JPH11113583A - New compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new compound consisting of a separated polypeptide containing an amino acid sequence having high identity to a specific amino acid sequence and useful e.g. for the treatment and diagnosis of hypercardia, cardiac failure, ischemia, arrhythmia, hypertension, atherosclerosis, cancer, etc. SOLUTION: This new polypeptide is a separated polypeptide containing an amino acid sequence having identity of >=70% to the amino acid sequence of the formula I and a separated polypeptide containing an amino acid sequence having identity of >=70% to the amino acid sequence of the formula II. It is useful e.g. for the treatment and diagnostic assay of hypercardia, cardiac failure, ischemia, arrhythmia, hypertension, atherosclerosis, restenosis, chronic and acute inflammation, cerebral attack, rheumatoid arthritis, disseminated sclerosis, intestinal disease, diabetes, cancer, etc. The polypeptide can be produced from a cDNA library originated from mRNA in a cell such as human brain, heart or fetus dura cell by preparing a gene from the library by standard cloning and screening technique and expressing the gene in a host cell by recombination technique.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a newly identified polypeptide and a polynucleotide encoding the polypeptide, an agonist, an antagonist and / or an inhibitor which may be useful in therapy and therapy. It relates to the use in the identification of compounds that may be and to the production of said polypeptides and polynucleotides.

[0002]

BACKGROUND OF THE INVENTION Currently, drug delivery methods are undergoing a fundamental revolution, including "functional genomics", i.e., high-throughput genomic or gene-based biology. This method of identifying genes and gene products as therapeutic targets has quickly replaced traditional methods based on "positional cloning". A phenotype that is a biological function or a hereditary disease is identified, which is then tracked for the causative gene based on its genetic map location. Functional genomics requires high throughput D to identify the gene sequence of interest from many currently available molecular biology databases.
It relies heavily on NA sequencing techniques and various bioinformatics tools. There is still a need to identify and characterize additional genes and their associated polypeptides / proteins as targets for drug discovery.

[0003]

SUMMARY OF THE INVENTION The present invention relates to CSB5, particularly CSB5 polypeptides and polynucleotides, recombinant materials, and methods for making them. In another aspect, the invention relates to, among others, hypertrophy, heart failure, ischemia, arrhythmia, hypertension, atherosclerosis, restenosis, chronic and acute inflammation, cerebral stroke, rheumatoid arthritis, multiple sclerosis The present invention relates to methods for using the polypeptides and polynucleotides, including the treatment of diseases, intestinal diseases, diabetes, and cancer (hereinafter referred to as “disease”). In a further aspect, the present invention provides a method comprising:
The present invention relates to a method for identifying agonists and antagonists / inhibitors using the material obtained according to the present invention, and a method for treating a condition associated with CSB5 imbalance with an identified compound. In a further aspect, the present invention relates to diagnostic assays for detecting diseases associated with inappropriate CSB5 activity or levels.

[0004]

DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention relates to a CSB5
Related to polypeptides. Such a peptide has SEQ ID NO: 2
Or at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably with the amino acid sequence of SEQ ID NO: 6 over the entire length of SEQ ID NO: 2 or SEQ ID NO: 6 Includes isolated polypeptides comprising amino acid sequences having at least 95% identity, most preferably at least 97-99% identity. Such polypeptides include polypeptides comprising the amino acids of SEQ ID NO: 2 or SEQ ID NO: 6. SEQ ID NO: 2 (CSB5) and SEQ ID NO: 6
By comparison of the polypeptides of (CSB5var), SEQ ID NO: 2 shows that amino acid residues 243 and 244 of SEQ ID NO: 6.
Has a 30 amino acid insertion compared to SEQ ID NO: 6 (this means that these two polypeptides are splice variants or at least one of the polypeptides of SEQ ID NO: 2 is unspliced) This suggests that the translation is due to translation of mRNA transcripts containing introns).

A further peptide of the invention has an amino acid sequence whose amino acid sequence is at least 70% identical, preferably at least 80%, over the entire length of SEQ ID NO: 2 or SEQ ID NO: 6 to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6.
Identity, more preferably at least 90% identity,
Even more preferably, includes isolated polypeptides having at least 95% identity, most preferably at least 97-99% identity. Such polypeptides include the polypeptides of SEQ ID NO: 2 and SEQ ID NO: 6. Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1 or SEQ ID NO: 5.

[0006] The polypeptides of the present invention are believed to be members of the guanine nucleotide exchange factor (GEF) family of polypeptides. Thus, they show that GEF proteins are useful, among others, for cytoskeletal tissues, cell growth and proliferation, cell migration, cell attachment, membrane trafficking, vesicle trafficking, mitosis, ADP-ribosylation, phospholipid metabolism and programmed cell metabolism. GTPas involved in death
It is of interest to regulate the RAS superfamily of se. These characteristics are hereinafter referred to as “CSB
5 activity "or" CSB5 polypeptide activity "or" CSB5 biological activity ". These activities also include the antigenic and immunogenic activities of the CSB5 polypeptide, particularly those of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6. Preferably, a polypeptide of the invention exhibits at least one CSB5 biological activity.

[0007] The polypeptides of the present invention may be in the form of the "mature" protein or may be part of a larger protein such as a fusion protein. It is advantageous to include additional amino acid sequences with sequences that facilitate purification such as secretory or leader sequences, pro-sequences, multi-histidine residues, or additional sequences for stability during recombinant production. There are many.

The present invention also provides a variant of the above-described polypeptide, ie, a conservative amino acid substitution, which differs from the control by:
Thus, polypeptides in which one residue is replaced with another residue having similar properties. Typically,
Such substitutions may be made between Ala, Val, Leu and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; between basic residues Lys and Arg; Or between the aromatic residues Phe and Tyr. Several, 5
-10, 1-5, 1-3, 2 or 1 amino acid is
Variants that are substituted, deleted or added in any combination are particularly preferred.

[0009] The polypeptide of the present invention can be prepared by any suitable method. Such a polypeptide is
Includes isolated native polypeptides, polypeptides obtained by recombinant techniques, polypeptides obtained synthetically, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

[0010] In a further aspect, the present invention provides a CSB
5 polynucleotides. Such a polynucleotide has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity over the entire length of SEQ ID NO: 2 or SEQ ID NO: 6 with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. And even more preferably an isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having at least 95% identity. In this regard, polypeptides having at least 97% identity are highly preferred, those with at least 98-99% identity are more preferred, and those with at least 99% identity are most preferred. Such polynucleotides include polynucleotides comprising the nucleotide sequences contained in SEQ ID NO: 5 encoding the polypeptide of SEQ ID NO: 2 and SEQ ID NO: 5 encoding the polypeptide of SEQ ID NO: 6.

[0011] A further polynucleotide according to the present invention has at least 70% identity, preferably at least 80% identity, over its coding region, with the nucleotide sequence coding for the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6, More preferably, it includes isolated polynucleotides comprising nucleotide sequences having at least 90% identity, even more preferably at least 95% identity. In this regard, polynucleotides having at least 97% identity are highly preferred, those with at least 98-99% identity are more preferred, and those with at least 99% identity are most preferred.

[0012] Further polynucleotides of the present invention may have at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 5 over the entire length of SEQ ID NO: 1 or SEQ ID NO: 5. Includes isolated polynucleotides comprising nucleotide sequences having at least 90% identity, even more preferably at least 95% identity. In this regard, polynucleotides having at least 97% identity are highly preferred, those with at least 98-99% identity are more preferred, and those with at least 99% identity are most preferred. Such polynucleotides include polynucleotides comprising the polynucleotides of SEQ ID NO: 1 or SEQ ID NO: 5 as well as polynucleotides of SEQ ID NO: 1 and SEQ ID NO: 5. The present invention also provides
Provide a polynucleotide that is complementary to any of the above-described polynucleotides.

The nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 5 show homology to human Trio (Debant, A.
Nat. Acad. Sci. USA 99, 5466-5471, 1966;
GenBankAccession No. U42390). The nucleotide sequence of SEQ ID NO: 1 is a cDNA sequence and includes a polypeptide of 580 amino acids, ie, a polypeptide-encoding sequence (nucleotides 88 to 1830) encoding a polypeptide of SEQ ID NO: 2. The nucleotide sequence of SEQ ID NO: 5 is a cDNA sequence and includes a polypeptide (SEQ ID NO: 6) encoding a polypeptide of 550 amino acids, ie, a polypeptide of SEQ ID NO: 6 (nucleotides 88 to 1740). The nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is SEQ ID NO: 1
Or a sequence other than the sequence contained in SEQ ID NO: 1 which encodes the polypeptide of SEQ ID NO: 2 or is the same as the sequence encoding the polypeptide contained in It may be something. Similarly, the nucleotide sequence encoding the polypeptide of SEQ ID NO: 6 is the same as the sequence encoding the polypeptide contained in SEQ ID NO: 5, or as a result of the redundancy (degeneracy) of the genetic code; It may be a sequence other than the sequence contained in SEQ ID NO: 5, which encodes the polypeptide of SEQ ID NO: 6. The polypeptides of SEQ ID NO: 2 and SEQ ID NO: 6 are structurally related to other proteins of the guanine nucleotide exchange factor (GEF) family that have homology and / or structural similarity to human Trio (Debant, A Proc. Nat. Acad. Sci. USA
99, 5466-5471, 1966; GenBank Accession No. U4239
0).

[0014] Preferred polypeptides and polynucleotides of the present invention are believed to have, inter alia, similar biological functions / properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least one CSB5 activity. The present invention also relates to the first identified partial or alternative polynucleotides and polypeptides before the corresponding full-length sequences of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 2 and SEQ ID NO: 6 have been determined. .

Thus, in a further aspect, the present invention provides a method comprising: (a) at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity over the entire length of SEQ ID NO: 3 with the nucleotide sequence of SEQ ID NO: 3; % Identity, even more preferably at least 95%
% Identity, more preferably at least 97-
Comprising a nucleotide sequence having 99% identity; (b) at least 75% identity, preferably at least 80% identity, more preferably at least 90% identity over the entire length of SEQ ID NO: 3 with the nucleotide sequence of SEQ ID NO: 3. % Identity, even more preferably at least 95%
% Identity, more preferably at least 97-
Having a nucleotide sequence having 99% identity; (c) a polynucleotide of SEQ ID NO: 3; or (d) at least 70% identity, preferably at least, over the entire length of SEQ ID NO: 4 with the amino acid sequence of SEQ ID NO: 4. 80% identity, more preferably at least 90
% Identity, even more preferably at least 95% identity, and even more preferably at least 97-99.
And a polynucleotide of SEQ ID NO: 3.

