KR20010005701A - Motilin homologs - Google Patents

Abstract

The present invention relates to polynucleotides, polypeptides and peptide fragments thereof and their use in novel cDNA sequences having homology to motilin. The tissue distribution of mRNA for this novel polypeptide is specific for the stomach, small intestine and pancreas. The invention further includes methods of increasing gastric motility using agonists, antagonists, antibodies, host cells expressing cDNA encoding the novel motiline homologues and novel molecules.

Description

Motilin homologues {MOTILIN HOMOLOGS}

Many regulatory peptides that are important for maintaining nutritional homeostasis are found in the gastrointestinal environment. These peptides can be synthesized in the digestive system and act locally, but can also be found in the brain. The reverse is also found. That is, peptides are synthesized in the brain but are found to regulate cells in the gastrointestinal tract. This phenomenon has been referred to as the "brain-intestinal axis" and is important for other physiological processes that signal satiety, regulate body temperature, and require feedback between the brain and intestines.

Intestinal peptide hormones include gastrin, cholecystokinin (CCK), secretin, gastric inhibitory peptide (GIP), intestinal polypeptide acting on blood vessels (VIP), motilin, somatostatin, pancreatic peptide (PP), substance P and neuropeptide Y (NPY), and they use a variety of different mechanisms of action. For example, gastrins, motilines and CCK act as endocrine and neuroendocrine hormones. Other hormones such as gastrin and GIP are believed to act exclusively in the endocrine modality. Other modes of action include a combination of endocrine, neuroendocrine and paracrine functions (somatostatin); Exclusive neurosecretory action (NPY); And a combination of neurosecretion and paracrine action (VIP and substance P). Most intestinal hormonal action is mediated by membrane-bound receptors and activates a second messenger system. For intestinal peptides see literature [Mulvihill et al., In Basic and Clinical Endocrinology, pp. 551-570, 4th edition Greenspan F. S. and Baxter, J. D. editors., Appleton & Lange: Norwalk, Connecticut, 1994].

Many of these enteric peptides are synthesized as inactive precursor molecules that require many peptide cleavage to be activated. The family known as the "glucagon-secretin" family, including VIP, gastrin, secretin, motiline, glucagon and galanine, are good examples of peptides that are regulated by multiple cleavage and post-translational modifications.

Motilin is a 22 amino acid peptide found in the intestinal tissue of mammalian species (Domschke, W., Digestive Disease 22 (5): 454-461, 1977). DNA and amino acid sequences have been identified for swine prepromotiline (US Pat. No. 5,006,469). Motiline has been identified as a factor that can increase gastric motility by stimulating pepsin secretion, affecting gastric secretory function [Brown et al, Canadian J. of Physiol. Pharmacol. 49: 399-405, 1971], recent evidence suggests that motilin plays a role in the regulation of myostatic in the stomach and small intestine. The increase in motiline in the circulatory system correlates with fasting contraction of phase III and duodenum of the EMG complex in the digestive system [Chey et al., In Gut Hormones, (eds.) Bloom, S. R., pp. 355-358, Edinburgh, Churchill Livingstone, 1978; Lee et al., Am. J. Digestive Diseases, 23: 789-795, 1978; And Itoh et al., Am. J. Digestive Diseases, 23: 929-935, 1978.]. Motiline and its analogs have been shown to cause contraction of gastrointestinal smooth muscle, but other types of smooth muscle cells fail to contract (Strunz et al., Gastroenterology 68: 1485-1491, 1975).

The present invention relates to a novel secreted protein having homology to motilin, which has been found to be transcribed in the gastrointestinal system. The discovery of these novel peptides is important for further explanation of how the body maintains nutritional homeostasis and development of therapies to modulate such processes, as well as other uses that will be apparent from the description herein.

Summary of the Invention

In one aspect, the invention provides a kit comprising: (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to nucleotide 111 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) orthologs of (a) and (b); And (d) encoding a polypeptide selected from the group consisting of degenerate nucleotide sequences of (a), (b) or (c).

In another aspect, the invention provides an antibody comprising: (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 37 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) an ortholog of (a) or (b).

In another aspect, the invention provides an operably linked component comprising: a transcriptional promoter; (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to nucleotide 111 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) the ortholog of (a) or (b); And (d) a DNA fragment selected from the group consisting of the degenerate nucleotide sequences of (a), (b) or (c); An expression vector comprising a transcription terminator is provided.

In another aspect, the invention provides an operably linked component comprising: a transcriptional promoter; (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to nucleotide 111 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) the ortholog of (a) or (b); And (d) a DNA fragment selected from the group consisting of the degenerate nucleotide sequences of (a), (b) or (c); Provided is a cultured cell into which an expression vector comprising a transcription terminator is introduced, wherein said cell expresses a polypeptide encoded by a DNA fragment.

In another aspect, the invention provides an antibody comprising (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 37 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) a purified polypeptide selected from the group consisting of the orthologs of (a) or (b) in combination with a pharmaceutically acceptable excipient.

In another aspect, the invention provides an antibody comprising (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 37 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) an antibody that binds to an epitope of a polypeptide selected from the group consisting of the orthologs of (a) or (b).

In another aspect, the invention provides an operably linked component comprising: a transcriptional promoter; (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to nucleotide 111 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) the ortholog of (a) or (b); And (d) a DNA fragment selected from the group consisting of the degenerate nucleotide sequences of (a), (b) or (c); There is provided a method for producing a zsig33 polypeptide comprising culturing a cell into which an expression vector comprising a transcription terminator is introduced, thereby allowing the cell to express a polypeptide encoded by a DNA fragment and recovering the polypeptide.

In another aspect, the invention provides an antibody comprising (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 37 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) a composition comprising an isolated polypeptide selected from the group consisting of orthologs of (a) or (b) in a pharmaceutically acceptable excipient, an amount sufficient to increase delivery time or gastric emptying of the ingested material; In another aspect, the present invention provides a method of stimulating gastric movement consisting of administering to a mammal in need thereof.

Before describing the invention in detail, it will be helpful to define the specific terms used herein.

The term “ortholog” refers to a polypeptide or protein obtained from any one species that is a functional counterpart of a polypeptide or protein derived from a different species. Sequence differences among the orthologs are the result of differentiation into new species.

A "paralog" refers to a distinct but structurally related protein made by an organism. Paralogs are thought to occur during gene copying. For example, α-globin, β-globin, and myoglobin are paralogs of each other.

The term “allelic variant” refers to any one of two or more surrogates of a gene that occupy the same chromosomal locus. Allelic variants occur naturally through mutations and can lead to phenotypic polymorphisms in a population. The gene mutation may be silent (no change in the encoded polypeptide) or may encode a polypeptide having an altered amino acid sequence. The term allelic variant is also used herein to refer to a protein encoded by an allelic variant of a gene.

The term “expression vector” refers to a linear or circular DNA molecule that contains a fragment encoding a polypeptide of interest that is operably linked with additional fragments that provide for its transcription. Such additional segments include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain both elements.

The term “isolated”, when applied to a polynucleotide molecule, removes the polynucleotide from its natural genetic environment and thereby is free of other adventitious or unwanted coding sequences and is suitable for use in genetically engineered protein production systems. It exists in form. Such isolated molecules are those that are isolated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention may contain naturally occurring 5 'and 3' untranslated regions such as promoters and terminators, although there are no other genes to which they were originally bound. Identification of bound regions will be apparent to those skilled in the art (see, eg, Dynan and Tijan, Nature 316: 774-778, 1985). When applied to a protein, the term “isolated” refers to the protein being found in conditions other than its natural environment, such as conditions separate from blood and animal tissue. An isolated protein is a form that is substantially free of other proteins, particularly other proteins of animal origin. The protein is preferably provided in a highly purified form, ie at least 95% purity, more preferably at least 99% purity.

The term “operably linked”, when applied to a DNA sample, means that the fragments are arranged to function in accordance with their intended purpose, such that transcription is initiated at the promoter and arranged through the coding fragments to reach the terminator. .

The term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotides or ribonucleotides that is 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 term “complement of a polynucleotide molecule” refers to a polynucleotide molecule having complementary base sequences and reverse orientation compared to the reference sequence. For example, the sequence 5 'ATGCACGGG 3' is complementary to 5 'CCCGTGCAT 3'.

The term “degenerate nucleotide sequence” refers to a sequence of nucleotides comprising one or more degenerate codons (as compared to a reference polynucleotide molecule encoding a polypeptide). Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residues (ie, the GAU and GAC triplets each encode Asp).

The term “promoter” refers to a portion of a gene containing a DNA sequence that provides for the binding of RNA polymerase and the initiation of transcription. Promoter sequences are common but are not always found in the 5 'non-coding region of a gene.

The term "secretory signal sequence" refers to a DNA sequence that encodes a polypeptide ("secretory peptide") that designates a larger polypeptide through the secretory pathway of the cell from which it is synthesized as a component of a larger polypeptide. Larger peptides are typically cleaved to remove secreted peptides in progress through the secretory pathway.

