KR20020011445A - Head trauma induced cytoplasmatic calcium binding protein - Google Patents

Abstract

ANIC-BP polypeptides and polynucleotides and methods of making such polypeptides by recombinant techniques are disclosed. Also disclosed are methods using ANIC-BP polypeptides and polynucleotides in diagnostic assays.

Description

HEAD TRAUMA INDUCED CYTOPLASMATIC CALCIUM BINDING PROTEIN}

Stroke and acute head trauma, multiple sclerosis and spinal cord injury are diseases that have never been treated as such. Stroke is the third leading cause of death and the burden on patients and social systems. For most stroke ischemic strokes, vascular blockage in the brain is the initial symptom. Head trauma is a major disease of young people in the West. Much fewer patients suffer from hemorrhagic stroke due to mechanical shock and arterial rupture. It is a common feature that the approved drugs are of little use. Recent available therapeutic approaches are based on abnormal physiological concepts derived from empirical studies of focal cerebral ischemia. This includes pharmacological strategies for arterial recombination, inhibition of infectious action and neuroprotection. In addition, studies using arterial reperfusion strategies are ongoing. Initial clinical studies with polymorphonuclear leukocyte-dependent endothelial receptor antagonists have been completed, but strategies have not yet emerged. However, any strategy that deals with just one step of the ischemic cascade is likely to offer only moderate benefits. Therefore, future therapies will most likely be based on combination therapies. The combination of low-dose acetylsalicylic acid (ASA) and modified-release dipyridamole has been shown to aid in secondary prevention of stroke [Tijssen et al., Int. J. Clin. Pract., Suppl. 91, 14-16, 1997]. Another combination proposed in recent clinical evaluations is tPA and an effective neuroprotective agent. However, the findings are not very effective. Therefore, in order to establish more commonly useful therapies for the treatment of patients with disorders such as multiple sclerosis and acute neuroinjury phenomena, and to provide drug targets that can be used to develop new drugs, further knowledge of the physiological mechanisms is needed. It is desirable to see.

The present invention focuses on Ca ²⁺ binding proteins which are important evidence for the role of Ca ²⁺ binding proteins in neuroprotection. Although the exact function of calcium binding protein (CaBP) is not clearly known, it has been suggested that CaBP acts to buffer intracellular Ca ²⁺ levels. Since the overload of Ca ²⁺ activates a biochemical process that causes proteolysis and mitochondrial dysfunction, the buffered dose of CaBP may have a protective effect against excitotoxic neuronal damage [Heizmann et al. , TINS, 15, 259-64, 1992]. Various evidence exists to support the proposed role of CaBP in neuroprotection and regulation of Ca ²⁺ levels.

The major Ca ²⁺ binding proteins (CaBP) (parvalbumin, calvindine-D28K, and caletinin) expressed in the central nervous system have very specific and selective expression patterns in various neuronal cell populations. Among the neurons expressing CaBP, few neurons express one or more of the major calcium binding proteins, while most express only one type. There is important evidence that the presence or absence of CaBP in certain cell types underlies a phenomenon known as selective vulnerability. Selective fragility is a characteristic of certain types of neurons that die in response to certain types of central nervous system (CNS) damage. For example, CA1 hippocampal neurons have selective susceptibility to pre-ischemia, cerebellar purkiniei cells have selective susceptibility to head trauma, stroke, and fatal alcohol exposure, while neurons in black matter have selective susceptibility to Parkinson disease have. Efforts have been made to link selective neuronal fragility with various CaBP expression patterns, and some authors report that high levels of CaBP are found in neuronal cell populations that are selectively susceptible to damage, while others are selective for damage. It has been reported that high levels of CaBP are found in resistant neuronal populations. For example, neurons expressing high levels of parvalbumin have been reported to be selectively vulnerable to AMPA-induced toxicity [Weiss et al., Neurol., 40, 1288-1292, 1990], while high levels of carbindine Cultured hippocampal neurons expressing -D28K have been reported to be selectively resistant to glutamate-induced toxicity (Baimbridge et al., TINS, 15, 303-8, 1992). Similarly, hippocampal neurons expressing high levels of calretinin are resistant to toxic doses of excitatory toxin glutamate, NMDA, catenate, and quisqualate [Winsky et al .: Novel Calcium-Binding Proteins, 277-300 , 1991].

CaBP also appears to have altered expression in various CNS disease states, but again the results are inconsistent whether CaBP expression is related to selective vulnerability or selective resistance to injury. Calvindine-D28K-expressing neurons in melanoma are not selectively susceptible to Parkinson's disease (Yamada et al., Brain Res., 526, 303-07, 1990), whereas neurons expressing Calvindine-D28K are Alzheimer's disease. lacopino et al., PNAS, 87, 4078-82, 1990, Hof et al., Exp. Neurol., 111, 293-301, 1991] and Huntington's disease (Kiyama et al., Brain Res., 526, 303-07, 1990) are reported to be selectively vulnerable. Another study suggests that parvalbumin-expressing hippocampal intermediate neurons are selectively vulnerable to Alzheimer's disease (Brady et al., Neurosci., 80, 1113-25, 1997), in the gerbil model of pre-ischemia. The presence of parvalbumin among certain hippocampal cell types has been shown to be positive with respect to survival [Tortosa et al., Neurosci., 1, 33-43, 1993].

Mice without the calvindine gene have functional defects that suggest severe dysfunction in the nerve cells, despite the morphologically normal appearance of neurons (eg cerebellar Purkinie cells) that normally express the CaBP ( For example, ataxia). The findings suggest that CaBP is important for cellular activity patterns (Airaksinen et al., PNAS, 94, 1488-93, 1997). In addition, retroviral infection of motor neurons with calvindine-D28K has been shown to have a neuroprotective effect on the toxicity induced by IgG from patients with amyotrophic lateral sclerosis [Ho et al., PNAS, 93, 6796]. -801, 1996], transfection with calvindine-D28k has been shown to protect PC12 cells from serum withdrawal, glutamate exposure, and toxicity due to neurotoxin MPP + [McMahon et al., Molec., Brain Res., 526, 303 -07, 1998].

In conclusion, there is considerable information regarding the role of calcium binding proteins in neurodegeneration. While it is clear that some CaBPs provide protection and others cause selective fragility, it is still unclear whether the expression of specific CaBP in different neuronal cell populations causes different functional responses of the given CaBP. Another observation is that the severity of different types of CNS damage may affect the apparent neuroprotective efficacy of CaBP, that is, one CaBP may provide resistance to a damage model involving mild damage, but more severe CNS damage. Is unable to buffer Ca ²⁺ increases. Thus, although there is considerable evidence that CaBP provides vulnerability as well as resistance to the CNS damaging process, the mechanisms and responses of the relationship are still under study.

A group of genes containing functionally unidentified genes (MO25) has recently been isolated from mouse induced cDNA libraries [Miyamoto et al., Mol. Reprod. Dev., 34, 1-7, 1993]. Libraries were constructed from RNA isolated from early fetal mice. The predicted amino acid sequence for Mo25 indicates that the MO25 gene may have structural homology with Ca ²⁺ binding protein and lacks a transmembrane domain, revealing that the protein may be involved in the cytoplasmic development of unfertilized eggs. However, the actual function of the protein is not yet known. Another Mo25-like gene was cloned from the Drosophila cDNA library (Nozaki et al., DNA Cell Biol., 15, 505-09, 1996). The deduced amino acid sequence of Mo25 cDNA is 69.3% identical to that of the mouse Mo25 mammal. The epithelium in Saccharomyces cerevisiae was encoded in an open reading frame near the calcineurin B subunit gene. Another most recent gene has been isolated from the Aspergillus hym A mutation [Karos et al., Mol. Gen Genet., 260, 510-521, 1999], yeast, plants, flies, worms, fish, mice, and humans. The cellular function for the Hym protein has not yet been defined in any of the described organisms. For many other proteins whose functional contributions are only partially known, the drug discovery process is currently undergoing a fundamental revolution involving "functional genomics", ie high throughput genome or gene-based biology. As one means of identifying genes and gene products as therapeutic targets, this approach is rapidly replacing the initial approach described in "position cloning". Phenotypes, ie biological functions or genetic diseases, will be identified, which will then be traced back to the causal gene based on their genetic map location.

