JP2001521761A - Secreted protein NSL4 - Google Patents

Abstract

  (57) [Summary]   Disclosed are NSL4 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques. Also disclosed are methods of using NSL4 polypeptides and polynucleotides for therapy, and diagnostic assays for such uses.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application claims the benefit of priority to US Provisional Application No. 60 / 065,139, filed Nov. 12, 1997, the disclosure of which is incorporated herein by reference in its entirety. Shall be.

[0002] The present invention relates to newly identified polypeptides, polynucleotides encoding the polypeptides, agonists, antagonists and / or inhibitors that may be effective in therapy and in therapy. The invention relates to the use of the polypeptides and polynucleotides in identifying compounds, and to a method of producing the polypeptides and polynucleotides.

[0003] The drug discovery process is currently undergoing a radical change. This is because it extends to "functional genomics", ie, high-throughput (high-efficiency) genomic or gene-based biology. This approach is rapidly replacing a relatively early approach based on “positional cloning”. A phenotype, a biological function or genetic disease, has been identified,
Subsequently, the etiological gene was identified using the position of the genetic map as a clue.

[0004] Genomics functionality relies heavily on various bioinformatics tools to identify gene sequences that may be of interest from the many molecular biology databases currently available. There remains a need to identify and characterize additional genes and their related polypeptides / proteins as targets for drug discovery.

[0005] Proteins and polypeptides that are naturally secreted into blood, lymph and other bodily fluids, or into cell membranes, are of primary interest in pharmaceutical research and development. The reason for this interest is the relative ease of targeting the protein therapeutic to its site of action (body fluid or cell membrane). The natural pathway of protein secretion into the extracellular space is the endoplasmic reticulum in eukaryotic cells and the inner membrane in prokaryotic cells (Palade, 1975, Science, 189, 347; Milstein, Brownlee, Harrison,
and Mathews, 1972, Nature New Biol., 239, 117; Blobel and Dobberstein,
1975, J. Cell. Biol., 67, 835). On the other hand, the natural pathway for transporting proteins from the outside of the cell to the cytoplasm is not known (except for pinocytosis, which is the mechanism of entry of snake venom toxins into cells). Therefore, targeting protein therapeutics intracellularly is very difficult.

[0006] Secretory proteins and membrane-bound proteins include all peptide hormones and their receptors (insulin, growth hormone, chemokines, cytokines, neuropeptides, integrins, kallikreins, lamins, melanins, sodium excretion-promoting hormones, neuropsin, neurons Tropins, pituitary hormones, pleiotropin, prostaglandins, secretogranins, selectins, thromboglobulins, thymosins), breast and colon cancer gene products, leptin, Obesity gene protein and its receptor, serum albumin, superoxide dismutase, spliceosome protein, 7TM (seven transmembrane type) protein also called G protein-coupled receptor, immunoglobulin Phosphorus, some serine proteinase family (of the blood coagulation cascade proteins, including digestive enzymes, but not limited to), but the like DNase I, but are not limited to.

[0007] Therapeutic agents based on secreted and membrane-bound proteins that are approved by the FDA or foreign authorities include insulin, glucagon, growth hormone,
Coriogonadotropin, follicle-stimulating hormone, luteinizing hormone, calcitonin,
Adrenocorticotropic hormone (ACTH), vasopressin, interleukin, interferon, immunoglobulin, lactoferrin (a variety of products are available from several companies), tissue-type plasminogen activator (Genentech's Alteplase), hyaurolin Hyaulorindase (Wyeth-Ayerst Wydase), Dornase
Alpha (Pulmozyme from Genentech), chymodiactin (Knoll c
hymopapain), alglucerase (Ceredase from Genzyme), streptokinase (Kabikinase from Pharmacia) (Streptase from Astra), and the like, but are not limited thereto.