The term “receptor” refers to a cell-bound protein that binds to a bioactive molecule (ie a ligand) and mediates its effect on the cells of the ligand. Membrane-bound receptors are characterized by having a multi-domain structure comprising an extracellular ligand-binding domain and an endogenous effector domain typically involved in signal transduction. Binding of the ligand to the receptor results in a morphological change of the receptor causing an interaction between the effector domain and other molecule (s) in the cell. This interaction continues to induce alterations in cell metabolism. Metabolic events associated with receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increased cyclic AMP production, immobilization of cellular calcium, immobilization of membrane lipids, cell fixation, and hydrolysis of inositol lipids. And hydrolysis of phospholipids. Most nuclear receptors also exhibit a multi-domain structure comprising amino-terminus, transactivating domains, DNA binding domains and ligand binding domains. In general, receptors may be membrane bound, cytosolic, or nuclear; It may be a monomer (eg thyroid stimulating hormone receptor, beta-adrenergic receptor) or a multimer (eg PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor And IL-6 receptor).

The term “complement / anticomplement pair” refers to unequal moieties that form a stable pair that is non-covalently bound under appropriate conditions. For example, biotin and avidin (or streptavidin) are circular (sample) members of the complement / anticomplement pair. Other exemplary complement / anticomplement pairs include receptor / ligand pairs, antibody / antigen (or hapten or epitope) pairs, sense / antisense polynucleotide pairs, and the like. If degradation of the complement / anticomplement pair is then desired, the complement / anticomplement pair preferably has a binding affinity of less than 10 ⁹ M ⁻¹ .

All references cited herein are incorporated by reference in their entirety.

The present invention is based, in part, on the discovery of novel human DNA sequences encoding novel secreted polypeptides homologous to motiline, which is most closely homologous to pig motiline (SEQ ID NO: 3 and 4). Motilin is a member of the polypeptide family that regulates the physiology of the stomach. Families of polypeptides important for gastrointestinal regulation to which motilines belong include glucagon, gastrin, galanine, and vasoactive intestinal peptides (VIPs). These polypeptides are synthesized in precursor forms that require multiple steps to be processed in the active form. Of particular relevance to the polypeptides of the invention are motilins, VIPs and galanines, the treatment of which involves the removal of signal sequences followed by cleaving one or more accessory peptides to release the active peptide. The resulting active peptides are generally small (10-30 amino acids) and may require additional post-translational modifications such as amidation, sulphation or pyrrolidane carbonyl acid modification of glutamic acid residues.

Analysis of the tissue distribution of mRNA corresponding to this novel DNA showed the highest levels of expression in the stomach and decreased expression levels in the small and pancreas. EST is also present in lung cDNA libraries. The polypeptide is designated zsig33.

The novel zsig33 polynucleotides and polypeptides of the present invention were initially identified by questioning the EST database for sequences with putative secretion signals. As a result, the EST sequence was discovered and expected to be related to the motilin family. EST sequences were derived from the fetal pancreatic library.

The new polypeptide encoded by full length cDNA is 117 amino acids. The expected signal sequence is 23 amino acid residues (amino acid residues 1-23 of SEQ ID NO: 2). The active peptide is expected to be 16 amino acid residues (amino acid residues 24 to 41 of SEQ ID NO: 2) and is expected to be cleaved at the C-terminus following amino acid residue 41 (Ser) of SEQ ID NO: 2. However, many entero-brain peptides require multiple cleavage. For example, the progastrin peptide is 101 amino acids and is cleaved at the N-terminus resulting in smaller peptides (G34, G17 and G14) sequentially [Sugano et al., J. Biol. Chem. 260: 11724-11729, 1985. Other peptides that require multiple treatment steps include cleavage of C-terminal cleavage resulting in glucagon-like peptide 1 and glucagon-like peptide 2, and cleavage of C-terminal peptide, whose treatment is known as GMAP. There is galanin included. Therefore, additional peptides based on cleavage after amino acid 37 (Gln) of SEQ ID NO: 2 were synthesized, resulting in 14 amino acid peptides (amino acid residues 24 (Gly) to amino acids of amino acid sequence SEQ ID NO: 2). Residue 37 (Gln)) was generated.

C-terminal peptides (amino acids 42 to 117 of SEQ ID NO: 2) may also have some specialized activity. Treatment of an active peptide against motiline (shown in SEQ ID NO: 4) results in the C-terminal of 70 amino acids from amino acid residues 50 (Ser) to amino acid residue 119 (Lys), known as motiline-binding peptide (MAP). The peptide was released. Adelman et al. Assumed that MAP would play a role in the regulation of digestion, appetite and nutrient absorption (Adelman et al., US Pat. No. 5,006,409).

Highly conserved amino acids of polypeptide zsig33 can be used as a tool to identify new family members. For example, reverse transcriptase polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding motifs conserved from RNA obtained from various tissue sources. Two such conserved domains have been identified using sequences from the present invention. The first domain was found at amino acid residues 31 to 36 of SEQ ID NO: 2, where the identified motif was Glu X Gln Arg X Gln (X is all amino acid residues) (shown in SEQ ID NO: 5), two The first domain was found at amino acid residues 78 to 84 of SEQ ID NO: 2, wherein the identified motif was Ala Pro X Asp X Gly Ile (X is all amino acid residues) (shown in SEQ ID NO: 6). In particular, highly degenerate primers designed from these sequences are useful for this purpose.

Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, significant sequence variation is possible between these polynucleotide molecules encoding SEQ ID NO: 2, including all RNA sequences replacing U instead of T. As such, zsig33 polypeptide-encoding polynucleotides and their RNA equivalents are contemplated by the present invention. Table 1 below shows the one letter code used to indicate the degenerate nucleotide position. "Resolution" is a nucleotide indicated by a code letter. "Complement" refers to the code for complementary nucleotide (s). For example, the code Y represents C or T, its complement R represents A or G, A is complementary to T, and G is complementary to C.

Degenerate codons, including all possible codons for a given amino acid, are shown in Table 2 below.

Those skilled in the art will appreciate that some ambiguity is introduced in determining the degenerate codons representing all possible codons encoding each amino acid. For example, degenerate codons for serine (WSN) can in some cases encode arginine (AGR) and degenerate codons for arginine (MGN) can encode serine in some cases (AGY). Similar relationships exist between codons encoding phenylalanine and leucine. As such, some polynucleotides comprised by degenerate sequences can encode variant amino acid sequences, but one of ordinary skill in the art can readily identify such variant sequences by referring to the amino acid sequence of SEQ ID NO: 2. Variant sequences can be easily tested for functionality as described herein.

Isolated polynucleotides within preferred embodiments of the invention will hybridize to regions of similar size of SEQ ID NO: 1, or to sequences complementary thereto, under stringency conditions. In general, stringency conditions are selected to be about 5 ° C. lower than the thermal melting point (T _m ) for specific sequences at defined ionic strengths and pH. T _m is the temperature (at defined ionic strength and pH) at which 50% of the target sequence hybridizes to the exactly matched probe. Typical stringency conditions are salt concentrations of at least about 0.02M, pH 7 and temperatures of at least about 60 ° C.

As mentioned above, isolated polynucleotides of the present invention include DNA and RNA. Methods of separating DNA and RNA are known in the art. In general, although DNA may be prepared using RNA from other tissues or may be isolated as genomic DNA, RNA is preferably isolated from the stomach. Total RNA can be prepared using guanidine HCl extraction followed by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18: 52-94, 1979). Poly (A) ⁺ RNA is prepared from total RNA using the methods of Aviv and Leather [Aviv and Leder, Proc. Natl. Acad. Sci. USA 69: 1408-1412, 1972. Complementary DNA (cDNA) is prepared from poly (A) ⁺ RNA using known methods. The polynucleotide encoding the zsig33 polypeptide is then identified and separated, such as by hybridization or PCR.

The invention further provides corresponding polypeptides and polynucleotides from other species (orthologs). Of particular interest are zsig33 polypeptides obtained from other mammalian species, including murine, rat, pig, sheep, bovine, canine, feline, horse and other primate proteins. Orthologs of human proteins can be cloned using the information and composition provided by the present invention in combination with conventional cloning techniques. For example, cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses a protein. Suitable sources of mRNA can be identified by probing the northern blot with a probe designed from the sequences disclosed herein. The library is then prepared from the mRNA of the positive tissue of the cell line. The zsig33 ortholog-encoded cDNA can then be separated by various methods, such as by probing using complete or partial human cDNA or using one or more degenerate probe sets based on the disclosed sequences. cDNA can also be cloned using primers designed from the sequences disclosed herein, using polymerase chain reaction, or PCR [Mullis, US Pat. No. 4,683,202]. Within further methods, cDNA libraries can be used to transform host cells (via cells or viruses), and expression of cDNA of interest can be detected using an antibody against zsig33. Similar techniques can also be applied to isolate genomic clones.

Those skilled in the art will appreciate that the sequence shown in SEQ ID NO: 1, and the polypeptides encoded thereby, represent a single allele of the human zsig33 gene and polypeptide, and allelic variation and replacement splicing are expected. Allelic variants can be cloned by probes of 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 silent mutations and those in which the mutation caused amino acid sequence changes, are those products of which the protein is an allelic variation of SEQ ID NO: 2 It is included within the scope of the present invention.