Functional genomics relies heavily on high-output DNA sequencing techniques and various means of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases currently available. As a target of drug discovery, there is a continuing need to identify and characterize other genes and related polypeptides / proteins.

Summary of the Invention

The present invention relates to ANIC-BP, in particular ANIC-BP polypeptides and ANIC-BP polynucleotides, recombinants and methods for their preparation. Such polypeptides and polynucleotides are of interest in the treatment of certain diseases, including but not limited to stroke and acute head trauma, multiple sclerosis and spinal cord injury, referred to hereinafter as the "disease of the invention." In another aspect, the present invention relates to methods of identifying agonists and antagonists (eg, inhibitors) using materials provided by the present invention, and treatment conditions associated with ANIC-BP imbalance with the compounds identified. In another aspect, the present invention relates to diagnostic assays for detecting diseases associated with inappropriate ANIC-BP activity or levels.

Description of the invention

In a first aspect, the present invention relates to an ANIC-BP polypeptide.

Such polypeptides include:

(a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1;

(b) an isolated polypeptide comprising a polypeptide sequence of at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequence of SEQ ID NO: 2;

(c) an isolated polypeptide comprising the polypeptide sequence of SEQ ID NO: 2;

(d) an isolated polypeptide having at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequence of SEQ ID NO: 2;

(e) the polypeptide sequence of SEQ ID NO: 2; And

(f) an isolated polypeptide having or comprising a polypeptide sequence having an identity index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the polypeptide sequence of SEQ ID NO: 2;

(g) fragments and variants of the polypeptides in (a) to (f).

The polypeptides of the present invention are considered to be members of the ^* calcium binding protein group of polypeptides. Thus they are of interest because they can act as novel drug targets.

Biological properties of ANIC-BP are referred to hereinafter as "biological activity of ANIC-BP" or "ANIC-BP activity". Preferably, the polypeptides of the present invention exhibit one or more biological activities of ANIC-BP.

Polypeptides of the invention also include variants of the aforementioned polypeptides, including all allelic forms and splice variants. Such polypeptides are modified from reference polypeptides by insertions, deletions and substitutions which may be conservative or non-conservative or any combination thereof. Particularly preferred variants are those in which several, for example 50 to 30, 30 to 20, 20 to 10, 10 to 5, 5 to 3, 3 to 2, 2 to 1 or 1 amino acids are inserted, substituted in any combination Or fruitful ones.

Preferred fragments of the polypeptides of the invention include an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQ ID NO: 2, or 30, 50 or 100 from an amino acid sequence of SEQ ID NO: 2 Isolated polypeptides include amino acid sequences in which at least two contiguous amino acids have been cleaved or deleted. Preferred fragments are biologically active fragments which mediate the biological activity of ANIC-BP, including those with similar or improved activity, or reduced undesirable activity. Also preferred are fragments that are antigenic or immunogenic in animals, especially humans.

Fragments of the polypeptides of the present invention can be used to prepare corresponding full-length polypeptides by peptide synthesis, and thus the variants can be used as intermediates for preparing full-length polypeptides of the present invention. Polypeptides of the invention may be in the form of "mature" proteins or may be part of larger proteins such as precursors or fusion proteins. Often, it is beneficial to include additional amino acid sequences, including secretory or leader sequences, pro-sequences, assisting sequences in purification, such as multiple histidine residues, or additional sequences for stability during the recombination process. .

Polypeptides of the invention may be isolated from, for example, natural sources, from genetically engineered host cells, including expression systems (see below), or chemical synthesis using, for example, automated peptide synthesizers, or of such methods. By combination in any suitable manner. Means for making such polypeptides are well known in the art.

In another aspect, the present invention relates to an ANIC-BP polynucleotide. The polynucleotides include:

(a) an isolated polynucleotide comprising a polynucleotide sequence of at least 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence of SEQ ID NO: 1;

(b) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1;

(c) an isolated polynucleotide that is at least 95%, 96%, 97%, 98% or 99% identical to the polynucleotide of SEQ ID NO: 1;

(d) an isolated polynucleotide of SEQ ID NO: 1;

(e) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence of at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequence of SEQ ID NO: 2;

(f) an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2;

(g) an isolated polynucleotide having a polynucleotide sequence encoding a polypeptide sequence of at least 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequence of SEQ ID NO: 2;

(h) an isolated polynucleotide encoding a polypeptide of SEQ ID NO: 2;

(i) an isolated polynucleotide having or comprising a polynucleotide sequence having an identity index of 0.95, 0.96, 0.97,0.98 or 0.99 compared to the polynucleotide sequence of SEQ ID NO: 1;

(j) an isolated polynucleotide having or comprising a polynucleotide sequence encoding a polypeptide sequence having an identity index of 0.95, 0.96, 0.97, 0.98 or 0.99 compared to the polypeptide sequence of SEQ ID NO: 2; And polynucleotides that are fragments and variants of the above-mentioned polynucleotides or are complementary to the above-mentioned polynucleotides over their entire length.

Preferred fragments of the polynucleotides of the invention include an isolated polynucleotide comprising a nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1, or 30, 50 from the sequence of SEQ ID NO: 1 Or isolated polynucleotides comprising sequences in which at least 100 contiguous nucleotides have been cleaved or deleted.

Preferred variants of the polynucleotides of the invention include splice variants, allelic variants, and polymorphs, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).

The polynucleotides of the invention also encode polypeptide variants comprising the amino acid sequence of SEQ ID NO: 2, and include several, for example 50 to 30, 30 to 20, 20 to 10, 10 to 5, 5 to 3, 3 Polynucleotides in which 2 to 2, 2 to 1 or 1 amino acid are substituted, deleted or added in any combination are included.

In another aspect, the invention provides polynucleotides that are RNA transcripts of the DNA sequences of the invention. Thus, RNA polynucleotides and complementary RNA polynucleotides are provided that are characterized by the following:

(a) comprises an RNA transcript of a DNA sequence encoding a polypeptide of SEQ ID NO: 2;

(b) an RNA transcript of a DNA sequence encoding a polypeptide of SEQ ID NO: 2;

(c) comprises an RNA transcript of the DNA sequence of SEQ ID NO: 1; or

(d) is an RNA transcript of the DNA sequence of SEQ ID NO: 1.

The polynucleotide sequences of SEQ ID NO: 1 are described in Hym A [Nozaki et al., DNA cell Biol., 15, 505-09, 1996] and Mo25 [Karos et al., Mol. Gen Genet., 260, 510-521, 1999]. The polynucleotide sequence of SEQ ID NO: 1 is a cDNA sequence encoding the polypeptide of SEQ ID NO: 2. The polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 may be identical to the polypeptide coding sequence of SEQ ID NO: 1 or as a result of excess (degeneracy) of the genetic code and also encodes the polypeptide of SEQ ID NO: 2 , SEQ ID NO: 1 may be a sequence. The polypeptide of SEQ ID NO: 2 is associated with other proteins of the ^* calcium binding protein family, having homology and / or structural similarities with Hym A and Mo25 proteins.