The present invention further provides: (a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity over the entire length of SEQ ID NO: 4 with the amino acid sequence of SEQ ID NO: 4.
% Comprising an amino acid sequence having even more preferably at least 95% identity, most preferably at least 97-99% identity; (b) the amino acid sequence of SEQ ID NO: 4 and the entire length of SEQ ID NO: 4. At least 70%, preferably at least 80%, more preferably at least 90%,
Even more preferably, having an amino acid sequence that is at least 95% identical, most preferably at least 97-99% identical; (c) comprising the amino acid of SEQ ID NO: 4; and (d) the polypeptide of SEQ ID NO: 4. A polypeptide, and a polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 3.

The nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are represented by EST (Expression Sequence Tag)
Derived from the sequence. One of ordinary skill in the art can understand that errors in reading a certain nucleotide sequence in the EST sequence are inevitable (see Adams, MD, et al., Nature 377 (supp) 3, 1995). Thus, the nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are subject to the same inherent limitations in sequence accuracy. Furthermore,
The peptide sequence encoded by SEQ ID NO: 3 is the closest homologous or structurally similar protein;
It includes regions of identity or close homology and / or close structural similarities (eg, conserved amino acid differences). .

The polynucleotides of the present invention may comprise c-derived mRNA from human brain, heart, fetal dura, testis, Hodgkin's lymphoma II, pancreatic islet cell tumor or uterine cells.
Expression sequence tag (EST) analysis from DNA libraries using standard cloning and screening techniques (Adams, MD et al., Science 252: 1651-1656 (1991); Ad
ams, MD et al., Nature 355: 632-634 (1992); Adams, M.
D. et al., Nature 377 Supp: 3-174 (1995)). The polynucleotide of the present invention comprises a genome D
Can also be obtained from natural sources such as NA libraries,
Alternatively, they can be synthesized using well-known and commercially available methods.

When the polypeptide of the present invention is produced by a recombinant method using the polynucleotide of the present invention, the polynucleotide may be a coding sequence for a mature polypeptide, or a coding sequence for a mature polypeptide; The coding sequence for the mature polypeptide may be in a reading frame with other coding sequences, such as coding sequences for the pro or prepro protein sequence, or other fusion protein portions. For example, it can encode a marker sequence that facilitates purification of the fusion polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is pQ
E Vector (Qiagen, Inc.); Gentz et al., Pr.
Natl. Acad. Sci. USA (1989) 86: 821-824, or a hexa-histidine peptide;
Or it is an HA tag. A polynucleotide may also have non-coding 5 'and 3' sequences, such as transcriptional, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.

A further embodiment of the invention has the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 6 and comprises several, for example 5
-10, 1-5, 1-3, 1 or 2 or 1
Includes polynucleotides encoding polypeptide variants in which the amino acid residues have been substituted, deleted or added in any combination.

A polynucleotide identical or sufficiently identical to the nucleotide sequence contained in SEQ ID NO: 1 or SEQ ID NO: 5 can be used as a hybridization probe for cDNA and genomic DNA, or for nucleic acid amplification (eg, PCR) reactions. Using as primers, the full-length cDNA and genome encoding the polypeptides of the present invention were isolated and other genes with high sequence similarity to SEQ ID NO: 1 or SEQ ID NO: 5 (paralogs from human sources and non-human CDNA and genomic clones (including genes encoding orthologs and paralogs from species) can be isolated. Typically, these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, and most preferably 95% identical to the control. Probes or primers generally comprise at least 15 nucleotides, preferably at least 30 nucleotides, and may have at least 50 nucleotides. Particularly preferred probes have 30 to 50 nucleotides. Particularly preferred probes have 20 to 25 nucleotides.

Polynucleotides encoding polypeptides of the present invention, including homologues from non-human species, may be labeled under stringent hybridization conditions with the sequence of SEQ ID NO: 1, SEQ ID NO: 5, or fragments thereof. Screening an appropriate library with a probe; and isolating a full-length cDNA and a genomic clone having the polynucleotide sequence. Such hybridization techniques are well-known to those skilled in the art. Preferred stringent hybridization conditions include 50% formaldehyde, 5 × SSC (150 mM NaCl, 15 mM
M trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution,
Incubate overnight at 42 ° C. in a solution comprising 10% dextran sulfate and 20 μg / ml denatured cut salmon sperm DNA; then wash the filters with 0.1 × SSC at about 65 ° C. Thus, the present invention also encompasses polynucleotides obtained by screening an appropriate library with a labeled probe having the sequence of SEQ ID NO: 1, SEQ ID NO: 5, or a fragment thereof, under stringent hybridization conditions. .

It will be appreciated by those skilled in the art that in many cases the isolated cDNA sequence is incomplete and the region encoding the polypeptide is short at the 5 'end of the cDNA. This is because reverse transcriptase, a genetically low "processability" enzyme that cannot complete DNA replication of an mRNA template during initial cDNA strand synthesis (enzyme binding to the template during the polymerization reaction). (A measure of the ability to retain).

To obtain a full-length cDNA or a short c
Methods for extending DNA that are available and well known to those skilled in the art, such as rapid amplification of cDNA ends (R
ACE) (eg, Frohman et al., PNA
S USA 85, 8998-9002, 1988). Marathon ^TM
A recent variant of the method, shown in the ® method (Clontech Laboratories Inc.), for example, has greatly simplified the search for long cDNAs. In the Marathon ^TM method,
CDNA is prepared from mRNA extracted from the selected tissue and an "adapter" sequence ligated to each end. Next, nucleic acid amplification (PCR) is performed and the cDNA is "deleted" using a combination of gene-specific and adapter-specific oligonucleotide primers.
Amplify the 5 'end. Then "nested" primers,
That is, a primer designed to anneal into the amplification product (typically, further 3 ′ of the adapter sequence)
The PCR reaction is repeated using an adapter-specific primer that anneals the primer and a gene-specific primer that also anneals 5 'of the known gene sequence. The reaction product is analyzed by DNA sequencing and the product is directly ligated to the existing cDNA to obtain the complete sequence, or another full-length PCR is performed using new sequence information regarding the design of the 5 'primer. By doing so, the full-length cD
Build NA.

The recombinant polypeptide of the present invention can be prepared from a genetically engineered host cell containing an expression system by a method well known in the art. Thus, in a further aspect, the present invention relates to expression systems comprising the polynucleotide (s) of the invention, to host cells genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques. . RNA from DNA construct of the invention
Cell-free translation systems can also be used to produce such proteins using.

When producing recombinants, host cells can be genetically engineered to incorporate an expression system having a polynucleotide of the present invention, or a portion thereof. Davis et al., Bas
ic Methods in Molecular Biology (1986) and Sambr
ook et al., Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor Laboratory Press, ColdSpr
Polynucleotides can be introduced into host cells by methods described in many standard laboratory manuals, such as Ining Harbor, NY (1989). Such preferred methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. I do. Representative examples of suitable hosts include streptococci, staphylococci, E. coli, streptomyces, and Bacillus subtilis.
bacterial cells such as tilis cells; fungal cells such as yeast cells and Aspergillus cells; Drosophila S2 and Spodoptera
optera) insect cells such as Sf9 cells; CHO, CO
S, HeLa, C127, 3T3, BHK, HEK29
3 and animal cells such as Bowes melanoma cells; and plant cells.

A very wide variety of expression systems, such as those derived from chromosomes, episomes and viruses, such as those derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, inserted elements, yeast chromosomal elements, baculovirus, SV40 Vectors derived from viruses such as papovavirus, vaccinia virus, adenovirus, chicken poxvirus, pseudorabies virus and retrovirus, and vectors derived from plasmid and bacteriophage genetic elements such as cosmids and phagemids, and combinations thereof. And vectors derived from E. coli. The expression system may have regulatory regions that control as well as cause expression. Generally, any system or vector that can maintain, amplify or express a polynucleotide and produce a polypeptide in a host may be used. Suitable nucleotide sequences are described, for example, in Sambrook.
Et al., Molecular Cloning: A Laboratory Manual (supra)
May be inserted into the expression system by any of a variety of well-known conventional techniques, such as the technique set forth in US Pat. Appropriate secretion signals may be incorporated into the desired polypeptide to secrete the translated protein into the endoplasmic reticulum lumen, periplasmic space or extracellular environment. These signals may be intrinsic to the polypeptide or they may be heterologous signals.

When a polypeptide of the present invention is expressed for use in a screening assay, it is preferred that the polypeptide be produced on the surface of cells. In this case, the cells may be harvested before use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered to recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide can be recovered. The polypeptides of the invention can be prepared by known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. It can be recovered and purified from recombinant cell culture by methods. Most preferably, high performance liquid chromatography is used for purification. If the polypeptide denatures during cell-free synthesis, isolation and / or purification, it can be reconstituted into an active conformation using well known methods for regenerating proteins.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic reagent. Diseases resulting from underexpression, overexpression, or spatial or temporal changes in expression of a gene by detecting a mutant form of the gene characterized by the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 5 involved in dysfunction. Alternatively, a diagnostic means can be provided in addition to or capable of identifying a suspected disease. Individuals having a mutation in the gene can be detected at the DNA level by various methods.

The nucleic acid to be used for diagnosis can be obtained from a subject cell, for example, from blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA can be used for direct detection or can be amplified enzymatically by using PCR or other amplification methods before subjecting it to analysis. RN
A or cDNA can be used in the same way. Changes in the size of the amplification product can be used to detect deletions and insertions as compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to a labeled CSB5 nucleotide sequence. Perfectly matched sequences are obtained by RNase digestion or by differences in melting temperatures.
Can be distinguished from mispaired double helix. DNA sequence differences can also be caused by changes in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct D
Can be detected by NA sequencing (eg, Myers et al., Sci.
ence, 230: 1242 (1985)). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S1 protection or chemical cleavage methods (eg, Cotton et al.,
Proc Natl Acad Sci USA 85: 4397-4401 (1985)). In another embodiment, an array of oligonucleotide probes comprising a CSB5 nucleotide sequence or a fragment thereof can be constructed, for example, to provide effective screening for genetic variation. Array techniques are well known and have general applicability and can be used to address various issues in molecular genetics, including gene expression, genetic linkage and genetic variability (eg, : M. Chee et al., Science, Vol.
274, pages 610-613 (1996)).