The invention also provides an isolated zsig33 polypeptide that is substantially homologous to the polypeptides of SEQ ID NO: 2 and their orthologs. The term “substantially homologous” herein refers to a polypeptide having a sequence identity of 50%, preferably 60%, more preferably at least 80% with the sequence set forth in SEQ ID NO: 2 or an ortholog thereof To be used. Such polypeptides will preferably be at least 90%, most preferably at least 95% identical to SEQ ID NO: 2 or an ortholog thereof. % Sequence identity is determined by conventional methods. See, eg, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992]. Briefly, two amino acid sequences were used to record sequence progression using a gap opening penalty of 10, a gap extension penalty of 1, and Heinkov and Heincorp's "bloom 62" history matrix, as shown in Table 3 below. Arranged for optimization (amino acids are represented by standard single letter codes).

Then% identity is calculated as:

Sequence identity of the polynucleotide molecules is measured by a similar method using the proportions as described above.

Substantially homologous proteins and polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably of insignificant nature, ie conservative amino acid substitutions (see Table 4 below) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; Small deletions, typically one to about 30 amino acids; And small amino- or carboxyl-terminal extensions, such as amino-terminal methionine residues, small linker peptides of about 20 to 25 residues, or small extensions (affinity tags) that facilitate purification, such as polyhistidine tracks, 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), maltose binding protein [Kellerman and Ferenci, Methods Enzymol. 90: 459-463, 1982; Guan et al., Gene 67: 21-30, 1987], thioredoxin, ubiquitin, cellulose binding protein, T7 polymerase, or other antigenic epitope or binding domain [Ford et al., Protein Expression and Purification 2: 95-107, 1991]. DNAs encoding affinity tags are commercially available (eg Pharmacia Biotech, Piscataway, NJ; New England Biolabs, Beverly, Mass.).

Conservative amino acid substitutions Basic Arginine lysine histidine acid Glutamic Acid Aspartic Acid polarity Glutamine asparagine Hydrophobic Leucine isoleucine valine directional Phenylalanine tryptophan tyrosine Small size Glycine Alanine Serine Threonine Methionine

In addition to the twenty standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovalin and α-methylserine) may also be substituted for the amino acid residues of zsig33. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can be substituted instead of zsig33 amino acid residues. "Non-natural amino acids" have been modified after protein synthesis and / or have a chemical structure different from that of standard amino acids in their side chain (s). Non-natural amino acids may be chemically synthesized or are preferably commercially available, for example pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethyl There is proline.

Essential amino acids of the zsig33 polypeptide of the invention can be identified following procedures known in the art such as site specific mutagenesis or alanine-scanning mutagenesis [Cunningham and Wells, Science 244: 1081-1085, 1989]. . In this latter technique, a single alanine mutation is introduced at every residue of the molecule, and the resulting mutant molecule is biologically active (eg, stimulates gastrointestinal cell contractility, modulates nutritional uptake, to identify amino acid residues important for the activity of the molecule). And / or secretion of digestive enzymes) (Hilton et al., J. Biol. Chem. 271: 4699-4708, 1996. Sites of ligand-receptor interactions can also be determined by physical analysis of the structure in combination with putative mutations of putative contact site amino acids, eg, as measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling. De Vos et al., Science 255: 306-312, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. Identity of essential amino acids can also be deduced from analysis of homology with related members of the glucagon-secretin family of intestinal-brain peptide hormones.

Multi-amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those published in the literature by Reidhar-Olson and Sauer or Bowie and Sauer. Reidhaar-Olson and Sauer, Science 241: 53-57, 1988; Bowie and Sauer, Proc. Natl. Acad. Sci. USA 86: 2152-2156, 1989]. In brief, these authors have presented a method for randomizing two or more positions of a polypeptide simultaneously, selecting for functional polypeptides, and then sequencing the mutated polypeptide to determine the spectra that allow substitution at each position. . Other methods that can be used include phage display (eg, Lowman et al., Biochem. 30: 10832-10837, 1991; Ladner et al., US Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204 and region-specific mutagenesis [Derbyshire et al., Gene 46: 145, 1986; Ner et al., DNA 7: 127, 1988].

Mutagenesis as mentioned above can be combined with large throughput automated screening methods to detect the activity of cloned and mutated polypeptides in a host cell. Mutated DNA molecules encoding active polypeptides (such as stimulation of gastrointestinal cell contractility, regulation of nutrient uptake and / or secretion of digestive enzymes) can be recovered from the host cell using modern equipment and rapidly sequenced. These methods make it possible to quickly determine the importance of the individual amino acid residues of a polypeptide of interest and to apply it to a polypeptide of unknown structure.

Using the methods mentioned above, those skilled in the art will be able to identify and / or prepare a variety of polypeptides that are substantially homologous to residues 24 to 37 of SEQ ID NO: 2 or to allelic variants thereof and retain the properties of the wild type protein. Can be. Such polypeptides may also include additional polypeptide fragments, such as Wawak, as generally discussed.

Polypeptides of the invention can be produced in host cells genetically treated according to conventional techniques, including full length proteins and fragments thereof. Suitable host cells are those of those cell types that can be transformed with exogenous DNA and grown in culture, including, for example, bacteria, fungal cells, and cultured higher eukaryotic cells. Preferred are eukaryotic cells, especially cultured cells of multinucleated organisms. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into various host cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., (Eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987].

In general, the DNA sequence encoding the zsig33 polypeptide of the invention is operably linked to other genetic components, such as transcriptional promoters and terminators, required for its expression in an expression vector. Those skilled in the art will recognize that selectable markers within a particular system may be provided on a separate vector, and that replication of exogenous DNA may be provided by integration into the host cell genome, but the vector is also typically one or more It will contain a selectable marker and one or more origins of replication. The selection of promoters, terminators, selectable markers, vectors and other components is a matter of basic design within conventional levels in the art. Many such components are described in the literature and are available from commercial suppliers.

To specify a zsig33 polypeptide in 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 secretory signal sequence may be of zsig33 polypeptide, or may be derived from other secreted proteins (eg t-PA) or newly synthesized. Secretory signal sequences are typically located 5 'of the DNA sequence encoding the propeptide of interest, although the particular signal sequence may be located elsewhere in the DNA sequence of interest (eg, Welch et al., US Pat. 5,037,743; Holland et al., US Pat. No. 5,143,830.

Cultured mammalian cells are also preferred hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transformation [Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973], electroporation [Neumann et al., EMBO J. 1: 841-845, 1982], DEAE-dextran mediated transformation [Ausubel et al., Eds. , Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987], liposome-mediated transformation [Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993], and viral vectors [A. Miller and G. Rosman, Biotechniques 7: 980-990, 1989; Q. Wang and M. Finer, Nature Med. 2: 714-716, 1996. The preparation of recombinant polypeptides in cultured mammalian cells is also disclosed (see, eg, Levinson et al., US Pat. No. 4,713,339; Hagen et al., US Pat. No. 4,784,950; Palmiter et al., US Pat. No. 4,579,821; And Ringold, US Patent 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 Chinese hamster ovary cell lines (eg CHO-K1, ATCC No. CCL 61). Additional suitable cell lines are known in the art and are available from publicly deposited institutions such as the American Type Culture Collection (Rockville, Maryland, USA). In general, strong transcriptional promoters, such as those from SV-40 or cytomegalovirus, are preferred (see US Pat. No. 4,956,288). Other suitable promoters include those derived from metallothionein genes (US Pat. Nos. 4,579,821 and 4,601,978) and adenovirus major late promoters.

Drug selection is generally used to select cultured mammalian host cells into which foreign DNA is inserted. Such cells are commonly referred to as "transformants". Cells cultured in the presence of a selection agent and capable of passage of the gene of interest to their offspring are referred to as "stable transformants". Preferred selectable markers are genes encoding resistance to the antibiotic neomycin. The selection is carried out in the presence of neomycin-type drugs such as G-418 and the like. Selection systems can also be used to increase the expression level of the gene of interest, which process is referred to as "amplification." Amplification is performed by culturing the transformant in the presence of low levels of the selection agent and then increasing the amount of selection agent to select cells that produce high levels of the product of the introduced gene. Preferred amplifiable selectable markers are dihydrofolate oxidoreductases that confer resistance to methotrexate. Other drug resistance genes such as hygromycin resistance, multi-drug resistance, puromycin acetyltransferase may also be used. FACS sorting from cells that have not been transformed with another marker that introduces an altered phenotype, such as a green fluorescent protein, or a cell surface protein such as CD4, CD8, class I MHC, placental alkaline phosphatase Or for sorting by means such as magnetic bead separation techniques.

Other higher eukaryotic cells, including plant cells, insect cells, and algal cells, can also be used as hosts. 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 the generation of foreign polypeptides therein are disclosed in the literature (Guarino et al., US Pat. No. 5,162,222 and WIPO Publication WO 94/06463). Insect cells can usually be infected with recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus (AcNPV). The DNA encoding the zsig33 polypeptide is inserted into the baculovirus genome in place of the AcNPV polyhedrin gene coding sequence by one of the following two methods. The first method is the traditional method of homologous DNA recombination between a wild type AcNPV and a transfer vector containing zsig33 flanked by an AcNPV sequence. Suitable insect cells, such as SF9 cells, are infected with a wild type AcNPV and transformed with a delivery vector comprising a zsig33 polynucleotide operably linked to the AcNPV polyhedrin gene promoter, terminator, and flanking sequences [King, L.A. and Possee, R.D., The Baculovirus Expression System: A Laboratory Guide, London, Chapman &Hall; O'Reilly, D.R. et al., Baculovirus Expression Vectors: A Laboratory Manual, New York, Oxford University Press., 1994; And Richardson, C.D., Ed., Baculovirus Expression Protocols. Methods in Molecular Biology, Totowa, NJ, Humana Press, 1995]. Natural recombination in insect cells will result in recombinant baculovirus containing zsig33 which is driven by the polyhedrin promoter. Recombinant viral stock is made by methods commonly used in the art.