SUMMARY OF THE INVENTION The present invention relates to NSL4, particularly NSL4 polypeptides and polynucleotides, recombinant materials, and methods for producing the same. In different embodiments, the invention relates to bacterial infections, fungal infections, protozoan and viral infections, especially infections caused by HIV-1 or HIV-2, pain, cancer, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute Heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, and anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and Huntington's disease or Jill Psychiatric and nervous system disorders, including motor abnormalities such as de la Tourette syndrome (hereinafter collectively referred to as "the disorders")
And methods for using said polypeptides and polynucleotides, including the treatment of In other aspects, the present invention relates to methods of identifying agonists and antagonists / inhibitors using the agents provided by the present invention, and treating conditions associated with NSL4 imbalance using the identified compounds. In yet another embodiment, the present invention relates to diagnostic assays for detecting diseases associated with inappropriate NSL4 activity or NSL4 levels.

Description of the Invention In one embodiment, the present invention relates to NSL4 polypeptides. This kind of peptide has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2. Comprises an isolated polypeptide comprising an amino acid sequence having at least 95% identity, most preferably at least 97-99% identity. Such polypeptides include polypeptides consisting of the amino acid sequence of SEQ ID NO: 2.

[0010] Other peptides of the present invention have at least 70% identity, preferably at least 80% identity, more preferably at least 70% 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, 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.

[0011] Further peptides of the invention include an isolated polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1. The polypeptides of the present invention are considered to be secreted and / or membrane-bound polypeptides. Secretory and membrane-bound proteins are of interest because they have an established and proven history as therapeutic targets. clearly,
There is a need to further identify and characterize secreted and membrane-bound proteins that can serve to prevent, ameliorate and cure dysfunction or disease. This dysfunction or disorder includes diabetes, breast, prostate and colon cancer and other malignancies, hypertension and hypotension, obesity, binge eating, anorexia, abnormal growth, asthma, manic depression, dementia,
Includes, but is not limited to delirium, mental retardation, Huntington's disease, Tourette's syndrome, schizophrenia, growth and developmental disorders, mental or sexual developmental disorders, and dysfunctions of the blood cascade leading to stroke is not. The protein of the present invention contains a signal sequence and is useful in further elucidating the mechanism of protein transport that is not yet completely understood, and therefore can be used as a research tool.

These properties are hereinafter referred to as “NSL4 activity” or “NSL4 polypeptide activity” or “NSL4 polypeptide activity”.
The biological activity of NSL4 ". These activities also include the antigenic and immunogenic activities of the NSL4 polypeptide, particularly the polypeptide of SEQ ID NO: 2. Preferably, the polypeptide of the invention exhibits at least one biological activity of NSL4.

[0013] The polypeptides of the 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 often advantageous to include additional amino acid sequences, such as secretory or leader sequences, prosequences, sequences that aid in purification such as multiple histidine residues, or during recombinant production. There are additional sequences to ensure stability.

[0014] Also, the present invention relates to a mutant of the above-mentioned polypeptide, that is, a polypeptide which differs from a reference polypeptide by conservative amino acid substitution (a certain residue is replaced by another residue having similar properties). include. Typical such substitutions are Ala, Val, Le
between u and Ile; between Ser and Thr; between the acidic residues Asp and Glu; between Asn and Gln; between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr. In particular, a mutant 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 is preferable.

[0015] 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.

[0016] In a further aspect, the present invention relates to NSL4 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, Preferably at least 9
An isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide having 5% identity is included. 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.

A further polynucleotide according to the present invention comprises at least 70% of the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 over its entire coding region.
Identity, preferably at least 80% identity, more preferably at least 9%
An isolated polynucleotide comprising a nucleotide sequence having 0% identity, more preferably at least 95% identity, is included. 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 those with at least 99% identity are most preferred. .

[0018] Additional polynucleotides of the present invention include SEQ ID NO: 1 over the entire length of SEQ ID NO: 1.
A nucleotide sequence having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity to a polynucleotide of Isolated polynucleotides are included. 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 those 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 cDNA sequence and includes a polypeptide coding sequence (nucleotide numbers 24-599) encoding a polypeptide consisting of 192 amino acids, which is the polypeptide of SEQ ID NO: 2. Although the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is identical to the polypeptide coding sequence contained in SEQ ID NO: 1, it is still the same as the polypeptide coding sequence of SEQ ID NO: 2 due to the redundancy (degeneracy) of the genetic code. And a sequence other than the sequence contained in SEQ ID NO: 1.