The diagnostic assay is performed using the CS
Provided is a method for diagnosing or determining a suspicion of a disease by detecting a mutation in the B5 gene. In addition, such diseases can be diagnosed by a method comprising measuring abnormally reduced or increased levels of polypeptide or mRNA from a sample from the subject. A decrease or increase in expression can be achieved, for example, by a nucleic acid amplification method,
Quantification of polynucleotides, such as RT-PCR, RNase protection, Northern blots and other hybridization methods, can be measured at the RNA level using methods well known in the art. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, and Western immunoassays.
Includes blot analysis and ELISA assays.

Thus, in another aspect, the present invention provides: (a) a polynucleotide of the present invention, preferably a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 5 or a fragment thereof, (b) a nucleotide of (a) A nucleotide sequence complementary to the sequence; (c) a polypeptide of the invention, preferably SEQ ID NO: 2,
A polypeptide of SEQ ID NO: 6 or a fragment thereof; or (d) a polypeptide of the invention, preferably SEQ ID NO: 2,
A diagnostic kit comprising an antibody against the polypeptide of SEQ ID NO: 6.

In such a kit, (a), (b),
It will be apparent that (c) or (d) contains substantial components. Such kits may include suspected disease or morbidity, particularly cardiac hypertrophy, heart failure, ischemia, arrhythmias, hypertension, atherosclerosis, restenosis, chronic and acute inflammation, cerebral stroke, rheumatoid arthritis, multiple sclerosis, It may be useful for diagnosing bowel disease, diabetes, and cancer.

[0034] The nucleotide sequences of the present invention are also useful for identifying chromosomes. The sequence can specifically target and hybridize to a particular location on an individual human chromosome. Mapping of chromosome-related sequences according to the present invention is an important first step in correlating the sequences with disease-associated genes. Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data.
Such data is available, for example, from V. McKusick, Mendelian
Inheritance in Man (John Hopkins University Welch
Available online from the Medical Library). Next, the relationship between the gene mapped to the same chromosomal region and the disease is identified using linkage analysis (joint inheritance of physically adjacent genes). Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be measured. If the mutation is observed in some or all affected individuals but not in normal individuals, then the mutation may be responsible for the disease.

The nucleotide sequence of the present invention is useful in tissue isolation. Using such a method, CSB5
Detection of mRNA encoding the polypeptide allows one to examine the expression pattern of CSB5 polypeptide in the tissue. These methods include in situ hybridization methods, for example, nucleotide amplification methods such as PCR. Such methods are well-known to those skilled in the art. The results of these studies can identify the normal function of the polypeptide in an organism. In addition, the normal expression pattern of CSB5 mRNA and mutant C
By comparing the expression pattern of the mRNA encoded by the SB5 gene, it is possible to effectively gain insight into the role of a mutant CSB5 polypeptide or the role of inappropriate expression of a normal CSB5 polypeptide in disease. Such inappropriate expression may be a temporal, spatial or simply quantitative feature.

Antibodies immunospecific for the polypeptide of the present invention can also be obtained using the polypeptide of the present invention, a fragment thereof, an analog thereof, or a cell expressing the same as an immunogen. The term "immunospecific" means that the antibody has a substantially greater affinity for the polypeptides of the present invention than for other related polypeptides in the prior art.

Antibodies raised against the polypeptide of the present invention can be obtained by administering a polypeptide or an epitope-carrying fragment, analog or cell to an animal, preferably a non-human animal, using a conventional protocol. Can be. For monoclonal antibody production,
Any method that provides antibodies obtained by continuous cell line culture can be used. For example, the hybridoma method (Kohler, G. and Milstein, C., Nature 256: 49).
5-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor et al., Immunology Today 4:72 (1983)) and EBV-hybridoma method (Cole et al., Monoclonal An).
tibodies and Cancer Therapy, 77-96, Alan R. Liss,
Inc., (1985)).

Applying the techniques for producing single-chain antibodies as described in US Pat. No. 4,946,778,
Single chain antibodies against the polypeptides of the present invention can be produced. Also, other organisms, including transgenic mice or other mammals, can be used to express humanized antibodies. Using the above-described antibodies, clones expressing the polypeptide may be isolated or identified, or the polypeptide may be purified by affinity chromatography. Antibodies to the polypeptides of the present invention may be used to treat, inter alia, diseases.

In a further aspect, the present invention provides a polypeptide of the invention or a fragment thereof, comprising various portions of the heavy or light chain immunoglobulin constant regions of various subclasses (IgG, IgM, IgA, IgE). And a genetically engineered soluble fusion protein comprising:
As an immunoglobulin, the constant part of the heavy chain of human IgG, especially IgG1, in which the fusion takes place in the hinge region, is preferred. Specifically, the Fc portion can be easily removed by incorporating a cleavage sequence cleavable by the blood coagulation factor (Xa). Furthermore, the invention relates to the production of these fusion proteins by genetic engineering and their use in drug screening, diagnosis and therapy. A further aspect of the invention also relates to a polynucleotide encoding such a fusion protein. Fusion protein technology is found, for example, in International Patent Applications WO 94/29458 and WO 94/22914.

Another aspect of the invention is a method of eliciting an immunological response in a mammal, comprising inoculating the mammal with an antibody and / or a polypeptide of the invention sufficient to generate a T cell immune response. And, inter alia, to a method comprising protecting the animal from the diseases mentioned above. Another aspect of the invention is a method of inducing an immunological response in a mammal, comprising administering a polypeptide of the invention to direct expression of a polynucleotide and encoding the polypeptide in vivo. And eliciting such an immunological response, producing antibodies, and protecting the animal from disease.

A further aspect of the present invention is an immunological / vaccine formulation (composition) which, when introduced into a mammalian host, induces an immunological response in the mammal to the polypeptide of the present invention. Wherein the composition comprises a polypeptide or polynucleotide of the invention. The vaccine formulation may further include a suitable carrier. Since the polypeptide may be destroyed in the stomach, it is preferably administered parenterally (eg, subcutaneously, intramuscularly, intravenously, intradermally). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the patient; It includes aqueous and non-aqueous sterile suspensions which may contain a thickening agent.
The formulations may be presented in single-dose or multi-dose containers, for example, sealed ampules and vials, or stored in a lyophilized state, which only requires the addition of a sterile liquid carrier immediately before use. Vaccine formulations may also include adjuvant systems to enhance the immunogenicity of the formulation, such as oil-in-water and other systems known in the art. The dose depends on the individual activity of the vaccine and can be easily determined by routine experimentation.

The polypeptides of the present invention are involved in many biological functions, including many pathologies, especially the diseases mentioned above. Therefore, it is desired to find a screening method for identifying a compound that stimulates or inhibits the function of the polypeptide. Thus, in a further aspect,
The present invention provides a screening method for identifying a compound that stimulates or inhibits the function of the polypeptide. Generally, the agonist or antagonist is
It is used for the purpose of treating or preventing the above-mentioned diseases. Compounds can be identified from a variety of sources, such as cells, cell-free preparations, chemical libraries and natural product mixtures. Agonists so identified,
An antagonist or inhibitor may optionally be a natural or modified substrate, ligand,
It may be a receptor, an enzyme or the like, or may be one that mimics its structure or function (Coligan
Et al., Current Protocols in Immunology 1 (2): Chapter
5 (1991)).

The screening method involves simply measuring the binding of a candidate compound to a polypeptide, a cell or membrane having the polypeptide, or a fusion protein thereof, using a label that is directly or indirectly bound to the candidate compound. Can be. Alternatively, the screening method may involve competition with a labeled competitor.
Furthermore, these screening methods can also use a suitable detection system for cells having the polypeptide to test whether the candidate compound produces a signal emitted by activation or inhibition of the polypeptide. .
In general, activation inhibitors are assayed in the presence of a known agonist and the effect of the agonist on activation in the presence of the candidate compound is observed. Using an essentially active polypeptide in a screening method for an inverse agonist or inhibitor by testing whether the candidate compound results in inhibition of activation of the polypeptide in the absence of the agonist or inhibitor Can be. In addition, the screening method briefly describes combining the candidate compound with a solution containing the polypeptide of the present invention to form a mixture, measuring the CSB5 activity in the mixture, and determining the CSB5 activity in the mixture. Comparing with the target.
Fusion proteins, such as those prepared from the Fc portion and the CSB5 polypeptide, described above, can also be used in high-throughput screening assays to identify antagonists of the polypeptides of the invention (D. Bennett et al., J Mol. R
ecognition, 8: 52-58 (1995); and K. Johanson et al., J.
BiolChem, 270 (16): 9459-9471 (1995)).

[0044] Using polynucleotides, polypeptides and antibodies to the polypeptides of the present invention, screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells can also be formed. For example, an ELISA assay may be configured to measure polypeptide secretion or cell binding levels using monoclonal and polyclonal antibodies by standard methods known in the art.
This assay can also be used to find agents (each of which is also referred to as an antagonist or agonist) that can inhibit or enhance the production of polypeptides from appropriately engineered cells or tissues.

The polypeptide can be used to identify membrane-bound or soluble receptors via standard receptor binding methods known in the art. These include, but are not limited to, labeling the polypeptide with a radioisotope (eg, ¹²⁵ I), chemically modifying (eg, biotinylated), or peptide sequences suitable for detection or purification And putative receptor sources (cells, cell membranes, cell supernatants, tissue extracts, body fluids)
And a ligand binding and crosslinking assay. Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods, if any, can also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptor. Standard methods for performing such assays are well-known in the art.