Preferred polypeptides and polynucleotides of the invention are particularly expected to have similar biological functions / properties to their homologous polypeptides and polynucleotides. In addition, preferred polypeptides and polynucleotides of the invention have one or more ANIC-BP activities.

Polynucleotides of the invention can be obtained using standard cloning and screening techniques from cDNA libraries derived from mRNA cells of the human central nervous system (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Polynucleotides of the invention can also be obtained from natural sources, such as genomic DNA libraries, or synthesized using well known and commercially available techniques.

When the polynucleotide of the present invention is used for the recombinant preparation of a polypeptide of the present invention, the polynucleotide may be a coding sequence of a mature polypeptide only, or a leader or secretory sequence, pre-, pro- or prepro-. ) The coding sequence of the mature polypeptide in a reading frame having a protein sequence, or other coding sequence, such as encoding another fusion peptide moiety. For example, marker sequences may be encoded that facilitate purification of the fusion polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is provided in a pQE vector (Qiagen, Inc.) and described as hexa-histidine described in Gentz et al., Proc Natl Acad Sci USA (1989) 86: 821-824. It is a peptide or an HA tag. Polynucleotides may also include non-coding 5 'and 3' sequences, such as sequences that are transcribed but not translated, splicing and polyadenylation signals, ribosomal binding sites, and sequences that stabilize mRNA.

Polynucleotides identical or sufficiently identical to the polynucleotide sequence of SEQ ID NO: 1 may be used as hybridization probes for cDNA and genomic DNA or as primers for nucleic acid amplification reactions (eg, PCR). The probes and primers isolate full-length cDNA and genomic clones encoding polypeptides of the invention and typically have at least 95% identical, high sequence similarity to other genes (para from human source) relative to SEQ ID NO: 1. Genomic clones and cDNAs of paralogs and genes encoding paralogs and orthologs from species other than humans. Preferred probes and primers will typically comprise at least 15 nucleotides, preferably at least 30 nucleotides, and may have at least 50, if not at least 100 nucleotides. Particularly preferred probes are between 30 and 50 nucleotides. Particularly preferred primers have 20 to 25 nucleotides.

Polynucleotides encoding polypeptides of the invention, having homology with species other than human, are subjected to stringent hybridization conditions using labeled probes having the sequence of SEQ ID NO: 1, or a fragment thereof, preferably of at least 15 nucleotides. Screen libraries; Full-length cDNA and genomic clones comprising the polynucleotide sequence can be obtained by a process comprising the step of isolating. Such hybridization techniques are well known to those skilled in the art. Preferred stringent hybridization conditions include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt solution, 10% dextran sulfate, and 20 μg incubated at 42 ° C. overnight in a solution containing / ml denatured, sheared salmon sperm DNA; Washing the filter in 0.1 x SSC at about 65 ° C. The present invention therefore also provides a screening library under stringent hybridization conditions, preferably at least 100 nucleotides, using a labeled probe having a sequence of SEQ ID NO: 1, or preferably a fragment thereof of at least about 15 nucleotides. Isolated polynucleotides having the sequence.

Those skilled in the art will recognize that in many cases an isolated cDNA sequence will be incomplete in that the coding region of the polypeptide does not always extend towards the 5 'end. This is the result of reverse transcriptase, an enzyme with an inherent low "progression" (a measure of the enzyme's ability to adhere to the template during the polymerization reaction), which fails to complete DNA copy of the mRNA template during first strand cDNA synthesis. .

There are several methods available that are well known to those skilled in the art for obtaining full-length cDNA, or for extending short cDNA, for example based on the rapid amplification (RACE) method of cDNA ends (eg , Frohman et al., Proc Nat Acad Sci USA 85, 8998-9002, 1988). A recent modification of technology, for example Marathon ™ technology (Clontech Laboratories Inc.), has significantly simplified the study of longer cDNAs. In Marathon® technology, cDNA was prepared from mRNA extracted from selected tissues and 'adapter' sequences ligated at each end. Nucleic acid amplification (PCR) was then performed to amplify the “deleted” 5 ′ ends of the cDNA using a combination of gene specificity and adapter specific oligonucleotide primers. The 'nested' primers, ie, primers designed to anneal in the amplified product (typically adapter specific primers that anneal further to 3 'of the adapter sequence and 5' in known gene sequences). The PCR reaction was repeated using a gene specific primer (annealed further). Subsequently, the product of the reaction can be DNA sequenced and bound to the existing cDNA directly to yield a complete sequence, or by performing individual full-length PCR using new sequence information for the design of the 5 'primer. Build a length cDNA.

Recombinant polypeptides of the invention can be prepared by methods well known in the art from genetically engineered host cells comprising an expression system. Thus, in another aspect, the invention relates to the production of an expression system comprising a polypeptide or polynucleotide of the invention, a host cell genetically engineered using the expression system, and a polypeptide of the invention by recombinant technology. . Cell-free translation systems can also be used to prepare these proteins using RNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for the polynucleotides of the invention. Polynucleotides can be introduced into host cells by the methods described in many standard experimental manuals, such as Davids et al., Basic Methods in Molecular Biology (1986) and Sambrook et al. (Same book, identical parts). Preferred methods of introducing polynucleotides into host cells include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, micro injection, cationic lipid-mediated transfection, electroporation, Transduction, scratch loading, ballistic introduction or infection.

Representative examples of suitable hosts include fungal cells such as Streptococci, Staphylococci, Escherichia coli, Streptomyces and Bacillus subtilis cells; Fungal cells such as yeast cells and Aspergillus cells; Insect cells such as Drosophila S2 and Spodoptera Sf9 cells; Animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; And plant cells.

A wide variety of expression systems, for example chromosomes, episomes and virus-derived systems such as fungal plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, Vectors derived from papova viruses such as SV40, vaccinia virus, adenovirus, avian pox virus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof, for example from plasmids and bacteriophage gene elements, eg For example cosmid and phagemid can be used. The expression system may include control regions that produce and regulate expression. Typically, any system or vector capable of maintaining, propagating or expressing polynucleotides may be used to produce the polypeptide in a host. Appropriate polynucleotide sequences can be inserted into the expression system by any of a variety of well-known and routine techniques, such as those described in Sambrook et al. (Same book). Appropriate secretion signals may be incorporated into the polypeptide of interest to secrete translated proteins into lumens of the transgenic network, cytoplasmic space, or extracellular environment. The signal may be endogenous to the polypeptide or they may be heterologous signals.

When polypeptides of the invention are expressed for screening assays, it is usually preferred that the polypeptides be prepared at the cell surface. In such cases, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium may be recovered for recovery and purification of the polypeptide. When prepared intracellularly, cells must be lysed before the polypeptide can be recovered.

Polypeptides of the invention are well known including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography Can be recovered and purified from recombinant cell culture. Most preferably, high performance liquid chromatography is used for purification. Well-known techniques for rebuilding proteins can be used to regenerate the active structure when the polypeptide is denatured during intracellular synthesis, isolation and / or purification.

The polynucleotide of the present invention can be used as a diagnostic reagent through detecting mutations in related genes. Detection of a variant form of a gene associated with dysfunction and characterized by the polynucleotide of SEQ ID NO: 1 in the cDNA or genomic sequence is a disease, or due to underexpressed, overexpressed or modified spatial or temporal expression of the gene, or Diagnostic tools will be provided to assist or define the diagnosis of disease susceptibility. Variations in genes can be detected at the DNA level by various techniques well known in the art.

