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on the contraction of the muscles can be evaluated by methods known in the art, such as those described by Smits and Lebebvre, J. Auton. Pharmacol. 14: 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 zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on the secretion of hormones can be evaluated by methods known in the art, such as those for the release of prolactin described by Henriksen et al., J. of Receiver & Signal Transduction Research 15 (1-4): 529-41, 1995, and the like. The impact of the zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on DNA synthesis or cell growth can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and the like. The impact of the zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on the production or rotation of the inositol phosphate can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341-52, 1993, and the like. Also, the impact of the zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on the release of the arachidonate can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341- 52, 1993, and the like. The impact of the polypeptide, fragment, fusion agent, agonist or antagonist of zsig39 on the activation of phospholipase-C can be evaluated by methods known in the art, such as those described by Dobrzanski et al., Regulatory Peptides 45: 341 -52, 1993, and the like. The impact of the polypeptide, fragment, fusing agent, agonist or antagonist of zsig39 on gastric emptying can be evaluated 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 zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on the activation of human neutrophils and the ability of ADCC can be evaluated 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 zsig39 polypeptide, fragment, fusion agent, agonist or antagonist on superoxide anion production can be evaluated by methods known in the art, such as those described by Wozniak et al., Immunology 78: 629-34, 1993, and similar. The present invention also provides zsig39 fusion proteins. For example, the fusion proteins of the present invention encompass (1) a polypeptide selected from the following: (a) a polypeptide comprising an amino acid residue sequence that is at least 80% identical in amino acid sequence to the residue of amino acids 19 to amino acid residue 243 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 16 to amino acid residue 243; (c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2 containing the collagen-like domain or a portion of the domain similar to the collagen capable of dimerization or oligomerization; e) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, which contains the globular domain or an active portion of the globular-like domain; or f) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, including the collagen-like domain and the globular domain; and (2) another polypeptide. The other polypeptide can be an alternative or additional globular domain, a domain similar to alternative or additional collagen, a signal peptide to facilitate the secretion of the fusion protein or the like. The globular domain of complement-binding IgG, therefore, the globular domain of the zsig39 polypeptide, fragment or fusion agent may have a similar role. The zsig39 polypeptides, ranging from amino acid 1 (Met) to amino acid 243 (Ala); the alternative mature zsig39 polypeptides, ranging from amino acid 16 (Ser) or amino acid 19 (Leu) to amino acid 243 (Ala); or the directing fragments of the alternative secretion thereof, such fragments ranging from amino acid 1 (Met) to amino acid 15 (Gly) or amino acid 18 (Pro), can be used in the study of the secretion of proteins from the cells. In the preferred embodiments of this aspect of the present invention, the mature polypeptides are formed as fusion proteins with the putative secretory signal sequences; the plasmids that support the 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 verified. In other preferred embodiments of this aspect of the present invention, the conductive fragments of the alternative secretion are formed as fusion proteins with alternative proteins selected for secretion; the plasmids carrying the regulatory regions capable of directing the expression of the fusion protein are introduced into the test cells; and the secretion of the protein is verified. Verification can be done by techniques known in the art, such as CLAR and the like.

The highly conserved amino acids, particularly those in the carboxy-terminal globular domain of the zsig39 polypeptide, can be used as a tool to identify new elements of the family. For example, the reverse transcription polymerase chain reaction (RT-PCR) can be used to amplify the sequences encoding the conserved radicals or motifs of RNA obtained from a variety of tissue sources. 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 121-126 of SEQ ID NO: 2 (corresponding to nucleotides 558-575 of the SEQ ID NO: l); 2) Amino acids 131-136 of SEQ ID NO: 2 (corresponding to nucleotides 588-605 of SEQ ID NO: 1); 3) Amino Acids 149-154 of SEQ ID NO: 2 (corresponding to nucleotides 642-659 of SEQ ID NO: 1); 4) Amino acids 202-207 of SEQ ID NO: 2 (corresponding to nucleotides 801-818 of SEQ ID NO: 1); and 5) Amino acids 226-231 of SEQ ID NO: 2 (corresponding to nucleotides 873-890 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 described above are also encompassed. Within the preferred embodiments of the invention, the isolated polynucleotides will hybridize to regions of similar size of SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, other probes specifically recited here or a complementary sequence for it, under strict conditions. In general, stringent conditions are selected to be about 5 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under a defined ionic strength and pH) at which 50% of the target or target sequence is hybridized to a perfectly matched probe. Typical stringent conditions are those in which the concentration of the salt is up to about 0.03 M at pH 7 and the temperature is at least about 60 ° C.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation between these polynucleotide molecules is possible. SEQ ID NO: 10 is a degenerate polynucleotide sequence that encompasses all polynucleotides that could encode the zsig39 polypeptide of SEQ ID NO: 2 (amino acids 1-243). Those skilled in the art will also recognize that the degenerate sequence of SEQ ID NO: 10 also provides all of the RNA sequences encoding SEQ ID NO: 2 by substituting U for T. Therefore, the polynucleotides encoding the zsig39 polypeptide they vary from nucleotide 1, 46 or 55 to nucleotide 729 of SEQ ID NO: 10 are contemplated by the present invention. Also contemplated by the present invention are fragments and fusions as described above with respect to SEQ ID NO: 1, which are formed from the analogous regions of SEQ ID NO: 10, wherein nucleotides 198 a 926 of SEQ ID NO: 1 correspond to nucleotides 1 to 729 of SEQ ID NO: 10. The symbols in SEQ ID NO: 10 are summarized in Table 1 below.

TABLE 1 Nucleotide Resolutions Complement Resolutions AATTCCGGGGCCTTAARA / GYC | TYC | TRA | GMA | CKG | TKG | TMA | CSC | GWA | TWA | TSC | GHA | C | TDA | G | TBC | G | TVA | C | GVA | C | GB CIGIT DA | G | THA | C | TN AICIGIT N AICIGIT The degenerate codons used in SEQ ID NO: 10, which encompass all possible codons for a given amino acid, are described in Table 2 below.

TABLE 2 AminoLetra Codons Acid codices Denegerated Cys C TGC TGT TGY Ser S AGC AGT TCA TCG TCT TCT WSN Thr T ACA 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 CAA 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 ATC ATT ATH Leu L CTA CTC CTG CTT 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 | Asp B RAY Glu | Glu Z SAR Any XX NNN Hollow A person with ordinary skill in the art will appreciate that some ambiguity is introduced in the determination of a degenerate codon, representative of the totality of possible codons that encode each amino acid. For example, the degenerate codon for serine (WSN), in some cases, can encode arginine (AGR), and the degenerate codon for arginine (MGN), in some circumstances, can encode serine (AGY). There is a similar relationship between the codons that encode phenylalanine and leucine. Accordingly, some polynucleotides encompassed by the degenerate sequence may have some incorrect amino acids, but one of ordinary skill in the art can easily identify such erroneous sequences by reference to the amino acid sequence of SEQ ID NO: 2. Another aspect of the present invention provides a pharmaceutical composition comprising the purified zsig39 polypeptide in combination with a pharmaceutically acceptable carrier. This pharmaceutical composition will be used to modulate the energy balance in mammals or to protect endothelial cells from damage. The expression configuration of the zsig39 polypeptide indicates expression in endothelial cell tissues. With respect to the protection of endothelial cells, the zsig39 polypeptide can be used in the preservation of organs, for cryopreservation, for surgical treatment to prevent damage due to ischemia and / or inflammation or in similar procedures. The high level of expression in the small intestine suggests that the zsig39 polypeptide may be an endogenous factor that protects the gastrointestinal tissue from the damage of ischemic reperfusion. The rat, rabbit and pig models of ischemic reperfusion injury are already known in the art and can be used to evaluate zsig39, agonists or antagonists thereof, antibodies, fusion proteins and fragments. For example, Golino et al., Nature Medicine, 2 (1): 35-40, 1996, describes a myocardial model of ischemic reperfusion injury using white New Zealand rabbits. White New Zealand rabbits have also been employed in (1) a model of central vein ischemic reperfusion in the ear and (2) a model of atherosclerotic femoral artery damage in which blood flow is restored by angioplasty of the ball See, for example, Winn et al., J. Clin. Invest. 92: 2042-7, 1993, and Jang et al., Circulation, 92 (10): 3041-50, 1995. A rat model of bowel ischemia can also be employed. For example, male Sprague Dawley rats weighing between 225 and 400 grams suffer three training sessions with respect to those who sit calmly in restrictive cages. Next, the rats undergo survival surgery, during which the jugular vein catheters are implanted. For survival surgery, the rats are anesthetized, and a catheter is implanted in the right jugular vein under selected conditions to maintain the opening. The rats are then placed in restrictive cages and receive administrations of the test composition or vehicle as described below. The rats were allowed to recover for 48 hours prior to a single intravenous bolus injection of 4 days (0.5 ml) per day of either the vehicle or the test composition. The rats are kept fasting, preferably for 16-24 hours, they are anesthetized, and an analgesic is administered prior to the fourth injection. Thirty minutes after the fourth injection, the abdomen of each rat is opened with a small incision, and the superior mesenteric artery is isolated and secured for one hour. The abdomen is closed with loose sutures during the period of consolidation, they are reopened for the removal of the tweezers or tongs and again closed with loose sutures. The rats are placed in retention cages resting on a heating pad at 37 ° C for a reperfusion period of two hours. Following the reperfusion period, the rats are sacrificed and jejunal intestinal segments are removed. Some excised intestinal segments are subjected to histological evaluation and others are analyzed to verify the activities of myeloperoxidase (MPO) and maltase. MPO is a measure of the amount of neutrophil infiltration in the tissue, whereas maltase activity is a measure of the integrity of the intestinal mucosa. Ischemic reperfusion injury is associated with increased levels of MPO and reduced levels of maltase activity. Accordingly, the improvement of damage by ischemic reperfusion is expected to lead to reduced MPO activity and increased maltase activity.

Also, the zsig39 polypeptide is expressed in the subthalamic nucleus, suggesting that the zsig39 polypeptide or the agonist thereof can be an endogenous suppressor of ballistic movement by providing an inhibitory stimulus to chronically active cells. Such ballistic movements result from the injury of the subthalamic nuclei. The evaluation of the zsig39 polypeptide, the agonists or antagonists thereof, the antibodies, the fusion proteins and the fragments to verify the effectiveness in the suppression of the ballistic movements, can be carried out using techniques that are known in the art. . For example, stereotactic instruments can be used for the injury of subthalamic nuclei; if the ballistic movement is observed, the zsig39 polypeptide, the agonists or antagonists thereof, the antibodies, the fusion proteins or the fragments are administered; and any modulation of the ballistic movement is noted. With respect to modulation of the energy balance, the zsig39 polypeptides modulate their cellular metabolic reactions. Such metabolic reactions include adipogenesis, gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake, protein synthesis, thermogenesis, utilization of oxygen and the like. Among other methods known in the art or described herein, the energy balance of the mammal can be evaluated by checking one or more of the metabolic functions mentioned above. These metabolic functions are verified by techniques (tests or animal models) known to a person skilled in the art, as described more fully below. For example, the glucurregulatory effects of insulin are predominantly exerted in the liver, the musculoskeletal system and adipose tissue. Insulin binds to its cellular receptor in these three tissues and initiates tissue-specific actions that lead, for example, to the inhibition of glucose production and the stimulation of glucose utilization. In the liver, insulin stimulates the absorption of glucose and inhibits gluconeogenesis and glycogenolysis. In skeletal muscle and adipose tissue, insulin acts to stimulate the absorption, storage and utilization of glucose. There are recognized methods in the art to verify all of the metabolic functions recited above. Accordingly, a person of ordinary skill in the art is able to evaluate the polypeptides, fragments, fusion proteins, antibodies, agonists and antagonists of zsig39 to verify metabolic modulation functions. Exemplary modulation techniques are described 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 inbred mice that are homozygous for an inactivation mutation in the ob (obese) site. Such ob / ob mice are hyperphagic and hypometabolic, and are thought to be deficient in the production of the circulating OB protein. The db / db mice are inbred mice that are homozygous for an inactivation mutation in the db (diabetes) site. The db / db mice exhibit a phenotype similar to that of the ob / ob mice, except that the db / db mice also exhibit a diabetic phenotype. Such db / db mice are believed to be resistant to the effects of the circulating OB protein. Also, several in vitro methods of evaluating these parameters are known in the art.

Insulin-stimulated lipogenesis, for example, can be verified by measuring the incorporation of 14 C-acetate into the triglyceride (Mackall et al., J.

Biol. Chem. 251: 6462-6464, 1976) or an accumulation of triglycerides (Kletzien et al., Mol. Pharmacol. 41: 393-398, 1992). The absorption of glucose can be evaluated, for example, in a test to verify the transport of glucose stimulated by insulin. The differentiated, non-transfected L6 myotubes (maintained in the absence of G418) are placed in DMEM containing 1 g / 1 glucose, 0.5 or 1.0% BSA, 20 mM Hepes, and 2 mm glutamine. After two to five hours of culture, the medium is replaced with glucose free DMEM, fresh, containing 0.5 to 0.1% BSA, 20 M Hepes, 1 mM pyruvate, and 2 M 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. The deoxyglucose labeled with 3 H or 14 C is added to a final concentration of 50 μM, and the cells are incubated for approximately 10-30 minutes. The cells are then rapidly rinsed with a cold buffer (e.g. PBS), then lysed with a suitable lysis agent (e.g., 1% SDS or 1 N NaOH). The lysate of the cells is then evaluated by counting in a scintillation counter. The radioactivity associated with the cell is taken as a measure of glucose transport after subtracting the non-specific binding as determined by the incubation of the cells in the presence of cytokinesin b, an inhibitor of glucose transport. Other methods include those described, for example, by Manchester et al., Am. J. Physiol. 266 (Endocrinol Metab 29): E326-E333, 1994 (glucose transport stimulated with insulin). The metabolism of fatty acids can also be verified by techniques known in the art. In particular, the absorption and metabolism of fatty acids by the heart. Suitable animal models are available and tissues are available. Cultured cells include cardiac fibroblasts and cardiac myocytes. Established cell lines include: NIH 3T3 fibroblast (ATCC No. CRL-1658), CHN-1 fish bait heart cells (ATCC No. CRL-1680), and heart myoblasts from the H9c2 rat (ATCC No. CRL-1446). It has been n that when cardiac cells age there is a shift in the metabolism of fatty acids to glucose metabolism (Sack et al., Circulation 94: 2837-42, 1996).

Protein synthesis can be evaluated, for example, by comparing the precipitation of proteins labeled with 35S-methionine following the incubation of test cells with 35S-methionine and 35S-methionine a putative modulator of protein synthesis . Thermogenesis can be evaluated as described by B. Stanley in The Biology of Neuropeptide and Related Peptides, W. Colmers and C. Wahlestedt (ed.), Humana Press, Ottawa, 1993, pp. 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., Am. J. Physiol. 252 (4 Pt 2): R661-7, 1987; and Heller et al., Am. J. Physiol. 245 (3): R321-8, 1983. Also, the metabolic rate, which can be measured by a variety of techniques, is an indirect measurement of thermogenesis. The use of oxygen can be evaluated as described by Heller et al., Pflugers Arch 369 (1): 55-9, 1977. This method also involved an analysis of the hypothalamic temperatures and the metabolic heat production. The use of oxygen and thermoregulation have also been evaluated in humans as described by Haskell et al., J. Appl. Physiol. 51 (4): 948-54, 1981.

Expression of the zsig39 polypeptide in the heart and brain tissue involved in involuntary function (ie, the medulla oblongata) suggests that the protein may modulate the release of acetylcholine and / or norepinephrine. Among other methods known in the art or described herein, the protection of the endothelial cell tissue of the mammal can be evaluated by verification of endothelial tissue function. For example, the function of the heart (aorta) can be evaluated by checking the release of acetylcholine, the release of norepinephrine or similar parameters. These parameters are verified by techniques (tests or animal models) known to a person with ordinary skill in the art, as described more fully below. The release of acetylcholine and norepinephrine can be verified by CLAR. Levy, Electrophysiology of The Sinotrial and Atrioventricular Nodes, Alan R. Liss, Inc., 187-197, 1998, describes the measurement of norepinephrine in the coronary sinus effluent. In addition, animals can be measured electrically, with verified results as described by Elsner, European Heart Journal 16 (Supplement N) 52-8, 1995, and Reiffel and Kuehnert, PACE 17 (Part 1): 349-65, 1994 .

