JP2002524025A - Human SBPSAPL gene with homology to the prosaposin family of neurotrophic factors - Google Patents

Abstract

  (57) [Summary] Disclosed are SBPSAPL polypeptides and polynucleotides and methods for producing these polypeptides by genetic engineering techniques. Also disclosed are methods of using SBPSAPL polypeptides and polynucleotides in therapy, and diagnostic assays therefor.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to newly identified polypeptides, polynucleotides encoding said polypeptides, agonists, antagonists and / or agonists which may be effective in or for the treatment of said polypeptides and polynucleotides. Or for use in identifying compounds that are inhibitors and methods for producing the polypeptides and polynucleotides.

[0002] currently in the background drug discovery process of the invention fundamentally very reduction has occurred. Because it extends to "functional genomics", that is, high-efficiency (high-throughput) genomic or gene-based biology. This approach as a means of identifying a gene or gene product as a therapeutic target is rapidly replacing a relatively early approach based on "positional cloning". The phenotype, ie, biological function or genetic disease, will be identified and the etiological gene will then be located based on its genetic map location.

[0003] Functional genomics is a highly efficient DNA sequencing technique and a variety of bioinformatics tools for identifying gene sequences of interest from many molecular biology databases currently available. Depends heavily on

SUMMARY OF THE INVENTION The present invention relates to SBPSAPL, particularly SBPSAPL polypeptides and SBPSAPL polynucleotides, recombinant materials, and methods for producing the same. In another embodiment, the invention provides a method for treating neurodegeneration, psychiatric disorders, neuropathy, developmental disorders, pain, chronic pain, rheumatoid arthritis pain, neuralgia, neuropathic pain, seizures,
The present invention relates to methods for using the polypeptides and polynucleotides, including the treatment of ischemia and Wolfram syndrome (hereinafter collectively referred to as "the disease").
In other aspects, the present invention relates to methods of identifying agonists and antagonists / inhibitors using the agents provided by the present invention, and to treating conditions associated with SBPSAPL imbalance using the identified compounds. In yet another embodiment, the invention is directed to a diagnostic assay for detecting a disease associated with inappropriate SBPSAPL activity or SBPSAPL levels.

[0005] In the description a first aspect of the invention, the present invention relates to SBPSAPL polypeptide. Such peptides have at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, and even more, to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Preferably, an isolated polypeptide comprising an amino acid sequence having at least 95% identity, most preferably at least 97-99% identity is included. Such polypeptides include those comprising the amino acid sequence of SEQ ID NO: 2.

[0006] Other peptides of the invention have at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2 Isolated polypeptides having at least 90% identity, even more preferably at least 95% identity, and most preferably at least 97-99% identity are included. Such polypeptides include polypeptides consisting of the amino acid sequence of SEQ ID NO: 2.

[0007] Further peptides of the invention include an isolated polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1.

[0008] The polypeptides of the present invention are believed to be members of the prosaposin family of polypeptides. Therefore, they are of deep interest. This is because both prosaposin and saposin C have neurotrophic activity and stimulate axonal growth in mouse neuroblastoma (NS20Y) cells (O'Brien et al., (1)
994) Proc. Natl. Acad. Sci, 91, 9593-9596). Prosaposin inhibits atrophy of motor neurons in the spinal cord and small sensory neurons in the spinal ganglia,
Promotes sciatic nerve regeneration in vivo (Kotani et al., (1996) J. Neurochem, 66 (5), 20
19-2025). Prosaposin is differentially expressed in the rat sciatic nerve following nerve crush (Gillen et al., (1995), J. Neurosci. Res., 42, 159-171). In addition, prosaposin has been shown to rescue hippocampal neurons from fatal ischemic damage (Sano et al. (1994) Biochem. Biophys. Res. Commun, 204 (2), 994-1000).
In Schwann cells, the 12 amino acid region of the saposin C peptide activates the MAP kinase cascade via a pathway mediated by pertussis-sensitive G tanks, resulting in enhanced myelin lipid synthesis and cell survival (Campana (1
998) FASEB J. 12, 307-314).

[0009] In addition, saposins A, B, C, and D, which are degradation products of prosaposin, are involved in controlling lipid metabolism. Mutations in human prosaposin increase the variants of Tay-Sachs disease, Gaucher disease and metachromatic leukodystrophy, which are disorders of human lipid metabolism. Hereinafter, these properties are referred to as "SBPSAPL activity", "SBPSAPL polypeptide activity" or "SBPSAPL polypeptide activity".
Biological activity of SBPSAPL ". Also included among these activities are the antigenic and immunogenic activities of the aforementioned SBPSAPL polypeptides, especially the antigenic and immunogenic activities of the polypeptide of SEQ ID NO: 2. Preferably, the polypeptide of the present invention exhibits at least one biological activity of SBPSAPL.

[0010] 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 precursor or a fusion protein. It is often advantageous to include additional amino acid sequences, such as secretory or leader sequences, prosequences, sequences useful for purification such as multiple histidine residues, or stable during recombinant production. There are additional arrangements to ensure the performance.

Variants of the above polypeptides, ie, polypeptides that differ from a reference polypeptide by conservative amino acid substitutions (where one residue is replaced by another having similar properties) are also contemplated by the present invention. included. Typical such substitutions are Ala, Val, Leu
Between Ile and Ile; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln;
Occurs between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr. In particular,
Variants in which several, 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1 amino acids are substituted, deleted or added in any combination are preferred.

[0012] The polypeptide of the present invention can be produced by any appropriate method. Such polypeptides include isolated natural polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. It is. The means for producing such polypeptides are well understood in the art.

[0013] In a further aspect, the present invention relates to SBPSAPL polynucleotides. Such a polynucleotide has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, More preferably at least
Included is an isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having 95% identity. In this regard, polypeptides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and those with at least 99% identity are most preferred. . Such polynucleotides include polynucleotides comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding the polypeptide of SEQ ID NO: 2.

[0014] A further polynucleotide of the invention includes at least 70% identity, preferably at least 80% identity over its entire coding region to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2. More preferably, an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity, even more preferably at least 95% identity is included. In this regard, polynucleotides having at least 97% identity are more preferred, but at least 98-
Even more preferred are those with 99% identity, and most preferred are polynucleotides with at least 99% identity.