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 NSL4 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 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 a nucleotide sequence having at least 90% identity, more preferably at least 95% identity, most preferably 97-99% identity; (b) relative to the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3 Nucleotides having at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, and most preferably 97-99% identity. (C) the polynucleotide of SEQ ID NO: 3, or (d) SEQ ID NO: 4 over the entire length of SEQ ID NO: 4 At least 70% identity to the amino acid sequence, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97-99% identity. A nucleotide sequence encoding a polypeptide consisting of an amino acid sequence having a sex property, and a polynucleotide of SEQ ID NO: 3.

In addition, the present invention provides (a) at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4 A polypeptide comprising an amino acid sequence having identity, more preferably at least 95% identity, most preferably 97-99% identity; (b) the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4; At least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity, and most preferably 97-99% identity. A polypeptide having an amino acid sequence; (c) a polypeptide comprising an amino acid of SEQ ID NO: 4; Polypeptide is a peptide, and polypeptide encoded by a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 3, to provide.

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). Thus, 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 may be in the same region as the protein with the highest homology or structural similarity, or with high homology and / or
Or regions of structural similarity (eg, conservative amino acid differences).

The polynucleotides of the present invention can be analyzed by standard cloning and screening techniques from a cDNA library derived from mRNA in human fetal hepatocytes by EST analysis (Adams, MD et al., Science (1991) 252: 1651). -1656; Adams, MD
Et al., Nature (1992) 355: 632-634; Adams, MD et al., Nature (1995) 377 Supp: 3-1
74). In addition, the polynucleotide of the present invention is genomic DN
It can be obtained from natural sources, such as the A library, or can be synthesized using commercially available, well-known techniques.

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). Sequence, sequence encoding a pre- or pro- or pre-pro-protein sequence, or other fusion peptide portion) 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 provided by the pQE vector (Qiagen, Inc.) and Gentz et al., Proc. Natl. Acad. Sci. USA
(1989) 86: 821-824, a hexa-histidine peptide, or an HA tag. The polynucleotide may also include 5 'and 3' non-coding sequences, for example, 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 sufficiently 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. To isolate cDNA and genomic clones of other genes with high sequence similarity to, including homologues from non-human species and genes encoding orthologs,
It can be used as a hybridization probe for DNA and genomic DNA, or as a primer for a nucleic acid amplification (PCR) reaction. Typically,
These nucleotide sequences are 70%, preferably 80%, of the reference nucleotide sequence.
%, More preferably 90%, and most preferably 95%. 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.

Polynucleotides encoding the polypeptides of the present invention (including homologs and orthologs from non-human species) can be stringent using a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof. It is obtained by a method comprising the steps of screening an appropriate library under hybridization conditions and isolating a full-length cDNA and a genomic clone containing the polynucleotide sequence. Such hybridization techniques are well-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 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 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 is 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 the polymerization reaction), and the mRNA template during first-strand cDNA synthesis. DNA copy cannot be completed.

There are several methods known and available to those skilled in the art for obtaining full-length cDNA or extending short-chain cDNA, 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 a gene-specific primer that anneals to the side. The product of this reaction is then analyzed by DNA sequencing and the product is directly linked to the existing cDNA to complete the sequence, or another full-length sequence using new sequence information for 5 'primer design. By performing PCR, a full-length cDNA can be constructed.

[0033] The recombinant polypeptides of the present invention can be produced from genetically engineered host cells containing the expression system using methods well known in the art. Thus, in a further aspect, the present invention relates to an expression system containing one or more polynucleotides of the present invention, a host cell engineered with said expression system, and the production of a polypeptide of the present invention by recombinant methods. 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, host cells are genetically engineered to incorporate the polynucleotide expression system of the present invention or portions 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, HEK
293, Bowes melanoma cells) and plant cells.

[0036] A wide variety of expression systems can be used. Such expression systems include, for example,
Systems derived from chromosomes, episomes and viruses, such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion factors, yeast chromosomal elements, viruses (eg, baculovirus, papovavirus such as SV40, vaccinia virus , Adenovirus, fowlpox virus,
Pseudorabies virus, retrovirus) and vectors derived from combinations thereof, for example, those derived from plasmid and bacteriophage genetic elements such as cosmids and phagemids. These expression systems may contain control regions that regulate as well as cause expression. Typically,
Any system or vector capable of maintaining, augmenting, and expressing a polynucleotide for production of a polypeptide in a host may be used. Sambrook et al., Molecu
The appropriate nucleotide sequence can be inserted into the expression system by any of the commonly used and well-known techniques, such as those described in the lar Cloning: 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 it is desired to express a polypeptide of the 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.