Zsig39 polypeptides may also find use as neurotransmitters or as modulators of neurotransmission, as indicated by the expression of the polypeptide in tissues associated with the parasympathetic or sympathetic nervous system. In this regard, the zsig39 polypeptides can find utility in the modulation of nutrient uptake, as demonstrated, for example, by the uptake of 2-deoxy-glucose in the brain or the like. Among other methods known in the art or described herein, the functions of neurotransmission can be evaluated by checking the absorption of 2-deoxy-glucose in the brain. This parameter is verified by the techniques (tests or animal models) known by a person with ordinary experience in the art, for example, autoradiography. Useful verification techniques are described, for example, by Kilduff et al., J. Neurosci. 10 2463-75, 1990, with the related techniques used to evaluate the "heart in hibernation" as described in Gerber et al., Circulation 94 (4): 651-8, 1996, and Fallavollita et al., Circulation 95 ( 7): 1900-1909, 1997. In addition, the zsig39 polypeptides, fragments, agonists or antagonists of the fusion thereof can be therapeutically useful for antimicrobial applications or modulated with a neurotransmitter. For example, the complement Clq component plays a role in the defense of the host against infectious agents, such as bacteria or viruses. Clq is known to exhibit several specialized functions. For example, Clq activates or triggers the complement cascade through interaction with bound antibody or C-reactive protein (CRP). Also, Clq interacts directly with certain bacteria, RNA viruses, mycoplasma, uric acid crystals, the lipid A component of bacterial endotoxin and the membranes of certain intracellular organelles. The binding of Clq to the Clq receptor is believed to promote phagocytosis. Clq also appears to improve the appearance of antibody formation of the host defense system. See, for example, Johnston, Pediatr. Infect. Dis. J. 12 (11): 933-41, 1993. Accordingly, soluble Clq-like molecules can be useful as antimicrobial agents, lysis promoters or phagocytosis of infectious agents. The zsig39 polypeptides of the present invention also exhibit a homology with respect to the portions that are believed to modulate neurotransmission. As shown in Figure 1, the zsig39 polypeptides are homologous with respect to the following proteins: HP25_TAMAS (SEQ ID NO: 5) (Takamatsu et al., Mol. Cell. Biol. 13: 1516-21, 1993 and Kondo and Kondo, J. Biol. Chem. 267: 473-8, 1992); HP27 TAMAS (SEQ ID NO: 6) (Takamatsu et al., And Kondo and Kondo referred to above) and CREL_RAT (SEQ ID NO: 7) (Wada and Ohtani, Brain Res. Mol. Brain Res. 9: 71-7, 1991 ). HP25 and HP27 are polypeptides found in the active serum (summer) of Siberian marmots in hibernation. CERL is present in the rat cerebellum. Accordingly, zsig39 polypeptides, fragments, fusions, agonists or antagonists can be useful in the modulation of neurotransmission for example, by binding to neutrotransmitters or receptors therefor. Hybrid radiation mapping 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 total knowledge of a gene sequence allows the design of PCR primers suitable for use with hybrid mapping panels by chromosomal radiation. Commercially available hybrid hybrid mapping panels, which cover 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 make possible chromosomal localizations and orderings of genes, based on PCR, rapid, sites labeled with the sequence / STSs), and other polymorphic and non-polymorphic markers within a region of interest. This includes the establishment of directly proportional physical distances between newly discovered genes of interest and previously mapped markers. Accurate knowledge of a gene position can be useful in a number of ways including: 1) determining whether a sequence is part of an existing contiguous and obtaining the additional surrounding genetic sequences in various forms such as the YAC-, BAC clones - or cDNA, 2) provide a possible candidate gene for a hereditary disease which shows the link to the same chromosomal region, and 3) for model cross-referenced organisms such as the mouse which may be beneficial in helping to determine which function I could have a particular gene. The results showed that maps 549.99 cR_3000 of the gene encode the zsig39 polypeptide from the top of the human chromosome 11 binding group on the hybrid WICGR radiation map. The near and far structure markers were AFMB048ZZA9 and FB17D4, respectively. The use of surrounding markers places the zsig39 gene in the Hq23.3 region on the map of integrated LDB chromosome 11 (The Genetic Location Database, University of Southhampton, WWW server: http: // cedar.genetics.soton.ac.uk / public_html /). The present invention also provides reagents which will find use in diagnostic applications. For example, the zsig39 gene, a probe comprising the zsig39 DNA or RNA or a subsequence thereof can be used to determine if the zsig39 gene is present on chromosome 11 or if a mutation has occurred. Chromosomal aberrations detectable at the zsig39 gene site include but are not limited to aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. In general, these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient, (b) incubating the genetic sample with a probe or polynucleotide primer as described above, under conditions where the polynucleotide will hybridize to a complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first product of the reaction with a product of the control reaction. A difference between the first product of the reaction and the product of the control reaction is indicative of 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, the complement of SEQ ID NO: 1, or an RNA equivalent thereof. Suitable assay methods in this regard include molecular genetic techniques known to those skilled in the art, such as restriction fragment length polymorphism analysis.

(RFLP), short series repeat (STR) analysis employing PCR techniques, the chain reaction of ligation (Barany, PCR Methods' and Applications 1: 5-16, 1991), protection assays of ribonuclease, and other genetic linkage analysis 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., Chapter 4) comprise the hybridization of an RNA probe to a sample of the patient's RNA, after which the product of the reaction ( RNA-RNA hybrid) is exposed to RNase. The hybridized regions of RNA are protected from digestion. Within PCR assays, a genetic sample from the patient is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in the size or quantity of the recovered product are indicative of mutations in the patient. Another PCR-based technique that can be employed is the analysis of single-strand conformational polymorphism (SSCP) (Hayashi, PCR Methods and Applications 1: 34-8, 1991). The zsig39 polypeptides can be used in the analysis of the energy efficiency of a mammal. The zsig39 polypeptides found in serum or tissue samples may be indicative of an ability of mammals to store food, with more highly efficient mammals tending to obesity. More specifically, the present invention contemplates methods for detecting the zsig39 polypeptide comprising: displaying a sample possibly containing the zsig39 polypeptide to an antibody bound to a solid support, wherein the antibody binds to an epitope of a polypeptide of zsig39; washing the immobilized antibody polypeptide to remove unbound contaminants; exposing the immobilized polypeptide-antibody to a second antibody directed to a second epitope of a zsig39 polypeptide, wherein the second antibody is associated with a detectable tag; and detect the detectable label. The concentration of the zsig39 polypeptide in the test sample appears to be indicative of the energy efficiency of a mammal. This information can help the nutritional analysis of a mammal. Potentially, this information may be useful in the identification and / or location as target of the energy-deficient tissue. As described in more detail below, mice receiving zsig39 were found to have reduced levels of serum free fatty acids and an increase in bone fat. Fatty acids are incorporated into triglycerides and stored as fats. Stored fat acts to isolate the body from heat loss and protects the internal organs. The fat also serves as a reservoir of stored energy. Fatty acids are released from triglycerides by lipases regulated by hormones for use in energy metabolism. The reduction in free fatty acid levels suggest that zsig39 have an effect on the absorption and metabolism of free fatty acids. Zsig39 can act to inhibit the release of fatty acids from fat stores, such as by inhibiting the action of hormonal lipases. Zsig39 can also act to improve fatty acid absorption, metabolism and storage. The zsig39 can act independently or in concert with other molecules, such as insulin, to inhibit lipolysis, improve the absorption of fatty acids and / or metabolism. As such, zsig39 could be useful in the regulation of energy metabolism. The invention therefore provides a method for modulating the metabolism of free fatty acids in individuals in need of such treatment by administering to such an individual a pharmaceutically effective dose of a zsig39 polypeptide. A "pharmaceutically effective amount" of a zsig39 polypeptide is an amount sufficient to induce a desired biological result. The result can be the relief of the signs, symptoms, or causes of a disease, or any other desired altion of a biological system. For example, an effective amount of a zsig39, agonist or antagonist polypeptide is that it provides either a subjective relief of the symptoms or an objectively identifiable improvement as will be observed by the physician or other qualified observer. In particular, such an effective amount of a zsig39 polypeptide leads to the reduction of free fatty acid levels or other beneficial effect. The effective amounts of the zsig39 polypeptides can vary widely depending on the disease or symptom to be treated. The amount of the polypeptide to be adminisd, and its concentration in the formulations, depends on the selected vehicle, the route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and the stability of the compound in the formulation . Accordingly, the physician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of the formulation adminisd, depending on clinical experience with the patient in question or with similar patients. Such amounts will depend, in part, on the particular condition to be treated, the age, weight, and general health of the patient, and other factors evident to those skilled in the art. Additional aspects of the invention provide antibodies or binding proteins synthesized (eg, those generated by the phage display, E. coli Fab, and the like) which specifically binds to the zsig39 polypeptides described above. Such antibodies are useful, among other uses as described herein, for the preparation of anti-idiopathic antibodies. The synthesized binding proteins can be produced by phage display using commercially available sets or sets, such as the Sets or Sets of the Ph.D. ™ Phage Display Peptide Library available from New England Biolabs, Inc. ( Beverly, Massachusetts). Phage display techniques are described, for example, in US Patent Nos. 5,223,409, 5,403,484 and 5,571,698. A further aspect of the present invention provides methods for identifying the agonists or antagonists of the zsig39 polypeptides described above, such agonists or antagonists may have valuable properties as described herein further. Within one embodiment, there is provided a method of identifying zsig39 polypeptide agonists, which comprises providing the cells responsible for this, culturing the cells in the presence of a test compound and comparing the cellular response with the cell cultured in the presence of the zsig39 polypeptide, and selecting the test compounds for which the cellular response is of the same type. Within another embodiment, there is provided a method of identifying antagonists of the zsig39 polypeptide, which comprises providing the cells responsible for a zsig39 polypeptide, culturing a first portion of the cells in the presence of the zsig39 polypeptide, culturing a second portion. of the cells in the presence of the zsig39 polypeptide and a test compound, and detect a decrease in a cellular response of the second portion of the cells when compared to the first portion of the cells. In addition to these assays described herein, samples can be tested for inhibition of zsig39 activity within a variety of assays designed to measure receptor binding or stimulation / inhibition of zsig39-dependent cellular responses. For example, zsig39 response cell lines can be transfected with a reporgene construct that is activated in response to a cell path stimulated by zsig39. Constructs of the reporgene of this type are already known in the art, and will generally comprise a DNA-zsig39 response element operably linked to a gene encoding a testable protein, such as luciferase. DNA response elements may include, but are not limited to, the cyclic AMP response elements (CRE), the hormone response elements (HRE), the insulin response element (IRÉ) (Nasrin et al. al., Proc. Nati, Acad. Sci. USA 87: 5273-7, 1990) and the serum response elements (SER) (Shaw et al., Cell 56: 563-72, 1989). The cyclic 7AMP response elements are reviewed in Roestler et al., J. Biol. Chem. 263 (19): 9063-6; 1988 and Habener, Molec. Endocrinol 4 (8): 1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56: 335-44; 1989. Candidate compounds, solutions, mixtures or extracts are tested to verify the ability to inhibit zsig39 activity on target or target cells as evidenced by a decrease in zsig39 stimulation of reporter gene expression . Tests of this type will detect compounds that directly block the binding of zsig39 to receptors on the cell surface, 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 to direct blockade of zsig39 binding to the receptor using zsig39 labeled with a detectable label (eg, 125I, biotin, horseradish peroxidase, FITC, and the like). Within as of this type, the ability of a test sample to inhibit the binding of labeled zsig39 to the receptor is indicative of the inhibitory activity, which can be confirmed by secondary as. The receptors used within the binding as may be cellular receptors or immobilized receptors, isolated. A further aspect of the invention provides a method for studying insulin. Such methods of the present invention comprise incubating the adipocytes in a culture medium comprising the zsig39 polypeptide, the monoclonal antibody, the agonist or the antagonist thereof, and the insulin and observing the changes in the secretion of the adipocyte protein. or differentiation. Antimicrobial agents can be direct acting or indirect acting. Such agents operate by means of membrane association or action pore formation mechanisms that are directly attached to offensive microbes. The antimicrobial agents can also act by means of an enzymatic mechanism, the breaking of the microbial protective substances or of the membrane / cell wall thereof. Antimicrobial agents, capable of inhibiting the proliferation of microorganisms or the action of altering the integrity of the microorganism by any mechanism described above, are useful in methods for preventing contamination in cell culture by microbes susceptible to this antimicrobial activity. Such techniques involve culturing the cells in the presence of an effective amount of the zsig39 polypeptide or an agonist or antagonist thereof. Also, zsig39 polypeptides or agonists thereof can be used as reagents for cell culture in in vitro studies of the infection of exogenous microorganisms, such as bacterial, viral or fungal infection. Such portions can also be used in animal models of infection in vivo. The present invention also provides methods for studying the cellular metabolism of the mammal. Such methods of the present invention comprise incubating the cells to be studied, for example, the human vascular endothelial cells, + the zsig39 polypeptide, the monoclonal antibody, the agonist or antagonist thereof and observing the changes in adipogenesis, gluconeogenesis , glycogenolysis, lipogenesis, glucose absorption, or the like. A further aspect of the invention provides a method for studying dimerization and oligomerization. Such methods of the present invention comprise the incubation of zsig39 polypeptides or fragments or fusion proteins thereof containing a collagen-like domain alone or in combination with other polypeptides carrying the collagen-like domains and observing the associations formed between domains similar to collagen. Therefore, both homomeric and heteromeric constructions can be studied in this way. Such associations are indicated by the CLAR, the circular dichroism or similar. As previously noted, isolated polynucleotides of the present invention include DNA and RNA. Methods for the isolation of DNA and RNA are well known in the art. It is generally preferred to isolate RNA from tumors of the brain, heart, placenta, adipose tissue and the like, although DNA can also be prepared using RNA from other tissues or isolated as genomic DNA. Total RNA can be prepared using the guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Nati, Acad. Sci. USA 69: 1408-1412, 1972). Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. The polynucleotides encoding the zsig39 polypeptides are then identified and isolated, for example, by hybridization or PCR. The present invention also provides polypeptides and counterpart polynucleotides from other species (orthologs). These species include, but are not limited to mammals, birds, amphibians, reptiles, fish, insects and other vertebrate and invertebrate species. Of particular interest are the zsig39 polypeptides of other mammalian species, including murine, rat, porcine, ovine, bovine, canine, feline, equine and other primates proteins. Orthologs of human proteins can be cloned using the information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using the 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 is then prepared from the mRNA of a positive tissue of the cell line. A cDNA encoding the zsig39 polypeptide can then be isolated by a variety of methods, such as by probing with a partial or complete 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 primers designed from the sequences described herein. Within a further method, the cDNA library can be used to transform or transfect the host cells, and the expression of the cDNA of interest can be detected with an antibody to the zsig39 polypeptide. Similar techniques can also be applied to the isolation of genomic clones. Those skilled in the art will recognize that the sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 represent a single allele of the human zsig39 DNA and protein and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures.

Allelic variants of the DNA sequence shown in SEQ ID NO: 1, including those containing the silent mutations and those in which the mutations lead to changes in the amino acid sequence, are within the scope of the present invention, as what are the proteins which are allelic variants of SEQ ID NO: 2. The cDNAs generated from the alternatively spliced mRNAs, which retain the properties of the zsig39 polypeptide, are included within the scope of the present invention, as are the polypeptides encoded by such cDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing the cDNA or the genomic libraries of different individuals or tissues according to standard procedures known in the art. The present invention also provides the isolated zsig39 polypeptides that are substantially homologous to the polypeptides of SEQ ID NO: 2 and their orthologs of the species. The term "substantially homologous" is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, identity of the sequence with respect to the sequences shown in SEQ ID NO: 2 or your orthologs. Such polypeptides will more preferably be at least 90% identical, and even more preferably 95% or more identical with respect to SEQ ID NO: 2 or their orthologs. The percent identity of the sequence 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 marks using a gap opening penalty of 10, a gap width penalty of 1, and the matrix of "blosum 62" labeling by Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by standard letter codes). The identity percentage is then calculated as: Total number of identical matches x 100 [length of the longest sequence plus the number of holes entered in the longest sequence to align the two sequences] H r-rH 1 = rH Ol ro rH 1 CO < * rH ro Ol l 1 1 1 c t ^ rH ro o; 1 I 1 1 faith O l J? -i ro rH 1 1 1 1 s? N O CM H H H H rH 1 1 1 1 1 ¡ai? ro rH O H ro l CN 1 1 1 1 1 1 J «i1 CN OJ o ro OJ rH í H rH m 1 1 1 1 1 1 < # CM ro rH o ro OJ rH rH ro 1 1 1 1 1 1 ri ctí K 00 ro rH rH í rH í J l ro 1 1 1 r 1 1 1 1 1 Ei ü U) J OJ ro ro JO l CM ro ro I 1 -1 1 1 1 1 1 1 1? ? n OJ O r ro rH OJ ro HO rH ro Ol I 1 1 1 1 1 1 1 1 1 1 s? n OJ OJ O R O R H O R H O R H O R H OJ 1 1 1 1 1 1 1 1 1 us \ ro ro rH H ro HJ ro HH OJ OJ rH 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Q kD 'ro O J H H ro H ro ro H H ro ro 1 1 1 1 1 1 1 1 1 1 1 1 1 & U3 H ro ooo tH ro ro o OJ ro Ol rH o? P OJ ro 1 1 1 1 1 1 1 1 to o o n o ro ro o o lu o ro J OJ rH ro O h H ro ro r 1 1 1 1 1 1 1 1 1 1 1 1 1 < ? < rH CN J O iH H o oi H rH H rH r r r o r o r OJ 1 1 1 1 1 1 1 1 1 1 1 < You're Q U? a .4 W 2 Cu C-J O EH & t »> The identity of the sequence of the polynucleotide molecules is determined by similar methods using a ratio as described above. Substantially, homologous proteins and polypeptides are characterized in that they have one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, which are the substitutions of conservative amino acids (see Table 4) and other substitutions that do not significantly affect the 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 amino-terminal methionine residue, a small linker peptide of up to about 20-25 r-residues, or a small extension that facilitates purification, an affinity tag. The polypeptides comprising the affinity tags may further comprise a proteolytic cleavage site between the zsig39 polypeptide and the affinity tag. Such preferred sites include thrombin cleavage sites and factor Xa cleavage sites.

Table 4 Substitutions of conservative amino acids Basics: argimna lysine histidine Acids; glutaric acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatics: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine The proteins of the present invention can also comprise amino acid residues that are not naturally present. Amino acids that are not naturally present include, without limitation, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxy-proline, trans-4-hydroxyproline, N-methyl-glycine, allo-threonine, methyl threonine, hydroxyethylcysteine, hydroxyethyl homocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethyl-proline, ter-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Various methods are known in the art for incorporating amino acid residues that are not naturally present in proteins. For example, an in vitro system can be employed wherein the antisense mutations are suppressed using the chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating the tRNA are already known in the art. The transcription and translation of the plasmids containing the antisense mutations are carried out in a cell-free system comprising an extract of E. coli S30 and commercially available enzymes and other reagents. The proteins are purified by chromatography. See, for example, Robertson et al., Am. Chem. Soc. 113: 2722, 1991; Ellman et al., Methods Enzymol. 202: 301, 1991; Chung et al., Science 259: 806-9, 1993; and Chung et al., Proc. Nati Acad. Sci. USA 90: 10145-9. 1993). In a second method, the translation is carried out in Xenopus oocytes by the microinjection of the mutated RNA and the chemically acylated 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 in the presence of the amino acid (s) that are not naturally present , desired (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The amino acid that is not naturally present is incorporated into the protein instead of its natural counterpart. See, Koide et al., Biochem. 33: 7470-6, 1994. The amino acid residues that are naturally present can be converted to species that are not naturally present by an in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand 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 are not naturally present, and non-natural amino acids can be substituted for the amino acid residues of zsig39. The essential amino acids in the polypeptides of the present invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning metagenesis (Cunningham and Wells, Science 244: 1081-5). , 1989, Bass et al., Proc. Nati, Acad. Sci. USA 88: 4498-502, 1991). In this latter technique, unique alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested to verify biological activity (eg, the ability to modulate the energy balance) as described below to identify the amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., J. Biol. Chem. 271: 4699-708, 1996. The ligand-receptor sites 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 photoaffinity labeling, in conjunction with the amino acid mutation of the putative contact site. See, for example, de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identities of the essential amino acids can also be inferred 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 selection, such as those described by Reidhaar-Olson and Sauer (Science 241: 53-7, 1988) or Bowie and Sauer (Proc. Nati. Acad. Sci. USA 86: 2152-6, 1989). Briefly, these authors describe methods for simultaneously randomly treating 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 (eg, Lowman et al., Biochem 30: 10832-7, 1991, Ladner et al., US Patent 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).Variants of the described zsig39 polypeptide and DNA sequences can be generated through intermixing of the DNA as described by Stemmer, Nature 370: 389-91, 1994, Stemmer, Proc. Nati Acad. Sci. USA 91: 10747-51, 1994 and the WIPO Application WO 97/20078. Briefly, the variant DNAs are generated by in vitro homologous recombination by the random fragmentation of an origin DNA followed by reassembly used PCR, leading to random mutations introduced point. This technique can be modified using a family of source DNAs, such as allelic variants or DNAs from different species, to introduce additional variability in the process. The selection or selection for the desired activity, followed by additional iterations of the mutagenesis and assay provide a rapid "evolution" of the sequences by selecting the desirable mutations while simultaneously selecting against the deleterious changes. Mutagenesis methods as described above can be combined with automated, high throughput screening methods to detect the activity of the mutagenized polypeptides cloned in the host cells. The mutagenized DNA molecules encoding the active polypeptides (e.g., the 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 described above, a person of ordinary skill in the art can identify and / or prepare a variety of polypeptides that are substantially homologous with respect to residues 19 to 243 of SEQ ID NO: 2 or the allelic variants thereof. and retaining modulation of the energy balance or other properties of the wild-type protein. Such polypeptides may include additional amino acids, such as additional collagen repeats of the Gly-Xaa-Pro or Gly-Xaa-Xaa type. Such polypeptides may also include the additional polypeptide segments as generally described above. The polypeptides of the present invention, including full length proteins, fragments thereof and fusion proteins, can be produced in host cells genetically engineered according to conventional techniques.