Diagnostic nucleic acids can be obtained from cells of a patient, 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, preferably RT-PCT, or other amplification techniques prior to analysis. RNA or cDNA can also be used in a similar manner. Deletion and insertion can be detected as a change in the size of the amplified product compared to normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled ANIC-BP nucleotide sequences. Perfectly aligned sequences can be distinguished from duplexes that are not fitted by RNase cleavage or differences in melting temperatures. DNA sequence differences can also be detected by changes in the electrophoretic mobility of DNA fragments in gels, with or without denaturants, or by direct DNA sequencing (eg, Myers et al., Science (1985) 230: 1242. Sequence changes at specific positions can also be indicated by nuclease protection assays, such as RNase and S1 protection or chemical cleavage (see, eg, Cotton et al., Proc Natl Acad). Sci USA (1985) 85: 4397-4401.

Alignment of oligonucleotide probes comprising ANIC-BP polynucleotide sequences or fragments thereof can be constructed, for example, by performing efficient screening of genetic variations. The alignment is preferably a high density alignment or a grating. Alignment technique methods are well known and commonly applicable and can be used to address a variety of questions in molecular genetics, including gene expression, gene binding, and gene diversity (eg, M. Chee et al., Science, 274). , 610-613 (1996) and other references cited therein).

Detection of abnormally reduced or increased levels of polypeptide or mRNA expression can also be used to diagnose or measure the patient's susceptibility to the disease of the invention. Reduced or increased expression is achieved at the RNA level using any polynucleotide quantification method well known in the art, such as nucleic acid amplification such as PCR, RT-PCR, RNase protection, northern blotting and other hybridization methods. It can be measured. In samples derived from a host, assay techniques that can be used to measure levels of proteins, such as polypeptides of the invention, are well known to those of skill in the art. Such assays include radioimmunoassays, competitive-binding assays, western blot assays and ELISA assays.

Thus, in another aspect, the present invention relates to a diagnostic kit comprising:

(a) a polynucleotide of the invention, preferably the nucleotide sequence of SEQ ID NO: 1, or a fragment or RNA transcript thereof;

(b) a nucleotide sequence complementary to (a);

(c) a polypeptide of the invention, preferably a polypeptide of SEQ ID NO: 2 or a fragment thereof; or

(d) an antibody against a polypeptide of the invention, preferably the polypeptide of SEQ ID NO: 2.

It will be appreciated that in any kit, (a), (b), (c) or (d) may constitute a substantial component. Such kits will be useful for diagnosing a disease, particularly susceptibility or disease to the disease of the present invention.

The polynucleotide sequences of the present invention are important for chromosome positioning studies. The sequence may specifically target and hybridize to a specific location on an individual human chromosome. The mapping of relevant sequences of chromosomes according to the invention is an important first step in the correlation of genetically linked diseases with these sequences. Once the sequence is mapped to the correct chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. The data is for example [V. McKusick, Mendelian Inheritance in Man] (available on-line through the Johns Hopkins University Welch Medical Library). Relationships between diseases and genes mapped to the same chromosomal region are then confirmed through chain analysis (co-genetics of physically contiguous genes). Accurate human chromosome localization of genomic sequences (gene fragments, etc.) is described by Radiation Hybrid (RH) Mapping [Walter, M. Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994). A method for constructing radiation can be determined using hybrid maps of whole genomes, Nature Genetics 7, 22-28. Many RH panels are described in Research Genetics (Huntsville, AL, USA), for example the GeneBridge4 RH panel [Hum Mol Genet 1996 Mar; 5 (3): 339-46 A radiation hybrid map of the human genome. Gyapay G, Schmitt K, Fizamens C, Jones H, Vega-Czarny N, Pillett D, Muselet D, Prud'Homme JF, Dib C, Auffray C, Morissette J, Weissenbach J, Goodfellow PN. In order to determine the chromosomal location of the gene using the panel, 93 PCR is performed using primers designed from the gene of interest on RH DNA. Each such DNA comprises a random human genomic fragment maintained in a hamster background (human / hamster hybrid cell line). The result of the PCR is 93 score indicating the presence or absence of the PCR product of the gene of interest. The scores are compared to the scores shown using PCR products from genomic sequences at known locations. The comparison is performed at http://www.genome.wi.mit.edu.

Polynucleotide sequences of the invention are also important tools for tissue expression studies. This study enables the determination of the expression pattern of a polynucleotide of the present invention that can direct the expression pattern of a encoded polypeptide in tissue by detecting the mRNA encoding it. The technique used is well known in the art and on a lattice, such as cDNA microalignment hybridization (Schena et al., Science, 270, 467-470, 1995 and Shalon et al., Genome Res, 6, 639-645, 1996). Nucleotide amplification techniques such as in situ hybridization techniques and PCR for aligned clones. Preferred methods use the TAQMAN ™ technology available from Perkin Elmer. The results from this study indicate normal function of polypeptides in organisms. In addition, a comparative study of the expression pattern of mRNA encoded by a modified form of the same gene (eg, a change in a polypeptide encoding a potential or regulatory variant) and the normal expression pattern of mRNA may serve as a polypeptide of the invention. , Or its improper expression in disease. Such inappropriate expression may be transient, spatial or simple quantitative nature. Polypeptides of the invention are expressed in the human brain.

Another aspect of the invention relates to an antibody. Polypeptides of the invention or fragments thereof, or cells expressing the same, can be used as an immunogen to produce antibodies that are immunospecific to the polypeptides of the invention. The term "immunogenicity" means that an antibody has substantially greater binding capacity to the polypeptide of the present invention than its binding capacity to other related polypeptides of the prior art.

Antibodies generated against polypeptides of the invention can be obtained by administering a polypeptide or epitope-containing fragment, or cell, to an animal, preferably a non-human animal, using conventional protocols. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line culture can be used. Examples include hybridoma technology (Kohler G. and Milstein, C., Nature (1975) 256: 495-497), trioma technology, human B-cell hybridoma technology [Kozbor et al., Immunology Today (1983) 4:72] and EBV-Hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).

Techniques for preparing single chain antibodies, such as those described in US Pat. No. 4,946,778, may also be employed to prepare single chain antibodies against the polypeptides of the invention. In addition, transgenic mice, or other organisms, including other mammals, can be used to express humanized antibodies.

The above-described antibodies can be used to isolate or identify clones expressing the polypeptide or to purify the polypeptide by affinity chromatography. Antibodies to the polypeptides of the invention can also be used, inter alia, to treat the diseases of the invention.

Polypeptides and polynucleotides of the invention can also be used as vaccines. Thus, in another embodiment, the present invention relates to antibodies and / or antibodies comprising, for example, cytokine-producing T cells or cytotoxic T cells to protect animals from disease, whether or not the disease has already been established in the individual. A method for inducing an immunological response in a mammal comprising inoculating a mammal of the invention suitable for generating a T cell immune response. The immunological response of mammals is also provided via a vector for encoding a polypeptide in vivo and expressing a polynucleotide to induce such an immunological response to produce an antibody for protecting said animal from a disease of the invention. It can be induced by a method comprising delivering the polypeptide. One method of administering a vector is by accelerating it to the cells of interest as a coating on the particles or as a coating. The nucleic acid vector may comprise DNA, RNA, modified nucleic acid, or DNA / RNA hybrid. To use a vaccine, a polypeptide or nucleic acid vector will typically be provided as a vaccine formulation (composition). The formulation may also contain a suitable carrier. Since the polypeptide can be degraded in the stomach, it is preferably administered parenterally (eg subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-sustainable sterile injectable solutions which may include antioxidants, buffers, bacteriostatics, and solutes that render the formulation isotonic with the recipient's blood; And aqueous and non-aqueous sterile suspensions which may include suspending or thickening agents. The formulations may be provided in single- or multi-dose containers, such as sealed ampoules and vials, and stored in freeze-drying conditions requiring only the addition of a sterile liquid carrier immediately before use. Vaccine formulations may also include adjuvant systems for enhancing the immunity of formulations such as oil-in-water and other systems known in the art. Dosage will vary depending on the specific activity of the vaccine and can be readily determined by routine experimentation.