Examples of potential polypeptide antagonists are antibodies or, in some cases, oligonucleotides or proteins, which in some cases are closely related to ligands, substrates, receptors, enzymes, etc. of the polypeptide, eg, Includes fragments such as ligands, substrates, receptors, enzymes, or small molecules that bind to a polypeptide of the invention but do not elicit a response such that the activity of the polypeptide is prevented.

Thus, in another aspect, the invention provides an agonist for the polypeptide of the invention,
A screening kit for identifying a compound that decreases or enhances production of such a polypeptide, which comprises: (a) a polypeptide of the present invention; (b) a kit of the present invention. A recombinant cell expressing the polypeptide; (c) a cell membrane expressing the polypeptide of the present invention; or (d) an antibody against the polypeptide of the present invention, wherein the polypeptide is preferably SEQ ID NO: 2 or SEQ ID NO: 6. A screening kit which is the polypeptide of In any such kit,
It will be apparent that (a), (b), (c) or (d) contains substantial components.

The polypeptide of the present invention may also comprise (a) the first
Determining the three-dimensional structure of the polypeptide; (b) estimating the three-dimensional structure for a similar reactive or binding site (s) of the agonist, antagonist or inhibitor; By synthesizing a candidate compound that is predicted to bind or react with a putative binding or reactive site, and (d) testing whether the candidate compound is in fact an agonist, antagonist or inhibitor An agonist of the polypeptide,
It will be apparent that antagonists or inhibitors may be used in the structure-based design methods. It will also be clear that this method is usually an interactive process.

[0049] In a further aspect, the present invention relates to any of the over- or under-expressed CSB5 polypeptide activities, eg, cardiac hypertrophy, heart failure, ischemia, arrhythmia, hypertension, atherosclerosis, restenosis, Chronic and acute inflammation, cerebral attack, rheumatoid arthritis, multiple sclerosis,
Provided is a method for treating an abnormal condition such as intestinal disease, diabetes, and cancer.

If the activity of the polypeptide is in excess, several methods can be used. One method is, for example,
The inhibitory compounds (antagonists) described above can be used in amounts effective to inhibit the function of the polypeptide by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by inhibiting another signal. Administering to a subject in need of such treatment, optionally in combination with a pharmaceutically acceptable carrier, thereby ameliorating the abnormal condition. In another method, a soluble form of the polypeptide that has the ability to compete with endogenous polypeptides and bind ligands, substrates, enzymes, receptors, etc., may be administered. Typical examples of such competitors include C
And fragments of the SB5 polypeptide.

In yet another alternative, expression of the gene encoding endogenous CSB5 may be inhibited using expression blocking techniques. Known such methods include the use of internally generated or separately administered antisense sequences (eg, Oligodeoxynucleotides as Antisense).
Inhibitors of Gene Expression, CRC Press, Boca Ra
ton, FL (1988), O'Connor, J. Neurochem 56: 560
(1991)). Alternatively, oligonucleotides can be provided that form a triple helix ("triplex") with the gene (eg, Lee et al., Nuc
leic Acids Res 6: 3073 (1979); Cooney et al., Science.
241: 456 (1988); Dervan et al., Science 251: 1360 (199).
See 1)). These oligomers themselves can be administered, or the relevant oligomers can be expressed in vivo. Synthetic oligomers or triple helix oligonucleotides may contain modified bases or backbones. Examples of the latter include methyl phosphonate,
Phosphorothioate or peptide nucleic acid (PNA) backbones. Such backbones are incorporated into antisense or triple helix oligonucleotides to protect them from nuclease degradation and are well known to those skilled in the art. The antisense and triple helix molecules or other modified backbones synthesized therein form part of the present invention.

In addition, expression of the CSB5 polypeptide is
The interference may be achieved by using a ribozyme specific for the CSB5 mRNA sequence. Ribozymes catalytically activate RNA, which can be natural or synthetic (see, eg, Usman, N et al., Curr. Opin. Struct. Biol (1
996) 6 (4), 527-33). Synthetic ribozymes can be designed to specifically cleave CSB5 mRNA at selective sites, thereby inhibiting translation of CSB5 mRNA into a functional polypeptide. Ribozymes may be synthesized with a natural ribosomal phosphate backbone and natural bases, as commonly found in RNA molecules. Alternatively, the ribozyme may be synthesized using a non-natural backbone such as, for example, 2'-O-methyl RNA, to protect it from ribonuclease degradation, and to contain modified bases.

[0053] Several methods are also available for treating abnormal conditions associated with an under-expression of CSB5 and its activity. One method comprises administering to a subject a therapeutically effective amount of a compound that activates a polypeptide of the present invention (ie, an agonist as described above) together with a pharmaceutically acceptable carrier, thereby ameliorating the abnormal condition. including.
Alternatively, gene therapy may be used to generate CSB5 endogenously by relevant cells in the subject. For example,
As noted above, the polynucleotides of the present invention may be engineered to be expressed in a replication defective retroviral vector. Then, isolating the retroviral expression construct,
The transfected cells may be introduced into a packaging cell transduced with an RNA-containing retroviral plasmid vector encoding the polypeptide of the present invention so that the packaging cell produces infectious virus particles containing the gene of interest. . These producer cells may be administered to a subject to manipulate the cells in vivo and to express the polypeptide in vivo. For an overview of gene therapy, see Human Molecular Genetics, T. Strachan an.
d AP Read, BIOS Scientific Publishers Ltd (199
6), Chapter 20, Gene Therapy and other Molecular
See Genetic-based Therapeutic Approaches (and references therein). Another method is to administer a therapeutic amount of a polypeptide of the present invention in combination with a suitable pharmaceutical carrier.

In a further embodiment, the invention provides a therapeutically effective amount of a polypeptide, eg, a soluble form of a polypeptide, agonist / antagonist peptide or small molecule compound of the invention, in a pharmaceutically acceptable carrier or excipient. There is provided a pharmaceutical composition in combination with the excipient. Such carriers include saline, buffered saline, dextrose,
It includes, but is not limited to, water, glycerol, ethanol, and combinations thereof. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more components of the composition of the invention as described above. The polypeptides and other compounds of the present invention may be used alone or in combination with other compounds, such as therapeutic compounds. The composition may be, for example, by systemic or oral administration.
It will be compatible with the route of administration. Preferred forms of systemic administration typically include injection by intravenous injection.
Other injection routes, such as subcutaneous, intramuscular or intraperitoneal, can also be used. Another means for systemic administration involves transmucosal or transdermal administration with penetrants or other surfactants, such as bile salts or fusidic acid. In addition, if the polypeptides or other compounds of the present invention can be formulated in enteric or capsule formulations, oral administration is also possible. Administration of these compounds may be topical and / or localized, in the form of salves, pastes, gels and the like.

The required dosage range depends on the choice of peptide or other compound of the invention, the route of administration, the nature of the formulation, the nature of the condition of the subject, and the judgment of the consulting physician. However, suitable doses are between 0.1 and 100 kg / subject.
μg range. However, in view of the different compounds available and the differing efficiencies of the various routes of administration, the required dosage will likely vary. For example, oral administration may require higher doses than intravenous injection. Variations in these dosages can be made using standard empirical routines for optimization well known in the art.

In the above-mentioned therapeutic methods, often referred to as "gene therapy", the polypeptide used for the treatment can be obtained endogenously in the subject. Thus, for example, a subject-derived cell may be engineered with a polynucleotide such as DNA or RNA using a retroviral plasmid vector, and the polypeptide may be encoded ex vivo. Next, the cells are introduced into the subject.

Polynucleotide and polypeptide sequences form a valuable source of information, with which additional sequences of similar homology can be identified. This involves recording the sequence on a computer readable medium and then using the recorded data to generate GCG and Lasergen.
This can be most easily performed by searching the sequence database using a known search means such as an e-software package. Thus, in a further aspect,
The present invention provides a computer-readable medium having recorded therein a polynucleotide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 5 and / or a polypeptide sequence encoded thereby.

The following definitions are provided to facilitate an understanding of certain terms commonly used above. As used herein, "antibody" encompasses polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including Fab products or other immunoglobulin expression libraries. "Isolated" means altered "by the hand of man" from its natural state. When an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal is "isolated"
The same polynucleotide or polypeptide that has not been, but is separated from, its naturally occurring co-existing material has been "isolated" as the term is used herein.

[0059] "Polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" refers to single-stranded and double-stranded D
NA, DNA that is a mixture of single- and double-stranded regions,
RNA that is single- and double-stranded RNA, and a mixture of single- and double-stranded regions, single-stranded or more typically double-stranded, or a mixture of single- and double-stranded regions. Including, but not limited to, hybrid molecules including DNA and RNA that may be used. In addition, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases, as well as DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications have been made to DNA and RNA. Thus, a “polynucleotide” is typically a chemically, enzymatically or metabolically modified form of a polynucleotide as found in nature, as well as DNA and RNA in chemical forms characteristic of viruses and cells. Is included. Also,
"Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

A "polypeptide" comprises a peptide or protein comprising two or more amino acids linked together by peptide bonds or two or more amino acids linked together by modified peptide bonds. Peptide or protein, ie
Refers to peptide isostere. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and long chains, referred to as proteins. Polypeptides may contain amino acids different from the 20 amino acids encoded by the gene. A “polypeptide” contains an amino acid sequence that has been modified by natural steps, such as post-translational processing, or by chemical modification techniques well known to those skilled in the art. Such modifications are well described in basic texts and in more detailed research articles, as well as in extensive research literature. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains and the amino or carboxyl terminus. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, branched or unbranched, and cyclic. Cyclic, branched and branched cyclic polypeptides may result from natural post-translational processes or may be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, sharing of phosphotidylinositol. Join,
Cross-linking, cyclization, disulfide bond formation, demethylation,
Cross-link covalent bond formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, sugar chain formation, GPI anchor formation, hydroxylation, iodination,
Includes transfer RNA-mediated amino acid addition to proteins, such as methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, arginylation, and ubiquitination ( For example, Proteins-Str
uctureand Molecular Properties, 2nd Ed., TE Cre
ighton, WH Freeman and Company, New York, 1993;
Wold, F., Post-translational Protein Modification
s: Perspectives and Prospects, pgs. 1-12 in Post-
translational Covalent Modification of Proteins,
BC Johnson, Ed., Academic Press, New York, 198
3; Seifter et al., "Analysis for protein modifications
and nonprotein cofactors ", Meth Enzymol (1990) 18
2: 626-646 and Rattan et al., "Protein Synthesis: Po
st-translational Modifications and Aging ", Ann NY
Acad Sci (1992) 663: 48-62).