Suitable host cells are those types of cells that can be transformed or transfected with the exogenous DNA and growing in the culture, and include bacteria, fungal cells, and higher, cultured eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating the cloned DNA molecules and introducing the exogenous DNA into a variety of host cells are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 / a. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. In general, a DNA sequence encoding a zsig39 polypeptide of the present invention is operably linked to other genetic elements required for their expression, which generally include a transcription promoter and 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 the selectable markers can be provided on separate vectors, and the replication of the exogenous DNA can be provided by the integration into the genome of the host cell. 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 such elements are described in the literature and are available through commercial providers. To digest a zsig39 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The sequence of the secretory signal may be that of the zsig39 polypeptide, or it may be derived from another secreted protein (eg, t-PA) or synthesized de novo. The sequence of the secretory signal is linked to "the DNA sequence of the zsig39 polypeptide in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell." Sequences of the secretory signal are commonly placed 5 'with respect to the DNA sequence encoding the polypeptide of interest, although certain sequences of the signal can be placed anywhere in the DNA sequence of interest (see, for example, Welch et al., US Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). Alternatively, the sequence of the secretory signal contained in the polypeptides of the present invention is used to direct other polypeptides towards the secretory pathway. The present invention provides such fusion polypeptides. A signal fusion polypeptide can be made wherein a sequence of the secretory signal derived from amino acid residues -1-15 or 1-19 of SEQ ID NO: 2 is to be operably linked to another polypeptide using the methods known in the art and described herein. The sequence of the secretory signal contained in the fusion polypeptides of the present invention is preferably amino-terminally fused to an additional peptide to direct the additional peptide towards the secretory pathway. Such 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. Such fusions can be used in vivo or in vitro to direct the peptides through the secretory pathway.

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 et al. Van der Eb, Virology 52: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), transfection mediated by dextran-DEAE (Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993), and viral vectors (Miller and Rosan, BioTechniques 7: 980-90, 1989; Wang and Finer, Nature Med. 2: 714-16, 1996). The production of the recombinant polypeptides in the cells of cultured mammals is described, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. 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 (ATCC No. CRL 1650), 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 the cell lines of the Chinese hamster ovary (eg CHO-Kl; ATCC No. CCL 61). Suitable cell lines are already known in the art and are available from public repositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters, such as the SV-40 or cytomegalovirus promoters, are preferred. See, for example, U.S. Pat. No. 4,956,288. Other suitable promoters include those of the metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the major anterior promoter of the adenovirus. The selection of the drug is generally used to select the cultured mammalian cells in which the foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and that are capable of passing the gene of interest to their progeny are referred to as "stable transfectants". A preferred selectable marker is a gene that codes for resistance to antibiotic neomycin. The selection is carried out in the presence of a drug of the neomycin type, such as G-418 or the like. The selection systems can also be used to increase the level of expression of the gene of interest, a process referred to as "amplification". The amplification is carried out by culturing the 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 of the introduced genes. A preferred selectable plificable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (eg, hygromycin resistance, multiple drug resistance, puromycin acetyltransferase) may also be used. Alternate markers that introduce an altered phenotype, such as green fluorescent protein, or cell surface proteins such as CD4, CD8, MHC Class I, placental alkaline phosphatase, can be used to classify transfected cells from cells not transfected by means such as the FACS classification or the separation technology of beads or magnetic ridges. Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and bird cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J.

Biosci. (Bangalore) 11: 47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is described by Guarino et al., U.S. Pat. No. 5,162,222 and the WIPO publication WO 94/06463. Insect cells can be infected with the recombinant baculovirus, commonly derived from the Autographa californica nuclear polyhedrosi virus (AcNPV). See, King and Possee, The Baculovirus Expression System: A Laboratory Guide, London, Chapman and Hall; O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Pres., 1994; and, Richardson, C.D., Ed. Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa NJ, Humana Press, 1995. A second method of manufacturing recombinant zsig39 baculovirus utilizes 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-Bac ™ game or set (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl ™ (Life Technologies) which contains a Tn7 transposon to move the DNA encoding the zsig39 polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid". See, Hill-Perkins and Possee, J. Gen. Virol. 71: 971-6, Bonning et al., J. Gen. Virol. 75: 1551-6, 1994; and, Chazenbalk and Rapoport, J. Biol. Chem. 270: 1543-9, 1995. Further, transfer vectors can include a frame fusion with DNA encoding an epitope tag at the C or N extremity of the polypeptide. of zsig39 expressed, for example, a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nati Acad. Sci. 82: 7952-4, 1985). Using a technique known in the art, a transfer vector containing zsig39 is transformed into E. coli, and selected for the bacmides which contain an interrupted lacZ gene indicative of the recombinant baculovirus. Bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, for example Sf9 cells. The recombinant virus expressing zsig39 is subsequently produced. The recombinant viral storage materials are made by the methods commonly used in the art. Recombinant virus is used to infect host cells, typically a cell line derived from the summer caterpillar, 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 FiveO ™ cell line.

(Invitrogen) derived from Trichoplusia ni (U.S. Pat. # 5,300,435). Commercially available free serum media are used to grow and maintain cells. The appropriate means are Sf900 II ™ (Life Technologies) or ESF 921 ™ (Expression Systems) for Sf9 cells; and Ex-cell405 ™ (JHR Biosciences, Lenexa, KS) or Express FiveO ™ (Life Technologies) for the T. ni cells. The cells were grown from an inoculation density of about 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells, at which time a recombinant viral storage material is added at a multiplicity of infections (MOI) of 0.1. to 10, more typically close to 3. The procedures used are generally described in the available laboratory manuals (King and Possee, ibid., O'Reilly et al., Richardson, ibid.). The subsequent purification of the zsig39 polypeptide from the supernatant can be accomplished using the 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 Sccharomyces cerevisiae, Pichia pastotis, and Pichia methanolica. Methods for transforming S. cerevisiae cells with the exogenous DNA and the production of recombinant polypeptides therefrom are described, for example, in Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al., U.S. Patent No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are selected by the phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A system of preferred vectors for use in Saccharomyces cerevisiae is the P0T1 vector system described by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows the transformed cells to be selected by growing the medium containing the glucose. Promoters and terminators suitable for use in yeast include those of the glycolytic enzyme genes (see, for example, Kawasaki, US Patent No. 4,599,311, Kingsman et al., US Patent No. 4,615,974, and Bitter, US Patent No. 4,977,092) and the alcohol dehydrogenase genes. See also U.S. No. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltose are already known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132: 3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells can be used according to the methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods for the transformation of Acremonium chrysogenum are described by Sumino et al., U.S. Pat. No. 5,162,228. Methods for the transformation of Neurospora are described by Lambowitz, U.S. Pat. No. 4,486,533. The use of Pichia methanolica as the host for the production of the recombinant proteins is described in WIPO Publications WO 97/17450, WO 97/17451, WO 9802536, and WO 98/02565. DNA molecules for use in the transformation of P.metanolica will commonly be prepared as circular, double-stranded plasmids, which are preferentially linearized prior to transformation. For the production of polypeptides in P. methanolica, it is preferred that the promoter and the terminator in the plasmid be those of a P. methanolica gene, such as a gene utilizing the alcohol of P. methanolica (AUG1 or AUG2). Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the complete expression segment of the plasmid flanked at both ends by the host DNA sequences. A preferred selectable marker for use in Pichia methanolica is a P. methanolica ADE2 gene, which encodes 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 the host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. For the production of the secreted proteins, the host cells deficient in the vacuolar protease genes (PEP4 and PRB1) are preferred. Electroporation is used to facilitate the introduction of a plasmid containing the DNA encoding a polypeptide of interest into the cells of P. methanolica. It is preferred to transform the P. methanolica cells by electroporation using a pulsing electric field, which decays exponentially, having a field strength of 2.5 to 4.5 kV / cm, preferably approximately 3.75 kV / cm, and a time constant (t) from 1 to 40 milliseconds, more preferably approximately 20 milliseconds. Prokaryotic host cells, including strains of the bacterium Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for the transformation of these hosts and the expression of foreign DNA sequences cloned therein are well known in the art (see, for example, Sambrook et al., Ibid.). When a zsig39 polypeptide is expressed in bacteria such as E. coli, the polypeptide can be retained in the cytoplasm, typically as the insoluble granules, or it can be directed to the periplasmic space by a bacterial secretion sequence. In the first case, the cells are used, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be redoubled and dimerized by diluting 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 altering the cells (for example, by subjecting them to the action of sound or osmotic shock) to release the content of the periplasmic space and recover the protein, by which obviates the need for denaturalization and withdrawal. The transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing the nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals. The media may also contain components such as growth factors or serum, when required. The growth medium will generally be selected for cells containing the exogenously added DNA, for example, by drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or cotransfected in the host cell.

The expressed recombinant zsig39 polypeptides (or the chimeric zsig39 polypeptides) can be purified using conventional methods and means of fractionation and / or purification. The precipitation of ammonium sulfate and the extraction with acid or chaotrope can be used for the fractionation of the samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse phase high performance liquid chromatography. Suitable anion exchange media include the dextrans derivatives, the agarose, the cellulose, the polyacrylamide, especially the silicas, and the like. Derivatives of PEI, DEAE, QAE and Q are preferred, with the DEAE Rapid Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred. Suitable chromatographic media include those media derived with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia), Toyoperl 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, styrene beads, crosslinked polyacrylamide resins and the like which are insoluble under the conditions in which they will be used. These supports can be modified with reactive groups that allow the fixation of the proteins by the amino groups, the carboxyl groups, the sulfhydryl groups, the hydroxyl groups and / or the carbohydrate moieties. Examples of the coupling chemicals include activation with cyanogen bromide, activation with N-hydroxysuccinimide, activation with epoxide, activation with sulfhydryl, activation with hydrazide, and the carboxyl and amine derivatives for the carbodiimide union. These and other solid media are well known and widely used in the art, and are available to commercial suppliers. Methods for attaching 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 medium. See, for example, Affinity Chro atography: Principies & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated by exploitation of their structural or binding properties. For example, metal ion adsorption chromatography (IMAC) can be used to purify proteins rich in histidine, or proteins that have His tags. Briefly, a gel is charged first with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem 3: 1-7, 1985). The proteins rich in histidine will be adsorbed in this matrix with different affinities, depending on the metal ion used, and will be eluted by competitive elution, lowering the pH or using strong chelating agents. Other purification methods include the purification of glycosylated proteins 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 the additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (eg, Glu-Glu affinity tags, FLAG tags, the maltose binding protein, an immunoglobulin domain) can be built to facilitate purification. Such purification methods are described in detail in the following Examples section. The methods of folding (and optionally reoxidation) of the proteins can be used advantageously. It is preferred to purify the protein up to > 80% purity, more preferably up to > 90% purity, even more preferably > 95%, and a pharmaceutically pure state, which is greater than 99.9% pure with respect to the contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents is particularly preferred. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin. Polypeptides or fragments of zsig39 thereof can also be prepared through chemical synthesis. Such zsig39 polypeptides can be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue. An in vivo approach to testing the proteins of the present invention involves the viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated viruses (AAV). Adenoviruses, a double-stranded DNA virus, is commonly the best-studied gene transfer vector for the delivery of heterologous nucleic acid (for review, see Becker et al., Meth Cell Biol. 43: 161-89, 1994; and Douglas and Curiel, Science &Medicine 4: 44-53, 1997). The adenovirus system offers several advantages: the adenovirus can (i) accommodate relatively large DNA inserts; (ii) growing to high concentrations; (iii) infect a wide range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because the adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By removing portions of the adenovirus genome, the larger inserts (up to 7 kb) of the heterologous 7? DN can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a cotransfected plasmid. In an exemplary system, the essential gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary).