The second method of producing recombinant baculovirus utilizes a transposon-based system described by Luxco et al. [Luckow, VA, et al., J. Virol. 67: 4566-4579, 1993. This system is commercially available as a Bac-to-Bac kit (Life Technologies, Rockville, MD). This system is pFastBacl ^™ (Life Technologies), a transfer vector containing a Tn7 transposon for transferring DNA encoding the zsig33 polypeptide into the baculovirus genome maintained in E. coli as a large plasmid called "bacmid". To utilize. The pFastBacl ^™ delivery vector utilizes the AcNPV polyhedrin promoter to induce the expression of the gene of interest in the case of zsig33. However, pFastBacl ^™ can be modified to a significant extent. The polyhedrin promoter can be removed and replaced with a baculovirus basic protein promoter (also known as the Pcor, p6.9 or MP promoter) that is initially expressed in baculovirus infection and is beneficial for expressing secreted proteins. Known [Hill-Perkins, MS and Possee, RD, J. Gen. Virol. 71: 971-976, 1990; Bonning, BC et al., J. Gen. Virol. 75: 1551-1556, 1994; And Chazenbalk, GD and Rapoport, B., J. Biol. Chem. 270: 1543-1549, 1995. In such delivery vector constructs, short or long versions of the basic protein promoter can be used. Moreover, delivery vectors can be constructed that replace native zsig33 secretion signal sequences with secretory signal sequences derived from insect proteins. For example, the secretory signal sequence obtained from ecsteroid glucosyltransferase (EGT), bee melittin (Invitrogen, Carlsbad, Calif.), Or baculovirus gp67 (PharMingen, San Diego, Calif.) Is a natural zsig33 secretion signal sequence. Can be used in constructs to replace In addition, the delivery vector may comprise in-frame fusion with DNA encoding an epitope tag, eg, a Glu-Glu epitope tag, at the C- or N-terminus of the expressed zsig33 polypeptide. Graussenmeyer, T et al., Proc. Natl. Acad. Sci. 82: 7952-7954, 1985. Using techniques known in the art, delivery vectors containing zsig33 are transformed into E. coli and screened for backmids containing blocked lacZ genes representing recombinant baculovirus. Backmid DNA containing the recombinant baculovirus genome is isolated using conventional techniques and used to transform Spodoptera frugiperda cells, such as Sf9 cells. Recombinant viruses expressing zsig33 continue to be produced. Recombinant virus stocks are made by methods commonly used in the art.

Recombinant viruses are used to infect host cells, typically cell lines derived from the fall beetle larva larvae, Spodothera prugiferda [Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, DC, 1994]. Another suitable cell line is the High Fiveo ^™ cell line (Invitrogen) derived from Trichoplusia ni (see US Pat. No. 5,300,435). Commercially available serum-free media is used to grow and maintain cells. Suitable medium is Sf900 II ^™ (Life Technologies) or ESF 921 ^™ (expression system) for Sf9 cells; For T. ni cells, it is Ex-cell0405 ^™ (JRH Biosciences, Lenexa, KS) or Express FiveO ^™ (Life Technologies). The cells were grown from an inoculation density of approximately 2-5 × 10 ⁵ cells to a density of 1-2 × 10 ⁶ cells, with recombinant virus stocks of 0.1-10, more typically near multiplicity of infection (MOI). ) Is added. Recombinant virus-infected cells typically produce recombinant zsig33 polypeptide 12-72 hours after infection and secrete it into the medium at varying efficiencies. Cultures are usually harvested 48 hours after infection. Cells are separated from the medium using centrifugation (supernatant). Supernatants containing zsig33 polypeptide are usually filtered through a 0.45 μm pore Millipore filter. General details of the procedures used are described in the available laboratory manuals [King, LA and Possee, RD, supra; O'Reilly, DR et al., Supra; Richardson, CD, same as above. Subsequently, the purification of the zsig33 polypeptide from the supernatant may be accomplished using the methods described herein.

Yeast cells, and especially fungal cells, including cells of the genus Saccharomyces and Peachia, may also be used within the present invention, for example, to prepare zsig33 fragments or polypeptide fusions. Methods for transforming yeast cells with exogenous DNA and preparing recombinant polypeptides therefrom are described, for example, in US Pat. No. 4,599,311 to Kawasaki; U.S. Patent No. 4,931,373 to Kawasaki et al .; US Patent No. 4,870,008 to Brake; US Patent No. 5,037,743 to Welch et al .; And US Pat. No. 4,845,075 to Murray et al. Transformed cells are selected by a selectable marker, typically a phenotype measured by drug resistance or the ability to grow in the absence of a particular nutrient (such as leucine). A preferred vector system for use in yeast is the POT1 vector system published by Kawasaki et al. (US Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing medium. Suitable promoters and terminators for use in yeast include those derived from glycolytic enzyme genes (see, eg, US Pat. No. 4,599,311 to Kawasaki; U.S. Patent No. 4,615,974 to Kingsman et al .; And US Pat. No. 4,977,092 to Bitter and those derived from alcohol dehydrogenase genes (US Pat. No. 4,990,446; 5,063,154; 5,139,936; And 4,661,454. Other yeasts such as Hansenula polymorpha, Shichizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis Transformation System for Ustilago maydis, Pichia pastoris, Pichia guillermondii, Pichia methnolica and Candida maltosa Also known in the art [Gleeson et al., J. Gen. Microbiol. 132: 3459-3465, 1986 and Cregg, US Pat. No. 4,882,279. Aspergillus cells can also be used according to the method of McKnight et al. (McKnight et al., US Pat. No. 4,935,349). A method for transforming Acremonium chrysogenum is described in US Pat. No. 5,162,228 to Sumino et al. A method for transforming Neurospora is disclosed in US Pat. No. 4,486,533 to Lamborwitz.

Transformed or transfected host cells are cultured in a culture medium containing nutrients and other ingredients necessary for the growth of the selected host cell according to conventional procedures. Various suitable media are known in the art, including defined and complex media, and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals. The medium may also contain ingredients such as growth factors or serum as needed. The growth medium selects cells containing foreign DNA added by drug selection or lack of essential nutrients, for example, carried out on expression vectors or complemented by selectable markers transformed together into host cells. Peachia metanolica cells are cultured at a temperature of about 25-35 ° C. in a medium containing a suitable carbon source, nitrogen source and trace amounts of nutrient source. Liquid cultures are provided with sufficient aeration by conventional means, such as by vibrating small flasks or sparging the fermentor. Preferred culture media for Peachia metanolica include YEPD (2% D-glucose, 2% Bacto ^™ peptone (Difco Laboratories, Detroit, MI), 1% Bacto ^™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

The expressed recombinant zsig33 polypeptide can be purified using fractionation and / or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of the sample. Exemplary purification steps include hydroxyapatite, size extraction, FPLC, and reversed phase high performance liquid chromatography. Suitable anion exchange media include derived dextran, agarose, cellulose, polyacrylamide, especially silica and the like. PEI, DEAE, QAE and Q derivatives are preferred, DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ) is particularly preferred. Exemplary chromatography media include phenyl, butyl, or octyl group-induced media such as phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl- Sepharose (Pharmacia) and the like; Or polyacrylic resins such as Amberchrom CG 71 (Toso Haas). Suitable solid supports include glass beads, silica-based resins, cellulose resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins, and the like. They are insoluble under the conditions in which they are used. These supports may be modified with reactive groups that allow the attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and / or carbohydrate moieties. Examples of coupling chemistries include carboxyl and amino derivatives for cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carbodiimide coupling chemistry. . These and other solid media are known in the art, widely used, and commercially available. Methods of binding the receptor polypeptide to the support medium are well known in the art. The choice of a particular method is a fundamental design issue and partly determined by the nature of the chosen support (eg Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).

Polypeptides of the invention can be isolated by the use of small size and low pi. For example, the polypeptides of the present invention can bind anion exchangers at low pH values. Other purification methods include 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. 529-539. Alternatively, a fusion and affinity tag (eg, polyhistidine, maltose-binding protein, immunoglobulin domain) of the polypeptide of interest can be configured to facilitate purification.

Protein refolding (and optionally reoxidation) procedures can be advantageously used. The protein is preferably at least 80% pure, more preferably at least 95% pure, and particularly preferably in a pharmaceutically pure state, i.e. at least 99.9% pure in terms of contaminating macromolecules, in particular other proteins and nucleic acids, and Purified to be free of infectious and pyrogenic pathogens. Preferably, the purified protein is substantially free of other proteins, especially other proteins of animal origin.

zsig33 polypeptide or fragments thereof may also be prepared via chemical synthesis. zsig33 polypeptide may be a monomer or a multimer; May or may not be glycosylated; May or may not be pegylated; May or may not be amidated; May or may not be sulfated; It may or may not include an initial methionine amino acid residue. For example, zsig33 polypeptides can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation methods or classical solution synthesis methods. Polypeptides are preferably prepared by solid phase peptide synthesis, for example as described in Merrifield, J. Am. Chem. Soc. 85: 2149, 1963. Synthesis is performed using amino acids protected at the alpha-amino terminus. Trifunctional amino acids with variable side chains are also protected with suitable groups to prevent unwanted chemical reactions that occur during assembly of the polypeptide. Alpha-amino protecting groups are optionally removed such that subsequent reactions occur at the amino-terminus. Conditions for the removal of the alpha-amino protecting group do not remove the protecting group of the side chain.