For recovering and purifying the polypeptide of the present invention from the recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Well-known methods can be used, including chromatography, hydroxylapatite chromatography and lectin chromatography. 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 well-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 of a gene characterized by a polynucleotide of SEQ ID NO: 1 associated with a dysfunction is useful for diagnosing a disease caused by under-expression, over-expression or altered expression of the gene or susceptibility to the disease. It will provide a diagnostic tool that can be added or diagnosed. Individuals with mutations in the gene can be found at the DNA level by various techniques.

Diagnostic nucleic acids can be obtained from cells of a subject, such as cells from 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. Deletions and insertions 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 NSL4 nucleotide sequence. Perfectly matched sequences and mismatched duplexes can be distinguished by RNase digestion or by differences in melting temperatures. Also,
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 (see, for example, 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 by chemical cleavage (Cotton et al., Pr.
oc. Natl. Acad. Sci. USA (1985) 85: 4397-4401). In another embodiment, an array of oligonucleotide probes containing the NSL4 nucleotide sequence or a fragment thereof can be constructed, eg, for efficient screening of gene mutations. Array techniques are well known, have general applicability, and have been used to solve various molecular genetics problems, including gene expression, genetic linkage and genetic variability (see, for example, M. Chee et al., Science, Vol. 274, p.
pp. 610-613 (1996)).

The diagnostic assay provides a method for diagnosing or determining susceptibility to the disease by detecting a mutation in the NSL4 gene by the method described above. 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 any method known in the art for quantifying polynucleotides, for example, nucleic acid amplification (eg,
PCR, RT-PCR), RNase protection, Northern blotting, and other hybridization methods. Assays that can be used to determine levels 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 assay and the like.

Thus, in another embodiment, the present invention provides: (a) a polynucleotide of the present 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 a kit may be a disease or a susceptibility to a disease, especially bacterial, fungal, protozoan and viral infections, especially infections caused by HIV-1 or HIV-2, pain, cancer, anorexia nervosa, bulimia, asthma , Parkinson's disease,
Acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcer, allergy, benign prostatic hypertrophy, and anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and Huntington's disease or It is useful in diagnosing psychiatric disorders, nervous system disorders, etc., including motor abnormalities such as Jill de la Tourette syndrome.

[0044] The nucleotide sequence of the present invention is also useful for chromosome identification. This sequence can specifically target a particular location on an individual human chromosome and hybridize to that particular 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, in V. McKusick, Mendelian Inheritance in Man (Johns Hopki
(available online at ns University 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).

[0045] 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 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.

Antibodies against the polypeptides of the present invention can be prepared in an animal (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 method (Kohler, G. and Milstein, C., Nature (
1975) 256: 495-497), trioma method, human B cell hybridoma method (Kozbor et al.,
Immunology Today (1983) 4:72) and the EBV-hybridoma method (Cole et al., Mo.
noclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss, Inc., 19
85).

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 above-mentioned antibodies, a clone expressing the polypeptide can be isolated and identified, or the polypeptide can be purified by affinity chromatography.

[0050] Antibodies to the polypeptides of the 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 constant regions of immunoglobulin H or L chains 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) which, when introduced into a mammalian host, elicit an immunological response against the polypeptide of the 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.

[0054] The polypeptides of the present invention are involved in a variety of biological functions, including a number of pathological conditions, particularly the aforementioned diseases. 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. In general,
Agonists or antagonists are used for the purpose of treating and preventing the above 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, and its structural or functional mimetic. (Coligan et al., C
urrent 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 NSL4 activity in the mixture, and comparing the NSL4 activity of the mixture to a standard. In high-throughput screening assays to identify antagonists of the polypeptides of the present invention, fusion proteins such as those made from the Fc portion and NSL4 polypeptides described above can also be used (D. Bennett et al., J. Mol. Recognition, 8: 52-58 (1995) and K.
See Johanson et al., J. Biol. Chem., 270 (16): 9459-9471 (1995)).

Further, 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.