When administered intravenously to intact animals, the adenovirus targets primarily the liver. If the adenoviral delivery system has a deletion of the gene, the virus can not replicate in the host cells. However, host tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. The secreted proteins will be introduced into the circulation in the highly vascularized liver, and the effects on the infected animal can be determined. The adenovirus system can also be used for the production of in vitro proteins. By culturing cells other than 293 infected with the adenovirus under conditions where the cells do not divide rapidly, the cells can produce proteins for extended periods of time. For example, BHK cells are grown to confluence in the cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum free conditions, which allow the infected cells to survive for several weeks without significant cell division. Alternatively, 293S cells infected with the adenovirus vector can be grown in the suspension culture at a relatively high density of the cells to produce significant amounts of protein (see Garnier et al., Cytotechnol 15: 145-55, 1994). With any protocol, a secreted heterologous protein, expressed, can be repeatedly isolated from the cell culture supernatant. Within the production protocol of infected 293S cells, non-secreted proteins can also be obtained effectively. A ligand binding polypeptide, such as a zsig39 polypeptide binding polypeptide, can also be used for the purification of the ligand. The polypeptide is immobilized on a solid support, such as agarose beads, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or similar materials that are stable under the conditions of use . Methods for binding polypeptides to solid supports are already known in the art, and include the chemistry of amines, the activation of cyanogen bromide, the activation of N-hydroxysuccinimide, the activation of epoxides, the activation of sulfhydryl, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and the fluids containing the ligand are passed through the column one or more times to allow the ligand to bind to the ligand binding polypeptide. The ligand is then eluted using changes in the concentration of the salt, the chaotropic agents (guanidine HCl), or the pH to alter receptor-ligand binding. An assay system using a ligand-binding receptor (or an antibody, an element of a complement / anticomplement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore ™, Pharmacia Biosensor, Piscataway, NJ) can be used advantageously. Such receptor, antibody, element of a complement / anticomplement pair or fragment is immobilized on the surface of a bit 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, element or fragment is covalently linked, using the chemistry of the amine or sulfhydryl, to dextran fibers that are attached to a gold film within the cell flow. A test sample is passed through the cell. If a ligand, epitope, or opposite element of the complement / anticomplement pair is present in the sample, it will bind to the immobilized receptor, antibody or element, respectively, causing a change in the refractive index of the medium, which is detected as a change in the resonance of the surface plasmon of the golden film. This system allows the determination of activation and disconnection speeds, from which the affinity of the union can be calculated, and the evaluation of the stoichiometry of the union. The ligand binding polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for the determination of binding affinity (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). As would be apparent to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice , hamsters, guinea pigs 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 the modified forms as well as in the cells of mammals and insects. The zsig39 polypeptides or a fragment thereof serve as an antigen (immunogen) to inoculate an animal or to produce an immune response. Suitable antigens could include the zsig39 polypeptide encoded by SEQ ID NO: 2 from amino acid residues 16-2243 of SEQ ID NO: 2, from amino acid residues 19-243 of SEQ ID NO. : 2, or a fragment thereof of contiguous amino acid residues 9-243. The immunogenicity of a zsig39 polypeptide can be increased by the use of an adjuvant, such as alum (aluminum hydroxide) or complete or incomplete Freund's adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zsig39 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 "like the hapten", such portion may be attached or advantageously linked to a macromolecular carrier (such as keyhole slip hemocyanin (KLH), serum albumin bovine (BSA) or tetanus (toxoid) for immunization. When used herein, the term "antibodies" includes polyclonal antibodies, polyclonal antibodies affinity purified monoclonal antibodies, and binding fragments of antigens, such as proteolytic fragments of F (ab ') 2 and Fab, such as chimeric antibodies, Fv fragments, antibodies, single chain and the like, as well as peptides and binding polypeptides antigen, synthetic, are also included. non-human antibodies may be humanized by grafting only non-human CRDs on human structure and constant regions, or incorporating non-human variable domains optionally "coating" them with a surface similar to the h it is used for the replacement of exposed waste, where the result is a "coated" antibody). In some cases, humanized antibodies can retain non-human residues within the domains of the variable region structure to improve the appropriate binding characteristics. Through the humanization of the antibodies, the biological half-life can be increased, and the potential for adverse immune reactions during administration to humans is reduced. Alternatives for generating or selecting antibodies useful techniques herein include in vitro exposure of lymphocytes to the protein or peptide of zsig39, and selection of display libraries of antibody phage or similar vectors (for instance, through the use of immobilized or labeled protein or zsig39 peptide). The antibodies are defined to be specific binding if: 1) they exhibit a threshold level of binding activity, and / or 2) they do not cross-react significantly with the molecules of the related polypeptides. First, the antibodies here bind specifically if they bind to a polypeptide of zsig39, peptide or epitope with a binding affinity (Ka) of 106 mol-1 or greater, preferably 107 mol-1 or greater, more preferably 108 mol-1 or greater, and even more preferably 109 mol "" 1 or greater. The binding affinity of an antibody can easily be determined by a person with ordinary skill in the art., for example, by Scatchard's analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-72, 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 zsig39 polypeptide but not the known related polypeptides using a standard Western blot analysis (Ausubel et al., Ibid.). Examples of known related polypeptides are orthologs, proteins of the same species that are elements of a family of proteins such as Acrp30 (SEQ ID NO: 8), polypeptides shown in alignment in Figure 1, polypeptides of zsig39 human mutants, and the like. In addition, the antibodies can be "selected against" the known related polypeptides to isolate a population that specifically bind to the polypeptides of the invention. For example, enhanced antibodies to human zsig39 polypeptides are adsorbed to the related polypeptides adhered to the insoluble matrix; antibodies specific for human zsig39 polypeptides will flow through the matrix under the appropriate buffer conditions. Such selection allows the isolation of polyclonal and monoclonal antibodies that do not cross-react with closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995). The selection 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 JW (eds.), Academic Press Ltd., 1996, Benjamin et al., Ann. Rev. Immunol., 2: 67-101, 1984). Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), spot spotting or Western spotting, inhibition or competition assay, and the sandwich test. Genes encoding polypeptides having potential zsig39 polypeptide binding domains, "binding proteins", can be obtained by screening the targeted or randomized peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli. The nucleotide sequences encoding the polypeptides can be obtained in various ways, such as through random mutagenesis and random polynucleotide synthesis. Alternatively, restricted phage display libraries can also be produced. These peptide display libraries can be used to select the peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a macromolecule, or organic or inorganic substances. Techniques for creating and selecting such peptide display libraries are already known in the art. (Ladner et al., US Patent No. 5,223,409, Ladner et al., US Patent No. 4,946,778, Ladner et al., US Patent No. 5,403,484 and Ladner et al., US Patent No. 5,571,698) and the exhibition libraries. of the peptides and sets or sets to select such 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). Peptide display libraries can be selected using the zsig39 sequences described herein to identify proteins which bind to zsig39. These binding proteins which interact with the zsig39 polypeptides can be used essentially in an antibody-like manner to target the cells; for isolating the homologous polypeptides by affinity purification; conjugated directly or indirectly with respect to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for the selection of expression libraries and the neutralizing activity. The binding proteins can also be used for diagnostic assays to determine the circulating levels of the polypeptides; to detect or quantify soluble polypeptides as the marker of the underlying disease or disease. To increase the half-life of these binding proteins, they can be conjugated. Their biological properties can be modified by dimerization or multimerization for use as agonists or antagonists. The binding peptides can be selected against the known related polypeptides as described above. Antibodies and binding proteins for zsig39 can be used to target cells expressing zsig39; for the isolation of zsig39 by affinity purification; for diagnostic assays to determine the circulating levels of the zsig39 polypeptides; to detect or quantify soluble zsig39 as a marker of the underlying disease or disease; in the analytical methods that use FACS; to select the expression libraries; to generate anti-idiopathic antibodies; and as neutralizing antibodies or as antagonists to block the energy balance modulating activity of the zsig39 polypeptide or a similar activity in vitro and in vivo. Suitable labels or direct signs include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like; Indirect labels or signs can characterize the use of biotin-avidin or other complement / anti-complement pairs as intermediates. In addition, antibodies to zsig39 or fragments thereof can be used in vitro to detect denatured zsig39 or fragments thereof in assays, eg, Western blots or other assays known in the art. The antibodies or binding proteins herein can also be conjugated directly or indirectly with the drugs, toxins, radionuclides and the like, and these conjugates used for diagnostic or therapeutic applications in vivo. For example, the polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (receptor or antigen, respectively, for example). More specifically, zsig39 polypeptides or anti-zsig39 antibodies, or bioactive fragments or portions thereof, can be coupled or linked to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule. Suitable detectable molecules can be attached directly or indirectly to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent labels, chemiluminescent labels, magnetic particles and the like. Suitable cytotoxic molecules can be attached directly or indirectly to the polypeptide or antibody, and include bacterial or plant toxins (eg, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin, and the like), as well as therapeutic radionuclides, such such as iodine-131 rhenium-188 or yttrium-90 (either fixed directly to the polypeptide or antibody, or indirectly fixed through the media of a chelating portion, for example). The polypeptides or antibodies can also be conjugated with cytotoxic drugs, such as adria icine. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with an element of a complementary / anti-complementary pair, wherein the other element is linked to the polypeptide or antibody portion. For these purposes, biotin / streptavidin is an exemplary complementary / anti-complementary pair. In another embodiment, the polypeptide-toxin fusion proteins or the toxin-antibody fusion proteins can be used for the inhibition or ablation of the targeted tissue or cell (e.g., to treat cancer cells or tissues). ). Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a target or target domain), a fusion protein that includes only the target location domain may be suitable to direct a detectable molecule, a cytotoxic molecule or a molecule complementary to a cell or tissue type of interest. In cases where the domain only of the fusion protein includes a complementary molecule, the anti-complementary molecule can be conjugated with a detectable or cytotoxic molecule. Such fusion proteins of the complementary molecule-domain thus represent a generic target location vehicle for the specific delivery of the cell / tissue of the conjugates of the cytotoxic / detectable-anticomplementary, generic molecule. The bioactive polypeptide or the antibody conjugates described herein can be delivered intravenously, intraarterially, intraductally with DMSO, intramuscularly, subcutaneously, intraperitoneally, also by transdermal methods, by electroblotting, orally or by means of an inhalant. The polynucleotides encoding the zsig39 polypeptides are useful within the applications of gene therapy where it is desired to increase or inhibit the activity of zsig39. If a mammal has a mutated or absent zsig39 'gene, the zsig39 gene can be introduced into the mammalian cells. In one embodiment, a gene encoding a zsig39 polypeptide is introduced in vivo into a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and similar. Defective viruses, which completely or almost completely lack viral genes, are preferred. A defective virus is not infectious after introduction into a cell. The use of defective viral vectors allows administration to cells in a specific localized area, regardless of which vector can infect other cells. Examples of the particular vectors include, but are not limited to, a defective herpes simplex 1 virus vector (HSV1) (Kaplitt et al., Molec. Cell Neurosci.2: 320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J. Clin. Invest. 90: 626-30, 1992; and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61: 3096-101, 1987; Samulski et al., J. Virol. 63: 3822-8, 1989). In another embodiment, a zsig39 gene can be introduced into a retroviral vector, for example, as described in Anderson et al., U.S. Pat. No. 5,399,346; Mann et al. Cell 33: 153, 1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62: 1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; WIPO publication WO 95/07358; and Kuo et al., Blood 82: 845, 1993. Alternatively, the vector can be introduced by lipofection in vivo using the liposomes. Synthetic cationic lipids can be used to prepare liposomes for the in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Nati, Acad. Sci. USA 84: 7413-7, 1987; Mackney et al., Proc. Nati, Acad. Sci. USA 85: 8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. The location as a target of liposome molecules with respect to specific cells represents an area of benefit. More particularly, directing transfection to particular cells represents an area of benefit. For example, directing transfection to particular cell types could 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 target location. Target or target peptides (e.g., hormones or neutrotransmitters), proteins such as antibodies, or molecules other than peptides can be chemically linked to liposomes. It is possible to remove target or target cells from the body; to introduce the vector as a naked DNA plasmid; and then reimplant the transformed cells in the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, eg, transfection, electroporation, macroinjection, transduction, cell fusion, DEAE dextran, the precipitation of calcium phosphate, the use of a gene gun or the 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 zsig39 gene, such as to inhibit the proliferation of cells in vivo. Polynucleotides that are complementary to a segment of a zsig39 coding polynucleotide (eg, a polynucleotide described in SEQ ID NO: 1) are designed to bind to the mRNA encoding zsig39 and to inhibit the translation of such mRNA. Such antisense polynucleotides are used to inhibit the expression of the genes encoding the zsig39 polypeptide in the cell culture or in a subject. Transgenic mice, designed to express the zsig39 gene, and mice that exhibit a complete absence of zsig39 gene function, referred to as "unconscious mice" (Snowaert et al., Science 257: 1083, 1992), also they can be generated (Lowell et al., Nature 366: 740-42, 1993). These mice can be used to study the zsig39 gene and the protein encoded by it in an in vivo system. For pharmaceutical use, the proteins of the present invention are formulated for parenteral administration, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion during a typical period of one to several hours. In general, the pharmaceutical formulations will include a zsig39 protein in combination with a pharmaceutically acceptable carrier, such as a saline solution, buffered brine, 5% dextrose in water or the like. The formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent loss of protein on the surfaces of the vial, 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, 19 / a. ed., 1995. The therapeutic doses in general will be determined by the doctor according to accepted standards, taking into account the nature and severity of the condition to be treated, the traits or qualities of the patient, 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 polynucleotides encoding the novel zsig39 polypeptides of the present invention were initially identified by selecting an EST from an EST database, predicting a protein sequence based thereon, and searching for known sequence databases for the secreted protein. which is more homologous with respect to the predicted protein based on the EST. ESTs that potentially encode proteins that have a biologically interesting homology to known secreted proteins were identified for further study. A single EST sequence was discovered and predicted to be homologous with respect to the adipocyte-specific protein. See, for example, Scherer et al., J. Biol. Chem. 270 (45): 26746-9, 1995. To identify the corresponding 7α DNc, a clone that is considered to probably contain the entire coding sequence is used. for sequencing. Using a set or kit Miniprep Invitrogen S.N.A.P. ™ (Invitrogen, Corp., San Diego, CA) according to the manufacturer's instructions, an overnight culture of 5 ml in LB + 50 μg / ml ampicillin was prepared. The model was sequenced on a ABIPRISM ™ Model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Ct.) Using the Reaction Set or Set Ready for Sequencing of the ABI PRISM ™ Dye Terminator Cycle (Perkin-Elmer Corp.) according to the manufacturer's instructions. Oligonucleotides ZC447 (SEQ ID NO: 11), ZC976 (SEQ ID NO: 12) up to M13 and the lacZ promoters on the vector containing the clone were used as sequencing primers. Oligonucleotides ZC14707 (SEQ ID NO: 13), ZC14708 (SEQ ID NO: 14), ZC14760 (SEQ ID NO: 15), ZC14758 (SEQ ID NO: 16) and ZC14759 (SEQ ID NO: 17) were used to complement the sequence of the clone. The sequencing reactions were carried out in a Hybaid OmniGene Temperature Cycling System (National Labnet Co., Woodbridge, NY). The analysis program of SEQUENCHER ™ 3.1 sequences was used for the analysis of the data. The resulting 1347 bp sequence is described in SEQ ID NO: 1. Comparison of the EST sequence originally derived with the sequence depicted in SEQ ID NO: 1 showed that there were differences of 27 base pairs which led to 11 amino acid differences between the deduced amino acid sequences. Note that 22 of the base pair differences were from the unknown "N" residues in the EST sequence with respect to the residues known in SEQ ID NO: 1, which led to the "presumed" amino acid changes.

Example 2 Fabric Distribution The Northerns were made using Clontech Multi-Human Tissue Staining (Palo Alto, CA). An approximately 1347 bp DNA probe, which corresponds to a sequence spanning a polynucleotide encoding the full-length zsig39 polypeptide, was degenerated by the EcoRI-Notl digestion of the plasmid DNA. The resulting fragment was gel purified for use as a probe. The DNA probe was radiolabelled with 32P using the REDIPRIME® DNA labeling system (Amersham, Arlington Heigts, Illinois) according to the manufacturer's specifications. The probe was purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA). An EXPRESSHYB solution (Clontech, Palo Alto, CA) was used for prehybridization and as a hybridization solution for Northern blots. Hybridization was carried out overnight at 65 ° C, and the spottings were then washed in 2X SSC and 0.1% SDS at room temperature, followed by a wash in 0.1X SSC and 0.1% SDS at 65 ° C. A size of the transcript was observed at approximately 1.2 kb. The intensity of the signal was higher for the small intestine and the heart, with relatively less intense signals in the pancreas, the musculoskeletal system, the kidney and the thyroid, and with signals of lower intensity in the placenta, the lungs, the liver, the spleen, the prostate, the ovaries, the colon, the stomach, the spinal cord, the lymph nodes, the trachea, the adrenal gland and the bone marrow. The analysis of additional Northern Spotting was done using a Northern Tissue Spotting of the Intestine. Spotting was prepared using mRNA from the SW480 cell line of human colorectal adenocarcinoma. (Clontech, Palo Alto, CA). Human small intestine tissue (Clontech), human stomach tissue (Clontech), human intestinal smooth muscle cell line (Hism, ATCC No. CRL-1692, American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD) , the normal human colon cell line (FHC; ATCC No. CRL-1831; American Type Culture Collection) and the human fetal small intestine cell line (FHs74 Int, ATCC No. CCL241, American Type Culture Collection). The total RNAs were isolated from Hism, FHC and FHs74 Int by the acid guanidium method (Cheomczinski et al., Anal. Biochem. 162: 156-9, 1987). The polyA + RNAs were selected by eluting the total RNA through a column that retains the polyA + RNAs (Aviv et al., Proc. Nat. Acad. Sci. 69: 1408-12, 1972). 2 μg of the polyA + RNA from each sample were separated into a 1.5% agarose gene in 2.2 M formaldehyde and the phosphate buffer. The RNAs were transferred onto the Nytran membrane (Schleicher and Schuell, Keene, NH) in 20X SSC overnight. Spotting was treated on the Stratalinker 2400 UV (Stratagene, La Jolla, CA) at 0.12 Joules. The roll was then baked at 80 ° C for one hour. The Northern blots were probed with the zsig39 PCR fragment (described later in Example 4) which encodes the mature zsig39 polypeptide, which was radiolabelled with 32Pt dCTP using a set or set of Rediprime pills (Amersham, Arlington Heigts, IL ) according to the manufacturer's specifications. The spotting was hybridized at EXPRESSHYB (Clontech) at 56 ° C overnight. The spotting was washed at room temperature in 2X SSC and 0.1% SDS, then in 2X SSC and 0.1% SDS at 65 ° C, and finally at 65 ° C in 0.1X SSC and 0.1% SDS. The results showed that zsig39 hybridized to all tissues except the HISM of the human intestinal smooth muscle cell line.

Example 3 Chromosomal Mapping of the Zsig39 Gene The gene encoding the zsig39 polypeptide was mapped to chromosome 11 using the "GeneBridge 4 Radiation Hybrid Panel "commercially available (Research Genetics, Inc., Huntsville, AL). The panel Radiation Hybrid GeneBridge 4 contains the DNAs that can be subjected to PCR from each of the 93 hybrid radiation clones, plus two control DNAs (the HFL donor and the A23 receptor). A publicly available WWW server (http: // www-genome-wi-mit .edu / cgi-bin / contig / rhmapper.pl) allows relative mapping with respect to the Whitehead Institute / MIT Center for the hybrid Genome radiation map Research of the human genome (the hybrid radiation map "WICGR) which was built with the Hybrid Radiation Panel GeneBridge 4. For the mapping of the zsig39 gene with the" Panel GeneBridge 4 RH ", the 20 μl reactions were placed in a 96-well microtiter plate that can be treated with PCR (Stratagene, La Jolla, CA) and used in a thermal recycling machine" RoboCycler Gradient 96"(Stratagene) Each of the 95 PCR reactions consisted of 2 μl 10X of the KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 μl of the dNTPs mixture (2.5 mM each, PERKIN). -ELMER, Foster City, CA), 1 μl of the sense primer, ZC15003 (SEQ ID NO: 19), 2 μl of RediLoad (Research Gerietics, Inc., Huntsville, AL), 0.4 μl of the 50X Advantage Polymerase Blend KlenTaq (Clontech Laboratories, Inc.), 25 ng of DNA from a single hybrid or control clone and ddH20 for a total volume of 20 μl .The reactions were deposited with an equal amount of mineral oil and sealed. The recycling machine were as follows: a denaturation of 5 minutes e 1 initial cycle at 95 ° C, 40 cycles of a denaturation of 1 minute at 95 ° C, 1 minute of annealing at 64 ° C and 1.5 minutes of extension at 72 ° C, followed by an extension of 1 final cycle of 7 minutes at 72 ° C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD). The results showed that maps 549.99 cR_3000 of the gene encode the zsig39 polypeptide from the top of the human chromosome binding group 11 on the hybrid radiation map of WICGR. The near and far structure markers were AFMB048ZA9 and FB17D4, respectively. The use of the surrounding markers places the zsig39 gene in region llq23.3 on the integrated LDB chromosome 11 map (The Genetic Location Database, University of Southhampton, WWW server: http: // cedar.genetics.soton-ac -uk / public html /).

Example 4 Construction of the Mammal Expression Vectors of zsig39 zsig39CEE / pZP9 and zsig39NEE / pZP9 Two expression vectors were prepared for the zsig39 polypeptide, zsig39CEE / pZP9 and zsig39NEE / pZP9, wherein the constructs are designed to express a zsig39 polypeptide with an N-terminal Glu-Glu tag (SEQ ID NO: 20).

Zsig39NEE / pZP9 A zsig39 DNA fragment generated by 690 bp PCR was created using ZC15037 (SEQ ID NO: 21) and ZC15038 (SEQ ID NO: 22) as the PCR primers and colonies described above as a model. An N-terminal Glu-Glu tag and the Bam Hl and Xba I restriction sites are added. Amplification of the zsig39 fragment was 94 ° C for 90 seconds, 5 cycles of 94 ° C for 10 seconds, 34 ° C for 20 seconds, 74 ° C for 40 seconds followed by 25 cycles at 94 ° C for 10 seconds, 68 ° C for 20 seconds, 72 ° C for 40 seconds, followed by an extension of 5 minutes at 72 ° C. A band of the predicted size, 690 bp, was visualized by electrophoresis of 1% agarose gel, excised and the DNA was purified from the gel with a QIAQUICK® column (Qiagen) according to the manufacturer's instructions. The DNA was digested with the restriction enzymes Bam Hl and XBA I, followed by extraction and precipitated. The excised DNA was subcloned into the plasmid pZP9 which has been cut with Bam Hl and Xba I. The zsig39NEE / pZP9 expression vector incorporates the TPA leader and the Glu-Glu epitope (SEQ ID NO: 2) is set at the limb N as an aid for purification. Plasmid pZP9 (deposited in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, ATCC No. 98668) is a mammalian expression vector that contains an expression cassette having the mouse metallothionein-1 promoter, multiple restriction sites for the insertion of coding, a stop codon and a terminator of human growth hormone. The plasmid also has a replication E. coli origin, a mammalian selectable marker expression unit having an SV40 promoter, an enhancer and the origin of replication, a DHFR gene and the SV40 terminator. 30 ng of the N-terminal restriction digested Glu-glu-zsig39 insert and 48 ng of the digested vector were ligated overnight at 16 ° C. One microliter of each ligation reaction was electroporated independently in the competent cells of DH10B (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions and placed on plates on LB plates containing 50 mg / ml ampicillin, and incubated overnight. Colonies were selected by PCR using the primers ZC13006 (SEQ ID NO: 23) and ZC13007 (SEQ ID NO: 24). The PCR selection was made at 94 ° C for 4 minutes, 25 cycles of 94 ° C for 30 seconds, 64 ° C for 20 seconds, 72 ° C for 1 minute, followed by a 10 minute extension at 72 ° C. Positive clones were placed on LB Amp plates as above. The insert sequence of the positive clones was verified by sequence analysis. A large scale plasmid preparation was made using a QIAGEN® Maxi prep kit or set according to the manufacturer's instructions.