[0015] Additional polynucleotides of the present invention include SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
At least 70% identity, preferably at least 80
Isolated polynucleotides comprising nucleotide sequences having a percent identity, more preferably at least 90% identity, even more preferably at least 95% identity. In this regard, polynucleotides having at least 97% identity are more preferred, while those with at least 98-99% identity are even more preferred, and polynucleotides with at least 99% identity are most preferred. . Such polynucleotides include a polynucleotide comprising the polynucleotide of SEQ ID NO: 1 and a polynucleotide of SEQ ID NO: 1.

The present invention also provides polynucleotides that are complementary to all of the above polynucleotides.

The nucleotide sequence of SEQ ID NO: 1 is a human prosaposin cDNA (KA Kretz et al., PNAS
87: 2541-2544, 1990). The nucleotide sequence of SEQ ID NO: 1 is cD
A polypeptide consisting of 521 amino acids, that is, an NA sequence, ie, SEQ ID NO: 2
Polypeptide coding sequence (nucleotides 1-1563) encoding the polypeptide of
including. Even though the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is identical to the polypeptide coding sequence contained in SEQ ID NO: 1, the polypeptide of SEQ ID NO: 2 is still duplicated due to the redundancy (degeneracy) of the genetic code. It may be a sequence other than the sequence contained in SEQ ID NO: 1 that encodes. The polypeptide of SEQ ID NO: 2 is structurally related to other proteins of the prosaposin protein family and has homology to human prosaposin cDNA (KA Kretz et al., PNAS 87: 2541-2544, 1990) and / or Or have structural similarity.

Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions / properties as the polypeptides and polynucleotides homologous thereto. Further, preferred polypeptides and polynucleotides of the present invention have at least one SBPSAPL activity.

The present invention also relates to partial or other polynucleotides and polypeptides initially identified prior to the determination of the corresponding full length sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

Thus, in a further aspect, the present invention provides: (a) at least 70% identity, preferably at least 80% identity, more preferably at least 80% identity to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 Is an isolated polynucleotide comprising a nucleotide sequence having at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97-99% identity; At least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity to the nucleotide sequence of SEQ ID NO: 3 over the entire length of # 3 And even more preferably an isolated polynucleotide having a nucleotide sequence having at least 97-99% identity. Reochido or (c) SEQ ID NO: 3 isolated polynucleotides, provides.

The nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby are derived from an Expressed Sequence Tag (EST) sequence. One skilled in the art will appreciate that there will necessarily be some nucleotide sequence reading errors in the EST sequence (Adams, MD et al., Nature 37).
7 (supp) 3, 1995). Accordingly, the nucleotide sequence of SEQ ID NO: 3 and the peptide sequence encoded thereby suffer from the same inherent limitations in sequence accuracy. In addition, the peptide sequence encoded by SEQ ID NO: 3 contains regions identical to the protein with the highest homology or structural similarity, or regions with high homology and / or structural similarity (eg, conservative amino acid differences). In.

The polynucleotides of the present invention can be prepared using standard cloning and screening techniques (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spr.
ing Harbor Laboratory Press, Cold Spring Harbor, NY (1989)) in human adipocytes, fetal stomach, stomach, lung, thymus, fetal thymus, melanoma, colorectal adenocarcinoma, melanocytes, uterus, brain and fetal heart cells. Can be obtained from a cDNA library derived from the mRNA of The polynucleotide of the present invention can be obtained from a natural source such as a genomic DNA library, and can also be synthesized using a commercially available known technique.

When a polynucleotide of the present invention is used for recombinant production of a polypeptide of the present invention, the polynucleotide may include the coding sequence for the mature polypeptide alone, or another coding sequence (eg, a leader or secretory sequence). , Pre-, pro- or pre-pro-protein sequence, or other fusion peptide moiety)
And the coding sequence of the mature polypeptide in the same reading frame. For example, a marker sequence that facilitates purification of the fusion polypeptide can be encoded. In a preferred embodiment of this aspect of the invention, the marker sequence is a pQE vector
(Qiagen, Inc.) and Gentz et al., Proc. Natl. Acad. Sci. USA (19
89) 86-821-824, hexa-histidine peptide, or HA
Tag. Also, the polynucleotide may have 5 'and 3' non-coding sequences, e.g.,
It may include transcribed but not translated sequences, splicing and polyadenylation signals, ribosome binding sites, and mRNA stabilizing sequences.

Further specific examples of the present invention include several, for example, 5 to 10, 1 to 5, 1 to 3,
There is a polynucleotide encoding a polypeptide variant comprising the amino acid sequence of SEQ ID NO: 2, wherein one, two, or one amino acid residue is substituted, deleted or added in any combination. .

A polynucleotide that is identical or substantially identical to the nucleotide sequence contained in SEQ ID NO: 1 can be used to isolate full-length cDNA and genomic clones encoding a polypeptide of the present invention. CDNAs of other genes with high sequence similarity to 1 (including paralogs of human origin and genes encoding orthologs and paralogs from non-human species) And for isolating genomic clones, as hybridization probes for cDNA and genomic DNA, or nucleic acid amplification (PCR)
It can be used as a primer for a reaction. These nucleotide sequences are typically 70%, preferably 80%, more preferably 90%, and most preferably 95% identical to the reference nucleotide sequence. Probes or primers usually contain 15 or more nucleotides, preferably 30 or more, and may have 50 or more nucleotides. Particularly preferred probes are those having a range of 30 to 50 nucleotides. Particularly preferred primers are those having a range of 20 to 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention (including a homologue derived from a species other than human) can be subjected to stringent hybridization using a labeled probe having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof. Screening a suitable library under conditions, the full length cDN containing the polynucleotide sequence
A and a genomic clone. Such hybridization techniques are known to those skilled in the art. Preferred stringent hybridization conditions are 50% formamide, 5 × SSC (150
mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),
Incubate overnight at 42 ° C. in a solution containing 5 × Denhardt solution, 10% dextran sulfate and 20 μg / ml of denatured sheared salmon sperm DNA, then wash the filters at about 65 ° C. in 0.1 × SSC. Thus, the present invention also includes a polynucleotide obtained by screening an appropriate library under stringent hybridization conditions using a labeled probe having the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof.