[0057] If membrane-bound or soluble receptors are present, the polypeptides of the invention can be used to identify such receptors 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 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 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 of designing agonists, antagonists or inhibitors of the polypeptide based on its 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 reactive 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.

In a further embodiment, the present invention relates to infections of, for example, bacterial, fungal, protozoan and viral, especially HIV-1 or HIV-2, associated with either over- or under-expression of NSL4 polypeptide activity. Infections, pain, cancer, anorexia nervosa, bulimia, asthma, Parkinson's disease, acute heart failure, hypotension, hypertension, urinary retention, osteoporosis, angina, myocardial infarction, ulcers, allergies, benign prostatic hyperplasia, and anxiety Schizophrenia, manic depression, delirium, dementia, severe mental retardation and Huntington's disease or Jill
Provided are treatments for abnormal conditions such as mental illness and nervous system diseases, including motor abnormalities such as de la Tourette 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 an NSL4 polypeptide.

[0063] In yet another approach, expression of the gene encoding endogenous NSL4 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, Ol
igodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Pre
ss, Boca Raton, FL (1988) O'Connor, J. Neurochem (1991) 56: 560). Alternatively, oligonucleotides that form a triple helix with this gene can be provided (eg, Lee et al., Nucleic Acids Res (1979) 6:
3073; Cooney et al., Science (1988) 241: 456; Dervan et al., Science (1991) 251: 136
0). These oligomers can be administered as such,
Related oligomers can also be expressed in vivo.

For treating abnormal conditions related to an under-expression of NSL4 and its activity, several approaches are also available. 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 produce NSL4 endogenously 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 the R encoding the polypeptide of the invention is isolated.
It is introduced into packaging cells transduced with a retroviral plasmid vector containing NA. As a result, the packaging cells will produce infectious viral particles containing the gene of interest. In vivo cell manipulation and in vivo
These producer cells are administered to a patient for polypeptide expression. For an overview of gene therapy, see Human Molecular Genetics, T Strachan and APR.
ead, Chapter 20, Gene Therapy a in BIOS Scientific Publishers Ltd (1996)
See nd 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 together with 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, 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 is facilitated by storing such sequences in a computer readable medium and then using the stored data to search a sequence database with known search tools such as GCG.
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 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 other products of an immunoglobulin expression library.

“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” refers to 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 called a peptide, oligopeptide or oligomer) and a long chain (commonly called 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, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond. , Cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, 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).

“Variant” as used herein 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 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. A variant of a 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 the affinity between two or more such sequences, as determined by comparing the polypeptide or polynucleotide sequences. In the art, "identity" also means the degree of sequence similarity between polypeptide or polynucleotide sequences, as determined by the match between the strands of such sequences. "Identity" and "similarity" can be calculated without difficulty by a known method, and examples of such a method include, for example, Computational Molecular Biolo
gy, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing:
Informatics and Genome Projects, Smith, DW, Academic Press, New Yo
rk, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and
Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in M
olecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysi
s Primer, Gribskov, M. and Devereux, J. Ed., M Stockton Press, New York,
1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073
(1988), but not limited thereto. Preferred methods for determining identity are designed to give the largest match between the sequences considered. Methods for determining identity and similarity have been compiled into publicly available computer programs. A preferred computer program method for determining the identity and similarity between two sequences is the GCG program package (Deve
reux, J. et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BL
ASTN and FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403-410 (
1990)), but not limited to these. The BLAST X program is generally available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI
NLM NIH Bethesda, MD 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-410.
(1990)). The well-known Smith Waterman algorithm can also be used to determine identity.

[0076] Preferred parameters for comparing polypeptide sequences include:
1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970)
Comparative matrix: BLOSSUM62, Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA, 89: 10915-10919 (1992) Gap penalty: 12 Gap length penalty: 4

A useful program with these parameters is the Genetics Computer Group
(Madison WI) is generally available as a "gap" program. The above parameters are the default parameters for peptide comparison (default paramet
er) (no end gap penalty).

Preferred parameters for comparing polynucleotide sequences include the following: 1) Algorithm: Needleman & Wunsch, J. Mol. Biol. 48: 443-453 (1970); Comparison matrix: match = + 10, Mismatch = 0 Gap penalty: 50 Gap length penalty: 3 A useful program with these parameters is Genetics Computer Group (M
adison WI) as a "gap" program. These are the default parameters for nucleic acid comparison.