Polypeptides of the invention have one or more biological functions appropriate in one or more disease states, in particular the diseases of the invention mentioned above. Thus, it is useful to identify compounds that stimulate or inhibit the function or level of a polypeptide. Thus, in another aspect, the present invention provides a screening method for identifying compounds that stimulate or inhibit the function or level of a polypeptide. The method identifies agents or antagonists that can be used for the treatment and prophylaxis of the above-mentioned diseases of the invention. Compounds can be identified from a variety of sources, such as cells, cell-free preparations, chemical libraries, aggregates of chemical compounds, and natural product mixtures. Such agents or antagonists thus identified include, but are not limited to, natural or modified substrates of ligands, ligands, receptors, enzymes, and the like; Structural or functional mimics thereof (see, eg, Colingan et al., Current Protocols in Immunology 1 (2): Chapter 5 (1991)) or small molecules.

Screening methods can simply measure the binding of a candidate compound to a polypeptide, or a cell or membrane with the polypeptide, or a fusion protein thereof, using a label that is directly or indirectly associated with the candidate compound. Alternatively, the screening method may comprise measuring or detecting (quantitatively or quantitatively) the competitive binding of the candidate compound to the polypeptide to the labeled competition (eg, agonist or antagonist). In addition, the screening method can test whether a candidate compound raises a signal generated by activation or inhibition of the polypeptide, using a detection system appropriate for the cell having the polypeptide. Activation inhibitors are commonly analyzed in the presence of known agents, and effects on activation by antagonists by the presence of candidate compounds are observed. In addition, the screening method simply mixes a solution comprising the polypeptide of the present invention with a candidate compound to form a mixture, measures the ANIC-BP activity in the mixture, and controls the ANIC-BP activity of the mixture to a control mixture that does not contain the candidate compound. And comparing with.

The polypeptides of the invention can be used in conventional low dose screening methods, and also in high throughput screening (HTS) formats. The HTS format includes well-established 96- and more recently 384-well microtiter plates as well as recent methods such as the nanowell method described in Schullek et al., Anal Biochem., 246, 20-29 (1997). do.

Fusion proteins, such as those prepared from ANIC-BP polypeptides and Fc moieties as described above, can also be used in high throughput screening assays to identify antagonists for the polypeptides of the invention (D. Bennett et al., J Mol Recognition, 8: 52-58 (1995) and K. Johanson et al., J. Biol Chem, 270 (16): 9459-9471 (1995).

Screening technology

Antibodies to polynucleotides, polypeptides, and polypeptides of the invention can also be used to form screening methods for detecting the effect of added compounds on the preparation of intracellular mRNAs and polypeptides. For example, ELISA assays can be constructed to measure secreted or cellular related levels of polypeptides using monoclonal and polyclonal antibodies by standard methods known in the art. It can be used to find agents (also called antagonists or agents, respectively) that can inhibit or enhance the production of polypeptides from cells or tissues that have been properly treated.

The polypeptides of the invention can be used to identify membrane binding or soluble receptors, where possible, through standard receptor binding techniques known in the art. This means that the polypeptide is labeled with a radioactive isotope (eg ¹²⁵ I), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and the source of putative receptor (cell , Ligand binding and crosslinking assays incubated with cell membranes, cell supernatants, tissue extracts, body fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. The screening method can also be used to identify antagonists and agonists of polypeptides, where possible, that compete with binding of the polypeptide to its receptor. Standard methods for performing such assays are well known in the art.

Examples of antagonists of polypeptides of the invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to ligands, substrates, receptors, enzymes, etc. of the polypeptides, such as fragments such as ligands, substrates, receptors, enzymes; Or small molecules that bind to the polypeptide of the present invention but do not elicit a reaction, thereby preventing the activity of the polypeptide.

Screening methods may also include transformation techniques and the use of ANIC-BP genes. Construction techniques for transgenic animals are well established. For example, the ANIC-BP gene may be a genetically modified embryonic stem cell, such as by microinjection of fertilized oocytes into the male germline, retroviral migration into pre- or post-transplant embryos, or electroporation. Can be introduced via injection into a host cyst. Particularly useful transgenic animals are so-called "knock-in" animals in which animal genes are substituted with human equivalents in the animal's genome. Knock-in transgenic animals are useful in the drug discovery process for target validation, where the compound is specific for human targets. Another useful transgenic animal is a so-called "knock-out" animal in which the expression of the animal orthologs of the polypeptide of the invention is partially or completely invalidated, coated by an endogenous DNA sequence in the cell. Gene knock-out may be targeted to specific cells or tissues and may occur only in specific cells or tissues as a result of limitations in technology, or may occur in all, or substantially all, cells of an animal. Transgenic animal techniques also provide whole animal expression-cloning systems in which the introduced genes are expressed to yield large amounts of the polypeptides of the invention.

Screening kits for use in the methods described above form another aspect of the present invention. The screening kit includes:

(a) a polypeptide of the invention;

(b) recombinant cells expressing a polypeptide of the invention;

(c) cell membranes expressing polypeptides of the invention; or

(d) an antibody against a polypeptide of the invention;

Wherein the polypeptide is preferably a polypeptide of SEQ ID NO: 2.

It will be appreciated that in any of the above kits, (a), (b), (c) or (d) may constitute a substantial component.

Glossary of Terms

The following definitions are provided to facilitate understanding of certain terms frequently used above.

As used herein, “antibody” includes Fab fragments, including products of polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, and Fab or another immunoglobulin expression library.

"Isolated" means altered from its natural state "by human hands", ie, if it occurs in nature, changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living organism is not "isolated", but the same polynucleotide or polypeptide that is separated from its natural co-existing material as the term is used herein is "isolated". will be. In addition, polynucleotides or polypeptides introduced into an organism by transformation, genetic engineering or any other recombinant method, even when still present in the organism, are "isolated" whether the organism is alive or not.

"Polynucleotide" typically means any polyribonucleotide (RNA) or polydeoxyribonucleotide (DNA), which can be unmodified or modified RNA or DNA. "Polynucleotide" includes single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA, single-stranded, which is a mixture of single- and double-stranded regions, Or more typically hybrid molecules comprising DNA and RNA, which may be double-stranded or a mixture of single- and double-stranded regions. "Polynucleotide" also refers to RNA or DNA or triple-stranded regions comprising both RNA and DNA. The term "polynucleotide" also includes DNA or RNA comprising one or more modified bases, and DNA or RNA having a backbone that has been modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases such as inosine and specific bases. Various modifications can be made to DNA and RNA, such that a "polynucleotide" refers to the DNA of viruses and cells as well as chemically, enzymatically or metabolically modified forms of polynucleotides typically found in nature. Chemical forms of RNA properties. "Polynucleotides" also include relatively short polynucleotides and are often referred to as oligonucleotides.