Alpha-amino protecting groups are known to be useful in staged polypeptide synthesis techniques. Such groups include acyl protecting groups (such as formyl, trifluoroacetyl, acetyl), aryl protecting groups (such as biotinyl), aromatic urethane protecting groups (such as benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and 9-fluorenylmethyloxycarbonyl (Fmoc), aliphatic urethane type protecting group (such as t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl type protecting group (such as benzyl Triphenylmethyl). Preferred protecting groups are tBoc and Fmoc.

The selected side chain protecting group must remain free during coupling and must not be removed during deprotection of the amino-terminal protecting group or while the coupling is in progress. Side chain protecting groups should also be able to be removed when synthesis is complete using reaction conditions that will not alter the final polypeptide. In tBoc chemistry, the side chain protecting groups for trifunctional amino acids are mostly based on benzyl. Side chain protecting groups in Fmoc chemistry are mostly based on tert-butyl or trityl.

In tBoc chemistry, preferred side chain protecting groups are tosyl groups for arginine, cyclohexyl groups for aspartic acid, 4-methylbenzyl groups (and acetamidomethyl groups) for cysteine, and benzyl groups for glutamic acid, serine and threonine Benzyloxymethyl group (and dinitrophenyl group) for histidine, 2-Cl-benzyloxycarbonyl group for lysine, formyl group for tryptophan, and 2-bromobenzyl group for tyrosine. In Fmoc chemistry, preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or 2,2,4,6,7-pentamethyldihydrobenzofuran for arginine -5-sulfonyl (Pbf), trityl for asparagine, cysteine, glutamine and histidine, tert-butyl group for aspartic acid, glutamic acid, serine, threonine and tyrosine and tBoc group for lysine and tryptophan.

For the synthesis of phosphopeptides, direct or post-assembly integration of phosphate groups is used. In direct integrated strategy, the phosphate groups on serine, threonine or tyrosine can be protected by methyl, benzyl, or tert-butyl groups in Fmoc chemistry and by methyl, benzyl or phenyl groups in tBoc chemistry. Direct integration of phosphotyrosine without phosphate protection can also be used in Fmoc chemistry. In post-assembly integrated strategy, deprotected hydroxyl groups of serine, threonine or tyrosine are induced on the solid phase with di-tert-butyl-, dibenzyl- or dimethyl-N, N'-diisopropylphosphoramidite. And then oxidized by ter-butylhydroperoxide.

Solid phase synthesis is usually performed by coupling the alpha-amino protected (side chain protected) amino acid from the carboxy-terminus to the appropriate solid support. When the attachment to the chloromethyl, chlorotrityl or hydroxymethyl resin is complete, an ester linkage is formed and the resulting polypeptide will have free carboxyl groups at the C-terminus. Alternatively, if an amide resin (for tBoc chemistry) such as benzhydrylamine or p-methylbenzhydrylamine resin and Rink amide or PAL resin are used (for Fmoc chemistry), an amide bond is formed and The resulting polypeptide will have a carboxyamide group at the C-terminus. These resins are commercially available, with or without handles or linkers, with or without the first amino acid attached, whether they are polystyrene- or polyamide-based or polyethylene glycol-grafted, Steewart et al., "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce Chemical Co., Rockford, IL, 1984) and Bayer & Rapp Chem. Pept. Prot. 3: 3 (1986); and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford, 1989].

The C-terminal amino acids protected at the side chain and at the alpha-amino group, if necessary, are dicyclohexylcarbodiimide (DCC), N, N'-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). Various activators are used to attach to the hydroxymethyl resin. It may be attached in the form of its cesium tetramethylammonium salt either directly or to chloromethyl or chlorotrityl in the presence of triethylamine (TEA) or diisopropylethylamine (DIEA). The first amino acid attachment to the amide resin is identical to the amide bond formation during the coupling reaction.

After attachment to the resin support, various reagents (eg, tBoc, Fmoc) are used to remove the alpha-amino protecting groups, depending on the protective chemistry. The degree of removal of Fmoc can be monitored at 300-320 nm or by conductive cells. After removal of the alpha-amino protecting group, the remaining protecting amino acids are stepwise coupled to obtain the desired sequence.

Various activators can be used in the coupling reaction, for example DCC, DIPCDI, 2-chloro-1,3-dimethylimide hexafluorophosphate (CIP), benzotriazol-1-yl-oxy-tris- ( Dimethylamino) -phosphonium hexafluorophosphate (BOP) and its pyrrolidine analogue (PyBOP), bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP), O- (benzotriazole- 1-yl) -1,1,3,3-tetramethyl-uronium hexafluorophosphate (HBTU) and its tetrafluoroborate analogue (TBTU) or its pyrrolidine analogue (HBPyU), O- ( 7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU) and its tetrafluoroborate analogue (TATU) or pyrrolidine analogue thereof (HAPyU) is mentioned. The most common catalyst additives used in coupling reactions are 4-methylaminopyridine (DMAP), 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazole (HODhbt), N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). Each protective amino acid is used in excess (> 2.0 equiv) and the coupling is usually carried out in N-methylpyrrolidone (NMP) or DMF, CH ₂ Cl ₂ or mixtures thereof. The degree of completion of the coupling reaction is described, for example, by Kaiser et al., Anal. Biochem. The ninhydrin reaction as described in 34: 595, 1970 can be monitored at each step.

After all the desired peptides are assembled, the peptide-resin is cleaved by reagent with a suitable scavenger. Fmoc peptides are typically cleaved and deprotected by TFA with scavengers (eg, H ₂ O, ethanedithiol, phenol and thioanisole). tBoc peptides are typically cleaved and deprotected with liquid HF at −5 ° C. to 0 ° C. for 1-2 hours, which cleaves the polypeptide from the resin and removes most of the side chain protecting groups. Typically, scavengers such as anisole, dimethylsulfide and p-thiocresol are used in combination with liquid HF to prevent cations formed during cleavage from alkylating and acylating amino acid residues present in the polypeptide. The formyl group of tryptophan and the dinitrophenyl group of histidine need to be removed, respectively, by piperidine and thiophenyl in DMF prior to HF cleavage. The acetamidomethyl group of cysteine can be removed by mercury (II) acetate and optionally by iodine, thallium trifluoroacetate (III) or silver tetrafluoroborate, which simultaneously oxidize cysteine to cystine. Other strong acids used for cleavage and deprotection of tBoc peptides include trifluoromethanesulfonic acid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).

The activity of the molecules of the present invention can be measured using various assays that measure stimulation of gastrointestinal cell contraction, regulation of nutrient absorption and / or secretion of digestive enzymes. Of particular importance is the change in the contractile properties of smooth muscle cells, for example, the contractile response of smooth muscle tissue sections of the duodenum or other gastrointestinal tract of mammals (Depoortere et al., J. Gastrointestinal Motility 1: 150-159, 1989, here Incorporated by reference). Exemplary in vivo measurements use ultrasonic micrometers to readily measure longitudinal size changes between cross-connections and to the face of the valve base (Hansen et al., Society of Thoracic Surgeons 60: S384-390, 1995).

Gastric motion is generally measured as the time required for gastric emptying in a clinical setting and the subsequent delivery time through the gastrointestinal tract. Gastric excretion scanning is well known to those skilled in the art and, in short, involves the use of oral contrast agents such as barium or radiolabelled meals. Solids and liquids can be measured independently. Test foods or liquids are radiolabelled with isotopes (e.g., ^99m Tc), and after ingestion or administration, transport time and gastric emptying through the gastrointestinal tract are measured by visualizing with a gamma camera (Meyer et al., Am. J. Dig. Dis. 21: 296, 1976; Collins et al., Gut 24: 1117, 1983; Maughan et al., Diabet. Med. 13 9 Supp. 5: S6-10, 1996 and Horowitz et al., Arch.Intern.Med. 145: 1467-1472, 1985). These studies can be performed before or after administration of an accelerator to quantify the efficacy of the medicament.

Assays to measure the ability of zsig33 polypeptides to affect proliferation or differentiation of cells are well known in the art. For example, measurements of proliferation include measurements such as chemistry for neutral red dyes (Cavanaugh et al., Investigational New Drugs 8: 347-354, 1990, incorporated herein by reference). ), Incorporation of radiolabeled nucleotides (Cook et al., Analytical Biochem. 179: 1-7, 1989, incorporated herein by reference), 5-bromo-2'-deoxyuridine into DNA of proliferating cells (BrdU) (Porstmann et al., J. Immunol. Methods 82: 169-179, 1985, incorporated herein by reference) and the use of tetrazolium salts (Mosmann, J. Immunol. Methods 65: 55-63, 1983; Alley et al., Cancer Res. 48: 589-601, 1988; Marshall et al., Growth Reg. 5: 69-84, 1995; and Scudiero et al., Cancer Res. 48: 4827-4833, 1988 , All incorporated herein by reference). Assays to measure differentiation include, for example, measuring cell surface markers associated with phase specific expression, enzymatic activity, functional activity or morphological changes in tissues (Watt, FASEB, 5: 281-284, 1991). Francis, Differentiation 57: 63-75, 1994; Raes, Adv. Anim. Cell Biol. Technol.Bioprocesses, 161-171, 1989, all incorporated herein by reference).