By way of example, the polynucleotide sequence of the invention is identical to the reference sequence of SEQ ID NO: 1, ie has 100% identity to the reference sequence, or has a constant It may contain up to an integer number of nucleotide variations. Such mutations are selected from the group consisting of deletions, substitutions (including transitions and transversions) or insertions of at least one nucleotide, such mutations being at the 5 ′ or 3 ′ terminal position of the reference nucleotide sequence, or It may be anywhere between terminal positions, interspersed individually between nucleotides in the reference sequence, or as one or more contiguous groups within the reference sequence. The number of nucleotide mutations
By multiplying the total number of nucleotides of SEQ ID NO: 1 by the absolute ratio of each percent identity value (value divided by 100), and subtracting the product from the total number of nucleotides of SEQ ID NO: 1, ie, n _n ≦ x _n - it can be obtained by (x _n · y). Wherein, n _n is the number of nucleotide alterations, x _n is the total number of nucleotides in SEQ ID NO: 1, y is about 0.85,90% for 0.80,85% for 0.70,80% for 70% e.g. Is 0.90, 95% is 0.95
And the non-integer product of _xn and y is rounded down to the closest integer before subtracting the product from _xn . The alteration of the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 results in a nonsense, missense or frameshift mutation in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such mutation. be able to.

Similarly, a polypeptide sequence of the invention is identical to the reference sequence of SEQ ID NO: 2,
That is, it may have 100% identity to the reference sequence or may include up to a fixed integer number of amino acid mutations to the reference sequence such that the percent identity is less than 100%. Such mutations are selected from the group consisting of deletions, substitutions (including conservative and non-conservative amino acid substitutions) or insertions of at least one amino acid, wherein the mutations are at the amino or carboxy terminal position of the reference polypeptide sequence. , Or anywhere between these terminal positions, interspersed 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 is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by the absolute ratio of each percent identity (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2, It is obtained by the formula. n _a ≦ x _a - in (x _a · y) formula, n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2, y 0.70,80% is about 70% for example 0.80 for 85% for 0.8%
And the non-integer product of x _a and y is rounded down to the closest integer before subtracting the product from x _a .

“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.

Examples Example 1: Identification of signal sequence The polynucleotide sequence of SEQ ID NO: 1 was obtained by modifying a signal peptide selection system (based on Klien et al., PNAS 93: 7108-7113, 1996) from yeast ( Saccharomyces cerevi
siae) using a positive growth screen. Briefly,
This selection takes advantage of the yeast requirement for the enzyme invertase for fermentative growth when sucrose is the sole carbon source. Usually, the invertase enzyme is secreted outside the plasma membrane via its N-terminal signal sequence (amino acids 1-20). When secreted, invertase catalyzes the hydrolysis of sucrose into sugars that yeast can metabolize under fermentation conditions. Yeast strains that lack the SUC2, a gene encoding invertase, are unable to utilize sucrose and therefore do not grow under these conditions. In the signal peptide selection screen, a truncated SUC2 gene lacking the initiation methionine codon (ATG) and signal coding sequence is provided to a yeast strain that otherwise lacks the SUC2 gene. Yeast with such a plasmid will not express or secrete invertase and will therefore not grow. However, the initiation codon and a functional signal sequence
Propagation will occur if given in-frame upstream of truncated SUC2. A library of human cDNA immediately upstream of truncated SUC2 was constructed,
By culturing the yeast containing the NA library on sucrose under fermentation conditions, clones supplied with these signals by cDNA can be selectively grown.

To perform this screening, the yeast strain deficient in the SUC2 gene was
Modified by deletion of the R1 gene (Baudin et al., Nucleic Acids Res. 21; 3329-33
30,1993), the PMR1 gene has been shown to cause high-level secretion of several heterologous proteins (Moir and Davidow, Methods in Enzymol. 194: 491-507,199).
1). The obtained yeast strain SSY13 was used as a host in the screening.