"Polypeptide" refers to any polypeptide, ie, peptide isoster, comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains commonly referred to as peptides, oligopeptides or oligomers, and long chains commonly referred to as proteins. Polypeptides may comprise amino acids other than twenty gene-encoded amino acids. A "polypeptide" includes amino acid sequences modified by natural processes, such as post-translational processes, or by chemical modification techniques well known in the art. Such modifications are well described in basic texts and more detailed monographs as well as in several volumes of research literature. Modifications can occur anywhere in the polypeptide including the peptide backbone, amino acid side chains and amino or carboxyl termini. It will be appreciated that the same type of modification may be present at the same or varying degrees at several sites in a given polypeptide. In addition, certain polypeptides may include many types of modifications. Polypeptides may be branched as a result of ubiquity, and they may be branched or unbranched cyclic. Cyclic, branched and unbranched cyclic polypeptides may arise from post-translational natural processes or may be prepared synthetically. Modifications include acetylation, acylation, ADP-ribose, amidation, biotinylation, covalent attachment of flavins, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, phosphatidyl Covalent attachment of crosslinks, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchors T) of amino acids for proteins such as formation, hydroxylation, iodide, methylation, myristoylation, oxidation, proteolysis, phosphorylation, prenylation, racemization, selenoylation, sulphation, arginylation RNA mediated addition, and ubiquity (see, eg, Proteins-Structure and Molecular Properties, 2nd Edition, TE Creighton, WH Freeman and Company, New York, 1993; Wold, F., Post-translational Protein Mo difications: Perspectives and Prospects, 1-12, in Post-translational Covalent Modification of Proteins, BC Johnson, Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol, 182 , 626-646, 1990, and Rattan et al., "Protein Synthesis: Post-translational Modifications and Aging", Ann Ny Acad Sci, 663, 48-62, 1992).

A “fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but retains the same biological function or activity as the reference polypeptide.

"Fragment" of a polynucleotide sequence refers to a polynucleotide sequence that is shorter than the reference sequence of SEQ ID NO: 1.

A “variant” refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains its essential properties. Typical variants of polynucleotides differ in reference polynucleotides and nucleotide sequences. Changes in the nucleotide sequence of the variant may or may not modify the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as discussed below. Typical variants of polypeptides differ in reference polypeptide and amino acid sequences. Typically, modifications are defined such that the sequences of the reference polypeptide and the variant are very similar throughout and, in many regions, identical. Variants and reference polypeptides may differ in any combination of one or more substitutions, insertions, deletions in the amino acid sequence. The substituted or inserted amino acid residue may or may not be encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And Phe and Tyr. Variants of the polynucleotide or polypeptide may be natural, such as alleles, or may be variants known to be non-natural. Non-natural variants of polynucleotides and polypeptides can be prepared by mutation techniques or by direct synthesis. Variants also include polypeptides having one or more post-translational modifications, such as glycosylation, phosphorylation, methylation, ADP ribose, and the like. Embodiments include methylation of N-terminal amino acids, phosphorylation of serine and trionine and modification of C-terminal glycine.

"Allele" refers to one of two or more optional forms of genes that occur at a given location in the genome.

"Polymorphism" refers to a modification of a nucleotide sequence (and encoded polypeptide sequence, if appropriate) at a location in the genome in a population.

"Single nucleotide polymorphism" (SNP) refers to the occurrence of nucleotide diversity at a single nucleotide position in the genome within a population. SNPs can occur within genes or within intergenic regions of the genome. SNPs can be analyzed using allele specificity amplification (ASA). Three or more primers are required for the process. Conventional primers are used for the reverse complement to the polymorphism being analyzed. The conventional primer may be 50 to 1500 bp from the polymorphic base. The other two (or more) primers are identical to each other except that the final 3 'base tends to correspond to one of the two (or more) alleles that make up the polymorph. Two (or more) PCR reactions are then performed on the sample DNA using conventional primers and one allele specific primer, respectively.

As used herein, “splice variant” refers to a cDNA molecule that is generated from an RNA molecule initially transcribed from the same genomic DNA sequence but has undergone alternative RNA splicing. Alternative RNA splicing occurs when the primary RNA transcript is subjected to splicing for removal of introns, typically resulting in the production of one or more mRNA molecules, each of which can encode a different amino acid sequence. The term splice variant also refers to a protein encoded by the cDNA molecule.

"Identity" reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing sequences. Typically, identity refers to the exact nucleotide to nucleotide or amino acid to amino acid match of two polynucleotides or two polypeptide sequences, respectively, over the length of the sequence being compared.

"% Identity"-For sequences that do not exactly match, "% identity" can be measured. Typically, two sequences to be compared are aligned to provide maximum correlation between the sequences. This may include insertion "gaps" in one or both sequences to enhance the degree of alignment. The percent identity can be determined over the entire length (so-called full alignment), particularly suitable for the same or very similar length of each sequence being compared, or over a shorter, defined length (so-called local alignment) more appropriate for non-matching sequences. Can be.

"Similarity" is an even more complex measure of the relationship between two polypeptide sequences. Typically, "similarity" refers not only to the exact correspondence between pairs of residues from each sequence being compared (with respect to identity), but also to the fact that, if not exactly, evolutionary based residues are appropriate substitutes for the other, By amino acid is meant a comparison between amino acids of two polypeptide chains based on residues versus residues. This possibility has an associated "score" in which the% similarity of the two sequences can be determined.

Methods of comparing the identity and similarity of two or more sequences are well known in the art. For example, the program available in Wisconsin Sequence Analysis Package, Version 9.1 [available from Devereux J et al., Nucleic Acids Res, 12, 387-395, 1984, Genetics Computer Group, Madison, Wisconsin, USA], for example BESTFIT and GAP can be used to determine% identity between two polynucleotides and% identity and% similarity between two polypeptide sequences. BESTFIT uses Smith and Waterman's “local homogeneity” algorithms (J Mol Biol, 147, 195-197, 1981, Advances in Applied Mathematics, 2, 482-489, 1981), and the single best region of similarity between the two sequences Find it. BESTFIT is more suitable for comparing two polypeptide sequences or two polynucleotides that are not similar in length, and the program assumes that shorter sequences represent longer portions. GAP, on the other hand, aligns two sequences looking for "maximum similarity" according to Neddleman and Wunsch's algorithm [J Mol Biol, 48, 443-453, 1970]. GAPs are more suitable for comparing sequences of approximately the same length and expect alignment over the entire length. Preferably, the parameters "gap weight" and "length weight" used in each program are 50 and 3 for the polynucleotide sequence and 12 and 4 for the polypeptide sequence, respectively. Preferably,% identity and similarity are measured when the two sequences being compared are optimally aligned.

Other programs for determining identity and / or similarity between sequences are also known in the art and include, for example, programs in the BLAST group [Altschul SF et al., J Mol Biol, 215, 403-410, 1990, Altschul SF et al., Nucleic Acids Res., 25: 389-3402, 1997, available from the National Center for Biotechnology Information (NCBI), Bethesda, Maryland, USA, accessible through NCBI's homepage at www.ncbi.nlm.nih.gov] And FASTA [Pearson WR, Methods in Enzymology, 183, 63-99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85, 2444-2448, 1988, available as part of the Wisconsin Sequence Analysis Package.

Preferably, the BLOSUM62 amino acid substitution matrix [Henikoff S and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992 are used for polypeptide sequence comparisons including where the nucleotide sequence is first translated into an amino acid sequence before comparison.