Assays can be used to measure other cellular responses, including chemotaxis, adhesion, changes in ion channel entry, control of second messenger levels, and release of neurotransmitters. Such assays are well known in the art (eg, "Basic & Clinical Endocrinology Ser., Vol. 3", Cytochemical Bioassays: Techniques & Applications, Chayen; Chayen, Bitensky, eds., Dekker, New York, 1983 Reference).

In terms of tissue distribution observed for zsig33, agonists (including natural ligands / substrates / carriers, etc.) and antagonists have great potential for both in vitro and in vivo applications. Compounds identified as zsig33 agonists are useful for stimulating gastrointestinal cell contraction, regulating nutrient absorption and / or promoting the secretion of digestive enzymes in vivo or in vitro. For example, agonist compounds are useful as components of certain cell culture media and regulate the uptake of nutrients and thus the G cells of the stomach, duodenum, proximal jejunum, sinus and base, enterochrome affinity cells and It is useful for specifically promoting growth and / or development of gastrointestinal tract cells such as epithelial mucosa.

Intestinal-brain peptides have been associated with neurological and CNS functions. For example, NPY, a peptide with receptors in both the brain and intestinal tract, has been shown to stimulate appetite when administered to the central nervous system (Gehlert, Life Sciences 55 (6): 551-562, 1994). The immunoreactivity of motilin has been identified in the brain, particularly in various parts of the cerebellum and in the pituitary gland (Gasparini et al., Hum. Genetics 94 (6): 671-674, 1994). Motiline was found to coexist with γ-aminobutyric acid, a neurotransmitter in the cerebellum (Chan-Patay, Proc. Sym. 50th Anniv.Meet.Br. Pharmalog. Soc.:1-24, 1982). In physiological studies, some evidence has been provided that motilin affects eating behavior (Rosenfield et al., Phys. Behav. 39 (6): 735-736, 1989), bladder control, and pituitary growth hormone release. Other intestinal-brain peptides such as CCK, enkephalin, VIP and secretin have been found to be involved in blood pressure, heart rate, behavior and pain control in addition to being active in the digestive system. Thus, one could expect zsig33 or some portion thereof to be neurologically related.

Site-specific changes in the amino acid or DNA sequences of the present invention can be used to prepare analogs that are antagonists, agonists or partial agonists (Macielay et al., Peptides: Chem. Struct. Biol. Pp.659, 1996 ). Antagonists are useful for clinical diseases associated with gastrointestinal hyperplasia such as diarrhea and Crohn's disease. Antagonists are also useful as research agents for characterizing the site of ligand-receptor interaction.

zsig33 ligand binding polypeptides can also be used for purification of ligands. The polypeptide is immobilized on a solid support such as agarose, crosslinked agarose, glass, cellulose based resin, silica based resin, polystyrene, crosslinked polyacrylamide or beads of similar material that is stable under the conditions of use. Methods of linking polypeptides to solid supports are well known in the art and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation and hydrazide activation. The resulting medium is generally configured in the form of a column and the ligand containing fluid is passed through the column one or more times to bind the ligand to the receptor polypeptide. The ligand is then eluted using a change in salt concentration, chaotropic agent (guanidine HCl) or pH to break down the ligand-receptor bond.

It may be advantageous to use a measurement system using a ligand binding receptor (or a member of an antibody, complement / anticomplement) or binding fragment thereof, and a commercially available biosensor instrument (BIAcore ^™ , Pharmacia Biosensor, Piscataway, NJ). . A member or fragment of the receptor, antibody, complement / anticomplement pair is immobilized on the surface of the receptor chip. The use of such instruments is described in Karlsson, J. Immunol. Methods 145: 229-40, 1991 and Cunningham and Wells, J. Mol. Biol. 234: 554-63, 1993. The receptor, antibody, member or fragment is covalently attached to the dextran fiber using amine or sulfhydryl chemistry and the textan fiber is attached to the gold film in the flow cell. Pass the test sample through the cell. If the opposite member of a ligand, epitope or complement / anticomplement pair is present in the sample, it is bound to the immobilized receptor, antibody or member, respectively, to change the refractive index of the medium, which changes the surface plasmon resonance of the gold film. Is detected as. This system enables the measurement of the opening and closing rate and the binding stoichiometry to calculate the binding affinity.

Ligand binding receptor polypeptides can also be used within other measurement systems known in the art. Such systems include Scatchard assays (see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetry (Cunningham et al., Science 253: 545-48, 1991) to determine binding affinity; Cunningham et al., Science 245: 821-25, 1991).

The zsig33 polypeptide may also be used to prepare antibodies that specifically bind to a zsig33 epitope, peptide or polypeptide. Methods of preparing polyclonal or monoclonal antibodies are well known in the art (eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, See JGR, Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, FL, 1982. These are incorporated herein by reference). As will be apparent to those skilled in the art, polyclonal antibodies can be generated from a variety of warm blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice and rats.

Immunogenicity of the zsig33 polypeptide can be increased through the use of an adjuvant or incomplete adjuvant such as alum (aluminum hydroxide) or Freud complex. Polypeptides useful for immunization also include fusion polypeptides such as fusions of zsig33 or a portion thereof with an immunoglobulin polypeptide or maltose binding protein. Polypeptide immunogens may be full-length molecules or portions thereof. If the polypeptide moiety is “similar to heptane,” this moiety favors macromolecular carriers (eg, keyhole limpet hemocyanin (KHL), bovine serum albumin (BSA) or tetanus toxoid for immunization). It can be combined or connected.

The term “antibody” as used herein includes polyclonal antibodies, affinity purified polyclonal antibodies, monoclonal antibodies and antigen binding fragments such as F (ab ′) ₂ and Fab proteolytic fragments. Also included are genetically engineered complete antibodies or fragments such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as antigen binding synthetic peptides and polypeptides. A nonhuman antibody grafts a nonhuman CDR into a human framework and a region or incorporates all of the various domains of the nonhuman (optionally "substitutes" exposed residues to "cloak" them to a surface such as a human, The resultant can be humanized by " veneered " antibodies. In some cases, humanized antibodies can maintain non-human residues within the framework domains of various regions of humans to enhance proper binding properties. Humanized antibodies can increase biological half-life and reduce the likelihood of adverse immune reactions when administered to humans. Alternative methods of generating or selecting antibodies useful in the invention include exposing lymphocytes to zsig33 protein or peptide in vitro and selecting an antibody-labeled library in a phage or similar vector (eg, immobilized or labeled). Through the use of the zsig33 protein or peptide).

The antibody has _a zsig33 polypeptide of binding affinity (K _a ) of at least 10 ⁶ M ⁻¹ , preferably at least 10 ⁷ M ⁻¹ , more preferably at least 10 ⁸ M ⁻¹ , most preferably at least 10 ⁹ M ^−1. When bound to, it is defined as specifically bound. The binding affinity of the antibody can be readily determined by one skilled in the art (eg by Scatchard assay).

Various assays known to those of skill in the art can be used to detect antibodies that specifically bind to zsig33 protein or peptide. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include co-immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked antibody (ELISA), dot blot or western blot assay, inhibition or competition assay, and sandwich assay. Antibodies can also be screened for binding to wild type versus mutant zsig33 protein or peptide.

Antibodies to zsig33 use FACS to isolate zsig33 by affinity purification, for diagnostic assays to measure the circulating level of zsig33 polypeptide, and to detect or quantify it as a marker of a condition or disease inherent in soluble zsig33. The assay can be used to screen expression libraries, generate genotype antibodies, and tag cells expressing zsig33 as antagonists that block zsig33 activity in vitro and in vivo or as neutralizing antibodies. Suitable tags or labels per suitable include radionuclides, enzymes, substrates, carriers, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles, and the like, and indirect tags or labels may be biotin-avidin or other complement / anticomplement pairs. It may be characterized by using as a medium. Antibodies in the present invention can also be conjugated directly or indirectly to drugs, toxins, radionuclides and the like.

Molecules of the invention can be used to identify and isolate receptors that mediate the function of zsig33. For example, the proteins and peptides of the present invention can be immobilized on membranes and on membrane preparations flowing over columns (Immobilized Affinity Ligand Techniques, Hermanson et al., Eds., Academic Press, San Diego, CA, 1992, pp. 195-202). Proteins and peptides may also be used as radiolabels (Methods in Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or photoaffinity labels (Brunner). et al., Ann. Rev. Biochem. 62: 483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33: 1167-1180, 1984) and specific cell surface proteins can be identified.

Polypeptides, nucleic acids and / or antibodies of the invention include diseases associated with contraction of gastrointestinal cells, secretion of digestive enzymes and acids, gastrointestinal motility, recruitment of digestive enzymes; Inflammation, especially inflammation affecting the gastrointestinal system; Treatment of reflux disease; And nutrient absorption. Specific conditions that benefit from treatment with the molecules of the invention include diabetic gastric palsy, postoperative gastric palsy, vagus nerve cutting, chronic idiopathic bowel obstruction, and; Gastroesophageal reflux disease, but is not limited to this. Further uses may include gastric emptying, gallbladder contractions, and antectomy for radiological studies.