"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not be different from the amino acid sequence of the polypeptide encoded by the control polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the control polypeptide and the variant are closely similar overall and, in many regions, identical. Variant and control polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a natural product, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides are
It can be produced by mutagenesis techniques or by direct synthesis.

As is known in the art, "identity" is determined by comparing the sequences, between two or more polypeptide sequences or two or more polynucleotide sequences. Relationship. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, when measuring pairing between chains of such sequences. I do. "Identity" and "similarity" include, but are not limited to, Computational Molecular Biology, L.
esk, AM, ed., Oxford University Press, New York,
1988; Biocomputing: Informatics and Genome Projec
ts, Smith, DW, ed., Academic Press, New York, 19
93; Computer Analysis of Sequence Data, Part I, Gr
iffin, AM, and Griffin, HG, eds., Humana Pres
s, New Jersey, 1994; Sequence Analysis in Molecula
r Biology, von Heinje, G., Academic Press, 1987; a
ndSequence Analysis Primer, Gribskov, M. and Dever
eux, J., eds., M Stockton Press, New York, 1991;
And Carillo, H., and Lipman, D., SIAM J. Appli
ed Math., 48: 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. A preferred computer program method for determining identity and similarity between two sequences is the GCG program package (Devereux, J. et al., Nuc.
leic Acids Research 12 (1): 387 (1984)), BLASTP, B
LASTN, FASTA (Atschul, SF et al., J. Molec. Biol. 215: 4
03-410 (1990)). The BLAST X program is available from NCBI and other sources (BLSAT Manual, Altschul, S. et al., NCBI NLM NIHBethesd
a, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 403.
-410 (1990)). In addition, the well-known Smit
h Identity may be measured using the Waterman algorithm.

Preferred parameters for comparing polypeptides include the following parameters: 1) Algorithm: Needleman and Wunsch, J. Mol Bio
l. 48: 443-453 (1970) Comparison matrix: Hentikoff and Hentikoff, Proc. N
atl. Acad. Sci. USA. 89: 10915-10919 (1992) BLOSSU
M62 Gap Penalty: 12 Gap Length Penalty: 4 A useful program with these parameters is "ga" from the Genetics Computer Group, Madison, Wiscosin.
Publicly available as "p" program. The above parameters are the defaults for comparing polypeptides (defaul
t) Parameter (end gap has no penalty)
It is.

The parameters for comparing polynucleotides include the following parameters: 1) Algorithm: Needleman and Wunsch, J. Mol Bio
l. 48: 443-453 (1970) Comparison matrix: match = +10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3 A useful program to use these parameters is:
Genetics Computer Group, Madison, Wiscosin
More publicly available as a "gap" program. The above parameters are default parameters for comparing polynucleotides.

By way of example, the polynucleotide sequence of the present invention may be identical to the control sequence of SEQ ID NO: 1 or SEQ ID NO: 5,
That is, they may be 100% identical, or the nucleotide sequences include those in which up to a certain number of nucleotides have been changed as compared to a control sequence. Such changes are selected from the group comprising deletions, transitions and transversions comprising at least one nucleotide, or insertions, wherein the changes are at the 5 'or 3' end of the control nucleotide sequence, or at these ends. It may occur anywhere between positions and may be interspersed between nucleotides of the control sequence or individually in one or more adjacent groups within the control sequence. The number of nucleotide changes is the percentage of identity (100
Multiplied by the number) divided by either subtracting that product from said total number of nucleotides in SEQ ID NO: 1 or SEQ ID NO: 5, _{or: n n ≦ X n - (} X n · y) ( where, n _n of nucleotide changes _Xn is the total number of nucleotides of SEQ ID NO: 1 or SEQ ID NO: 5, for example, y is 0.70 at 70% and 0.8 at 80%.
It is 0.85 at 0, 85%, 0.90 at 90%, 0.95 at 95%, etc., before subtracting the product of _Xn and y from _Xn , the fraction is rounded down to the nearest integer. ). A change in the polynucleotide encoding the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6 forms a nonsense, missense or frameshift mutation in the coding sequence, and the polypeptide encoded by the polynucleotide following such change Bring change.

Similarly, the polypeptide sequence of the present invention may be identical, ie, 100% identical, to the control sequence of SEQ ID NO: 2 or SEQ ID NO: 6, or the polypeptide sequence may be compared to a control sequence. Up to a certain number of amino acids are varied, including those with less than 100%% identity. Such alterations are selected from the group comprising deletions, substitutions, including conservative and non-conservative substitutions, or insertions of at least one amino acid, wherein the alterations are at the amino or carboxy terminus of the control polypeptide sequence, or at those termini. It may occur anywhere between positions and may be interspersed between amino acids of the control sequence or individually in one or more adjacent groups in the control sequence. Predetermined%
The number of amino acid changes in identity is calculated by multiplying the total number of amino acids in SEQ ID NO: 2 or SEQ ID NO: 6 by the percentage of percent identity (number divided by 100), respectively, and multiplying the product by SEQ ID NO: 2 or SEQ ID NO: 6. total (X _a · y) (where, n _a is the number of amino acid changes, amino acids and X _a SEQ ID NO: 2 or SEQ ID NO: 6 - n _a ≦ X _a: if subtracted from the total number of amino acids, or And y is 7
It is 0.70 at 0%, 0.80 at 80%, and 0.7 at 85%.
85 and the like, before subtracting the product of X _a and y from X _a, the nearest integer by truncating the fractional) is determined by.

“Homolog” is used in the art,
Genetic term meaning a polynucleotide or polypeptide having a high degree of sequence relatedness to a subject sequence.
Such relationships may be quantified by examining the degree of identity and / or similarity between the compared sequences as described herein above. This genetic term includes "orthologs" which refer to polynucleotides or polypeptides that are functional equivalents of the polynucleotide or polypeptide in different species, and sequences that are functionally similar when considered among the same species There is a term “paralog” which means Thus, for example, in the art, many members of the serotonin receptor family are paralogs of other members of the rat serotonin receptor family.

“Fusion protein” refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. In one example, EP-A-0464 discloses fusion proteins combining various portions of the constant region of an immunoglobulin molecule with other human proteins or portions thereof.
In many cases, the use of an immunoglobulin Fc region as part of a fusion protein is therapeutically or diagnostically advantageous, for example, resulting in improved pharmacokinetic properties [see, for example, EP-A 0232262. ].
On the other hand, for some uses, it is desirable to be able to remove the Fc portion after the fusion protein has been expressed, detected, and purified. All publications, including but not limited to the patents and patent applications listed herein, are limited and individually described herein, even if fully disclosed. , But are incorporated herein by reference.

Sequence information SEQ ID NO: 1

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SEQ ID NO: 2

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SEQ ID NO: 3

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SEQ ID NO: 4

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SEQ ID NO: 5

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SEQ ID NO: 6

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[0075]

[Sequence List] (1) General Information (i) Applicant: SmithKline Beecham (ii) Title of Invention: New Compound (iii) Number of Sequences: 6 (iv) Mailing Address (A) Name: SmithKline Beecham (B) Street: New Horizons Court, Great West Road (C) City: Brenford (D) State: (E) Country: United Kingdom (F) Postal Code: TW8 9EP (v) Computer Readable form: (A) Media type: Diskette (B) Computer: IBM compatible (C) Operating system: DOS (D) Software: Windows version 2.0
FastSEQ for