Preferably, the program BESTFIT can be used to determine the percent identity of the polynucleotide or polypeptide sequence in question with respect to the reference polynucleotide or polypeptide sequence, wherein the sequence in question and the reference sequence are optimally aligned and programmed. The parameter of is set to the default value as described above.

An "identity index" is a measure of sequence relationship that can be used to compare candidate sequences (polynucleotides or polypeptides) and reference sequences. Thus, for example, a candidate polynucleotide sequence having an identity index of 0.95 compared to the reference polynucleotide sequence may be a reference sequence except that the candidate polynucleotide sequence may comprise an average of up to 5 differences per 100 nucleotides of the reference sequence. Is the same as The difference is selected from the group consisting of substitutions or insertions including one or more nucleotide deletions, transitions and transversions. The difference may occur at the 5 'or 3' end position of the reference polynucleotide sequence or anywhere between the terminal positions, either individually in the nucleotides in the reference sequence or interspersed with one or more contiguous groups in the reference sequence. In other words, in order to obtain a polynucleotide sequence having an index of identity of 0.95 relative to the reference polynucleotide sequence, an average of up to five of every 100 nucleotides of the reference sequence will be deleted, substituted or inserted or any combination as described above. Can be. The same applies by making the necessary changes to other values of the identity index, for example 0.96, 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, a candidate polypeptide sequence having an identity index of, for example, 0.95 compared to the reference polypeptide sequence, except that the polypeptide sequence may comprise an average of up to five differences per 100 amino acids of each of the reference sequences. Identical to the sequence. The difference is selected from the group consisting of one or more amino acid deletions, substitutions including conservative and non-conservative substitutions, or insertions. The difference may occur at the amino- or carboxy-terminal position of the reference polypeptide sequence or anywhere between the terminal positions, either individually in the amino acids in the reference sequence or interspersed in one or more contiguous groups in the reference sequence. In other words, to obtain a polypeptide sequence having an identity index of 0.95 compared to a reference polypeptide sequence, an average of up to five of every 100 amino acids in the reference sequence, as described above, may be deleted, substituted, inserted, or any combination thereof. Can be. The same applies with the necessary changes to other values of identity indexes, for example 0.96, 0.97, 0.98 and 0.99.

The relationship between the number of nucleotide or amino acid differences and the identity index can be represented by the following equation:

_{n a ≤x a - (x a} · I),

Food:

n _a is the number of nucleotide or amino acid differences,

x _a is the total number of nucleotides or amino acids in SEQ ID NO: 1 or SEQ ID NO: 2, respectively,

I is the identity index,

· Is a symbol for a multiplication operator,

At this time, any non-integer result of x _a and I is rounded down to the nearest integer before subtracting from x _a .

"Animal" is a general term used in the art to indicate a polynucleotide or polypeptide sequence that has a high degree of sequence relevance to a reference sequence. The association can be quantified by measuring the degree of identity and / or similarity between the two sequences as defined above. The general terms include the terms "ortholog" and "paralog". "Ortholog" refers to a polynucleotide or polypeptide that is a functional equivalent of a polynucleotide or polypeptide in another species. "Paralog" refers to a polynucleotide or polypeptide within the same species that is functionally similar.

"Fusion protein" refers to a protein encoded by two, unrelated, fusion genes or fragments thereof. Examples are disclosed in US 5541087, US 5726044, EP 574395, EP 493418 and EP 0232 262. In the case of Fc-ANIC-BP, the use of immunoglobulin Fc regions as part of the fusion protein is used to improve the pharmacokinetic properties of the fusion protein when used in therapy, and to generate the dimer Fc-ANIC-BP. It is beneficial to carry out functional expression of Fc-ANIC-BP. The Fc-ANIC-BP DNA construct contains a secretion cassette, ie, a signal sequence that allows it to exit out from mammalian cells, DNA encoding an immunoglobulin Fc region fragment as a fusion partner, and Fc-ANIC-BP in the 5 'to 3' direction. DNA that encodes. In some applications, it is desirable to be able to alter endogenous functional properties (complementary binding, Fc-receptor binding) by altering functional Fc aspects while leaving the rest of the fusion protein intact, or to delete the Fc portion after expression is complete. .

All publications and references, including but not limited to patents and patent applications, cited herein, are incorporated by reference as if each individual publication or reference were specifically and individually described fully herein. As indicated, the entirety of which is incorporated herein by reference. Any patent application to which this application claims priority is also incorporated herein by reference in its entirety in the manner described above for the publications and references.

Description of the drawing:

1

Representative mRNA difference display gel with differentially regulated bands in cerebellar hemispheres contralateral to the injury side. Arrows indicate differentially expressed bands.

2

RT-PCR using 270 bp gene fragment of rat Mo25 in cerebellum after TBI in cycles 5, 10, 15, 20, 25 and 30, respectively. Note the strong expression in L2 lanes in cycles 20, 25 and 30.

L2: cDNA of TBI rat flanked with intact; R2: cDNA of flawed TBI rats.

3

Point blot analysis using radioactive 32P labeled 270 bp gene fragment of rat Mo25 with rat cerebellar mRNA after TBI. Six individual rats were tested, three with TBI and three with false treatments.

4

Multiple tissue northern blot using radioactive ³² P labeled 270 bp gene fragment of rat Mo25 hybridized to 8 rat tissues (Clontech Laboratories Inc, Palo Alto, CA, USA)

5

In situ hybridization of rat brain sections. Sense and antisense probes specific for SICCBP and Mo25 were used. Strong signals indicative of myelinated neural stems (brain volume, sising, whisker middle nerve, cerebellar anterior crosslinking) were monitored using antisense probes.

6

Real time TaqMAn PCR expression of ANIC-BP in rats 7 days after external brain injury. The intact cortical flank, damaged cortex flank and cerebellum were compared with brain tissue from falsely treated rats and brain tissue from rats sacrificed 7 days after the experiment. The error bar is the standard error of the mean (S.E.M.).

Another embodiment

Example 1

External brain damage model

Identification of genes up or down regulated in response to external brain injury (TBI) was studied in rats using the lateral fluid method. Appropriate TBI centered on the right parietal cortex was induced by lateral percussion in male Sprague-Dawley rats. After 5 days of TBI, rats were sacrificed and dissected brain tissue was analyzed by difference display. Trauma was confirmed by RT-PCR upregulation of protein in the cerebellum of the brain.

Control rats were anesthetized and the temporal muscles contracted but no craniotomy was performed. After 5 days of survival, all rats were anesthetized, killed and brain dissected and frozen in liquid nitrogen.

A second series of TBI rats was used for histochemical and immunohistochemical staining. After one week of TBI, the rats were anesthetized and perfused with saline followed by 3% paraformaldehyde. Brains were cut into 30 μm coronal sections on frozen microtome.

Example 2

Immunohistochemistry

Cerebellar cross sections were labeled with monoclonal antibodies against calcium binding proteins. Immune complexes were visualized by the avidin-biotin / DAB method. Calvindine-D (28 kD) was used as a positive control as a reliable marker of cerebellar purkinie cells.

Example 3

In-situ Hybridization

Oligodeoxynucleotides (sense, antisense) selected from SEQ ID NO: 1 were designed according to standard methods apparent to those skilled in the art. The probes were used for in situ hybridization in rat brain tissue cross sections according to methods known in the art. Radiographs were visualized after 5 days of exposure on Kodak BioMax-Film.