Due to the motility and neurological effects of the molecules of the present invention, the molecules of the present invention are useful in the treatment of other metabolic disorders in which obesity and neurological feedback modulate the absorption of nutrients. Molecules of the present invention are useful for modulating satiety, glucose uptake and metabolism, and neuropathy-related stomach and disorders.

The molecules of the present invention are also useful as additives for antihyperglycemic preparations containing glucose and as adsorbents for oral pharmaceuticals requiring rapid action of nutrients. In addition, the molecules of the present invention can be used to stimulate insulin release induced by glucose.

For pharmaceutical use, the proteins of the invention can be formulated for parenteral, nasal aspiration, in particular intravenous or subcutaneous delivery, according to conventional methods. Intravenous administration may be by bolus injection or infusion over a typical period of 1 to several hours. In general, pharmaceutical preparations include zsig33 protein with pharmaceutically acceptable vehicles such as saline, buffered saline, 5% textose in water, and the like. The formulation may further comprise one or more excipients, preservatives, solubilizers, buffers, albumin, etc. to prevent loss of protein in the vial surface. Formulation methods are well known in the art and are described, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mark Publishing Co., Easton PA, 1990, which is incorporated herein by reference. Therapeutic dosages are generally from 0.1 to 100 μg per kg of patient's body weight per day, preferably 0.5-20 μg / kg per day, and the exact dosage is determined by taking into account the nature and severity of the disease to be treated by the clinician and the characteristics of the patient Determined according to accepted standards. Determination of dosage is within the level of ordinary skill in the art. The protein may be administered for acute treatment over a period of up to one week, often one to three days, or may be used for chronic treatment over months or years. For example, a therapeutically effective amount of zsig33 is an amount sufficient to produce a clinically significant change in the parameters used to measure changes in gastric motility and nutrient uptake. Certain tests for making these measurements are known to those skilled in the art.

Example 1

Scanning the cDNA database for cDNA containing secretion sequences revealed that the expressed sequence tag (EST) had homology to motilin. cDNA was obtained from the pancreatic cDNA library of human fetus.

The EST sequence was confirmed by analyzing the sequence of cDNA from which EST was derived. This cDNA contained a plasmid and was excised using a cloning site. As a result, the cDNA contained the entire coding region of the DNA encoding zsig33.

Example 2

Northern was performed using Human Multiple Tissue Blots and Human RNA Master dot blot by Clontech (Palo Alto, Calif.). The probe was about 40 bp oligonucleotide ZC12,494 (SEQ ID NO: 7). This probe was endlabeled using T4 polynucleotide kinase (Life Technologies, Inc., Gaithersburg, MD) and T4 polynucleotide kinase forward buffer (Life Technologies, Inc.). Probes were purified using NUCTRAP push columns (Stratagene, La Jolla, Calif.). EXPRESSHYB (Clontech) solution was used for hybridization and as a hybridization solution for northern blots. Hybridization took place at 42 ° C. and blots were washed at room temperature with 2 × SSC and 0.05% SDS followed by 71 × C with 1 × SSC and 0.1% SDS. About 600 bp of the transcript was observed as a strong signal in the stomach and weak in the pancreas and small intestine.

Example 3

Two male Sprague-Dawley rats (Harlan, Indianapolis, Ind.), About 12 weeks old, were anesthetized with urethane and their stomachs were slightly incised to expose the stomach. Two 2.4 mm transduction crystals (Sonometrics, Ontario, Canada) were placed in the upper part of the stomach so that cyclic contraction could be monitored as a change in the spacing between the two crystals. Crystals were attached by VETBOND TISSUE ADHESIVE (3M, St. Paul, MN).

10 μl of 1 μM acetylcholine was applied locally over the two crystals, and the spacing between the two crystals increased rapidly but temporarily. 10 μl of 1M norepinephrine (NE) reduced the spacing between the two crystals. The amplification induced by NE was about 50% of the interval increase induced by acetylcholine. Both reactions were temporary.

10 μl of phosphate buffer (PBS), a negative control applied locally between the crystals, had no effect.

A 14 amino acid zsig33 peptide (obtained from amino acid residues 24 (Gly) to amino acid residues 37 (Gln) of SEQ ID NO: 2) was dissolved in PBS and 10 μl was applied locally to a final concentration of 1 μg, 10 μg or 100 μg. . 1 μg of zsig33 increased and decreased the crystal spacing continuously and periodically. This effect appeared to be dose dependent and showed an improved response in both rate and amplification when tested at concentrations of 10 μg and 100 μg.

Example 4

Eight female ob / ob mice (Jackson Labs, Bar Harbor, ME), approximately six weeks old, were acclimated for two weeks on a dietary schedule of four hours per day. After 2 weeks of this dietary schedule, immediately after their diet (postprandial) mice were treated with 100 μg of 14 amino acid amino zsig33 peptide (SEQ ID NO: amino acid residue 24 (Gly)) to 100 μl of sterile 0.1% BSA. Amino acid residue 37 (obtained from Gln) was administered by oral feeding. After 30 minutes, mice were dosed orally with 0.5 ml volume of 25% glucose. Post-orbital bleeding occurred to measure serum glucose levels. Blood was drawn before zsig33 administration, before oral administration of glucose, and at 1, 2, 4 and 20 hours after glucose administration.

When the oral administration of 100 μg of the zsig33 peptide 30 minutes before oral administration of glucose showed an improvement in postprandial glucose uptake.

Example 5

Zsig33-1, a peptide corresponding to amino acid residues 24 (Gly) to amino acid residue 37 (Gln) of SEQ ID NO: 2, was subjected to solid phase peptide using a model 431A peptide synthesizer (Applied Biosystems / Perkin Elmer, Foster City, CA). Synthesized by synthesis. Fmoc-glutamine resin (0.63 mmol / g; Advanced Chemtech, Louisville, KY) was used as the initial support resin. A 1 mmolol amino acid cartridge (Anaspec, Inc. San Jose, Calif.) Was used for the synthesis. 2 (1-benzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), 2m N, N-diiso A mixture of propylethylamine, N-methylpyrrolidone, dichloromethane (all from Applied Biosystems / Perkin Elmer) and piperidine (Aldrich Chemical Co., St. Louis, Mo.) was used as a synthetic reagent.

Peptide companion software (Peptides International, Louisville, KY) was used to predict aggregation levels and difficulty levels for zsig33-1 peptide synthesis. Synthesis was performed according to the manufacturer's instructions using a single coupling program.

Peptides were cleaved from the solid phase according to standard TFA cleavage (according to the Peptide Cleavage Manual, Applied Biosystems / Perkin Elmer). Purification of the peptide was performed by RP-HPLC using a C18, 100 μm semi-preparation column (Vydac, Hesperial, Calif.). Fractions eluted from the column were taken and analyzed for correct mass and purity by electrospray mass spectrometry. Two pools of eluted material were taken. As a result of this mass spectrometry, it was found that the two pools contained a purified form of zsig33 with a mass of 1600 Daltons. This was the expected mass, so the pools were combined and lyophilized.

Example 6

zsig33 was mapped to chromosome 3 using the commercially available "GeneBridge 4 Radiation Hybrid Panel" (Research Genetics, Inc., Huntsville, AL). The GeneBridge 4 Radiation Hybrid Panel contained DNA from 93 radiation hybrid clones and DNA from two control DNAs (HFL donor and A23 receptor), respectively. The publicly available WWW server (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl), the Whitehead Institute / MIT Center for the Human Genome, composed by the GeneBridge 4 Radiation Hybrid Panel A mapping to Genome Research's radiation hybrid map ("WICGR" radiation hybrid map) is possible.

To map zsig33 by the "GeneBridge 4 RH Panel", 20 μl of the reaction was made in a 96-well microtiter plate (Stratagene, La Jolla, Calif.) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene). 95 PCR reactions each contain 2 μl of 10 × KlenTag PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.), 1.6 μl of dNTP mixture (2.5 mM each, Perkin-Elmer, Foster City, CA), 1 μl Sense primer, ZC13,166 (SEQ ID NO: 8), 1 μl antisense primer, ZC13,167 (SEQ ID NO: 9), 2 μl of "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 μl 50 × Advantage KlenTaq polymerase mixture (Clontech Laboratories, Inc.), 25 ng of DNA from individual hybrid clones or controls and ddH ₂ O to bring the total volume to 20 μl. An equal amount of mineral oil was applied to the reaction and sealed. PCR cycle conditions were as follows. Ie initial 1 cycle: 5 min denaturation at 95 ° C., 35 cycles: 1 min denaturation at 95 ° C., 1 min annealing at 64 ° C. and 1.5 min extension at 72 ° C., then last 1 cycle: 7 min extension at 72 ° C. The reactions were separated by electrophoresis on 3% NuSieve GTG agarose gel (FMC Bioproducts, Rockland, ME).

As a result, it was demonstrated that zsig33 maps 10.43 cR_3000 from framework marker AFMA216ZG1 on the WICGR chromosome 3 radiation hybrid map. Proximal and distal framework markers were AFMA216ZG1 and D3S1263, respectively. Surround markers were used to position zsig33 in the 3p26.1 region on the integrated LDB chromosome 3 map (The Genetic Location Database, University of Southhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/ ).