(2) Information on SEQ ID NO: 1 (i) Sequence characteristics: (A) Sequence length: 2178 base pairs (B) Sequence type: nucleic acid (C) Number of chains: 1 (D) topology: the type of linear (ii) molecular: wherein the cDNA (xi) SEQ: SEQ ID NO 1: ACGAGCCCGG CGCCGTCCCC GCCCCGCCCC CCGTGATTCC CCCTGCATGG CCGGCCCGGG 60 TGGGGGGCGC GGGGGGGCCC GGGCGCCATG CGGGGGGGGC ACAAAGGGGG TCGCTGTGCC 120 TGTCCCCGTG TGATCCGAAA AGTGCTGGCA AAATGCGGCT GCTGCTTCGC CCGGGGGGGA 180 CGTGAATCCT ATTCCATTGC GGGCAGTGAG GGGAGTATAT CGGCTTCTGC TGCCTCCGGT 240 CTGGCTGCCC CCTCTGGCCC CAGCTCTGGC CTCAGCTCTG GCCCCTGTTC CCCAGGCCCC 300 CCAGGGCCCG TCAGTGGCCT GAGGAGATGG TTGGATCATT CCAAACATTG TCTCAGTGTG 360 GAAACTGAGG CAGACAGTGG TCAGGCAGGA CCATATGAGA ACTGGATGTT GGAGCCAGCT 420 CTAGCCACAG GAGAGGAGCT GCCGGAACTG ACCTTGCTGA CCACACTGTT GGAAGGCCCT 480 GGAGATAAGA CGCAGCCACC TGAAGAGGAG ACTTTGTCCC AAGCCCCTGA GAGTGAGGAG 540 GAACAGAAGA AGAAGGCTCT GGAAAGGAGT ATGTATGTCC TGAGTGAAC T GGTAGAAACA 600 GAGAAAATGT ACGTTGACGA CTTGGGGCAG ATTGTGGAGG GTTATATGGC CACCATGGCT 660 GCTCAGGGGG TCCCCGAGAG TCTTCGAGGC CGTGACAGGA TTGTGTTTGG GAATATCCAG 720 CAAATCTATG AGTGGCACCG AGACTATTTC TTGCAAGAGC TACAACGGTG TCTGAAAGAT 780 CCTGATTGGC TGGCTCAGCT ATTCATCAAA CACGAGCGCC GGCTGCATAT GTATGTGGTG 840 TACTGTCAGA ATAAGCCCAA GTCAGAGCAT GTGGTGTCAG AGTTTGGGGA CAGCTACTTT 900 GAGGAGCTCC GGCAGCAGCT GGGGCACCGC CTGCAGCTGA ACGACCTCCT CATCAAACCT 960 GTGCAGCGGA TCATGAAATA CCAGCTGCTG CTCAAGGATT TTCTCAAGTA TTACAATAGA 1020 GCTGGGATGG ATACTGCAGA CCTAGAGCAA GCTGTGGAGG TCATGTGCTT TGTGCCCAAG 1080 CGCTGCAACG ATATGATGAC GCTGGGGAGA TTGCGGGGAT TTGAGGGCAA ACTGACTGCT 1140 CAGGGGAAGC TCTTGGGCCA GGACACTTTC TGGGTCACCG AGCCTGAGGC TGGAGGGCTG 1200 CTGTCTTCCC GAGGTCGAGA GAGGCGCGTC TTCCTCTTTG AGCAAATCAT CATCTTCAGT 1260 GAAGCCCTGG GAGGAGGAGT GAGAGGTGGA ACACAGCCTG GATATGTATA CAAGAACAGC 1320 ATTAAGGTGA GCTGCCTGGG ACTGGAGGGG AACCTCCAAG GTGACCCTTG CCGCTTTGCA 1380 CTGACCTCCA GAGGGCCAGA GGGTGGGATC CAGCGCTATG TCCTGCAGGC TGCAGACCCT1440 GCTATCAGTC AGGCCTGGAT CAAGCATGTG GCTCAGATCT TGGAGAGCCA ACGGGACTTC 1500 CTCAACGCAT TGCAGTCACC CATTGAGTAC CAGAGACGGG AGAGCCAGAC CAACAGCCTG 1560 GGGCGGCCAA GAGGGCCTGG AGTGGGGAGC CCTGGAAGAA TTCGGCTTGG AGATCAGGCC 1620 CAGGGCAGCA CACACACACC CATCAATGGC TCTCTCCCCT CTCTGCTGCT GTCACCCAAA 1680 GGGGAGGTGG CCAGAGCCCT CTTGCCACTG GATAAACAGG CCCTTGGTGA CATCCCCCAG 1740 GCTCCCCATG ACTCTCCTCC AGTCTCTCCA ACTCCAAAAA CCCCTCCCTG CCAAGCCAGA 1800 CTTGCCAAGC TGGATGAAGA TGAGCTGTAA CTGGTGAAAA CCATGGGGGT GGTGCTGACT 1860 CAGCCGCCTA TTCCCCAAGG AGCTTCAGGG CAGTCCTTCT GGCACTGCTC CAGAATTCCT 1920 CCTTCTTGGT GTGTCTGGAG GGTGGGCAAG GCTGGGAGGG ATATCAACTT GGAGGAGAAC 1980 ACCTAGACCC AAGGACTTTT TTCTGCCCAA GGAACACAGT TTCCTTCAGC TCCCATCCCT 2040 ATGCATGCAT CATGGTCCCC CCAAAAGGAG GATATGTGGG TGGGTGGGAG GGCTGGGGCA 2100 GGGGCCAGAT AGAAATTATT GGTTTTGTTT TTTAATTTTG TTTTTCCTGT TTTCTGAGAA 2160 TAAAGGTTTT GTTATATC 2178

(2) Information on SEQ ID NO: 2 (i) Sequence characteristics: (A) Sequence length: 580 amino acids (B) Sequence type: amino acids (C) Number of chains: 1 (D) Topology : Linear (ii) Molecular type: protein (xi) Description of sequence: SEQ ID NO: 2: Met Arg Gly Gly His Lys Gly Gly Arg Cys Ala Cys Pro Arg Val Ile 1 5 10 15 Arg Lys Val Leu Ala Lys Cys Gly Cys Cys Phe Ala Arg Gly Gly Arg 20 25 30 Glu Ser Tyr Ser Ile Ala Gly Ser Glu Gly Ser Ile Ser Ala Ser Ala 35 40 45 Ala Ser Gly Leu Ala Ala Pro Ser Gly Pro Ser Ser Gly Leu Ser Ser 50 55 60 Gly Pro Cys Ser Pro Gly Pro Pro Gly Pro Val Ser Gly Leu Arg Arg 65 70 75 80 Trp Leu Asp His Ser Lys His Cys Leu Ser Val Glu Thr Glu Ala Asp 85 90 95 Ser Gly Gln Ala Gly Pro Tyr Glu Asn Trp Met Leu Glu Pro Ala Leu 100 105 110 Ala Thr Gly Glu Glu Leu Pro Glu Leu Thr Leu Leu Thr Thr Leu Leu 115 120 125 Glu Gly Pro Gly Asp Lys Thr Gln Pro Pro Glu Glu Glu Glu Thr Leu Ser 130 135 140 Gln Ala Pro Glu Ser Glu Glu Glu Gln Lys Lys Lys Ala Leu Glu Arg 145 150 155 160 Ser Met Tyr Val Leu Ser Glu Leu Val Glu Thr Glu Lys Met Tyr Val 165 170 175 Asp Asp Lep Glu Gly Gln Ile Val Glu Gly Tyr Met Ala Thr Met Ala Ala 180 185 190 Gln Gly Val Pro Glu Ser Leu Arg Gly Arg Asp Arg Ile Val Phe Gly 195 200 205 Asn Ile Gln Gln Ile Tyr Glu Trp His Arg Asp Tyr Phe Leu Gln Glu 210 215 220 220 Leu Gln Arg Cys Leu Lys Asp Pro Asp Trp Leu Ala Gln Leu Phe Ile 225 230 235 240 Lys His Glu Arg Arg Leu His Met Tyr Val Val Tyr Cys Gln Asn Lys 245 250 255 Pro Lys Ser Glu His Val Val Ser Glu Phe Gly Asp Ser Tyr Phe Glu 260 265 270 Glu Leu Arg Gln Gln Leu Gly His Arg Leu Gln Leu Asn Asp Leu Leu 275 280 285 Ile Lys Pro Val Gln Arg Ile Met Lys Tyr Gln Leu Leu Leu Lys Asp 290 295 300 Phe Leu Lys Tyr Tyr Asn Arg Ala Gly Met Asp Thr Ala Asp Leu Glu 305 310 315 320 Gln Ala Val Glu Val Met Cys Phe Val Pro Lys Arg Cys Asn Asp Met 325 330 335 Met Thr Leu Gly Arg Leu Arg Gly Phe Glu Gly Lys Leu Thr Ala Gln 340 345 350 Gly Lys Leu Leu Gly Gln Asp Thr Phe Trp Val Thr Glu Pro Glu Ala 355 360 365 Gly Gly Leu Leu Ser Ser Arg Gly Arg Glu Arg Arg Val Phe Leu Phe 370 375 380 Glu Gln Ile Ile Ile Phe Ser Glu Ala Leu Gly Gly Gly Val Arg Gly 385 390 395 400 Gly Thr Gln Pro Gly Tyr Val Tyr Lys Asn Ser Ile Lys Val Ser Cys 405 410 415 Leu Gly Leu Glu Gly Asn Leu Gln Gly Asp Pro Cys Arg Phe Ala Leu 420 425 430 Thr Ser Arg Gly Pro Glu Gly Gly Ile Gln Arg Tyr Val Leu Gln Ala 435 440 445 Ala Asp Pro Ala Ile Ser Gln Ala Trp Ile Lys His Val Ala Gln Ile 450 455 460 Leu Glu Ser Gln Arg Asp Phe Leu Asn Ala Leu Gln Ser Pro Ile Glu 465 470 475 480 Tyr Gln Arg Arg Glu Ser Gln Thr Asn Ser Leu Gly Arg Pro Arg Gly 485 490 495 Pro Gly Val Gly Ser Pro Gly Arg Ile Arg Leu Gly Asp Gln Ala Gln 500 505 510 Gly Ser Thr His Thr Pro Ile Asn Gly Ser Leu Pro Ser Leu Leu Leu 515 520 525 Ser Pro Lys Gly Glu Val Ala Arg Ala Leu Leu Pro Leu Asp Lys Gln 530 535 540 Ala Leu Gly Asp Ile Pro Gln Ala Pro His Asp Ser Pro Pro Val Ser 545 550 555 560 Pro Thr Pro Lys Thr Pro Pro Cys Gln Ala Arg Leu Ala Lys Leu Asp 565 570 575 Glu Asp Glu Leu 580

(2) Information on SEQ ID NO: 3 (i) Sequence characteristics: (A) Sequence length: 705 base pairs (B) Sequence type: nucleic acid (C) Number of strands: single (D) Topology: linear (ii) Molecular type: cDNA (xi) Description of sequence: SEQ ID NO: 3: GACTTGGGGC AGATTGTGGA GGGTTATATG GCCACCATGG CTGCTCAGGG GGTCCCCGAG 60 AGTCTTCGAG GCCGTGACAG GATTGTGTTT GGGAATATCC AGCAAATCGA TGA TGA GTC TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA TGA ATGA TGA TGA TGA TGA TGA TGA ATGA TGA GAATAAGCCC 240 AAGTCAGAGC ATGTGGTGTC AGAGTTTGGG GACAGCTACT TTGAGGAGCT CCGGCAGCAG 300 CTGGGGCACC GCCTGCAGCT GAACGACCTC CTCATCAAAC CTGTGCAGCG GATCATGAAA 360 TACCAGCTGC TGCTCAAGGA TTTTCTCAAG TATTACAATA GAGCTGGGAT GGATACTGCA 420 GACCTAGAGC AAGCTGTGGA GGTCATGTGC TTTGTGCCCA AGCGCTGCAA CGATATGATG 480 ACGCTGGGGA GATTGCGGGG ATTTGAGGGC AAACTGACTG CTCAGGGGAA GCTCTTGGGC 540 CAGGACACTT TCTGGGTCAC CGAGCCTGAG GCTGGAGGGC TGCTGTTTTC CCG AGGTCGA 600 GAGAGGCGCG TTTTCCTCTT TGAGCAAATC ATCATCTTCA GTGAAGCCCT GGGAGGAGGA 660 GTNAGAGGTG GAACAAAGCC TGGATATGTA TTACAAGACC AGCAT 705