As will be appreciated by those skilled in the art, the isolated cDNA sequence will often be incomplete, since in many cases the region encoding the polypeptide will be truncated at the 5 ′ end of its cDNA. Would. It is due to the reverse transcriptase, which originally has low "processivity" (a measure of the enzyme's ability to remain attached to the template during polymerization), and the presence of the mRNA template during first-strand cDNA synthesis. The DNA copy cannot be completed.

There are several methods known and available to those skilled in the art for obtaining full-length cDNAs or elongating short-chain cDNAs, for example the cDNA end rapid amplification method (R
ACE) (see, eg, Frohman et al., PNAS USA 85, 8998-9002,
1988). Recent improvements in the above techniques, as demonstrated, for example, by Marathon ^™ technology (Clontech Laboratories Inc.), have greatly simplified the search for longer cDNAs. With Marathon ^™ technology, mRNA extracted from a given tissue
And an "adapter" sequence is ligated to each end. Subsequently, nucleic acid amplification (PCR) is performed using a combination of gene-specific and adapter-specific oligonucleotide primers to amplify the "deleted" 5 'end of the cDNA. next,
"Nested" primers, ie, primers designed to anneal inside the amplification product (typically an adapter-specific primer that anneals further 3 'to the adapter sequence and an additional 5' to the known gene sequence The PCR reaction is repeated using the gene-specific primers. The product of this reaction is then analyzed by DNA sequencing and the product directly linked to the existing cDNA to give the complete sequence, or another full-length sequence using new sequence information for 5 'primer design. By performing PCR, a full-length cDNA can be constructed.

[0029] The recombinant polypeptides of the present invention can be produced from genetically engineered host cells containing the expression system using methods known in the art. Thus, in a further aspect, the invention relates to an expression system containing one or more polynucleotides of the invention, a host cell engineered with said expression system, and the production of a polypeptide of the invention by recombinant techniques. Cell-free translation systems can also be used to produce such proteins using RNA derived from the DNA constructs of the present invention.

For recombinant production, the host cell is genetically engineered to incorporate a polynucleotide expression system of the invention or a portion thereof. Introduction of polynucleotides into host cells is described in Davis et al., Basic Methods in Molecular Biology (1986) and Sambro.
ok et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbo
r Laboratory Press, Cold Spring Harbor, NY (1989) and many other standard laboratory manuals. Suitable such methods include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, bullet loading. Introduction (b
allistic introduction) or infection.

Representative examples of suitable hosts include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2) , Spodoptera Sf9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293)
, Bowes melanoma cells) and plant cells.

[0032] A wide variety of expression systems can be used. Such expression systems include, in particular, systems derived from chromosomes, episomes, and viruses, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion factors, yeast chromosomal elements, viruses (eg, baculovirus, SV40 Such as papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, and retrovirus), and vectors derived from combinations thereof, such as plasmids such as cosmids and phagemids and bacteriophage genetics. Some come from elements. These expression systems may contain control sequences that regulate as well as cause expression. Generally, any system or vector that can maintain, augment, and express a polynucleotide for the production of a polypeptide in a host can be used. Sambrook et al., Molecular Clon
ing: The appropriate nucleotide sequence can be inserted into the expression system by any of the commonly used techniques known in the art, as described in the A Laboratory Manual (supra). Appropriate secretion signals can be incorporated into the polypeptide of interest to secrete the translated protein into the endoplasmic reticulum lumen, into the periplasmic space, or into the extracellular environment. These signals may be endogenous to the polypeptide of interest or heterologous signals.

When trying to express a polypeptide of the present invention for use in a screening assay, it is generally preferred that the polypeptide be produced on the surface of cells. If so, the cells are harvested prior to use in the screening assay.
If the polypeptide is secreted into the medium, the medium is recovered to recover and purify the polypeptide. If produced intracellularly, it is necessary to first lyse the cell and then recover the polypeptide.

To recover and purify the polypeptide of the present invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Known methods including chromatography, hydroxylapatite chromatography, and lectin chromatography can be used. Most preferably, high performance liquid chromatography is used for purification. When the polypeptide is denatured during intracellular synthesis, isolation and / or purification, it is possible to restore the active conformation using known techniques for regenerating the protein.

The present invention also relates to the use of the polynucleotide of the present invention as a diagnostic. Detection of a variant form of the gene characterized by the polynucleotide of SEQ ID NO: 1 associated with a dysfunction may result in a disease resulting from under-expression, over-expression or spatially or temporally altered expression of the gene or a disease thereof. It will provide a diagnostic tool that can be added to or make a diagnosis of susceptibility. Individuals with mutations in the gene can be found at the DNA level by various techniques.

The nucleic acid for diagnosis can be obtained from a subject's cells, such as blood, urine, saliva, tissue biopsy or autopsy material. Genomic DNA may be used directly for detection, or may be enzymatically amplified using PCR or other amplification methods prior to analysis. RNA or cDNA can be used in a similar manner. Deletion and insertion mutations can be detected by a change in the size of the amplified product when compared to the normal genotype. Point mutations can be identified by hybridizing the amplified DNA to a labeled SBPSAPL nucleotide sequence. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of the DNA fragments on gels with or without denaturing agents, or by direct DNA sequencing (eg, Myers et al., Science (1985)). 230: 1242). Sequence changes at specific positions can also be confirmed by nuclease protection assays (eg, RNase and S1 protection) or chemical cleavage methods (Cotton et al.,
Proc. Natl. Acad. Sci. USA (1985) 85: 4397-4401). In another embodiment, for example, to perform efficient screening for gene mutations, SBPSAPL
An array of oligonucleotide probes comprising a nucleotide sequence or a fragment thereof (a
rray) can be constructed. Array techniques are known and have general applicability and have been used to solve various molecular genetics problems, including gene expression, genetic linkage and genetic variability (eg, M Chee et al., Science, Vol.
274, pp. 610-613 (1996)).