Next, a human cDNA library was constructed using standard protocols (Gerard
Et al., Focus 14: 491-507, 1991). Size selection (0.4-1.0 kb) by preparing human fetal liver cDNA and increasing the random primer concentration in the reverse transcription reaction
Was done. The cDNA was subcloned into the NotI site of pST20. The pST20 vector is a truncated yeast SUC lacking a DNA sequence encoding amino acids 1-20.
Code 2 This region usually consists of the translation initiation site (ATG) of the gene and the signal sequence, which are necessary for the expression and secretion of the protein product, respectively. The deleted SUC2 DNA sequence was identified by standard site-directed mutagenesis techniques.
Replaced with a tI restriction site.

[0086] The plasmid cDNA library was introduced into SSY13, and URA3 + prototrophy was selected.
The pooled transformants were collected and cultured on YEP sucrose medium supplemented with 1 μg / ml antimycin A. Proliferative yeast colonies were isolated, and the human cDNA sequence was amplified by PCR and sequenced.

The cDNA sequence of one positive clone is reported herein. To determine that growth on sucrose was dependent on the sequence of this plasmid, plasmid DNA from this clone was isolated and used to transform E. coli. Plasmid DNA was isolated from E. coli and introduced into SSY13. SSY13 colonies containing this plasmid
Proliferation is seen on YEP sucrose / antimycin, but the yeast containing the parental pST20 plasmid is not. This result indicates that the NSL4 cDNA sequence is required for growth in the aforementioned yeast selection, and that its function as a signal sequence in human cells is therefore predicted. The peptide sequence of SEQ ID NO: 2 is
Evaluation was performed using the SignalP method. The SignalP program predicts that the peptide sequence of SEQ ID NO: 2 can function as a signal sequence.

Example 2 Cloning of Full Length Gene Cloning of the full length NSL4 gene is performed using standard molecular biological hybridization techniques using one or more DNA sequences derived from the sequence shown in SEQ ID NO: 1. . There are several ways to obtain full-length cDNA, two of which are outlined below.

1) The 3 ′ end can be obtained by using the cDNA end rapid amplification (RACE) method. Frohma
See, et al., Proc. Nat. Acad. Sci. USA 85,8998-9002. Briefly, a specific oligonucleotide is annealed to the mRNA and used to initiate synthesis of the cDNA strand. After removing the mRNA using RNaseH, a poly-C anchor sequence was added to the 3 ′ end of the cDNA, and the resulting fragment was ligated using a `` nested '' NSL1 antisense primer set and an anchor sequence primer. Amplify. The amplified fragment is cloned into a suitable vector and subjected to restriction digest analysis and sequencing analysis. 2) Using the polymerase chain reaction, the 3 'end of cDNA can be amplified from a human cDNA library by sequential rounds of "nested" PCR using two primer sets. One set of antisense primers is specific for the 3 'end of the partial cDNA and the other set anneals to the vector-specific sequence. Amplification products are cloned into a suitable vector and subjected to restriction digest analysis and sequencing analysis.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12N 1/15 C12N 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 33/53 D G01N 33/15 33/566 33/50 C12N 15/00 ZNAA 33/53 A61K 37/02 33/566 C12N 5/00 A (72) Inventor Stockwell, Simon, UK 235 CM, Hertfordshire, Bishop's Stortford, Goldings 19 (72) Inventor McCrawlin, Megan, M. United States 19026 Pennsylvania, Drexel Hill, Mansfield Avenue 2505 (72) Inventor Gallagher, Kathleen, Teresa United States 19090 Pennsylvania, Willow Grove, North Hills Avenue 1812 (72) Inventor Kickley, Christine, Cay United States 19468 Linn, Pennsylvania Field, Limerick Center Road 499

Claims (14)