Example 7

The effect of locally applied zsig33 peptide (amino acids 24-37 of SEQ ID NO: 2) on the passage of phenol red through the stomach of fasted male Sprague-Dawley rats (Harlan, Indianapolis, IN) was evaluated. Rats (6 rats, 8 weeks old) were fasted 24 hours before anesthesia with urethane (0.5 ml / 100 g 25% solution). After anesthesia, animals were orally administered 1 ml of phenol red solution (50 mg / ml in 2% methylcellulose solution).

The stomach of each animal was exposed by a small incision in the abdomen and a localized amino acid control of 1 μg of zsig33 peptide or scrambled sequence peptide was applied locally to the stomach 5 minutes after feeding. The amount of phenol red remaining in the stomach was measured by measuring the optical density of the gastric contents discharged 30 minutes after feeding.

The zsig33 peptide reduced the amount of phenol red remaining in the stomach by about 25% compared to the scrambled peptide, indicating that the zsig33 peptide enhances gastric emptying in these rats.

Example 8

Sixteen week old female ob / ob mice (Jackson Labs, Bar Harbor, ME) were acclimated for two weeks on a dietary schedule of four hours per day. The animals were randomly fed daily from 7:30 am to 11:30 am. After two weeks of this dietary schedule, mice were divided into two 8 groups. In one group, 1.0 μg per mouse of zsig33-1 (14 amino acid peptide) and other vehicle (14 amino acid scrambled sequence peptide) in 100 μl of sterile 0.1% BSQA immediately before feeding and at the end of the 4 hour feeding period. Administration was by oral feeding. Mice were administered twice daily for 14 days, during which time food uptake and body weight were measured. Immediately after the second oral ingestion of the zsig33-1 peptide on day 14, mice were dosed orally with 25% glucose in a volume of 0.5 ml. Serum glucose levels were measured by post-orbital bleeding immediately prior to administration of the zsig33-1 peptide or vehicle (t = 30 minutes) and 0, 1, 2 and 4 hours after the administration of glucose.

As a result, it was found that the oral administration of zsig33-1 peptide by 1 μg per mouse did not affect the daily weight or food absorption or glucose removal rate measured on day 14.

Example 9

A. Northern Blot of the Intestinal Tissue

Northern blots were prepared using mRNA from the following sources.

1. RNA from Human Colorectal Andenocarcinoma cell line SW480 (Clontech, Palo Alto, CA)

2. RNA from human small intestine tissue (Clontech)

3. RNA from human stomach tissue (Clontech)

4. Human intestinal smooth muscle cell line (Hism; ATCC No. CRL-1692; American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD)

5. Colon Cell Line of Normal Person (FMC; ATCC No. CRL-1831; American Type Culture Collection)

6. Small intestine cell line of human normal fetus (FHs74 Int .; ATCC No. CCL241; American Type Culture Collection).

Total RNA was isolated from Hism, FHC and FHs74 Int. By sanguanidine method (Chomczynski et al., Anal. Biochem. 162: 156-159, 1989). Eluting total RNA through a column that retention of the poly A ⁺ RNA was selected from poly ^{A + RNA (Aviv et al,} Proc Nat Acad Sci 69:..... 1408-1412, 1972). 2 μg of polyA ⁺ RNA from each sample was isolated on 1.5 M agarose gel in 2.2 M formaldehyde and phosphate buffer. RNA was transferred overnight to a Nytran membrane (Schleicher and Schuell, Keene, NH) in 20 × SSC. Blots were treated with 0.12 Joules in UV Stratalinker 2400 (Stratagene, La Jolla, Calif.). The blot was then baked at 80 ° C. for 1 hour.

Using a full-length cDNA amplified by PCR (shown in SEQ ID NO: 1), a Radiprime pellet kit (Amersham, Arlington Heights, IL) was prepared using approximately 50 ng of zsig33 DNA and 42.5 μl of water according to the manufacturer's instructions. Radiolabeled with ³² P dCTP. Blots were hybridized overnight at 55 ° C. in EXPRESSHYB (Clontech). The blot was washed with 2 × SSC and 0.1% SDS at room temperature, then with 2 × SSC and 0.1% SDS at 65 ° C. and finally with 0.1 × SSC and 0.1% SDS at 65 ° C. As a result, it was demonstrated that zsig33 hybridized to the above RNA but not to RNA from other tissues.

B. Northern Blot of Tumors

Northern Territory ^TM -Human Tumor Panel Blot II (Invitrogen, San Diego, CA) and Northern Territory ^TM -Human Stomach Tumor Panel Blot (Invitrogen) for expression of zsig33 RNA Was analyzed.

Human tumor panel blots contained 20 μg of total RNA per lane and flowed onto a 1% denaturing formaldehyde gel. This blot contained RAN from esophageal tumor, normal esophagus, rectal tumor and normal rectum. Human gastric tumor panel blots contained total RNA isolated from normal tissues of four independent donors. 20 μg of RNA was used for the lanes of each sample and the lanes were alternating normal and tumor sets from each donor.

A probe with an oligonucleotide ZC12,494 (SEQ ID NO: 7) of about 40 bp was prepared. This probe was end labeled using T4 polynucleotide kinase (Life Technologies, Inc., Gaithersburg, MD) and T4 polynucleotide kinase forward buffer (Life Technologies, Inc.). Probes were purified using NUCTRAP push columns (Stratagene, La Jolla, Calif.). Both tumor blots and stomach blots were treated in the same manner. EXPRESSHYB (Clontech) solution was used for prehybridization and as a hybridization solution for northern blots. Hybridization took place at 42 ° C. and blots were washed with 0.1 × SSC and 0.01% SDS at 60 ° C. followed by 0.1 × SSC and 0.1% SDS at 70 ° C. As a result, it can be clearly seen that zsig33 is exclusively expressed in normal tissue of the stomach in both human tumor panels and human intestinal tumor panels.

While specific embodiments of the invention have been described herein for purposes of illustration, it will be appreciated from the above that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (18)

(a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to nucleotide 111 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) orthologs of (a) and (b); And (d) the degenerate nucleotide sequence of (a), (b) or (c) An isolated polynucleotide molecule encoding a polypeptide selected from the group consisting of. (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to nucleotide 120 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) the ortholog of (a) or (b); And (d) the degenerate nucleotide sequence of (a), (b) or (c) An isolated polynucleotide molecule encoding a polypeptide selected from the group consisting of. (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to 351 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) the ortholog of (a) or (b); And (d) the degenerate nucleotide sequence of (a), (b) or (c) An isolated polynucleotide molecule encoding a polypeptide selected from the group consisting of. (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 1 to nucleotide 111 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) the ortholog of (a) or (b); And (d) the degenerate nucleotide sequence of (a), (b) or (c) An isolated polynucleotide molecule encoding a polypeptide selected from the group consisting of. The isolated polynucleotide molecule of claim 4, wherein the polynucleotide molecule further comprises a nucleotide sequence of nucleotides 1 to 351 shown in SEQ ID NO: 1. 6. The isolated polynucleotide of claim 1, wherein the polynucleotide is DNA. The following operatively connected components: Transcriptional promoters; (a) a polynucleotide molecule comprising the nucleotide sequence of nucleotides 70 to nucleotide 111 shown in SEQ ID NO: 1; (b) allelic variants of (a); (c) the ortholog of (a) or (b); And (d) the degenerate nucleotide sequence of (a), (b) or (c) DNA fragments selected from the group consisting of; Warrior terminator Expression vector comprising a. A cultured cell in which the expression vector according to claim 7 has been introduced, wherein said cell expresses the polypeptide encoded by the DNA fragment. (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 37 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) the ortholog of (a) or (b) An isolated polypeptide selected from the group consisting of. (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 41 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) the ortholog of (a) or (b) An isolated polypeptide selected from the group consisting of. (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 117 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) the ortholog of (a) or (b) An isolated polypeptide selected from the group consisting of. (a) a polypeptide molecule comprising the amino acid sequence of residues 1 to 37 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) the ortholog of (a) or (b) An isolated polypeptide selected from the group consisting of. 10. The isolated polypeptide of claim 9, wherein said polypeptide molecule further comprises the amino acid sequence of residues 1 to 117 shown in SEQ ID NO: 2. A pharmaceutical composition comprising the purified polypeptide of claim 9 together with a pharmaceutically acceptable vehicle. (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 117 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) the ortholog of (a) or (b) An antibody that binds to an epitope of a polypeptide selected from the group consisting of: As a method for preparing a zsig33 polypeptide, Culturing the cells into which the expression vector according to claim 7 has been introduced, thereby allowing said cells to express the polypeptide encoded by the DNA fragment, A method comprising recovering the polypeptide. As a stimulation method of gastric movement, (a) a polypeptide molecule comprising the amino acid sequence of residues 24 to 37 shown in SEQ ID NO: 2; (b) allelic variants of (a); And (c) the ortholog of (a) or (b) A pharmaceutical composition comprising an isolated polypeptide selected from the group consisting of: administered to a mammal in need of stimulation of gastric motility in an amount sufficient to increase delivery time or gastric emptying of the ingested material. 18. The method of claim 17, wherein the delivery time or gastric emptying is measured using a radiolabelled material.