Example 4

RNA-Isolation from TBI Rats

mRNA difference displays have been developed as a method for identifying and analyzing altered gene expression at mRNA levels in any eukaryotic cells [Liang and Pardee, Science 257, 967, 1992]. In the present invention, to obtain better insight into the molecular effects of CNS damage, the method was used to study genes that are up- or down-regulated in response to external brain damage [den Daas et al .; Meeting of the American Neuroscience Society Washington, D.C., USA; 1998]. The animal model for external brain injury was called a lateral percussion model and was performed as follows: Moderate external brain injury centered on the right parietal cortex of the brain was induced by the lateral shock method in male Sprague-Dawley rats. Control rats were anesthetized and the temporal muscles contracted but no craniotomy was performed. After 5 days of survival, rats were anesthetized, killed and brain dissected and frozen in liquid nitrogen. Whole brain was homogenized and total RNA was isolated (Sambrook et al., 1989).

Difference display

The obtained RNA was analyzed by RNA difference display. Reverse transcription of mRNA was performed by starting the reaction at the poly (A) terminus using oligo-dT primers with two additional nucleotides (downstream primer; 13 mere) in all possible combinations. Amplification of the cDNA was performed using the same 3 'primer and second decamer arbitrary 5' primer (upstream primer). Amplification products were analyzed on an unmodified 10% polyacrylamide gel (Amersham Pharmacia Biotech, Germany). DNA was visualized by silver staining. After staining, the gel was dried at room temperature for 1 hour (FIG. 1). Differently adjusted bands were cut from the gel. DNA was eluted, reamplified and subcloned with pCR2.1 vector (Invitrogen, USA). Subcloned fragments were sequenced by Sanger method [Sanger F., et al., PNAS, USA 74, 5463-5467] and the sequences were compared to genomic databases. Confirmation of the obtained gene fragment was carried out by reverse transcription PCR (RT-PCR). For identification of otherwise expressed rat Mo25, sequences were analyzed by RT-PCR using a Titan One tube RT-PCR system (Boehringer Mannheim, Germany) using specific 19 and 21 mer primers. 1 μg total RNA from control and TBI animals was used for RT-PCR. More gene identification was performed using the dot blot technique using probes from rat Mo25 and Northern blot analysis performed using probes from human Mo25.

Reverse Transcription PCR (RT-PCR)

For identification of differently expressed stroke induced calcium binding proteins, sequences were analyzed by RT-PCR using a Titan One Tube RT-PCR system (Boehringer Mannheim, Germany) using specific 19 and 21 primers. 1 μg total RNA from control and TBI animals was used for RT-PCR.

Real time PCR

The distribution of ANIC-BP in the rat brain after head trauma was tested by Real Time TaqMan PCR technique in an ABI Prism 7700 sequence detection system (PE, Applied Biosystems, Germany). Using this technique, the absolute concentration of mRNA can be measured with high sensitivity. Specific primers of 25 and 29 bp length and 32 mer TaqMan probes (reporter dye: FAM / quench dye: TAMRA) were designed.

Ca ²⁺ bond

Proteins isolated by SDS-PAGE were blotted onto nitro cellulose membranes, Maruyama, K. et al. Biochem. 95: 511-519, 1984, was used for the binding of radiolabeled Ca ²⁺ . After incubation, the remaining isotopes were washed away and the membranes were exposed to Kodak XOMAT.TM film for an appropriate time. A band was developed at the location of the binding protein. The presence of the binding protein was confirmed by using an antibody specific for the binding protein and applying the antibody by Western blot methods well known in the art.

The present invention is a human acute nerve cell induced calcium-binding protein (A cute euronal I nduced N C alcium- B inding P rotein: ANIC-BP) relates to a polynucleotide encoding a view and, and said protein. The invention also provides expression vectors, host cells and antibodies. The present invention also provides a method of preparing a protein and treating or preventing a disorder associated with the expression of the protein. The invention also relates to the inhibition or activation of the action of said polynucleotides and polypeptides.

Claims (11)

Isolated polypeptide selected from the group consisting of: (a) an isolated polypeptide encoded by a polynucleotide comprising the sequence of SEQ ID NO: 1; (b) an isolated polypeptide comprising a polypeptide sequence that is at least 95% identical to the polypeptide sequence of SEQ ID NO: 2; (c) an isolated polypeptide at least 95% identical to the polypeptide sequence of SEQ ID NO: 2; And (d) the polypeptide sequence of SEQ ID NO: 2, and (e) fragments and variants of the polypeptides in (a) to (d). The isolated polypeptide of claim 1 comprising the polypeptide sequence of SEQ ID NO: 2. The isolated polypeptide of claim 1, wherein the polypeptide is SEQ ID NO: 2. Isolated polynucleotides selected from the group consisting of: (a) an isolated polynucleotide comprising a polynucleotide sequence that is at least 95% identical to the polynucleotide sequence of SEQ ID NO: 1; (b) an isolated polynucleotide that is at least 95% identical to the polynucleotide of SEQ ID NO: 1; (c) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide sequence that is at least 95% identical to the polypeptide of SEQ ID NO: 2; (d) an isolated polynucleotide having a polynucleotide sequence that encodes a polypeptide sequence that is at least 95% identical to the polypeptide sequence of SEQ ID NO: 2; (e) an isolated polynucleotide having a nucleotide sequence of at least 100 nucleotides obtained by screening the library under stringent hybridization conditions using a labeled probe having a sequence of SEQ ID NO: 1 or a fragment thereof having at least 15 nucleotides; (f) a polynucleotide that is an RNA equivalent of the polynucleotides of (a) to (e); Or a polynucleotide sequence complementary to said isolated polynucleotide And a polynucleotide that is a variant and fragment of the above-mentioned polynucleotide, or is complementary to the above-mentioned polynucleotide over its entire length. The isolated polynucleotide of claim 4, wherein the polynucleotide is selected from the group consisting of: (a) an isolated polynucleotide comprising the polynucleotide of SEQ ID NO: 1; (b) an isolated polynucleotide of SEQ ID NO: 1; (c) an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2; And (d) an isolated polynucleotide encoding a polypeptide of SEQ ID NO: 2. An expression system comprising a polynucleotide capable of producing the polypeptide of claim 1 when the expression vector is present in a compatible host cell. A recombinant host cell or membrane thereof comprising the expression vector of claim 6, wherein the polypeptide of claim 1 is expressed. A method of making a polypeptide of claim 1 comprising culturing a host cell as defined in claim 7 under conditions sufficient to produce the polypeptide and recovering the polypeptide from the culture medium. A fusion protein consisting of an immunoglobulin Fc-region and any one polypeptide of claim 1. An antibody that is immune specific for the polypeptide of any one of claims 1 to 3. A screening method for identifying a compound that stimulates or inhibits the level or function of a polypeptide of claim 1, comprising a method selected from the group consisting of: (a) quantitatively or qualitatively measuring or detecting binding of a candidate compound to a polypeptide (or cell or membrane expressing the polypeptide) or a fusion protein thereof, using a label directly or indirectly associated with the candidate compound; (b) measuring the competition of binding of the candidate compound to the polypeptide (or cell or membrane expressing the polypeptide) or a fusion protein thereof in the presence of a labeled competitor; (c) using a detection system appropriate for the cell or cell membrane that expresses the polypeptide to test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide; (d) mixing the solution comprising the polypeptide of claim 1 with the candidate compound to form a mixture, measuring the activity of the polypeptide in the mixture and comparing the activity of the mixture to a control mixture that does not contain the candidate compound; or (e) determining the effect of the candidate compound on the production of said polypeptide or mRNA encoding said polypeptide, for example using an ELISA assay, and (f) preparing said compound according to biotechnology or chemical standard techniques.