[Claims] 1. An isolated polypeptide selected from the group consisting of: (i) at least (a) relative to the amino acid sequence of SEQ ID NO: 2 over the entire length of SEQ ID NO: 2 An isolated amino acid sequence comprising an amino acid sequence selected from the group having 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity. Polypeptide,
(ii) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or (iii) an isolated polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 2. An isolated polynucleotide selected from the group consisting of the following (i) to (vi), or a nucleotide sequence complementary to the isolated polynucleotide: (i) SEQ ID NO: 2 At least (a) 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity to the amino acid sequence of SEQ ID NO: 2 over its entire length. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide having: (ii) at least (a) 70% identity over its entire length to the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 (B) an isolated polynucleotide comprising a nucleotide sequence having 80% identity, (c) 90% identity, or (d) 95% identity; (iii) SEQ ID NO: 1. Sequence number over the entire length of A nucleotide sequence having at least (a) 70% identity, (b) 80% identity, (c) 90% identity, or (d) 95% identity to the nucleotide sequence of No. 1. (Iv) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, (v) an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO: 1 Polynucleotide,
And (vi) an isolated polynucleotide obtained by screening a suitable library with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof under stringent hybridization conditions. 3. An antibody immunospecific for the polypeptide of claim 1. 4. A method of treating a subject, comprising: (i) when the subject requires increased activity or expression of the polypeptide of claim 1, (a) treating the polypeptide. Administering to said subject an effective amount of an agonist, and / or (b) producing an isolated polynucleotide comprising a nucleotide sequence encoding said polypeptide in vivo. And (ii) when the subject requires suppression of the activity or expression of the polypeptide of claim 1, (a) the polypeptide Administering to the subject a therapeutically effective amount of an antagonist to the subject, and / or (b) administering to the subject a nucleic acid molecule that suppresses expression of the nucleotide sequence encoding the polypeptide. And / or (c) administering to the subject a therapeutically effective amount of the polypeptide that competes with the polypeptide for its ligand, substrate, or receptor. how to. 5. A method for diagnosing a disease or susceptibility of a subject related to the expression or activity of the polypeptide according to claim 1 in the subject, the method comprising: (a) analyzing the genome of the subject; Examining for the presence of a mutation in the nucleotide sequence encoding the polypeptide of and / or (b) analyzing the presence or amount of expression of the polypeptide in a sample obtained from the subject A method comprising: 6. 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 a polypeptide carrying the polypeptide); Measuring the binding of the candidate compound to the polypeptide (or the polypeptide) or to the fusion protein thereof, or by using a label directly or indirectly bound to the candidate compound. Measuring the binding to a cell or its membrane) or its fusion protein in the presence of a labeling competitor, and (c) the candidate compound provides a signal resulting from activation or inhibition of the polypeptide. Or not, using a detection system suitable for cells or cell membranes carrying the polypeptide, (d) a candidate compound, Combining a solution containing the polypeptide and a solution containing the polypeptide to determine the activity of the polypeptide in the mixture and comparing the activity of the mixture to a standard; or A method selected from the group consisting of detecting the mRNA encoding the polypeptide and the effect on the production of the polypeptide using, for example, an ELISA assay. 7. An agonist or antagonist of the polypeptide according to claim 1. 8. An expression system comprising a polynucleotide capable of producing the polypeptide of claim 1 when said expression system is present in a compatible host cell. 9. The host cell, under suitable culture conditions, produces a 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. A method for producing a recombinant host cell, comprising transforming or transfecting a cell with the expression system according to claim 8. 10. A recombinant host cell obtained by the method according to claim 9. 11. The recombinant host of claim 10, which expresses a 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. Cell membrane. 12. The method of claim 11, wherein the host cell is cultured under conditions sufficient to produce the polypeptide, and the polypeptide is recovered from the culture medium. Production method. 13. An isolated polynucleotide selected from the group consisting of: (a) at least 70% of the nucleotide sequence of SEQ ID NO: 3 over the entire length of SEQ ID NO: 3, An isolated polynucleotide comprising a nucleotide sequence having 80%, 90%, 95%, 97% identity; (b) an isolated polynucleotide comprising a polynucleotide of SEQ ID NO: 3; c) at least 70%, 80%, 90%, 95%, 97-99% identity to the polynucleotide of SEQ ID NO: 3, or (d) the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4 An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide. 14. A polypeptide selected from the group consisting of: (a) to (e): (a) at least 70%, 80%, 90% of the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4. (B) at least 70%, 80%, 90% relative to the amino acid sequence of SEQ ID NO: 4 over the entire length of SEQ ID NO: 4; A polypeptide having an amino acid sequence having 95%, 97-99% identity; (c) a polypeptide comprising the amino acid of SEQ ID NO: 4, (d) a polypeptide consisting of the polypeptide of SEQ ID NO: 4, e) A polypeptide encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 3.