Zsig39CEE / pZP9 A zsig39 DNA fragment generated by 744 bp PCR was created according to the procedure described above using ZC15609 (SEQ ID NO: 2) and ZC15232 (SEQ ID NO: 26) as the PCR primers to add the Glu- Glu C-terminal and the restriction sites of Eco IR and Bam Hl. The PCR amplification was done at 94 ° C for 3 minutes, 5 cycles of 94 ° C for 30 seconds, 30 ° C for 20 seconds, 72 ° C for 1 minute, 25 cycles at 94 ° C for 30 seconds, 64 ° C for 20 seconds, 72 ° C for 1 minute, followed by an extension of 5 minutes at 72 ° C. The purified PCR fragment was digested with the restriction enzymes Eco IR and Bam Hl, followed by extraction and precipitation. The cleaved zsig39 DNA was subcloned into the plasmid pZP9 which has been cut with Eco IR and Bam Hl. The zsig39CEE / pZP9 expression vector uses the peptide of the wild-type zsig39 signal and binds the Glu-Glu tag (SEQ ID NO: 20) to the C-terminus of the polynucleotide sequence encoding the zsig39 polypeptide. Thirty-four ng of the C-terminal restriction-digested Glu-glu-zsig39 insert and 48 ng of the corresponding vector were ligated into the DH10B cells and the positive colonies were selected as described above. Positive clones were plated on the LB Amp plates as above. The insert sequence of the positive clones was verified by the sequence analysis. A large-scale plasmid preparation was made using a QIAGEN® Maxi Prep kit or set (Qiagen) according to the manufacturer's instructions.

Example 5 Transfection and Expression of zsig39NEE and CEE Polypeptides BHK 570 cells (ATCC No. CRL-10314) were plated on 10 cm tissue culture dishes and allowed to grow to a confluence of approximately 50 to 70% overnight at 37 ° C, 5% C02, in the DMEM / FBS medium (DMEM, BRL / DMEM Elevated Glucose, (Gibco BRL, Gaithersburg, MD), 5% fetal bovine serum (Hyclone, Logan, TU) , 2 μM L-glutamine (JRH Biosciences, Lenexa, KS), 1 μM sodium pyruvate (Gibco BRL) .The cells were then transfected with the plasmid zsig39NEE / pZP9 (N-terminal Glu-Glu tag) or zsig39CEE / pZP9 (C-terminal Glu-Glu tag), using Lipofectamine® (Gibco BRL), in a serum free (SF) medium formulation (DMEM, Gibco / BRL High in Glucose, (Gibco BRL, Gaithersburg, MD), 2 mM of L-glutamine, 2 mM 'of sodium pyruvate, 10 μg / ml of transferrin, 5 μg / ml of insulin, 10 μg / ml of fetuin and 2 ng / ml of selenium). e zsig39NEE / pZP9 and 16 μg of zsig39CEE / pZP9 were separately diluted in 15 ml tubes to a final total volume of 640 μl of SF medium. In separate tubes, 35 μl of Lipofectamine ™ (Gibco BRL) is mixed with 605 μl of the SF medium. Lipofectamine ™ was added to the DNA mixture and allowed to incubate approximately 30 minutes at room temperature. Five milliliters of the SF medium were added to the DNA mixture: Lipectamine ™. The cells were rinsed once with 5 ml of SF medium, aspirated and the DNA mixture Lipofectamine was added. The cells were incubated at 37 ° C for five hours, then add 6.4 ml of DMEM medium / 10% FBS, 1% PSN to the plate. The plate is incubated at 37 ° C overnight and the DNA mixture: Lipofectamine ™ is replaced with the FBS / DMEM medium the next day. On day 2 after 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 refed on day 5 after transfection with the fresh selection medium.

Selection of colonies Approximately 10-12 days after transfection, a 150-mm culture dish of the methotrexate-resistant colonies is chosen from each transfection, the medium aspirated, the plates washed with 10 ml of serum free ESTEP 2 medium (668.7 g / 50 L DMEM (Gibco), 5.5 g / 50 L pyruvic acid, 96% sodium salt (Mallinckrodt), 185.0 g / 50 L NaHCO 3 (Mallinkrodt), 5.0 mg / ml, 25 ml / 50 L insulin, 10.0 mg / ml and 25 ml / 50 L of transferrin). The washed medium was aspirated and replaced with 5 ml of serum free ESTEP 2. A sterile Teflon mesh (Spectrum Medical Industries, Los Angeles, CA), pre-blotted in serum free ESTEP 2 was then placed on the cells. In sterile nitrocellulose filter pre-refreshed in serum free ESTEP 2 was then placed on the mesh. The orientation marks on the nitrocellulose were transferred to the culture disk. The plates were then incubated for 5-6 hours in a 5% C02 incubator at 37 ° C. Following the incubation, the filter was removed, and the medium was aspirated and replaced with DMEM / 5% FBS medium, IX PSN (Gibco BRL). The filter was then placed in a sealable bag containing 50 ml of the buffer solution (25 mM Tris, 25 mM glycine, 5 mM β-mercaptoethanol) and incubated in a 65 ° C water bath for 10 minutes. The filters were blocked in a medium of PBS / 10% non-fat dry milk, 0.1% PBS (Sigma) for 15 minutes at room temperature on a rotary shaker. The filter was then incubated with an HRP-anti-Glu-Glu antibody conjugate at a dilution of 1: 1000 in 10% non-fat dry milk, 0.1% PBS, 0.1% TWEEN, overnight at 4 ° C on a rotary shaker. The filter is then washed three times at room temperature in PBS plus 0.1% Tween 20, 5-15 minutes per wash. The filter was developed with the ECL reagent (Amersham Corp., Arlington Heigths, IL) according to the manufacturer's instructions and exposed to the film (Hyperfilm ECL, Amersham) for approximately 35 seconds. The film was aligned with the plate containing the colonies. Using the film as a guide, the appropriate colonies were selected. The sterile 3-mm colonization discs (PGC Scientific Corp., Frederick, MD) were steeped in trypsin and placed on the colonies. Twelve colonies for each construct were transferred to 200 μl of the selection medium in a 96-well plate. A series of seven two-fold dilutions was carried out for each colony. The cells were grown for a week at 37 ° C at which time the cavities that received the lowest dilution of the cells which are now at the optimum density were selected, treated with trypsin and transferred to a 12 cavity plate which contains the means of selection. The 150 mm culture dish was also treated with trypsin and the rest of the cells were pooled and subjected to Western blot analysis and mycoplasma test. The grouped material was frozen for storage. The clones were expanded directly from the 12-well plate to two bottles of T-75 each. One vial or container was retained to continue cell growth, the second vial was grown in serum free E? TEP 2 which was collected for Western blot analysis. The clones of each of the expression constructs, based on Western blot analysis, were selected, pooled and transferred to a large scale culture.

Scale 6 Expression of the Large Scale Mammal of the zsig39CEE and zsig39NEE A vial or container of T-162, containing confluent cells expressing zsig39CEE and one containing zsig39NEE obtained from the expression procedure described above, was expanded into six flasks or containers of T-162 each. One of the six flasks or resulting containers was used to freeze four cryopresults, and the other five bottles or containers were used to generate a Nunc cell factory. Cells from the five T-162 vessels of zsig39CEE and zsig39NEE were used to independently seed two Nunc cell factories (10 layers, commercially available from VWR). Briefly, the cells from the T-162 bottles or containers described above were disunited or removed using trypsin, pooled, and added to 1.5 liters of ESTEP1 medium (668.7 g / 50 L DMEM (Gibco), 5.5 g / 50 L pyruvic acid, 96% sodium salt (Mallinckrodt), 185.0 g / 50L of NaHCO3 (Mallinkrodt), 5.0 mg / ml and 25 ml / 50L insulin (JRH Biosciences), 10.0 mg / ml and 25 ml / 50L of transferrin ( JHR Biosciences), 2.5L / 50L of fetal bovine serum (characterized) (Hyclone), 1 μM MTX, with the pH adjusted to 7.05 +/- 0.05) preheated to 37 ° C. The medium containing the cells was then poured into the Nunc cell factories by means of a funnel. The cell factories were placed in a 5.0% C02 / 37 ° C incubator. At a confluence of 80-100%, a visual contamination test (change of phenol red color) was carried out on the content of the Nunc cell factories. Since no contamination was observed, the supernatant from the confluent factories was poured into a small collection container, sampled and discarded. The adherent cells were then washed once with 400 ml of PBS. To disunite the cells from the factories, 100 ml of trypsin was added to each and they were removed and the cells were then incubated for 2 or 5 minutes in the residual trypsin. The cells were collected in two 200 ml washes with the ESTEP1 medium. To each of the ten bottles containing the ESTEP1 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 fill a Nunc factory. Each cell factory was placed in a 5% C02 / 37 ° C incubator. At a confluence of 80-90%, a visual contamination test (change of the phenol red color) was carried out on the Nunc cell factories. Since no contamination was observed, the supernatant from the confluent factories was poured into a small collection receiver, sampled and disposed of. The cells were then washed once with 400 ml of PBS. 1.5 liters of medium ESTEP2 (668.7g / 50L of 'DMEM (Gibco), 5.5 g / 50L of pyruvic acid, 96% sodium salt (Mallinckrodt), 185.0 g / 50L of NaHCO3 (Mallinkdrodt), 25 ml / 50L of insulin, 10.0 mg / ml and 25 ml / 50L of transferrin) were added to each Nunc cell factory. The cell factories were incubated at 37 ° C / 5.0% C02. Approximately after 48 hours a visual contamination test (change of phenol red color) was carried out on the Nunc cell factories. The supernatant from each factory was poured into small collection containers. Fresh serum free medium (1.5 liters) was poured into each Nunc cell factory, and the factories were incubated at 37 ° C / 5.0% C02. One ml of the supernatant collected for each construct was transferred to a microscope slide, and subjected to a microscopic analysis to verify contamination. The contents of the small collection containers for each construction were added and filtered immediately. A second collection was then made, substantially as described above at 48 hours and the cell factories were discarded after this. An assembled filter train apparatus was used for the aseptic filtration of the collected supernatant (conditioned medium). The assembly was as follows; the pipe was wire-secured to an Opti-Cap filter (Millipore Corp., Bedford, MA) and to a Gelman Supercap 50 filter (Gelman Sciencies, Ann Arbor, MI). The Supercap 50 filter was also fixed to a sterile, capped container located on a cover; the tubing placed upstream of the Millipore Opti-cap filter was inserted into a peristaltic pump; and the free end of the pipe is placed in large collection container. The peristaltic pump is operated between 200 and 300 rpm, until all the conditioned medium was passed through the final 0.22 μm filter into a sterile collection container. The filter was placed in a cold room at 4 ° C during purification. Samples of the medium saved at various points of time were concentrated 10X with a 5 kDa Millipore cut-off concentrator (Millipore Corp., Bedford, MA) according to the manufacturer's instructions and was subjected to Western blot analysis. The variation in the mobility of the standards is probably responsible for the evident size differences between the two preparations.

Zsig39CEE 5 Flasks T-162 = > 0.125 mg / L, 28 kDa; 1 Factory, FBS = > 0.125 mg / L, 28 kDa; 10 Factories, FBS = > 0'.125 mg / L, 28 kDa; 10 Factories (# 1), SF = > 0.125 mg / L, 28 kDa; and 10 Factories (# 2), SF = > 0.125 mg / L, 28 kDa Zsig39NEE: 5 Flasks T-162 = > 0.14 mg / L, 38 kDa; 1 Factory, FBS = > 1.39 mg / L, 38 kDa; 10 Factories, FBS = > 0.14 mg / L, 38 kDa; 10 Factories (# 1), SF = > 1.39 mg / L, 38 kDa; and 10 Factories (# 2), SF = > 1.39 mg / L, 38 kDa.

Example 7 Purification conditions for the zsig39 NEE and CEE Unless stated otherwise, all operations were carried out at 4 ° C. The following procedure was used for the purification of zsig39 containing the N-terminal or C-terminal Glu-Glu labels (EE). A total of 25 liters of the conditioned medium of the baby hamster kidney cells (BHK) are sequentially filtered through sterilization through a Millipore Opticap capsule filter (Bedford, MA) of 0.2 mM, 10.16 cm (4 inches) and a Gelman Supercap 50 (Ann Arbor, MI) 0.2 M. The material is then concentrated to approximately 1.3 liters using a Millipore ProFlux A30 tangential flow concentrate with an Amicon (Bedford, MA) S10Y3 membrane of 3000 kDa cutoff. The concentrated material was filtered again under sterile conditions with the Gelman filter as described above. A mixture of protease inhibitors was added to the conditioned conditioned medium to the final concentrations of 2.5 mM acetylenediamintetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, IN), pepstatin. 0.001 mM (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 shaken on a Wheaton rotary cultivator (Millville, NJ) for 18.0 h at 4 ° C. C. The mixture is then poured into an Econo-Column of 5.0 x 20.0 cm (Bio-Rad, Laboratories, Hercules, CA) and the gel is washed with 30 volumes of the column of salted solution buffered with phosphate (PBS). The through fraction of the non-retained flow was discarded. Once the absorbance of the effluent at 280 nM was less than 0.05, the flow through the column was reduced to zero and the anti-SE Sepharose gel was washed in batch mode with 2.0 volumes of the PBS column that it contains 0.4 mg / ml of the EE peptide (AnaSpec, San José, CA). The peptide used has the sequence Glu-Tyr-Met-Pro-Val-Asp (SEQ ID NO: 27). After 1.0 h at 4 ° C, the flow was resumed and the eluted protein was collected. This fraction was referred to as elution of the peptide. The anti-SE Sepharose gel was then washed with 2.0 volumes of the 0.1 M glycine column, 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 future analysis if necessary. The peptide solution 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 elution of the concentrated peptide was separated from the free peptide by chromatography on a Sephadex G-50 column (Pharmacia, Piscataway, NJ) of 1.5 x 10 cm equilibrated in PBS at a flow rate of 1.0 ml / min using a CLAR from BioCad Sprint (PerSeptive BioSystems, Framingham, MA). Two ml fractions were collected and the absorbance was verified at 280 nM. The first peak of the material that is absorbed at 280 nM and that elutes near the empty volume of the column was collected. This fraction was zsig39 NEE and zsig39 CEE pure. Pure material was concentrated as described above, analyzed by SDS-PAGE and treated with Western blot with anti-Glu-Glu antibodies, and samples were taken for amino acid analysis and N-terminal sequencing. The rest of the sample was aliquoted, and stored at -80 ° C in accordance with our standard procedures. The concentration of purified zsig39 NEE protein was 0.65 mg / ml. The concentration of zsig39 CEE protein was 0.3 mg / ml.

The electrophoresis of zsig39 NEE on SDS-PAGE gels in the absence of reducing agents showed two bands, present in approximately equimolar amounts, on gels stained with Coomassie blue of apparent molecular weights of -50,000 and -29,000. On Western blots these bands showed a cross-reactivity with the anti-EE antibodies. Three of other apparent molecular weight bands of -150,000, -80,000, and -60,000 were also observed on Western blots under these conditions. In the presence of the reducing agent, the only band observed on the gels stained with Coomassie Blue migrated with an apparent molecular weight of 30,000. The intensity of this band was increased in relation to any band observed on the non-reducing gels. The 30,000 'kDa band also showed cross-reactivity with anti-EE antibodies on Western blots and was the only cross-reactive protein present. In addition, the intensity of this band increased in relation to the intensity of the band under non-reducing conditions. Virtually identical results are obtained for zsig39 CEE by SDS-PAGE and Western blotting with anti-EE antibodies.

Preparation of the anti-SE Sepharose A 100 ml bed volume of the G-Sepharose protein (Pharmacia, Piscataway, NJ) was washed three times with 100 ml of PBS containing 0.02% sodium azide using a 0.45 micron filter unit of 500 ml Nalgene . The gel was washed with 6.0 volumes of the 200mM triethanolamine, pH 8.2 (TEA, Sigma Co.) and an equal volume of the ES antibody solution containing 900 mg of the antibodies. After an overnight incubation at 4 ° C, unbound antibodies were removed by washing the resin with 5 volumes of 200 mM TEA as described above. The resin was resuspended in 2 volumes of TEA, transferred to a suitable vessel, and dimethylpimylimidate-2HCl (Pierce), dissolved in TEA, was added to a final concentration of 36 mg / ml of the gel. The gel was stirred at room temperature for 45 minutes and the liquid was removed using the filter unit as described above. The non-specific sites of the gel were then blocked by incubation for 10 minutes at room temperature with 5 volumes of 20 mM ethanolamine in 200 mM TEA. The gel is washed with 5 volumes of PBS containing 0.02% sodium azide and stored in this solution at 4 ° C.

Example 8 Construction of Yeast Expression Vectors Labeled with Glu-Glu Amino Terminal and Glu-Glu Carboxy Terminal The expression of zsig39 in Pichia methanolica uses the expression system described in the co-assigned WOIPO publication WO 97/17450. An expression plasmid containing all or part of a polynucleotide encoding zsig39 is constructed by homologous recombination. An expression vector was constructed from pCZR204 to express zsig39 polypeptides labeled with C-terminal Glu-Glu (CEE). The pCZR204 vector contains the AUG1 promoter, followed by the direct sequence of aFpp, followed by a Sma I restriction site with blunt ends, a carboxy-terminal peptide tag (Glu-Glu), a translational stop codon, followed by the AUG1 terminator, the ADE2 selectable marker, and finally the 3 'non-translated region of AUGl. Also included in this vector are the URA3 and CEN-ARS sequences required for selection and replication in S. cerevisiae, and the AmpR and colEl ori sequences required for selection and replication in E. coli. A second expression vector was constructed from ZCZR204 to express zsig39 polypeptides labeled with N-terminal Glu-Glu (NEE). The zCZR204 expression vector is as described above, which has an amino terminal Glu-Glu tag. The sequence of zsig39 inserted into these vectors starts at residue 19 (Leu) of the amino acid sequence of zsig39 (SEQ ID NO: 2). For each construction two linkers are prepared, and in the company of zsig39, were recombined homologously in the expression vectors of the yeast described above. The unlabeled N-terminal linker (SEQ ID NO: 28) extends up to 70 base pairs of the prepro coding sequence of alpha factor (aFpp) on one end and binds thereto at the 70 base pairs of the coding sequence of the amino terminal from the mature zsig39 sequence over the other. The linker labeled with NEE (SEQ ID NO: 29) binds to the Glu-Glu tag (SEQ ID NO: 20) between the aFpp coding sequence and the zsig39 sequence. The unlabeled C-terminal linker (SEQ ID NO: 30) extends up to about 70 base pairs of the carboxy terminus encoding the zsig39 sequence on one end with 70 base pairs of an AUGI terminator sequence. The linker labeled CEE (SEQ ID NO: 31) inserts the Glu-Glu tag (SEQ ID NO: 20) between the C-terminal end of zsig39 and the terminator region of AUG1.