The diagnostic assay is performed by detecting a mutation in the SBPSAPL gene by the method described above.
A method is provided for diagnosing or determining susceptibility to the disease. In addition, the disease can be diagnosed by a method of measuring an abnormal decrease or increase in the level of polypeptide or mRNA from a sample obtained from a subject. Decreased or increased expression can be determined by methods known in the art for quantifying polynucleotides, such as nucleic acid amplification (
Examples: PCR, RT-PCR), RNase protection, Northern blotting, and other hybridization methods. Assays for measuring the level of a protein, such as a polypeptide of the present invention, in a sample obtained from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, western blot analysis, ELISA assays and the like.

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

It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component. Such kits are particularly useful for diseases or disorders such as neurodegeneration, psychiatric disorders, neuropathy, developmental disorders, pain, chronic pain, rheumatoid arthritis pain, neuralgia, neuropathic pain, seizures, ischemia and Wolfram syndrome. It is useful in diagnosing susceptibility to illness.

[0040] The nucleotide sequence of the present invention is also useful for chromosome identification. This sequence can target a specific location on an individual human chromosome and hybridize to that specific location. Creating a chromosomal map of related sequences according to the present invention is an important first step in correlating these sequences with gene-related diseases. Once a sequence has been mapped to a precise chromosomal location, the physical location of that sequence on that chromosome can be correlated with genetic map data. This type of data is described, for example, by V. McKusick, Mendelian Inheritance in Man (Johns Hopkins Unive
rsity (available online from the Welch Medical Library). Thereafter, the relationship between the gene mapped to the same chromosomal region and the disease is confirmed by linkage analysis (co-inheritance of physically adjacent genes).

[0041] Differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation may be responsible for the disease.

The gene of the present invention maps to human chromosome 4p16D4S394.

[0043] The nucleotide sequences of the present invention are also useful for tissue localization. Such techniques enable the determination of the expression pattern of a human SBPSAPL polypeptide in a tissue by detecting the mRNA encoding the polypeptide. These techniques include in situ hybridization techniques and nucleotide amplification techniques such as, for example, PCR. Such techniques are known to those skilled in the art. The results of these studies provide an indication of the normal function of the polypeptide in an organism. further,
A comparative study of the normal expression pattern of human SBPSAPL mRNA with the expression pattern of mRNA encoded by the human SBPSAPL gene has shown that mutant human SBPS
It provides useful insight into the role of APL polypeptides or of inappropriate expression of normal human SBPSAPL polypeptides. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature.

The polypeptide of the present invention, a fragment or an analog thereof, or a cell expressing the same can also be used as an immunogen for producing an antibody immunospecific for the polypeptide of the present invention. By "immunospecific" is meant that the antibody exhibits a substantially higher affinity for the polypeptides of the present invention than its affinity for other related polypeptides in the prior art.

[0045] Antibodies to the polypeptides of the present invention can be prepared in animals (using conventional protocols).
(Preferably a non-human animal) by administering a fragment, analog or cell containing the polypeptide or epitope. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. For example, the hybridoma technique (Kohler, G. and Milstein, C., Nature
(1975) 256: 495-497), trioma technique, human B cell hybridoma technique (Kozb
or et al., Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Co
le et al., Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss,
Inc., 1985).

To generate single chain antibodies to a polypeptide of the invention, US Pat. No. 4,946,
Methods for preparing single chain antibodies as described in US Pat. No. 778 can be adapted. Also, transgenic mice or other organisms, including other mammals, can be used to express humanized antibodies.

Using the antibody described above, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography.

[0048] Antibodies to the polypeptides of the present invention may be used, inter alia, for the treatment of the aforementioned diseases.

In a further aspect of the present invention, the present invention provides a polypeptide of the present invention or a fragment thereof and a variety of H or L chain constant regions of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). And a genetically engineered soluble fusion protein comprising: The immunoglobulin is preferably the constant part of the heavy chain of human IgG, especially IgG1, in which case the fusion takes place in the hinge region. In certain instances, the Fc portion can be easily removed by incorporating a cleavage sequence that can be cleaved by blood coagulation factor Xa. Furthermore, the invention relates to methods for the genetic engineering of these fusion proteins and their use in drug screening, diagnosis and therapy. A further aspect of the present invention relates to a polynucleotide encoding such a fusion protein. Examples of fusion protein technology are described in International Patent Application WO94 / 2945
8 and WO94 / 22914.

A further aspect of the invention relates to a method of eliciting an immunological response in a mammal,
The method comprises inoculating a mammal with an antibody and / or a polypeptide of the invention sufficient to generate a T cell immune response, particularly to protect the animal from the disease. Yet another aspect of the invention directs the expression of a polynucleotide encoding a polypeptide of the invention in vivo to elicit an immunological response that produces antibodies that protect the mammal from the disease. A method of eliciting an immunological response in a mammal, comprising providing the polypeptide via a vector.

A further aspect of the present invention relates to immunological / vaccine formulations (compositions) that, when introduced into a mammalian host, elicit an immunological response against the polypeptide of the present invention in the mammal. Contains a polypeptide or polynucleotide of the invention. The vaccine formulation may further comprise a suitable carrier. Since the polypeptide may be broken down in the stomach, it is preferably administered parenterally (eg, by subcutaneous, intramuscular, intravenous or intradermal injection). Formulations suitable for parenteral administration include sterile aqueous and non-aqueous injections, which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and suspensions. There are aqueous and non-aqueous sterile suspensions which may contain agents or thickeners. Such formulations may be presented in single or multi-dose containers (eg, sealed ampules and vials) and may also be stored in a lyophilized condition which requires only the addition of a sterile liquid carrier immediately before use. it can. The vaccine formulation may include an adjuvant system to enhance the immunogenicity of the formulation, such as an oil-in-water adjuvant system or other adjuvant systems known in the art. The dose varies with the specific activity of the vaccine, but can be easily determined by routine experimentation.

[0052] The polypeptides of the invention are involved in one or more biological functions, including one or more pathological conditions, in particular the diseases mentioned above. Therefore, it is desirable to develop screening methods that identify compounds that stimulate or suppress the function of this polypeptide. Thus, in a further aspect, the present invention provides a method of screening compounds to identify compounds that stimulate or suppress the polypeptide. Generally, agonists or antagonists are used for the purpose of treating and preventing the above-mentioned diseases. Compounds can be identified from a variety of sources, such as mixtures of cells, cell-free preparations, chemical libraries and natural products. The agonist, antagonist or inhibitor so identified may optionally be a natural or modified substrate, ligand, receptor, enzyme, etc. of the polypeptide;
It may also be its structural or functional mimetic (Coligan et al., Cur
rent Protocols in Immunology 1 (2): Chapter 5 (1991)).