(2) Information on SEQ ID NO: 4 (i) Sequence characteristics: (A) Sequence length: 235 amino acids (B) Sequence type: amino acids (C) Number of chains: one (D) Topology : Linear (ii) Molecular type: peptide (xi) Description of sequence: SEQ ID NO: 4: Asp Leu Gly Gln Ile Val Glu Gly Tyr Met Ala Thr Met Ala Ala Gln 1 5 10 15 Gly Val Pro Glu Ser Leu Arg Gly Arg Asp Arg Ile Val Phe Gly Asn 20 25 30 Ile Gln Gln Ile Tyr Glu Trp His Arg Asp Tyr Phe Leu Gln Glu Leu 35 40 45 Gln Arg Cys Leu Lys Asp Pro Asp Trp Leu Ala Gln Leu Phe Ile Lys 50 55 60 His Glu Arg Arg Leu His Met Tyr Val Val Tyr Cys Gln Asn Lys Pro 65 70 75 80 Lys Ser Glu His Val Val Ser Glu Phe Gly Asp Ser Tyr Phe Glu Glu 85 90 95 Leu Arg Gln Gln Leu Gly His Arg Leu Gln Leu Asn Asp Leu Leu Ile 100 105 110 Lys Pro Val Gln Arg Phe Met Lys Tyr Gln Leu Leu Leu Lys Asp Phe 115 120 125 Leu Lys Tyr Tyr Asn Arg Ala Gly Met Asp Thral Ala Asp Leu Glu Gln 130 135 140 Ala Va l Glu Val Met Cys Phe Val Pro Lys Arg Cys Asn Asp Met Met 145 150 155 160 Thr Leu Gly Arg Leu Arg Gly Phe Glu Gly Lys Leu Thr Ala Gln Gly 165 170 175 Lys Leu Leu Gly Gln Asp Thr Phe Trp Val Thr Glu Pro Glu Ala Gly 180 185 190 Gly Leu Leu Phe Ser Arg Gly Arg Glu Arg Arg Val Phe Leu Phe Glu 195 200 205 Gln Ile Ile Ile Phe Ser Glu Ala Leu Gly Gly Gly Val Arg Gly Gly 210 215 220 Thr Lys Pro Gly Tyr Val Leu Gln Asp Gln His 225 230 235

(2) Information on SEQ ID NO: 5 (i) Sequence characteristics: (A) Sequence length: 2088 base pairs (B) Sequence type: nucleic acid (C) Number of strands: 1 (D) topology: the type of linear (ii) molecular: wherein the cDNA (xi) SEQ: SEQ ID NO 5: ACGAGCCCGG CGCCGTCCCC GCCCCGCCCC CCGTGATTCC CCCTGCATGG CCGGCCCGGG 60 TGGGGGGCGC GGGGGGGCCC GGGCGCCATG CGGGGGGGGC ACAAAGGGGG TCGCTGTGCC 120 TGTCCCCGTG TGATCCGAAA AGTGCTGGCA AAATGCGGCT GCTGCTTCGC CCGGGGGGGA 180 CGTGAATCCT ATTCCATTGC GGGCAGTGAG GGGAGTATAT CGGCTTCTGC TGCCTCCGGT 240 CTGGCTGCCC CCTCTGGCCC CAGCTCTGGC CTCAGCTCTG GCCCCTGTTC CCCAGGCCCC 300 CCAGGGCCCG TCAGTGGCCT GAGGAGATGG TTGGATCATT CCAAACATTG TCTCAGTGTG 360 GAAACTGAGG CAGACAGTGG TCAGGCAGGA CCATATGAGA ACTGGATGTT GGAGCCAGCT 420 CTAGCCACAG GAGAGGAGCT GCCGGAACTG ACCTTGCTGA CCACACTGTT GGAGGGCCCT 480 GGAGATAAGA CGCAGCCACC TGAAGAGGAG ACTTTGTCCC AAGCCCCTGA GAGTGAGGAG 540 GAACAGAAGA AGAAGGCTCT GGAAAGGAGT ATGTATGTCC TGAGTGAAC T GGTAGAAACA 600 GAGAAAATGT ACGTGGACGA CTTGGGGCAG ATTGTGGAGG GTTATATGGC CACCATGGCT 660 GCTCAGGGGG TCCCCGAGAG TCTTCGAGGC CGTGACAGGA TTGTGTTTGG GAATATCCAG 720 CAAATCTATG AGTGGCACCG AGACTATTTC TTGCAAGAGC TACAACGGTG TCTGAAAGAT 780 CCTGATTGGC TGGCTCAGCT ATTCATCAAA CACGAGCTCC GGCAGCAGCT GGGGCACCGC 840 CTGCAGCTGA ACGACCTCCT CATCAAACCT GTGCAGCGGA TCATGAAATA CCAGCTGCTG 900 CTCAAGGATT TTCTCAAGTA TTACAATAGA GCTGGGATGG ATACTGCAGA CCTAGAGCAA 960 GCTGTGGAGG TCATGTGCTT TGTGCCCAAG CGCTGCAACG ATATGATGAC GCTGGGGAGA 1020 TTGCGGGGAT TTGAGGGCAA ACTGACTGCT CAGGGGAAGC TCTTGGGCCA GGACACTTTC 1080 TGGGTCACCG AGCCTGAGGC TGGAGGGCTG CTGTCTTCCC GAGGTCGAGA GAGGCGCGTC 1140 TTCCTCTTTG AGCAAATCAT CATCTTCAGT GAAGCCCTGG GAGGAGGAGT GAGAGGTGGA 1200 ACACAGCCTG GATATGTATA CAAGAACAGC ATTAAGGTGA GCTGCCTGGG ACTGGAGGGG 1260 AACCTCCAAG GTGACCCTTG CCGCTTTGCA CTGACCTCCA GAGGGCCAGA GGGTGGGATC 1320 CAGCGCTATG TCCTGCAGGC TGCAGACCCT GCTATCAGTC AGGCCTGGAT CAAGCATGTG 1380 GCTCAGATCT TGGAGAGCCA ACGGGACTTC CTCAACGCAT TGCAGTCACC CATTGAGTAC1440 CAGAGACGGG AGAGCCAGAC CAACAGCCTG GGGCGGCCAA GAGGGCCTGG AGTGGGGAGC 1500 CCTGGAAGAA TTCGGCTTGG AGATCAGGCC CAGGGCAGCA CACACACACC CATCAATGGC 1560 TCTCTCCCCT CTCTGCTGCT GTCACCCAAA GGGGAGGTGG CCAGAGCCCT CTTGCCACTG 1620 GATAAACAGG CCCTTGGTGA CATCCCCCAG GCTCCCCATG ACTCTCCTCC AGTCTCTCCA 1680 ACTCCAAAAA CCCCTCCCTG CCAAGCCAGA CTTGCCAAGC TGGATGAAGA TGAGCTGTAA 1740 CTGGTGAAAA CCATGGGGGT GGTGCTGACT CAGCCGCCTA TTCCCCAAGG AGCTTCAGGG 1800 CAGTCCTTCT GGCACTGCTC CAGAATTCCT CCTTCTTGGT GTGTCTGGAG GGTGGGCAAG 1860 GCTGGGAGGG ATATCAACTT GGAGGAGAAC ACCTAGACCC AAGGACTTTT TTCTGCCCAA 1920 GGAACACAGT TTCCTTCAGC TCCCATCCCT ATGCATGCAT CATGGTCCCC CCAAAAGGAG 1980 GATATGTGGG TGGGTGGGAG GGCTGGTTCATTGATTTTGG TTGG

(2) Information on SEQ ID NO: 6: (i) Sequence characteristics: (A) Sequence length: 550 amino acids (B) Sequence type: amino acids (C) Number of chains: one (D) Topology : Linear (ii) molecular type: peptide (xi) description of sequence: SEQ ID NO: 6: Met Arg Gly Gly His Lys Gly Gly Arg Cys Ala Cys Pro Arg Val Ile 1 5 10 15 Arg Lys Val Leu Ala Lys Cys Gly Cys Cys Phe Ala Arg Gly Gly Arg 20 25 30 Glu Ser Tyr Ser Ile Ala Gly Ser Glu Gly Ser Ile Ser Ala Ser Ala 35 40 45 Ala Ser Gly Leu Ala Ala Pro Ser Gly Pro Ser Ser Gly Leu Ser Ser 50 55 60 Gly Pro Cys Ser Pro Gly Pro Pro Gly Pro Val Ser Gly Leu Arg Arg 65 70 75 80 Trp Leu Asp His Ser Lys His Cys Leu Ser Val Glu Thr Glu Ala Asp 85 90 95 Ser Gly Gln Ala Gly Pro Tyr Glu Asn Trp Met Leu Glu Pro Ala Leu 100 105 110 Ala Thr Gly Glu Glu Leu Pro Glu Leu Thr Leu Leu Thr Thr Leu Leu 115 120 125 Glu Gly Pro Gly Asp Lys Thr Gln Pro Pro Glu Glu Glu Glu Thr Leu Ser 130 135 140 Gln Al a Pro Glu Ser Glu Glu Glu Gln Lys Lys Lys Ala Leu Glu Arg 145 150 155 160 Ser Met Tyr Val Leu Ser Glu Leu Val Glu Thr Glu Lys Met Tyr Val 165 170 175 Asp Asp Leu Gly Gln Ile Val Glu Gly Tyr Met Ala Thr Met Ala Ala 180 185 190 Gln Gly Val Pro Glu Ser Leu Arg Gly Arg Asp Arg Ile Val Phe Gly 195 200 205 Asn Ile Gln Gln Ile Tyr Glu Trp His Arg Asp Tyr Phe Leu Gln Glu 210 215 220 220 Leu Gln Arg Cys Leu Lys Asp Pro Asp Trp Leu Ala Gln Leu Phe Ile 225 230 235 240 Lys His Glu Leu Arg Gln Gln Leu Gly His Arg Leu Gln Leu Asn Asp 245 250 255 Leu Leu Ile Lys Pro Val Gln Arg Ile Met Lys Tyr Gln Leu Leu Leu Leu 260 265 270 Lys Asp Phe Leu Lys Tyr Tyr Asn Arg Ala Gly Met Asp Thr Ala Asp 275 280 285 Leu Glu Gln Ala Val Glu Val Met Cys Phe Val Pro Lys Arg Cys Asn 290 295 300 Asp Met Met Thr Leu Gly Arg Leu Arg Gly Phe Glu Gly Lys Leu Thr 305 310 315 320 Ala Gln Gly Lys Leu Leu Gly Gln Asp Thr Phe Trp Val Thr Glu Pro 325 330 335 Glu Ala Gly Gly Leu Leu Ser Ser Arg Gly Arg Glu Arg Arg Val Phe 340 345 350 Leu Ph e Glu Gln Ile Ile Ile Phe Ser Glu Ala Leu Gly Gly Gly Val 355 360 365 Arg Gly Gly Thr Gln Pro Gly Tyr Val Tyr Lys Asn Ser Ile Lys Val 370 375 380 Ser Cys Leu Gly Leu Glu Gly Asn Leu Gln Gly Asp Pro Cys Arg Phe 385 390 395 400 Ala Leu Thr Ser Arg Gly Pro Glu Gly Gly Ile Gln Arg Tyr Val Leu 405 410 415 Gln Ala Ala Asp Pro Ala Ile Ser Gln Ala Trp Ile Lys His Val Ala 420 425 430 Gln Ile Leu Glu Ser Gln Arg Asp Phe Leu Asn Ala Leu Gln Ser Pro 435 440 445 Ile Glu Tyr Gln Arg Arg Glu Ser Gln Thr Asn Ser Leu Gly Arg Pro 450 455 460 Arg Gly Pro Gly Val Gly Ser Pro Gly Arg Ile Arg Leu Gly Asp Gln 465 470 475 480 Ala Gln Gly Ser Thr His Thr Pro Ile Asn Gly Ser Leu Pro Ser Leu 485 490 495 Leu Leu Ser Pro Lys Gly Glu Val Ala Arg Ala Leu Leu Pro Leu Asp 500 505 510 Lys Gln Ala Leu Gly Asp Ile Pro Gln Ala Pro His Asp Ser Pro Pro 515 520 525 Val Ser Pro Thr Pro Lys Thr Pro Pro Cys Gln Ala Arg Leu Ala Lys 530 535 540 Leu Asp Glu Asp Glu Leu 545 550