Construction of Zsig39 plasmid labeled with NEE A zsig39 plasmid of NEE labeled was homologously recombining 100 ng of the pCZR204 acceptor vector digested with Smal, 1 μg of the zsig39 linker labeled with NEE (SEQ ID NO: 29) and 1 μg of the linker not labeled with C terminal ( SEQ ID NO: 30) in S. cerevisiae. The NEE-zsig39 linker was synthesized by a PCR reaction. At a final reaction volume of 100 μl, 1 ppmol of each of the linkers is added, ZC13731 (SEQ ID NO: 32) and ZC15268 (SEQ ID NO: 33), and 100 pmoles of each primer ZC13497 (SEQ ID NO: 34) and ZC15274 (SEQ ID NO: 35), 10 μl of 10X PCR buffer (Boehringer Mannheim), 1 μl of Pwo Polymerase (Boehringer Mannheim), 10 μl of the mixture of 0.25 mM nucleotide triphosphate (Perkin Elmer) and dH20. The PCR reaction was run 10 cycles at 30 seconds at 94 ° C, 1 minute at 50 ° C and 1 minute at 72 ° C, and was concluded with an extension of 6 minutes at 72 °. The resulting 144bp double-stranded NEE-labeled linker is described in SEQ ID NO: 29. The unlabeled Cs-terminal zsig39 linker was made by means of a PCR reaction as described using oligonucleotides ZC15273 (SEC ID NO: 36), ZC15724 (SEQ ID NO: 37), ZC15223 (SEQ ID NO: 38) and ZC13734 (SEQ ID NO: 39). The double-stranded, C-terminal, unlabeled linker of 147 bp is described in SEQ ID NO: 30.

Construction of plasmid CEE-zsig39 A CEE-zsig39 plasmid was made homologously recombining 100 ng of the pIZR204 acceptor vector digested with Sma I, 1 μg of the zsig39 cDNA donor fragment of Eco IR-Bam Hl, 1 μg of the non-labeled N-terminal zsig39 linker ( SEQ ID NO: 28) and 1 μg of the CEE tagged linker (SEQ ID NO: 31) in an S. cerevisiae. The unlabeled N-terminal zsig39 linker was made by means of a PCR reaction as described above using the oligonucleotides ZC14822 (SEQ ID NO: 40), ZC14821 (SEQ ID NO: 41), ZC15269 (SEQ ID NO: 42) ) and ZC15274 (SEQ ID NO: 43). The resulting 144 bp double-stranded N-terminal unlabeled linker is described in SEQ ID NO: 28.

The linker labeled with CEE was made by means of a PCR reaction as described above using ZC15273 (SEQ ID NO: 44), ZC15267 (SEQ ID NO: 45), ZC14819 (SEQ ID NO: 49) and ZC14820 (SEQ ID NO: 45). NO: 47). The resulting, double-stranded, EEC labeled linker of about 144 bp is described in SEQ ID NO: 31. One hundred microliters of the competent yeast cells (S. cerevisiae) were independently combined with 10 μl of various DNA mixtures of above and transferred to a 0.2 cm electroporation cell. Yeast / DNA mixes were subjected to electrical pulses at 0.75 kV (5 kV / cm), 8 ohms, 25 μF. To each cell is added 600 μl of 1.2 M sorbitol and the yeast is plated in two 300 μl aliquots on two URA D plates and incubated at 30 ° C. After approximately 48 hours, the Ura + yeast transformants from a single plate are resuspended in 2.5 ml of H20 and centrifuged briefly to convert the yeast cells into pill. The cell pill is resuspended in 1 ml of 2% Triton X-100 lysis buffer, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture are added to an Eppendorf tube containing 300 μl of washed glass beads and 200 μl of phenol-chloroform, vortexed at 1 minute intervals two or three times, followed by centrifugation 5 minutes in an Eppendorf centrifuge as the maximum speed. Three hundred microliters of the aqueous phase were transferred to a fresh tube and the DNA was precipitated with 600 μl of ethanol (EtOH), followed by centrifugation for 10 minutes at 4 ° C. The DNA pill was resuspended in 100 μl of H20. The transformation of the electrocompetent E. coli cells (DH10B, Gibco BRL) was done with 1 μl of prep yeast DNA and 50 μl of DH10B cells. The cells were subjected to electrical impulses at 2.0 kV, 25 μF and 400 ohms. Following electroporation, 1 ml of SOC (2% Triptona Bacto ™ (Difco, Detroit, MI), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) is plated in aliquots of 250 μl on four plates of LB AMP (LB Broth (Lennox), 1.8% Bacto ™ Agar (Dífco), 100 mg / L Ampicillin). Individual clones containing the construction of the correct expression for the zsig39 constructs labeled with NEE and CEE were identified by analysis of the sequences to verify the presence of the zsig39 insert and to confirm that the various DNA sequences have been correctly linked between yes. The insert of the positive clones was subjected to the analysis of the sequences. Plasmid DNA on a larger scale was isolated using the Qiagen Maxi set or set according to the manufacturer's instructions and the DNA was digested with Not I to release the Pichia-Zsig39 expression cassette from the vector backbone. The DNA fragment deferred with the Not I restriction was then transformed into the expression host of Pichia methanolica, PMAD16. This was done by mixing 100 μl of the competent PMAD16 cells prepared with 10 μg of the zsig39 digested with the Not I restriction and transferred to a 0.2 cm electroporation cell. The DNA / yeast mixture was subjected to electric impulses at 0.75 kV, 25 μF, infinite omh. 1 ml of Yeast IX Nitrogen Base is added to the tank and 500 μl aliquots are placed on two ADE DS plates (0.056% -Ade -Trp -Thr powder, 0.67% yeast nitrogen base without amino acids, 2% of D-glucose, 0.5% 200X tryptophan, threonine solution, and 18.22% D-sorbitol) for selection and incubated at 30 ° C. The clones were taken and selected by means of Western blotting for the expression of high-level zsig39. The resultant NEE-labeled zsig39 plasmid containing the yeast cells was designated PMAD16:: pCZR206.14.51 and 14.61 and the EEC-tagged zsig39 plasmid containing the yeast cells were designated PMAD16:: pCZR209 # l and # 2. The clones were then subjected to fermentation.

Example 9 Purification of zsig39CEE from a Pichia methanolica Conditioner Unless stated otherwise, all operations were carried out at 4 ° C. A mixture of protease inhibitors was added to a 3000 ml sample of the medium conditioned from Pichia cultures at final concentrations of 2.5 mM of ethylenediaminetetraacetic acid (EDTA, 'Signa Chemical Co.), 0.001 mM of leupeptin (Boehringer-Mannheim), 0.001 mM of pepstatin (Boehringer-Mannheim) and 0.4 M of Pefabloc (Boehringer-Mannheim). The pH of the medium was adjusted to 7.2 with a concentrated solution of NaOH (Sigma Chemical Co.) following the addition of potassium phosphate (Sigma Chemical Co.) to a final concentration of 0.05 M. The sample was centrifuged at 18,000 xg. for 30 minutes at 4 ° C in a Beckman JLA-10.5 rotor (Beckman Instruments) in a Beckman Avanti J25I centrifuge (Berckman Instruments) to remove debris from the cells. To the supernatant fraction is added a 50.0 ml sample of anti-SE Sepharose, prepared as described above, and the mixture was gently shaken on a Wheaton rotary cultivator (Millville, NJ) for 18.0 h at 4 ° C. The mixture was then processed as described above for zsig39CEE from the BHK cells. The pure material was concentrated as described above, analyzed by SDS-PAGE and Western spotted with anti-EE antibodies, and the samples were taken for amino acid analysis and N-terminal sequencing. The rest of the sample was aliquoted, and stored at -80 ° C according to standard procedures. On the SDS-PAGE gels stained with Coomassie Blue, the preparation contained two major bands of apparent molecular weights of 23,000 and 28,000 and two minor components of 21,000 and 45,000. The mobility of these bands was the same in the presence and absence of the reducing agents. The only band visible on Western blots with anti-EE antibodies in the absence of reducing agents was a protein of apparent molecular weight of 150,000 (probably the IgG that was eluted from the anti-SE Sepharose column). Western blotting with anti-EE antibodies in the presence of reducing agents, on the other hand, showed three bands of apparent molecular weights of 28,000, 24,000, and 23,000. The concentration of zsig39CEE from Pichia methanolica was 0.35 mg / ml.

Example 10 Antibodies of Zsig39 A polyclonal antibody was prepared by immunizing two female New Zealand white rabbits with the full-length zsig39 polypeptide (SEQ ID NO: 2). The polypeptide was derived from the purified BHK expressed material described above. The polypeptide was conjugated with the anti-slip limpet hemocyanin of the carrier protein (KLH) with gluteraldehyde. Rabbits were each given an initial intraperitoneal (ip) injection of 200 μg of the peptide in the Complete Freund Adjuvant followed by the ip booster injections of 100 μg of peptide in the Incomplete Freund's Adjuvant every three weeks. Seven to ten days after the administration of the third booster injection, the animals were subjected to bleeding and the serum was collected. The animals were then subjected to rercement injections and underwent bleeding every three weeks. The zsig39-specific antibody was purified from the serum using a Protein A Sepharose. The zsig39 antibody can be characterized by an ELISA titration check using the polypeptide of SEQ ID NO: 2 as a target of the antibody.

Example 11 In vivo administration of zsig39 by Adenoviral Supply Twenty-four male and 24 female mice C57B16 / J, approximately 12 'weeks of age (Jackson Labs, Bar Harbor, ME) were weighed, body temperature was measured and food intake was checked daily for four days prior to injection (days -4 to -1 ). On day 0, the mice were divided into three groups and received 0.1 ml of the virus (1.8x1O11 virus particles AdV-empty / 0.1 ml or 5x1o11 AdV-zsig39-CEE virus particles / 0.1 ml) by injection into the vein of the intravenous tail , or no injection in its entirety. The injection should lead to host liver infection and the expression of the virally supplied gene should begin within 24 hours and continue for 1 to 4 weeks. Three groups of mice were tested. Group 1, untreated, n = 8 each female and male. Group 2, AdV-Empty (empty virus), n = 8 each male and female. Group 3, AdV-zsig39 CEE, n = 8 each female and male. The production of the adenovirus containing the zsig39CEE was done according to the procedure of Becker et al., Meth. Cell Biol. 43: 161-89, 1994 using commercially available vectors. The body temperatures of the animals, the weights and the weight of the ingested food were checked during the three-week 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. The animals were devoid of blood and the tissues were collected for necropsy. The chemistry panel of the standard serum was made at the time of sacrifice. Liver, kidney and metabolic parameters were all within normal ranges. The total free fatty acids were evaluated on the remaining serum of each animal. A statistically significant difference in the levels of Free Fatty Acid of Serum is observed between both male and female mice (p <0.05 for both) receiving the empty virus and those receiving the zsig39 coding virus by the Dunn Multiple Comparisons Test. The zsig39 mice had lower levels. The liver, spleen, kidney, thymus, heart and brain were weighed after removal. These tissues and femurs were saved for histology. Histopathological analyzes of metaphyseal femoral bone marrow revealed a difference between the treatment groups. The average% of the value or brand of fat from the metaphyseal bone marrow of the female zsig39 mice was significantly higher (p <0.05% by the Dunn Multiple Comparisons Test) than that of the female mice receiving the 'adenovirus' empty No significant observations were made on the other tissues examined. From the foregoing, it will be appreciated that, although the specific embodiments of the invention have been described herein for purposes of illustration, modifications can be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.

LIST OF THE SEQUENCES < 110 > ZymoGenetics, Inc. < 120 > PROTEIN HOMOLOGOUS SPECIFIC FOR ADIPOCYTES < 130 > 97-49PC < 150 > 60 / 056,983 < 151 > 1997-08-26 < 160 > 47 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 1347 < 212 > DNA < 213 > Homo sapiens < 220 > < 221 > CDS < 222 > (198) ... (926) < 400 > gaattcggct cgagagggag cgaaccagga ctggggtgac ggcagggcag ggggcgcctg 60 gccggggaga agcgcggggg ctggagcacc accaactgga gggtccggag tagcgagcgc 120 cccgaaggag gccatcgggg agccgggagg ggggactgcg agaggacccc ggcgtccggg 180 ctcccggtgc cagcgct atg agg cea etc etc gtc ctg ctg etc ctg ggc 230 Met Arg Pro Leu Leu Val Leu Leu Leu Leu Gly January 5 10 ctg gcg gee ggc teg ecc cea ctg gac gac aac aag ate ecc age etc 278 Leu Ala Ala Gly Ser Pro Pro Leu Asp Asp Asn Lys He Pro Ser Leu 15 20 25 tgc ceg ggg drops ecc ggc ctt cea ggc acg ceg ggc falls cat ggc age 326 Cys Pro Gly His Pro Gly Leu Pro Gly Thr Pro Gly His His Gly Ser 30 35 40 cag ggc ttg ceg ggc cgc gat ggc cgc gac ggc cgc gac ggc gcg ecc 374 Gln Gly Leu Pro Gly Arg Asp Gly Arg Asp Gly Arg Asp Gly Ala Pro 45 50 55 ggg gct ceg gga gag aaa ggc gag ggc ggg agg ceg gga ctg ceg gga 42 Gly Wing Pro Gly Glu Lys Gly Glu Gly Gly Arg Pro Gly Leu Pro Gly 60 65 70 75 ect cga ggg gac ecc ggg ceg cga gga gag gcg gga ecc gcg ggg ecc 47 Pro Arg Gly Asp Pro Gly Pro Arg Gly Glu Wing Gly Pro Wing Gly Pro 80 85 90 ggg ect gee ggg gag tgc teg gtg ect ceg cga tec gee ttc age 51 Thr Gl? Pro Ala Gly Glu Cys Ser Val Pro Pro Arg Ser Ala Phe Ser 95 100 105 gee aag cgc tec gag age cgg gtg ect ceg tet gac gca ecc ttg 56 Wing L? S Arg Ser Glu Ser Arg Val Pro Pro Pro Ser Asp Wing Pro Leu 110 115 120 ecc ttc gac cgc gtg ctg gtg aac gag cag gga cat tac 'gac gee gtc 61 Pro Phe Asp Arg Val Leu Val Asn Glu Gln Gl? His Tyr Asp Ala Val 125 130 135 acc ggc aag ttc acc tgc cag gtg ect ggg gtc tac tac ttc gee gtc 66 Thr Gly Lys Phe Thr Cys Gln Val Pro Gly Val Tyr Tyr Phe Wing Val 140 145 150 155 cat gee acc gtc tac cgg gee age ctg cag ttt gat ctg gtg aag aat 71 His Wing Thr Val Tyr Arg Wing Being Leu Gln Phe Asp Leu Val Lys Asn 160 165 170 ggc gaa tec att gee tet ttc ttc cag ttt ttc ggg ggg tgg ecc aag 75 Gl? Glu Ser He Wing Being Phe Phe Gln Phe Phe Gly Gly Trp Pro Lys 175 180 185 cea gee teg etc teg ggg ggg gee atg gtg agg ctg gag ect gag gac 80 Pro Wing Ser Leu Ser Gly Gly Wing Met Val Arg Leu Glu Pro Glu Asp 190 195 200 ca gtg tgg gtg cag gtg ggt gtg ggt gac tac att ggc ate tat gee 85 Gln Val Trp Val Gln Val Gly Val Gly Asp Tyr He Gly He Tyr Ala 205 210 215 age ate aag ac gac age acc ttc tec gga ttt ctg gtg tac tec gac 90 Ser He Lys Thr Asp Ser Thr Phe Ser Gly Phe Leu Val Tyr Ser Asp 220 225 230 235 tgg falls age tec cea gtc ttt gct tagtgcccac tgcaaagtga gctcatgctc 95 Trp His Ser Ser Pro Val Phe Ala 240 tcactcctag aaggagggtg tgaggctgac aaccaggtca tccaggaggg ctggcccccc 101 tggaatattg tgaatgacta gggaggtggg gtagageact ctccgtcctg ctgctggcaa 107 ggaatgggaa cagtggctgt ctgcgatcag gtctggcagc atggggcagt ggctggattt 113 ctgcccaaga ccagaggagt gtgctgtgct ggcaagtgta agtcccccag ttgctctggt 119 ccaggagccc acggtggggt gctctcttcc tggtcctctg cttctctgga tcctccccac 125 cccctcctgc tcctggggcc ggcccttttc tcagagatca etcaataaac ctaagaaccc 131 tcaaaaaaaa aaaaaaaaaa agggcggccg c 134 < 210 > 2 < 211 > 243 < 212 > PRT < 213 > Homo sapiens < 400 > Met Arg Pro Leu Leu Val Leu Leu Leu Leu Gl? Leu Ala Ala Gl? Ser 1 5 10 15 Pro Pro Leu Asp Asp Asn L? S He Pro Ser Leu C? S Pro Gl? His Pro 20 25 30 Gl? Leu Pro Gl? Thr Pro Gl? His His Gl? Be Gln Gl? Leu Pro Gl? 35 40 45 Arg Asp Gl? Arg Asp Gl? Arg Asp Gl? Ala Pro Gl? Ala Pro Gl? Glu 50 55 60 L? S Gl? Glu Gl? Gl? Arg Pro Gl? Leu Pro Gl? Pro Arg Gl? Asp Pro 65 70 75 80 Gl? Pro Arg Gl? Glu Ala Gl? Pro Ala Gl? Pro Thr Gl? Pro Ala Gl? 85 90 95 Glu C? S Ser Val Pro Pro Arg Ser Wing Phe Ser Wing Lys Arg Ser Glu 100 105 110 Ser Arg Val Pro Pro Pro Ser Asp Wing Pro Pro Leu Pro Phe Asp Arg Val 115 120 125 Leu Val Asn Glu Gln Gl? His T? R Asp Ala Val Thr Gl? L? S Phe Thr 130 135 140 C? S Gln Val Pro Gl? Val T? R T? R Phe Ala Val His Ala Thr Val T? R 145 150 155 160 Arg Ala Ser Leu Gln Phe Asp Leu Val L? S Asn Gl? Glu Ser He Ala 165 170 175 To be Phe Phe Gln Phe Phe Gl? Gl? Trp Pro L? S Pro Ala Ser Leu Ser 180 185 190 Gl? Gl? Wing Met Val Arg Leu Glu Pro Glu Asp Gln Val Trp Val Gln 195 200 205 Val Gl? Val Gl? Asp Tyr He Gl? He T? R Ala Ser He L? S Thr Asp 210 215 220 Ser Thr Phe Ser Gl? Phe Leu Val T? R Ser Asp Trp His Ser Ser Pro 225 230 235 240 Val Phe Ala < 210 > 3 < 211 > 244 < 212 > PRT < 213 > Homo sapiens < 400 > Met Leu Leu Leu Gl? Wing Val Leu Leu Leu Leu Wing Leu Pro Gl? His 1 5 10 15 Asp Gln Glu Thr Thr Thr Gln Gl? Pro Gl? Val Leu Leu Pro Leu Pro . 25 30 L? S Gl? Ala Cys Thr Gl? Trp Met Ala Gl? He Pro Gl? His Pro Gl? 40 45 'His Asn Gl? Ala Pro Gl? Arg Asp Gl? Arg Asp Gl? Thr Pro Gl? Glu 50 55 60 L? S Gl? Glu L? S Gl? Asp Pro Gl? Leu He Gl? Pro L? S Gl? Asp He 65 70 75 80 Gl? Glu Thr Gl? Val Pro Gl? Ala Glu Gl? Pro Arg Gl? Phe Pro Gl? 85 90 95 He Gln Gl? Arg Lys Gl? Glu Pro Gl? Glu Gly Ala T? R Val Tyr Arg 100 105 110 Ser Ala Phe Ser Val Gl? Leu Glu Thr T? R Val Thr He Pro Asn Met 115 120 125 Pro He Arg Phe Thr L? S He Phe T? R Asn Gln Gln Asn His T? R Asp 130 135 140 Gl? Be Thr Gl? L? S Phe His C? S Asn He Pro Gl? Leu T? R T? R Phe 145 150 155 160 Ala T? R His He Thr Val T? R Met L? S Asp Val L? S Val Ser Leu Phe 165 170 175 L? S L? S Asp L? S Wing Met Leu Phe Thr T? R Asp Gln Tyr Gln Glu Asn 180 185 190 Asn Val Asp Gln Ala Ser Gl? Ser Val Leu Leu His Leu Glu Val Gl? 195 200 205 Asp Gln Val Trp Leu Gln Val Tyr Gl? Glu Gl? Glu Arg Asn Gl? Leu 210 215 220 T? R Wing Asp Asn Asp Asn Asp Ser Thr Phe Thr Gl? Phe Leu Leu T? R 225 230 235 240 His Asp Thr Asn < 210 > 4 < 211 > 245 < 212 > PRT < 213 > Homo sapiens < 400 > Met Gl u Gl? Pro Arg Gl? Trp Leu Val Leu C? S Val Leu Wing He Ser 1 5 10 15 Leu Al a Ser Met Val Thr Glu Asp Leu C? S Arg Ala Pro Asp Gl? L? S 25 30 L? S Gl? Glu Ala Gl? Arg Pro Gl? Arg Arg Gl? Arg Pro Gl? Leu L? S 40 45 Gl? Gl u Gln Gl? Glu Pro Gl? Ala Pro Gl? He Arg Thr Gl? He Gln 50 55 60 Gl? Leu L? S Gl? Asp Gln Gl? Glu Pro Gl? Pro Ser Gl? Asn Pro Gl? 65 70 75 80 L? S Val Gl? T? R Pro Gl? Pro Ser Gl? Pro Leu Gl? Ala- Arg Gl? I have 85 90 95 Pro Gly He L? S Gl? Thr L? S Gl? Be Pro Gl? Asn He L? S Asp Gln 100 105 110 Pro Arg Pro Wing Phe Be Wing He Arg Arg Asn Pro Pro Met Gl? Gl? 115 120 125 Asn Val Val He Phe Asp Thr Val He Thr Asn Gln Glu Glu Pro T 130 130 140 Gln Asn His Ser Gl? Arg Phe Val C? S Thr Val Pro Gl? T? R T? R T? R 145 150 155 160 Phe Thr Phe Gln Val Leu Ser Gln Trp Glu He C? S Leu Ser He Val 165 170 175 To Be Being Arg Gl? Gln Val Arg Arg Ser Leu Gl? Phe C? S Asp Thr 180 185 190 Thr Asn L? S Gl? Leu Phe Gln Val Val Ser Gl? Gl? Met Val Leu Gln 195 200 205 Leu Gln Gln Gl? Asp Gln Val Trp Val Glu L As Asp Pro L? S L? S Gl? 210 215 220 His He T? R Gln Gl? Ser Glu Ala Asp Ser Val Phe Ser Gl? Phe Leu 225 230 235 240 He Phe Pro Ser Wing 245 < 210 > 5 < 211 > 215 < 212 > PRT < 213 > Tamias sibricus < 400 > 5 Met Pro Ala Gln Arg Gl? Gl? Ala Leu Ser Met Gl? Ala Ala Gl? Phe 1 5 10 15 Trp He Leu Val Leu Ser He Thr Ser Ala Leu Ala Asp Ser Asn Asn 20 25 30 Gln Gl? Asn Ser Glu Pro C? S Gl? Pro Pro Gl? Pro Pro Gl? Pro Pro 35 40 45 Gl? He Pro Gl? Phe Pro Gl? Ala Pro Gl? Ala Leu Gl? Pro Pro Gl? 50 55 60 Pro Pro Gl? Val Pro Gl? He Pro Gl? Pro Gln Gl? Pro Pro Gl? Asp 65 70 75 80 Val Glu L? S C? S Ser Ser Arg Pro L? S Ser Ala Phe Ala Val L? S Leu 85 90 95 Ser Glu Arg Pro Pro Glu Pro Phe Gln Pro He Val Phe L? S Glu Ala 100 105- 110 Leu T? R Asn Gln Glu Gl? His Phe Asn Met Ala Thr Gl? Glu Phe Ser 115 120 125 Cys Val Leu Pro Gl? Val T? R Asn Phe Gl? Phe Asp He: Arg Leu Phe 130 135 140 Gln Ser Ser Val L? S He Arg Leu Met Arg Asp Gl? He Gln Val Arg 145 150 155 160 Glu L? S Glu Wing Gln Wing Asn Asp Being T? R L? S His Wing Met Gl? Ser 165 170 175 Val He Met Ala Leu Gl? L? S Gl? Asp L? S Val Trp Leu Glu Ser L? S 180 185 190 Leu L? S Gl? Thr Glu Ser Glu L? S Gl? He Thr His He Val Phe Phe 195 200 205 Gl? T? R Leu Leu T? R Gl? L? S 210 215 25 < 210 > 6 < 211 > 236 < 212 > PRT < 213 > Ta ias sibricus < 400 > 6 Met Tyr Glu Wing Gly Lys Arg Wing Being Phe Met Gly Gl? Ala Gl? He 1 5 10 15 Trp He Leu Ala Leu Ser Val Leu Met His Val Val Cys Ser Met T? R 20 25 30 10 Glu Ala Gl? L? S Arg Ala Being Phe Met Gl? Gl? Ala Gl? He Trp He 35 40 45 Leu Ala Leu Ser Val Leu Met His Val Val C? S Ser Asn Val Pro Gl? 50 55 60 Pro Gln Gl? Pro Pro Gl? Met Arg Gl? Pro Pro Gl? Thr Pro Gl? L? S 65 70 75 80 Pro Gl? Pro Pro Gl? Trp Asn Gl? Phe Pro Gl? Leu Pro Gl? Pro Pro 85 90 95 Gl? Pro Pro Gl? Met Thr Val Asn Cys- His Ser L? S Gl? Thr Ser Wing 100 105 110 Phe Wing Val L? S Wing Asn Glu Leu Pro Pro Wing Pro Ser Gln Pro Val 115 120 125 He Phe L? S Glu Ala Leu His Asp Ala Gln Gl? His Phe Asp Leu Ala 130 135 140 Thr Gl? Val Phe Thr Cys Pro Val Pro Gl? Leu T? R Gln Phe Gl? Phe 145 150 155 160 His He Glu Ala Val Gln Arg Ala Val Lys Val Ser Leu Met Arg Asn 165 170 175 Gl? Thr Gln Val Met Glu Arg Glu Wing Glu Wing Gln Asp Gl? T? R Glu 180 185 190 His He Ser Gl? Thr Ala He Leu Gln Leu Gl? Met Glu Asp Arg Val 195 200 205 Trp Leu Glu Asn L? S Leu Ser Gln Thr Asp Leu Glu Arg-Gl? Thr Val 210 215 220 25 Gln Ala Val Phe Ser Gl? Phe Leu He His Glu Asn 225 230 235 < 210 > 7 < 211 > 222 < 212 > PRT < 213 > Rattus norvegicus < 400 > Met Pro Pro Pro Gl? Arg Gl? Pro Arg Gl? Pro Leu Leu Ser Met Pro 1 5 10 15 Gl? Arg Arg Gl? Wing Leu Arg Glu Pro Wing Asp Phe Gl? Ser Ser Leu 20 25 30 10 Gl? Ala Ala Leu Ala Leu Leu Leu Leu Leu Leu Pro Ala C? S C? S Pro 35 40 45 Val L? S Met T? R Glu Ala Gl? L? S Arg Wing Being Phe Met Gly Gly Wing 50 55 60 Gly He Trp He Leu Wing Leu Being Val Leu Met His Val Val C? S Being 65 70 75 80 Gly He Ser Val Arg Ser Gl? Be Ala L? S Val Ala Phe Ser Ala Thr 85 90 95 Arg Ser Thr Asn His Glu Pro Ser Glu Met Ser Asn Arg Thr Met Thr 100 105 110 He T? R Phe Asp Gln Val Leu Val Asn He Gl? Asn His Phe Asp Leu 115 120 125 Wing Being Ser He Phe Val Wing Pro Arg L? S Gl? I Have To Be Ser Phe 130 135 140 Phe His Val Val L? S Val T? R Asn Arg Gln Thr He Gln Val Ser Leu 145 150 155 160 Met Gln Asn Gl? T? R Pro Val He Be Wing Phe Wing Gl? Asp Gln Asp 165 170 175 Val Thr Arg Glu Wing Wing Being Asn Gl? Val Leu Leu Leu Met Glu Arg 180 185 190 Glu Asp L? S Val His Leu L? S Leu Glu Arg Gl? Asn Leu Met Gly Gly 195 200 205 Trp L? S T? R Ser Thr Phe Ser Gl? Phe Leu Val Phe Pro Leu 210 215 220 25 < 210 > 8 < 211 > 247 < 212 > PRT < 213 > Homo sapiens < 400 > Met Leu Leu Leu Gln Leu Leu Leu Phe Leu Le Le Le Pro Pro His 1 5 10 15 Wing Gl u Asp Asp Val Thr Thr Thu Glu Glu Leu Al a Pro Al a Leu Val 25 30 Pro Pro Pro L? S Gl? Thr C? S Wing Gl? Trp Met Al a Gl? H e Pro Gl? 35 40 45 His Pro Gl? His Asn Gl? Thr Pro Gl? Arg Asp Gl? Arg Asp Gl? Thr 50 55 60 Pro Gl? Glu L? S Gl? Glu Lys Gl? Asp Al a Gl? Leu Leu Gl? Pro L? S 65 70 75 80 Gl? Glu Thr Gl? Asp Val Gly Met Thr Gl? Al a Gl u Gl? Pro Arg Gl? 85 90 95 Phe Pro Gln Thr Pro Gl? Arg L? S Gl? Gl u Pro Gl? Gl u Ala Ala T? R 100 105 110 Met Tyr Arg Ser Ala Phe Ser Val Gl? .Leu Gl u Thr Arg Val Thr Val 115 120 125 Pro Asn Val Pro He Arg Phe Thr L? S H e Phe T? R Asn Gln Gln Asn 130 135 140 His T? R Asp Gl? Be Thr Gl? L? S Phe T? R C? S Asn He Pro Gl? Leu 145 150 155 160 T? R T? R Phe Ser T? R His He Thr Val T? R Met L? S Asp Val L? S Val 165 170 175 Being Leu Phe L? S L? S Asp L? S Wing Val Leu Phe Thr T? R Asp Gln T? R 180 185 190 Gln Glu L? S Asn Val Asp Gln Ala Ser Gl? Ser Val Leu Leu His Leu 195 200 205 Glu Val Gl? Asp Gln Val Trp Leu Gln Val T? R Gl? Asp Gl? Asp His 210 215 220 Asn Gl? Leu T? R Wing Asp Asn Val Asn Asp Ser Thr Phe Thr Gl? Phe 225 230 235 240 Leu Leu T? R His Asp Thr Asn 245 < 210 > 9 < 211 > 4517 < 212 > 7ADN < 213 > Homo sapiens < 400 > ctgattccat accagagggg ctcaggatgc tgttgctggg agctgttcta ctgctattag ctctgcccgg gcatgaccag gaaaccacga ctcaagggcc cggagtcctg cttcccctgc 1 ccaagggggc ctgcacaggt tggatggcgg gcatcccagg gcatccgggc cataatgggg 1 ccccaggccg tgatggcaga gatggcaccc ctggtgagaa gggtgagaaa ggagatccag 2 gtcttattgg tcctaaggga gacatcggtg aaaccggagt acccggggct gaaggtcccc 30 gaggctttcc gggaatccaa ggcaggaaag gagaacctgg agaaggtgcc tatgtatacc 3 gctcagcatt cagtgtggga ttggagactt acgttactat ccccaacatg cccattcgct 4 ttaccaagat cttctacaat cagcaaaacc actatgatgg ctccactggt aaattccact 4 gcaacattcc tgggctgtac tactttgcct accacatcac agtctatatg aaggatgtga 5 aggtcagcct cttcaagaag gacaaggcta tgctcttcac ctatgatcag taccaggaaa 60 ataatgtgga ccaggcctcc ggctctgtgc tcctgcatct ggaggtgggc gaccaagtct 6 ggctccaggt gtatggggaa atggactcta ggagagcgta tgctgataat gacaatgact 7 ccaccttcac aggctttctt ctctaccatg acaccaactg atcaccacta actcagagcc 7 tcctccaggc caaacagccc caaagtcaat taaaggcttt cagtacggtt aggaagttga 8 ttattattta gttggaggcc tttagatatt attcattcat ttactcattc atttattcat 90 agtaacttta tcattcatca aaaaaatcat atgctatgtt cccagtcctg gggagcttca 9 caaacatgac cagataactg actagaaaga agtagttgac agtgctattt tgtgcccact 10 gtctctcctg atgctcatat caatcctata aggcacaggg aacaagcatt ctcctgtttt 10 tacagattgt atcctgaggc tgagagagtt aagtgaatgt ctaaggtcac acagtattaa 11 gtgacagtgc tagaaatcaa acccagagct gtggactttg ttcactagac tgtgcccttt 12 tatagaggta catgttctct ttggagtgtt ggtaggtgtc tgtttcccac ctcacctgag 12 agccattgaa tttgccttcc tcatgaatta aaacctcccc caagcagagc ttcctcagag 13 aaagtggttc tatgatgaag tcctgtcttg gaaggactac tactcaatgg cccctgcact 138 actctacttc ctcttaccta tgtcccttct catgcctttc cctccaacgg ggaaagccaa 14 ctccatctct aagtgctgaa ctcatccctg ttcctcaagg ccacctggcc aggagcttct 150 ctgatgtgat atccactttt tttttttttt gagatggagt ctcactctgt cacccaggct 15 ggagtacagt gacacgacct cggctcactg cagcctcctt ctcctgggtc caagcaatta 16 ttgtgcctca gcctcccgag tagctgagac ttcaggtgca ttccaccaca catggctaat 16 ttttgtattt ttagtagaaa tggggtttcg tcatgttggc caggctggtc tcgaactcct 17 ggcctaggtg atccacccgc ctcgacctcc caaagtgctg ggattacagg catgagccac 180 catgcccagt cgatatctca ctttttattt tgccatggat gagagtcctg ggtgtgagga 18 ccaggctaga acacctccca ggcaactgcc caggaaggac tgtgcttccg tcacctctaa 19 gatccttgat atcccttgca tgaagaccaa aaatgcctca cccatatcta tctcttgaat 19 cccagaatta actcc ATTCC agtctctgca tgtaatcagt tttatecaca gaaacatttt 2040 cattttagga aatccctggt ttaagtatca atccttgttc agctggacaa tatgaatctt 2100 ttccactgaa gttagggatg actgtgattt teagaacaeg tecagaattt ttcatcaaga 2160 aggtagcttg agcctgaaat gcaaaaccca tggaggaatt ctgaagccat tgtctccttg 2220 gggtcaggga agtaccaaca agactgggcc tcctgaattt attattgttc tttaagaatt 2280 acaggttgag gtagttgatg gtggtaaaca ttctctcagg agacaataac tccagtgatg 2340 attttagcaa tttttcaaag aatageatte aaacagagta tetatcaata tataaattta 2400 tttttgctta aaaaactatc cagttttaaa ttctgaacaa tttetettat atgtgtattg 2460 ctaatcatta aggtattatt ttttccacat ataaagcttt gtctttttgt tgttgttgtt 2520 gtttttaaga tggagtttcc ctctgttgcc aggctagagt gcagtggcat gatctcggct 2580 tactgcaacc tttgcctccc aggtttaagc gattettetg cctcagcctc cegagtaget 2640 gggaccacag gtgcctacca ccatgccagg ctaatttttg tatttttagt aaagacaggg 2700 tttcaccata ttggccaggc tggtctcgaa ctcctgacct tgtgatctgc ccgcctccat 2760 tttgtgagaa tgtgttgtta agatagatat gaggtttaga gagggatgaa gaggtgagag 2820 gttagtcaga taagccttgt actctgtgtt gtgaatgtca ttcacaacag aaaacccaaa 2880 aactactgta atattatgca agcaagaaaa ataaaggaaa aatggaaaca tttattcctt 2940 tgcataatag aaattaccag agttgttctg tetttagata aggtttgaac caaagetcaa 3000 aacaatcaag acccttttct gtatgtcctt ctgttctgcc ttccgcagtg taggctttac 3060 cctcaggtgc tacacagtat agttctaggg tttccctccc gatatcaaaa agactgtggc 3120 ctgcccagct ctcgtatccc caagccacac catctggcta aatggacatc atgttttctg 3180 gtgatgccca aagaggagag aggaagctct tgccccagca ctttcccaga agtgtaacct 3240 tgcatctcat tgctctggct gagttgtgtg cctgtttctg accaatcact gagtcaggag 3300 gatgaaatat tcatattgac ttaattgcag cttaagttag gggtatgtag aggtattttc 3360 aattgggaca cctaaagcaa aaataggaga ctgttatcag gtggatgata gatgcaaaat 3420 aatacctgtc cacaacaaac tettaatget gtgtttgagc tttcatgagt tteccagaga 3480 gacatagctg gaaaattcct attgattttc tctaaaattt caacaagtag ctaaagtctg 3540 cagtctcaca gctatgctca tctggtgggg gtgggctcct tacagaacac gctttcacag 3600 ttaccctaaa ctctctgggg cagggttatt accagaggca cctttgtgga cagagacagt 3660 caactgaggc ccaacagagg cctgag agaa actgaggtca agatttcagg attaatggtc 3720 ttgaagtaca ctgtgatgct attgtggatt tgtccaattc tctttagttc tgtcagcttt 3780 tgcttcatat attttagcgc tetattatta gatatataca tgtttagtat tatgtcttat 3840 tggtgcattt actetettat cattatgtaa tgtccttctt tatctgtgat aattttctgt 3900 gttctgaagt ctactttgtc taaaaataac atacgcactc aacttccttt tctttcttcc 3960 ttcctttctt tcttccttcc tttctttctc tctctctctt tccttccttc cttcctcctt 4020 ttctctctct ctctctctct ctctcttttc ttgacagact ctcgttctgt ggccctggct 4080 ggagttcagt ggtgtgatct tggctcactg ctacctctac catgagcaat tctcctgcct 4140 agtagctgga cagcctccca actacaggct catgccactg cgcccagcta atttttgtat 4200 ttttegtaga gacggggttt caccacattc gtcaggttgg tttcaaactc ctgactttgt 4260 gatccacccg cctcggcctc ccaaagtgct gggattacag gcatgagcca tcacacctgg 4320 tcaactttct tttgattagt gtttttgtgg tatatctttt tecatcatgt tactttaaat 4380 atatctatat tattgtattt aaaatgtgtt tettacagac tgcatgtagt tgggtataat 4440 ttttatccag tctaaaaata tctgtctttt aattggtgtt tagacaattt atatttaata 4500 atttaaa aaatggtgga 4517 <; 210 > 10 < 211 > 729 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > The nucleotide sequence encodes the zsig39 polypeptide of SEQ ID NO: 2 < 400 > 10 atgmgnccny tn? tngtnyt n? tn? tn? tn ggn? tngcng cnggnwsncc nccn? tnga? ga? aa to thccnwsnyt ntg? ccnggn ca? ccnggn? tnccnggnac nccnggnca? 1 ca? Ggnwsnc arggn? Tncc nggnmgnga? ggnmgngayg gn gngaygg ngcnccnggn 1 gcnccnggng araarggnga rggnggnmgn ccnggnytnc cnggnccnmg nggngayccn 2 ggnccnmgng gngargcngg nccngcnggn ccnacnggnc cngcnggnga rtg? wsngtn 3 ccnccnmgnw sngcntt? ws ngcnaarmgn wsngarwsn gngtnccncc nccnwsnga? 3 gcnccn? Tnc cntt? Ga? Mg ngtn? Tngtn aa? Garcarg gncaytayga ygcngtnacn 4 ggnaartt? A cntg? Cargt nccnggngtn ta? Ta? Tt? G cngtnca? Gc nacngtnta? 4 mgngenwsn? tncartt? ga ?? tngtnaar aa? ggngarw snathgcnws nttyttycar 5 tt? tt? ggng gntggccnaa rccngcnwsn ytnwsnggng gngcnatggt nmgnytngar 6 ccngarga? c argtntgggt ncargtnggn gtnggnga? t a? athggnat hta? gcnwsn 6 athaaracng a? wsnacntt? wsnggntt? ? tngtnta? w snga? tggca? wsnwsnccn 7 gtntt? gcn 7 < 210 > 11 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC44 < 400 > 11 taacaatttc acacagg 17 < 210 > 12 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC976 < 400 > 12 cgttgtaaaa cgacggcc 18 < 210 > 13 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14707 < 400 > 13 cccactggac gacaacaaga 20 < 210 > 14 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14 < 400 > 14 agcacactcc tctggtcttg 20 < 210 > 15 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14760 < 400 > 15 ccaatgtagt cacccacacc 20 < 210 > 16 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14 < 400 > 16 tggtgaacga gcagggacat 20 < 210 > 17 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14759 < 400 > 17 tccccagtct ttgcttagtg 20 < 210 > 18 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15002 < 400 > 18 agggaggtgg ggtagagc 18 < 210 > 19 < 211 > 18 < 212 > DNA < 213 > Artificial Sequence 5003 < 220 > < 223 > Oligonucleotide ZC 15003 < 400 > 19 tgggggactt acacttgc 18 < 210 > 20 < 211 > 6 < 212 > PRT < 213 > Artificial Sequence < 220 > < 223 > peptide with affinity tag Glu-Glu < 400 > 20 Glu Tyr Met Pro Val Asp 1 5 < 210 > 21 < 211 > 26 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15 < 400 > 21 actcattcta gactacagca aagact 26 < 210 > 22 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15038 < 400 > 22 atgtatggat ccctggacga caaca 25 < 210 > 23 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13 < 400 > 23 ggctgtcctc taagcgtcac 20 < 210 > 24 < 211 > 19 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13007 < 400 > 24 ftcaca gggatgcca 19 < 210 > 25 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15 < 400 > 25 ttgtgagaat tcatgaggcc actc 24 < 210 > 26 < 211 > 25 < 212 > DNA < 213 > S Artificial Seecuctance of Oligonucleotide ZC < 220 > < 223 > Oligonucleotide ZC15232 < 400 > 26 attcaaggat ccagcaaaga caggt 25 < 210 > 27 < 220 > < 223 > Peptide Glu-Glu < 400 > 27,000 < 210 > 28 < 211 > 144 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Linker not labeled N-terminal < 400 > 28 ttattgttta tcaatactac tattgctagc attgctgcta aagaagaagg tgtaagcttg 6 gacaagagag aactggacga caacaagatc cccagcctct gcccggggca ccccggcctt 12 ccaggcacgc cgggccacca tggc 14 < 210 > 29 < 211 > 144 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Linker with tag < 400 > 29 agcattgctg ctaaagaaga aggtgtaagc ttggacaaga gagaagaaga atacatgcca atggaaggtg gtctggacga caacaagatc cccagcctct gcccggggca ccccggcctt ccaggcacgc cgggccacca tggc < 210 > 30 < 211 > 147 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Linker does not tag < 400 > 30 agcatcaaga cagacagcag gttctccgga tttctggtgt actccgactg gcacagctcc 60 ccagtctttg cttagatttc ggctgcctgt ttggatattt ttataatttt tgagagtttg 120 ccaactaatg tttttctctt ctatgat 147 < 210 > 31 < 211 > 144 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Linker with tag < 400 > 31 agcatcaaga cagacagcac cttctccgga tttctggtgt actccgactg gcacagctcc 6 ccagtctttg ctggagggga ggagtatatg cctatggagt agaattccta gtattctagg 12 gctgcctgtt tggatatttt tata 14 < 210 > 32 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13731 < 400 > 32 ggtgtaagct tggacaagag agaagaagaa tacatgccaa tggaaggtgg t 51 < 210 > 33 < 211 > 62 < 212 > DNA < 213 > Artificial Sequence of Oligonucleotide ZC < 220 > < 223 > Oligonucleotide ZC15268 < 400 > 33 tgccccgggc agaggctggg gatcttgttg tcgtccagac caccttccat tggcatgtat 6 6 < 210 > 34 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13487 < 400 > 34 agcattgctg ctaaagaaga aggtgtaagc ttggacaaga gaga 4 < 210 > 35 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15273 < 400 > 35 catggtggcc cggcgtgcct ggaaggccgg ggtgccccgg gcagaggctg g 51 < 210 > 36 < 211 > 50 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15 < 400 > 36 catcaagaca gacagcacct tctccggatt tctggtgtac tccgactggc 50 < 210 > 37 < 211 > 65 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15724 < 400 > 37 tttctggtgt actccgactg gcacagctcc ccagtctttg cttagaattc ggctgcctgt ttgga < 210 > 38 < 211 > 63 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15 < 400 > 38 tggcaaactc tcaaaaatta taaaaatatc caaacaggca gccctagaat actaggaatt cta < 210 > 39 < 211 > 52 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC13734 < 400 > 39 atcatagaag agaaaaacat tagttggcaa actctcaaaa attataaaaa ta < 210 > 40 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14 < 400 > 40 acggtttatt gtttatcaat actactattg ctagcattgc < 210 > 41 < 211 > 62 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14821 < 400 > 41 tcaatactac tattgctagc attgctgcta aagaagaagg tgtaagcttg gacaagagag 60 aa 62 < 210 > 42 < 211 > 63 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15269 < 400 > 42 tgccccgggc agaggctggg gatcttgttg tcgtccagtt ctctcttgtc caagcttaca 60 ect 63 < 210 > 43 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15274 < 400 > 43 catggtggcc cggcgtgcct ggaaggccgg ggtgccccgg gcagaggctg g 51 < 210 > 44 < 211 > 50 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15 < 400 > 44 catcaagaca gacageaect tctccggatt tctggtgtac tccgactggc < 210 > 45 < 211 > 68 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC15 < 400 > 45 atttctggtg tactccgact ggcacagctc cccagtcttt gctggtggtg aagaatacat gccaatgg < 210 > 46 < 211 > 58 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucleotide ZC14819 < 400 > 46 aacaggcagc cctagaatac taggaattct attccattgg catgtattct tcaccacc 5 < 210 > 47 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Oligonucl * = ottid ZC14 < 400 > 47 attataaaaa tatccaaaca ggcagcccta gaatactag 3 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following

Claims (29)

1. An isolated polypeptide comprising a sequence of amino acid residues that is at least 80% identical to SEQ ID NO: 2, characterized in that the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128-130, 136 -139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO: 2.

2. An isolated polypeptide according to claim 2, characterized in that the polypeptide is at least 90% identical to SEQ ID NO: 23.

An isolated polypeptide according to claim 2, characterized in that the polypeptide comprises a collagen-like domain having at least 22 repeats of collagen.

4. An isolated polypeptide according to claim 2, characterized in that the polypeptide comprises residues 19-243 of SEQ ID NO: 2.

5. An isolated polypeptide according to claim 1, characterized in that it is covalently, amino terminally or carboxyterminally linked to a portion selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and flurophores.

6. An isolated polypeptide, characterized in that it is selected from the group consisting of: a) a polypeptide having a sequence of amino acid residues from the amino acid residue 30 to amino acid residue 95 of SEQ ID NO: 2; b) a polypeptide having an amino acid residue sequence from amino acid residue 30 to amino acid residue 96 of SEQ ID NO: 2; and c) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to 97 of SEQ ID NO: 2; d) a polypeptide having an amino acid residue sequence from amino acid residue 30 to amino acid residue 98 of SEQ ID NO: 2; e) a polypeptide having a sequence of amino acid residues from the amino acid residue 98 to amino acid residue 243 of SEQ ID NO: 2; f) a polypeptide having a sequence of amino acid residues from the amino acid residue 99 to amino acid residue 243 of SEQ ID NO: 2; g) a polypeptide having a sequence of amino acid residues from amino acid residue 30 to amino acid residue 243 of SEQ ID NO: 2; and h) a polypeptide having an amino acid residue sequence that is 90% identical in the amino acid sequence a), b), c), d), e), f), g) or).

7. A fusion protein consisting essentially of a first portion and a second portion linked by a peptide bond, the first portion comprises a polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is minus 80% identical to SEQ ID NO: 2, wherein the sequence comprises; beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO: 2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO. NO: 2 from amino acid residue 16 to amino acid residue 243; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2 containing the collagen-like domain or a portion of the collagen-like domain of dimerization or oligomerization; e) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, which contains the globular-like domain or the receptor binding portion of the globular-like domain; or f) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, which includes the collagen-like domain and the globular domain; and the second portion comprises 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 amino acid residue 30 to the amino acid residue 95 of SEQ ID NO: 2; b) a polypeptide having the sequence of amino acid residue 30 to the amino acid residue 96 of SEQ ID NO: 2; c) a polypeptide having the sequence of amino acid residue 30 up to the amino acid residue 97 of SEQ ID NO: 2; d) a polypeptide having the sequence from amino acid residue 30 to amino acid residue 98 of SEQ ID NO: 2; e) a polypeptide having the sequence from amino acid residue 30 to amino acid residue 243 of SEQ ID NO: 2; f) a polypeptide having the sequence of amino acid residue 98 to amino acid residue 243 of SEQ ID NO: 2; and g) a polypeptide having the sequence from amino acid residue 99 to the amino acid residue 243 of SEQ ID NO: 2.

9. A fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-15 or 1-18 of SEQ ID NO: 2, characterized in that the sequence of the secretory signal is operably linked to a additional polypeptide.

10. A pharmaceutical composition, characterized in that it comprises a polypeptide according to claim 1, in combination with a pharmaceutically acceptable carrier.

11. An antibody that specifically binds to an epitope of a polypeptide according to claim 1.

12. An isolated polynucleotide that encodes a polypeptide comprising an amino acid residue sequence that is at least 80% identical to SEQ ID NO: 2, characterized in that the sequence comprises: beta strands corresponding to amino acid residues 105-109, 128- 130, 136-139, 143-146, 164,171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by at least two amino acid residues; and a receptor binding domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO: 2.

13. An isolated polynucleotide according to claim 12, characterized in that the polypeptide is at least 90% identical to SEQ ID NO: 2.

14. An isolated polynucleotide according to claim 12, characterized in that the polypeptide comprises a collagen-like domain having at least 22 repeats of collagen.

15. An isolated polynucleotide according to claim 12, characterized in that the polynucleotide is DNA.

16. An isolated polynucleotide, characterized in that it is selected from the group consisting of: a) a nucleotide sequence from nucleotide 243 to nucleotide 962 of SEQ ID NO: 1; b) a nucleotide sequence from nucleotide 252 to nucleotide 962 of SEQ ID NO: 1; c) a nucleotide sequence from nucleotide 285 to nucleotide 482 of SEQ ID NO: 1; d) a nucleotide sequence from nucleotide 285 to nucleotide 485 of SEQ ID NO: 1; e) a nucleotide sequence from nucleotide 285 to nucleotide 488 of SEQ ID NO: 1; f) a nucleotide sequence from nucleotide 285 to nucleotide 491 of SEQ ID NO: 1; g) a nucleotide sequence from nucleotide 285 to nucleotide 926 of SEQ ID NO: 1; h) a nucleotide sequence from nucleotide 491 to nucleotide 926 of SEQ ID NO: 1; i) a polynucleotide encoding a polypeptide having a nucleotide sequence that is at least 80% identical in nucleotide sequence a), b), c), d), e), f), g) and h); j) the complementary nucleotide sequences for a), b), c), d), e), f), g), h) or i); and k) degenerate nucleotide sequences of a), b), c), d), e), g), h), i) or j).

17. An isolated polynucleotide encoding a fusion protein, characterized in that it consists essentially of a first portion and a second portion linked by a peptide bond, the first portion is selected from the group consisting of: a) a polypeptide comprising a residue sequence of amino acids that is at least 80% identical to SEQ ID NO: 2, wherein the sequence comprises; beta strands corresponding to amino acid residues 105-109, 128-130, 136-139, 143-146, 164-171, 176-182, 187-200, 204-210 and 226-231 of SEQ ID NO: 2, wherein the beta strands are separated by at least two amino acid residues; and a "receptor binding" domain comprising amino acid residues 111-135 and 170-174 of SEQ ID NO: 2; b) a polypeptide comprising an amino acid residue sequence as shown in SEQ ID NO: 2; NO: 2 from amino acid residue 16 to amino acid residue 243; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 243; d) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2 containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, which contains the globular-like domain or the active portion of the globular-like domain; or f) a portion of the zsig39 polypeptide as shown in SEQ ID NO: 2, which includes the collagen-like domain and the globular domain; and the second portion comprises another polypeptide.

18. An isolated polynucleotide encoding a fusion protein, characterized in that it comprises a secretory signal sequence having the amino acid sequence of amino acid residues 1-15 or 1-18 of SEQ ID NO: 2, wherein the sequence of the secretory signal is operably linked to an additional polypeptide.

19. An isolated polynucleotide, characterized in that it comprises the sequence from nucleotide 1 to nucleotide 729 of SEQ ID NO: 10.

20. An expression vector, characterized in that it comprises the following operatively linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1; and a transcription terminator.

21. An expression vector according to claim 20, characterized in that the DNA segment encodes a polypeptide that is at least 90% identical to SEQ ID NO: 2.

22. An expression vector according to claim 20, characterized in that the DNA segment encodes a polypeptide further comprising a collagen-like domain having at least 22 repeats of the collagen.

23. An expression vector according to claim 20, characterized in that the DNA segment encodes a polypeptide covalently linked, amino terminally or carboxy terminally to an affinity tag.

24. An expression vector according to claim 20, characterized in that the DNA segment encodes a sequence of the secretory signal operably linked to the polypeptide.

25. An expression vector according to claim 20, characterized in that the sequence of the secretory signal comprises residues 1-15 or 1-18 of SEQ ID NO: 2.

26. A cultured cell into which an expression vector has been introduced, characterized in that it comprises the following operatively linked elements: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1; and a transcription terminator; wherein the cell expresses the polypeptide encoded by the DNA segment.

27. A method of producing a polypeptide, characterized in that it comprises: culturing a cell in which an expression vector comprising the following operably linked elements has been introduced: a transcription promoter; a DNA segment encoding a polypeptide according to claim 1; and a transcription terminator; whereby the cell expresses the polypeptide encoded by the DNA segment; and recovering the expressed polypeptide.

28. An oligonucleotide probe or primer, characterized in that it comprises at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 10 or a sequence complementary to SEQ ID NO: 10.

29. A method for modulating the metabolism of free fatty acids by administering a pharmaceutically effective dose of a polypeptide according to claim 1.