In the screening method, binding of the candidate compound to the polypeptide of the present invention, or a cell or membrane carrying the polypeptide, or a fusion protein thereof is performed using a label directly or indirectly bound to the candidate compound. And easy to measure. Alternatively, the screening method may use competition with a labeled competitor.
Furthermore, such screening methods can test whether a candidate compound results in a signal resulting from activation or suppression of the polypeptide, using a detection system suitable for cells carrying the polypeptide. . Generally, inhibitors of activation are assayed in the presence of a known agonist to determine the effect of the presence of the candidate compound on activation by the agonist. Constitutively active polypeptides are used in screening methods for inverse agonists or inhibitors by examining whether a candidate compound inhibits activation of a polypeptide in the absence of an agonist or inhibitor. In addition, these screening methods
It simply involves mixing the candidate compound and a solution containing the polypeptide of the invention to form a mixture, measuring the SBPSAPL activity in the mixture, and comparing the SBPSAPL activity of the mixture to a standard. In high-throughput screening assays to identify antagonists of the polypeptides of the invention, fusion proteins such as those made from the Fc portion and SBPSAPL polypeptides described above can also be used (D. Bennett et al., J. Mol. Recognition, 8: 52-58 (1995) and K
Johanson et al., J. Biol. Chem., 270 (16): 9459-9471 (1995)).

In addition, using the polynucleotide, polypeptide or antibody against the polypeptide of the present invention, a screening method for detecting the effect of an additional compound on the production of mRNA or polypeptide in cells may be assembled. it can. For example, using a monoclonal or polyclonal antibody by standard methods known in the art to determine the level of polypeptide secretion or cell binding
ISA assays can be constructed, which can be used to search for substances (also referred to as antagonists or agonists, respectively) that suppress or enhance the production of polypeptides from appropriately engineered cells or tissues.

If a membrane-bound or soluble receptor is present, the polypeptide of the invention may be used to identify such receptor by standard receptor binding methods known in the art. Can be used. Such receptor binding methods include, but are not limited to, ligand binding assays and cross-linking assays, in which the polypeptide is labeled with a radioactive isotope (eg, ¹²⁵ I) or chemically modified (eg, ¹²⁵ I). Eg, biotinylated) or fused to a peptide sequence suitable for detection and purification and incubated with a putative receptor source (cells, cell membranes, cell supernatants, tissue extracts, body fluids, etc.). Other methods include biophysical methods such as surface plasmon resonance and spectroscopy. These screening methods can also be used to identify agonists or antagonists of the polypeptide that compete for binding to the polypeptide or its receptor, if present. Standard methods for performing screening assays are well understood in the art.

Examples of potential antagonists of the polypeptides of the present invention include antibodies, and in some cases, oligonucleotides or proteins (eg, oligonucleotides or proteins) that are closely related to the ligands, substrates, receptors, enzymes, etc., of the polypeptide. , Ligands, substrates, receptors,
Fragments, such as enzymes) or small molecules that bind to a polypeptide of the invention but do not elicit a response (and thus prevent the activity of the polypeptide).

Thus, in other aspects, the present invention identifies agonists, antagonists, ligands, receptors, substrates, enzymes, etc., of the polypeptides of the invention, or compounds that decrease or increase the production of such polypeptides. This kit comprises: (a) a polypeptide of the present invention, (b) a recombinant cell expressing the polypeptide of the present invention, and (c) expressing the polypeptide of the present invention. A cell membrane, or (d) an antibody against the polypeptide of the present invention, wherein said polypeptide is preferably the polypeptide of SEQ ID NO: 2.

It will be appreciated that in such a kit, (a), (b), (c) or (d) is a substantial component.

Those skilled in the art will readily appreciate that the polypeptides of the present invention can also be used in methods for designing agonists, antagonists or inhibitors of the polypeptides based on their structure. This method comprises the steps of (a) first analyzing the three-dimensional structure of the polypeptide, (b) assuming the three-dimensional structure of a likely reactive or binding site of an agonist, antagonist or inhibitor; Synthesizing a candidate compound that is expected to bind or react with the selected reaction site or binding site, and (d) determining whether the candidate compound is in fact an agonist, antagonist or inhibitor. It will be further appreciated that this is usually an interactive process.

[0060] In a further embodiment, the present invention relates to any of an over- and under-expression of SBPSAPL polypeptide activity, eg, neurodegeneration, psychiatric disorders, neuropathy,
Methods of treating abnormal conditions such as developmental disorders, pain, chronic pain, rheumatoid arthritis pain, neuralgia, neuropathic pain, stroke, ischemia and Wolfram syndrome.

If the activity of the polypeptide is in excess, several approaches are available. One approach is to suppress the function of the polypeptide, for example, by blocking the binding of ligands, substrates, receptors, enzymes, etc., or by reducing an abnormal condition by suppressing a second signal. Administering an inhibitor compound (antagonist) as described above to a patient in an amount effective to do so with a pharmaceutically acceptable carrier. In another approach, soluble forms of the polypeptide that are still capable of binding ligands, substrates, enzymes, receptors, etc., in competition with the endogenous polypeptide can be administered. A typical example of such a competitor is a fragment of the SBPSAPL polypeptide.

[0062] In yet another approach, expression of the gene encoding endogenous SBPSAPL polypeptide can be suppressed using expression blocking techniques. These known techniques require the use of antisense sequences produced in the body or administered separately (eg, Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (oligodeoxynucleotides as antisense inhibitors of gene expression), CRC
Press, Boca Raton, FL (1988), O'Connor, J. Neurochem (1991) 56: 560). Alternatively, oligonucleotides can be provided that form a triple helix ("triplex") with this gene (e.g., Le
e et al., Nucleic Acids Res (1979) 6: 3073; Cooney et al., Science (1988) 241: 456;
Dervan et al., Science (1991) 251: 1360). These oligomers can be administered as such, or the relevant oligomer can be expressed in vivo. Synthetic antisense or triple helix oligonucleotides may have modified bases or modified backbones. In the latter example,
Methyl phosphonate, phosphorothioate or peptide nucleic acid backbones are included. Such backbones are incorporated into antisense or triple helix oligonucleotides, which are known in the art, to protect against nuclease degradation. Antisense or triple helix molecules synthesized with these or other modified backbones also form part of the invention.

Further, expression of the human SBPSAPL polypeptide can be inhibited using a ribozyme specific for the human SBPSAPL mRNA sequence. Ribozymes are natural or synthetic RNAs having catalytic activity (eg, Usman, N et al., Curr. Opin. Struct. B
iol (1996) 6 (4), 527-33). Synthetic ribozyme is human SBPSAPL mRNA
Can be designed to be cleaved at selected locations and to prevent human SBPSAPL mRNA from being translated into a functional protein. Ribozymes can be synthesized using the natural ribose phosphate backbone and natural bases commonly found in RNA molecules. Alternatively, ribozymes may be synthesized using a non-natural backbone to protect against ribonuclease degradation (eg, 2'-O-methyl R
NA), and may contain modified bases.

For treating abnormal conditions related to an under-expression of SBPSAPL and its activity,
Several approaches can be taken. One approach involves administering to a patient a therapeutically effective amount of a compound that activates a polypeptide of the invention (ie, the agonist) together with a pharmaceutically acceptable carrier to alleviate the abnormal condition. It becomes. Alternatively, gene therapy can be used to endogenously produce SBPSAPL in the relevant cells of the patient. For example, a polynucleotide of the invention may be engineered for expression by a replication defective retroviral vector as described above. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. As a result, the packaging cells will produce infectious viral particles containing the gene of interest. In vivo cell manipulation and
These producer cells are administered to a patient for in vivo polypeptide expression. For an overview of gene therapy, see Human Molecular Genetics, T Strachan and AP Rea
d, Chapter 20, Gene Therapy and in BIOS Scientific Publishers Ltd (1996)
See other Molecular Genetic-based Therapeutic Approaches (and references therein). Another approach is to administer a therapeutic amount of a polypeptide of the invention together with a suitable pharmaceutical carrier.

In a further aspect, the invention provides a therapeutically effective amount of a polypeptide (eg, a soluble form of the polypeptide of the invention), an agonist or antagonist peptide, or a small molecule compound in a pharmaceutically acceptable carrier or Pharmaceutical compositions are provided that contain the excipients. Such carriers include, but are not limited to, saline, saline, dextrose, water, glycerol, ethanol, and combinations thereof. The present invention further provides a composition comprising one or more of the above-described compositions of the present invention.
It relates to a pharmaceutical pack and a kit comprising the above container. The polypeptides and other compounds of the present invention may be used alone or in conjunction with other compounds, eg, therapeutic compounds.

The pharmaceutical composition can be adapted to the route of administration, for example by a systemic or an oral route. A suitable form for systemic administration is infusion (injection), typically intravenous.
Other injection routes, such as subcutaneous, intramuscular or intraperitoneal, can also be used. Alternative means of systemic administration include transmucosal and transdermal administration using penetrants such as bile salts, fusidic acid, and other surfactants. In addition, oral administration is possible provided the polypeptide or other compound of the present invention can be formulated as an enteric coating or capsule. These compounds may be administered topically and / or localized in the form of an ointment, paste, gel or the like.

The required dosage range depends on the choice of peptide or other compound of the invention, the route of administration,
It depends on the nature of the formulation, the condition of the patient and the judgment of the physician. However, suitable dosages range from 0.1 to 100 μg / kg of patient weight. Given the variety of compounds available and the differing efficiencies of the routes of administration, it is expected that the required dosage will vary widely. For example, oral administration would be expected to require higher dosages than intravenous administration. Such variation in dosage level can be adjusted using standard empirical optimization procedures, as is well understood in the art.

The polypeptide used for the treatment can also be produced in the body of a patient in a treatment called “gene therapy” as described above. For example, cells from a patient
It is genetically engineered ex vivo by a polynucleotide such as DNA or RNA encoding the polypeptide, for example, using a retroviral plasmid vector. Thereafter, these cells are introduced into the patient.

[0069] Polynucleotide and polypeptide sequences provide a valuable source of information in identifying alternative sequences having similar homology. This involves storing such sequences in a computer readable medium and then using the stored data to perform GCC and LaC
Searching the sequence database is greatly facilitated by known search tools such as those in the sergene software package. Accordingly, in a further aspect, the present invention provides a computer readable medium having stored thereon a polynucleotide comprising the sequence of SEQ ID NO: 1 and / or a polypeptide sequence encoded thereby.

The following definitions are intended to facilitate understanding of terms often used in the above description.

As used herein, “antibody” includes polyclonal and monoclonal antibodies, chimeric antibodies, single-chain antibodies, humanized antibodies, as well as Fab fragments, including Fab or the products of other immunoglobulin expression libraries. It is.

“Isolated” means altered “by the hand of man” from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or separated from its original environment, or both.
For example, a polynucleotide or polypeptide naturally occurring in the body of a living animal is not "isolated", but a polynucleotide or polypeptide separated from its naturally occurring co-localized material is described herein. As used herein, "isolated".

“Polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which is unmodified RNA or DNA
Or modified RNA or DNA. “Polynucleotide” includes, but is not limited to, single- and double-stranded DNA, DNA in which single- and double-stranded regions are mixed, single- and double-stranded RNA, single-stranded RNA with mixed region and double-stranded region, hybrid molecule containing DNA and RNA (may be single-stranded or more typically double-stranded, where single-stranded and double-stranded regions are mixed May be included). In addition, "polynucleotide" may be RNA or D
NA or a triple-stranded region consisting of both RNA and DNA. The term "polynucleotide" also refers to DNA or RNA containing one or more modified bases.
And DNA having a backbone modified for stability or for other reasons
Also includes NA. "Modified" bases include, for example, tritylated bases and special bases such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides which are commonly found in nature, as well as DNA and RNA chemical forms characteristic of viruses and cells. . "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

“Polypeptide” means a peptide or protein comprising two or more amino acids linked by a peptide bond or a modified peptide bond (ie, a peptide isostere). A "polypeptide" is a short chain (usually referred to as a peptide, oligopeptide or oligomer) and a long chain (commonly referred to as a protein)
Refers to both. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences that have been modified by natural processes, such as post-translational processing, or by any of the chemical modification methods known in the art. Such modifications are elaborated in basic textbooks, more detailed scholarly articles and research literature. Modifications can be made anywhere in the polypeptide, including the peptide backbone, amino acid side chains, amino or carboxyl termini. 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 can be branched for ubiquitination or cyclic, with or without branching. Cyclic, branched or branched cyclic polypeptides can result from post-translation natural processes and can also be produced by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavin, covalent bonding of heme moieties, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, covalent bonding of phosphatidylinositol Crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslink formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination Transfer RNA-mediated addition of amino acids to proteins, such as, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulphation, arginylation, and ubiquitination (
For example, Proteins-Structure and Molecular Properties 2nd Ed., TE Cre
ighton, WH Freeman and Company, New York, 1993; Posttranslational Cova
lent Modification of Proteins, edited by BC Johnson, Academic Press, New York,
Wold, F., Post-translational Protein Modifications: Perspective in 1983
s and Prospects, pgs. 1-12; Seifter et al., “Analysis for protein modificati
ons and nonprotein cofactors ", Meth Enzymol (1990) 182: 626-646; and R
attan et al., “Protein Synthesis: Post-translational Modifications and Aging
", Ann NY Acad Sci (1992) 663: 48-62).

As used herein, a “variant” is 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 a reference polynucleotide. Changes in the nucleotide sequence of this variant
The amino acid sequence of the polypeptide encoded by the reference polynucleotide may or may not be changed. Nucleotide changes can result in amino acid substitutions, deletions, additions, fusions and truncations of the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from a reference polypeptide. In general, the sequences of the reference polypeptide and the variant are generally closely similar and limited to differences so that they will be identical in many regions. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, deletions, or additions in any combination. The amino acid residues to be substituted or added may or may not be those encoded by the genetic code. The variant of the polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be made by mutagenesis methods or by direct synthesis.

“Identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence similarity between polypeptide or polynucleotide sequences, which may optionally be determined by the match between rows of such sequences. . “Identity” and “similarity” can be easily calculated by known methods including, but not limited to, those described in the following documents. (Computational Molecular Biology, Lesk, AM
, Oxford University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, DW, Academic Press, New York, 1993; Comput
er Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG
, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology
, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer,
Gribskov, M. and Devereux, J. Ed., M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J Applied Math (1988) 48: 1073). Preferred methods of 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. Suitable computer programming methods for determining the identity and similarity between two sequences include the GCG program package (Deve
reux, J. et al., Nucleic Acids Research (1984) 12 (1): 387), BLASTP, BLASTN, FA
STA (Atschul, SF et al., J Molec Biol (1990) 215: 403-410), but are not limited thereto. The BLASTP X program is publicly available from NCBI and other sources (BLAST Manual, Altschl, S. et al., NCBI NLM NIH Bethesda, MD 20894: Aotschul
, S. et al., J. Mol. Biol. 215: 403-410 (1990)). The well-known Smith Waterman algorithm may be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: BLOSSUM62 (Hentikoff and Hentikoff, Proc. Natl. Acad.
Sci. USA. 89: 10915-10919 (1992)) Gap penalty: 12 Gap length penalty: 4 A useful program for these parameters is Genet as the "gap" program.
Available publicly from the ics Computer Group, Madison WI. The above parameters are default parameters for peptide comparisons (plus no penalty for terminal gaps).

Preferred parameters for polynucleotide comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) Comparison matrix: match = + 10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3 Available from Genetics Computer Group, Madison WI as a "gap" program.

These parameters are default parameters for nucleic acid comparison.

For example, the polynucleotide sequence of the invention is identical to the reference nucleotide sequence of SEQ ID NO: 1, ie has 100% identity to the reference nucleotide sequence, or an integer to the reference sequence. It can include up to nucleotide variations. Such mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) and additions of at least one nucleotide, wherein the mutations are at the 5 ′ or 3 ′ terminal position of the reference nucleotide sequence, or And may be interspersed individually between nucleotides in the reference sequence, or as one or more contiguous groups within the reference sequence. The number of nucleotide variations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the respective percent identity (divided by 100) value and subtracting the product from the total number of nucleotides in SEQ ID NO: 1, ie, It is obtained by the formula.

N _n ≦ x _n − (x _n × y), where _nn is the number of nucleotide mutations, x _n is the total number of nucleotides in SEQ ID NO: 1, and y is, for example, 70% 0.80, 85% for 0.70, 80%
Is 0.85 for 90%, 0.90 for 90%, 0.95 for 95%, etc., where the non-integer product of _xn and y is rounded down to the closest integer before subtracting the product from _xn . Modification of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 can result in nonsense, missense or frameshift mutations in the coding sequence, followed by alteration of the polypeptide encoded by the polynucleotide after such mutations. .

[0082] Similarly, the polypeptide sequence of the invention is identical to the reference sequence of SEQ ID NO: 2,
That is, it has 100% identity to the reference sequence or has a percent identity of 10
It may contain up to an integer number of amino acid mutations relative to the reference sequence so as to be less than 0%. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative amino acid substitutions) and additions of at least one amino acid, wherein the mutations are at the amino or carboxy terminal position of the reference polypeptide sequence, or It may be anywhere between these terminal positions and may be scattered individually between amino acids in the reference sequence or as one or more contiguous groups within the reference sequence.
The number of amino acid mutations for a given% identity is calculated by multiplying the total number of amino acids in SEQ ID NO: 2 by the respective percentage identity (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2. That is, it is obtained by the following equation.

N _a ≦ x _a − (x _a × y) where na is the number of amino acid mutations, x _a is the total number of amino acids in SEQ ID NO: 2, and y is 0.70 for 70%, for example. 0.80 for 80%, 0.85 for 85%, etc., and the non-integer product of x _a and y is rounded down to the closest integer before subtracting the product from x _a .

“Homolog” is a general term used in the art to indicate a polynucleotide or polypeptide sequence that has a high degree of sequence similarity to a subject sequence. Such affinity can be quantified by measuring the degree of identity and / or similarity between the compared sequences, as described herein above. Included within this generic term are the terms "ortholog", which refers to a polynucleotide or polypeptide that is a functional equivalent of another species of polynucleotide or polypeptide, and contemplates within the same species. There is a "paralog" that means a functionally similar sequence when

“Fusion protein” refers to a protein encoded by two, often unrelated, fused genes or fragments thereof. As an example, EP-A-0 4
64 describes a fusion protein comprising various portions of the constant region of an immunoglobulin molecule and other human proteins or portions thereof. Often, for use in therapy and diagnosis, immunoglobulin Fc as part of a fusion protein
The use of regions is advantageous, for example, to improve pharmacokinetic properties (see, for example, EP-A-0232262). On the other hand, for some uses, it may be desirable to remove the Fc portion after expressing, detecting and purifying the fusion protein.

All publications, including patents and patent application specifications, cited herein are incorporated by reference in their entirety, as if each publication was clearly and individually indicated. It shall be incorporated here.

Sequence information SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3

[Sequence list]

[Procedure amendment]

[Submission date] February 7, 2001 (2001.2.7)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 45/00 A61P 9/10 4C084 48/00 19/02 4C085 A61P 9/10 25/00 4C086 19/02 101 4H045 25/00 29/00 101 101 29/02 29/00 101 43/00 105 29/02 C07K 14/47 43/00 105 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/15 Z C12P 21/02 33/50 Z C12Q 1/68 33/566 G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A 33/566 A61K 37/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT SE), JP (72) Inventor Mikalovich, David C.E.M. 195 EDS Essex, Harrow, The Pinnacles, Cold Harbor Road, Smith Klein Beechham Pharmaceuticals (72) Inventor Evans, Joan, Rachel C.E.M. 19 5 ed Essex, Harlow, The Pinnacles, Cold Harbor Road, Smith Klein Beechham Pharmaceuticals F-term (reference) 2G045 AA25 AA40 DA36 FB01 FB03 4B024 AA01 AA11 BA80 CA04 DA02 EA02 GA11 HA12 HA15 4B063 QA19 QAQQ20 QR56 QS25 QS34 QX02 4B064 AG26 CA10 CA20 CC24 DA01 DA13 4B065 AA90X AA93Y AB01 BA01 CA24 CA44 CA46 4C084 AA02 AA06 AA13 AA17 BA01 BA08 BA22 BA23 MA01 NA14 ZA012 ZA022 ZA082 ZA152 AA12 ZA13 AA12 ZA32 ZA32 AA12 A12 MA01 MA04 NA14 ZA01 ZA02 ZA08 ZA15 ZA39 ZA96 ZB15 ZB21 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA75 EA20 EA21 EA50 FA72 FA74

Claims (18)

[Claims] 1. An isolated polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. 2. The method of claim 1, wherein said amino acid sequences have at least 95% identity.
An isolated polypeptide according to claim 1. 3. The polypeptide according to claim 1, which comprises the amino acid sequence of SEQ ID NO: 2. 4. The isolated polypeptide of SEQ ID NO: 2. 5. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 70% identity to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2, or said isolation. A nucleotide sequence that is complementary to the rendered polynucleotide. 6. An isolated polynucleotide comprising a nucleotide sequence having at least 70% identity to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 over its entire coding region, or the isolated polynucleotide. A nucleotide sequence that is complementary to the released polynucleotide. 7. An isolated polynucleotide comprising a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1, or Complementary nucleotide sequence. 8. The isolated polynucleotide according to any one of claims 5 to 7, wherein said identity is at least 95%. 9. The following (a) to (c): (a) a polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, (b) a polynucleotide of SEQ ID NO: 1, and (c) A polynucleotide obtained by screening a suitable library under stringent hybridization conditions using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, an isolated polynucleotide selected from the group consisting of: Or a nucleotide sequence complementary to the isolated polynucleotide. 10. An expression system comprising a polynucleotide capable of producing the polypeptide of claim 1 when the following expression system is present in a compatible host cell. 11. A host cell comprising the expression system of claim 10, or a membrane thereof expressing the polypeptide of claim 1. 12. A method comprising culturing the host cell of claim 11 under conditions sufficient to produce the polypeptide of claim 1, and recovering the polypeptide from the culture medium. A method for producing the polypeptide according to claim 1. 13. An antibody immunospecific for the polypeptide of claim 1. 14. A screening method for identifying a compound that stimulates or suppresses the function of the polypeptide according to claim 1, comprising: (a) a candidate compound and the polypeptide (or carrying the polypeptide); Measuring the binding to the candidate compound with the label directly or indirectly to the candidate compound and the polypeptide (or carrying the polypeptide). Measuring the binding to the cell or its membrane) or its fusion protein in the presence of a labeled competitor; and (c) determining whether the candidate compound results in a signal resulting from activation or suppression of the polypeptide. (D) examining the candidate compound and the polypeptide according to claim 1 using a detection system suitable for a cell or cell membrane carrying the polypeptide; Preparing a mixture, measuring the activity of the polypeptide in the mixture and comparing the activity of the mixture to a standard, or (e) determining whether the candidate compound Detecting the effect on the production of the encoded mRNA and the polypeptide, for example, using an ELISA assay, the screening method comprising a method selected from the group consisting of: An agonist or antagonist for the polypeptide according to any one of claims 1 to 4. 16. The following (a) to (c): (a) an agonist or antagonist to the polypeptide according to any one of claims 1 to 4, and (b) any one of claims 5 to 9. Or a nucleic acid molecule that modulates the expression of a nucleotide sequence encoding the polypeptide of claim 1. 17. A method for diagnosing a disease associated with the expression or activity of the polypeptide according to claim 1 or a susceptibility to said disease in a subject, comprising: (a) a method for diagnosing the disease in the genome of the subject. Determining whether there is a mutation in the nucleotide sequence encoding the peptide, and / or (b) analyzing the presence or amount of said polypeptide expression in a sample obtained from said subject. The method. 18. The following (a) to (c): (a) an isolated sequence comprising a nucleotide sequence having at least 70% identity to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 (B) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 3, or (c) a polynucleotide of SEQ ID NO: 3.