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 48/00 ABN A61K 48/00 ABN ACJ ACJ ADP ADP ADU ADU ACU C07K 14/47 C07K 14/47 16/18 16/18 19 / 00 19/00 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 5/10 C12P 21/02 C C12P 21/02 C12Q 1/02 C12Q 1/02 G01N 33/15 Z G01N 33/15 33/50 P 33/50 T 33/53 D 33/53 C12P 21/08 // C12P 21/08 C12N 5/00 BC (71) Applicant 591002957 SmithKline Beecham Corporation SMITHKLINE BEECHAM CORPORATION Swedland Row, King of Prussia, 19406-0939, PA, USA No. 709 (72) Inventor Antoine Michel Alain Brill 35762 Saint-Gregoire, Rue du Chesnay Beauregard No. 4, Bouute Postal 58, Smith Clin Beauchamp Laboratoire Falmatique (72) Inventor Thierry Paul Gierrard Carmel France 35762 Saint-Gregoire, Rue du Chesnay Beauregard No. 4, Boute Postal 58, SmithKline Beecham Laboratoire Falmasartique (72) Inventor Mark Mark Robert Haar United States 19406 King of Prussia, Pennsylvania, Swedland Road No. 709, Smith Klein Be Cham Pharmaceuticals (72) Inventor Isabel Marie-Leger France 35762 Saint-Gregoire, New du Chesnay Beauregard No.4, Boute Postal 58, SmithKline Beecham Laboratoire far Massautique (72) Inventor Michel Louis Suche France 35762 Saint-Gregoire, Le Du Chesnay Beauregard No. 4, Bourte Postal 58, SmithKline Beecham Laboratoire Fal-Masothik (72) Inventor Loranth Natalie Patricia to Louterrier France 35762 Saint-Gregoire, Rie du Chesnay Beauregard 4, Bourate・ Postal 58, SmithKline Beecham Laboratoire Fal Masatoik

Claims (22)

[Claims] 1. An isolated polypeptide comprising: (a) an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2; and (b) at least 70% identity to the amino acid sequence of SEQ ID NO: 6. An isolated polypeptide selected from the group consisting of an isolated polypeptide comprising an amino acid sequence having the same. 2. The isolated polypeptide of claim 1, wherein the amino acid sequences have at least 95% identity. 3. The polypeptide according to claim 1, comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6. 4. The isolated polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6. 5. A polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide having at least 70% identity over the entire length of SEQ ID NO: 2 with the amino acid sequence of SEQ ID NO: 2, and (b) a polynucleotide comprising: At least 70% over the entire length of the amino acid sequence and SEQ ID NO: 6
An isolated polynucleotide selected from the group consisting of a polynucleotide comprising a nucleotide sequence encoding a polypeptide having the above-mentioned identity, or a nucleotide sequence complementary to the isolated polynucleotide. 6. A polynucleotide comprising: (a) a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 and a nucleotide sequence having at least 70% identity over its coding region; and (b) the polypeptide of SEQ ID NO: 6. Or a polynucleotide selected from the group consisting of a polynucleotide comprising a nucleotide sequence having at least 70% identity over its entire coding region to a nucleotide sequence that encodes, or is complementary to, the isolated polynucleotide. Nucleotide sequence. 7. A polynucleotide comprising: (a) a nucleotide sequence having at least 70% identity over the entire length of SEQ ID NO: 1 with the nucleotide sequence of SEQ ID NO: 1; and (b) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 5.
An isolated polynucleotide selected from the group consisting of a polynucleotide comprising a nucleotide sequence having at least 70% identity over the entire length of, or a nucleotide sequence complementary to, the isolated polynucleotide. 8. The isolated polynucleotide according to claim 5, which has at least 95% identity. 9. A polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2; (b) a polynucleotide of SEQ ID NO: 1; and (c) an appropriate library under stringent hybridization conditions. Below, an isolated polynucleotide selected from a polynucleotide or a fragment thereof obtainable by screening with a labeled probe having the sequence of SEQ ID NO: 1, or a nucleotide sequence complementary to the isolated polynucleotide. 10. A polynucleotide comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 6; (b) a polynucleotide of SEQ ID NO: 5; and (c) a suitable library under stringent hybridization conditions. Below, an isolated polynucleotide selected from a polynucleotide or a fragment thereof obtainable by screening with a labeled probe having the sequence of SEQ ID NO: 5, or a nucleotide sequence complementary to the isolated polynucleotide. 11. When in a compatible host cell,
An expression system comprising a polynucleotide capable of producing the polypeptide of claim 1. 12. A host cell comprising the expression system according to claim 11, which expresses the polypeptide according to claim 1, or a membrane thereof. 13. The method of claim 12, comprising culturing the host cell of claim 12 under conditions sufficient to produce the polypeptide of claim 1, and recovering the polypeptide from the culture medium. Manufacturing method. 14. An antibody immunospecific for the polypeptide of claim 1. 15. A screening method for identifying a compound that stimulates or inhibits the function of the polypeptide according to claim 1, comprising: (a) a label that is directly or indirectly bound to the candidate compound; Measuring the binding between the compound and the polypeptide (or a cell or membrane having the polypeptide) or its fusion protein; (b) in the presence of a labeled competitor, the candidate compound and the polypeptide (or the polypeptide Cells or membranes)
Or measuring its binding to a fusion protein; (c) using a suitable detection system for cells or cell membranes containing the polypeptide to determine whether the candidate compound produces a signal resulting from activation or inhibition of the polypeptide. (D) mixing the candidate compound with the solution containing the polypeptide of claim 1 to form a mixture, measuring the activity of the polypeptide in the mixture, and standardizing the activity of the mixture. Or e) detecting in a cell the mRNA encoding the polypeptide and the effect of the candidate compound on the production of the polypeptide using, for example, an ELISA assay. Method. 16. An agonist or antagonist for the polypeptide according to claim 1. 17. A method for use in therapy, comprising: (a) an agonist or antagonist to the polypeptide according to claims 1 to 4; (b) an isolated polynucleotide according to claims 5 to 10; A compound that is a nucleic acid molecule that regulates the expression of a nucleotide sequence encoding the polypeptide according to Item 1. 18. A method for diagnosing a disease or susceptibility of a subject to a disease or susceptibility to a disease associated with the expression or activity of the polypeptide of claim 1 in the subject, comprising: (a) the polypeptide in the genome of the subject; And / or (b) analyzing the presence or amount of expression of said polypeptide in a sample from said subject. 19. An isolated polynucleotide comprising a nucleotide sequence having at least 75% identity over the entire length of SEQ ID NO: 3 with SEQ ID NO: 3; (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3 (C) a polynucleotide of SEQ ID NO: 3; or (d) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 70% identity over the entire length of SEQ ID NO: 4 with the amino acid sequence of SEQ ID NO: 4; An isolated polynucleotide selected from the group consisting of: 20. a polypeptide comprising an amino acid sequence having at least 70% identity over the entire length of SEQ ID NO: 4 with the amino acid sequence of SEQ ID NO: 4; A polypeptide having at least 70% identity over the entire length of SEQ ID NO: 4; (c) a polypeptide comprising the amino acid of SEQ ID NO: 4; (d) a polypeptide that is the polypeptide of SEQ ID NO: 4;
Or (e) a polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 3; a polypeptide selected from the group consisting of: 21. An isolated polynucleotide comprising a nucleotide sequence having at least 70% identity over the entire length of SEQ ID NO: 5 to SEQ ID NO: 5; (b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 5 (C) a polynucleotide of SEQ ID NO: 5; or (d) an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 70% identity over the entire length of SEQ ID NO: 6 with the amino acid sequence of SEQ ID NO: 6; An isolated polynucleotide selected from the group consisting of: 22. a polypeptide comprising: (a) an amino acid sequence having at least 70% identity over the entire length of SEQ ID NO: 6 with the amino acid sequence of SEQ ID NO: 6; (b) an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 6 A polypeptide having at least 70% identity over the entire length of SEQ ID NO: 6; (c) a polypeptide comprising the amino acid of SEQ ID NO: 6; (d) a polypeptide that is the polypeptide of SEQ ID NO: 6;
Or (e) a polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 5; a polypeptide selected from the group consisting of: