Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4728—Calcium binding proteins, e.g. calmodulin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to a human Acute Neuronal Induced Calcium-Binding Protein and polynucleotides (ANIC-BP) which identify and encode this protein.
• ANIC-BP Acute Neuronal Induced Calcium-Binding Protein and polynucleotides
• the invention as well provides expression vectors, host cells and antibodies.
• the invention provides methods for producing the protein and for treating or preventing disorders associated with the expression of the protein.
• the invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides.
• Stroke and acute head trauma multiple sclerosis and spinal cord injury are diseases for which so far there is no therapy available. Stroke is the third leading cause of death and the burden for the patients as well as for the social systems are enormous.
• ischaemic stroke which accounts for most of the strokes, blood vessel blockade in the brain is an initial event. Head trauma accidents are the leading disease of young people in the western world. A much smaller number of patients is affected by haemorhagic stroke caused by mechanical impact and artery rupture. It is a common feature, that approved pharmaceuticals are hardly available.
• Treatment approaches are based upon pathophysiologic concepts derived from experimental work with focal cerebral ischaemia. These include pharmacological strategies for arterial recanalisation, inhibition of inflammatory processes and neural protection.
• CaBPs calcium binding proteins
• CaBPs The major Ca 2+ -binding proteins (CaBPs) expressed in the central nervous system (parvalbumin, calbindin-D28K, and calretinin) have a very unusual and selective pattern of expression in various neuronal populations. Among neurons that do express CaBPs, most express only one type, although a small number of neurons express more than one of the major calcium binding proteins. There is growing evidence that the presence or absence of CaBPs in particular cell types underlies the phenomenon known as selective vulnerability. Selective vulnerability is a property of specific types of neurons to die in response to particular types of central nervous system (CNS) injury.
• CNS central nervous system
• CA1 hippocampal neurons are selectively vulnerable to global ischemia
• cerebellar Purkinje cells are selectively vulnerable to head trauma, stroke, and fetal alcohol exposure
• neurons in the substantia nigra are selectively vulnerable in Parkinson's Disease.
• neurons expressing high levels of parvalbumin are reported to be selectively vulnerable to AMPA-induced toxicity (Weiss et al., Neurol., 40, 1288-1292, 1990), whereas cultured hippocampal neurons expressing high levels of calbindin-D28K are reported to be selectively resistant to glutamate-induced toxicity (Baimbridge et al., TINS, 15, 303-8, 1992).
• hippocampal neurons expressing high levels of calretinin are resistant to toxic doses of the excitotoxins glutamate, NMDA, kainate, and quisqualate (Winsky et al., in: Novel
• CaBPs have also been shown to have altered expression in various CNS disease states, but again the results are inconsistent about whether CaBP expression is related to selective vulnerability or selective resistance to injury.
• Neurons expressing calbindin-D28K are reported to be selectively vulnerable in Alzheimer's Disease (lacopino et al. PNAS, 87, 4078-82, 1990, Hof et al. Exp. Neurol., 1 1 1 , 293-301 , 1991 ) and Huntington's Disease (Kiyama et al., Brain Res., 526, 303-07, 1990), although calbindin-D28K-expressing neurons in the substantia nigra are not selectively vulnerable in Parkinson's Disease (Yamada et al., Brain
• Alzheimer's Disease Brain et al., Neurosci., 80, 1 1 13-25, 1997.
• mice with a knockout of the calbindin gene show functional deficits (e.g. ataxia) that suggest severe dysfunction in neurons normally expressing this CaBP (e.g., cerebellar Purkinje cells) despite the fact that these neurons appear morphologically normal. This finding suggests that
• CaBPs are vital to cellular activity patterns (Airaksinen et al., PNAS, 94, 1488-93, 1997).
• retroviral infection of motoneurons with calbindin-D28k has been shown to have neuroprotective effects against toxicity induced by IgG from patients with amyotrophic lateral sclerosis (Ho et al., PNAS, 93, 6796-801 , 1996) and transfection with calbindin-
• D28k has been shown to protect PC12 cells from toxicity due to serum withdrawl, glutamate exposure, and the neurotoxin MPP+ (McMahon et al., Moiec. Brain Res. , 526, 303-07, 1998).
• CaBPs provide protection and others cause selective vulnerability, however it is not yet clear whether the expression of certain CaBPs within different neuronal populations results in different functional responses of a given CaBP. Another observation is that the severity of different types of CNS injury may affect the apparent neuroprotective efficacy of CaBPs - i.e., one CaBP may confer resistance in an injury model involving a mild injury, but may be unable to buffer Ca 2+ increases in more severe CNS injuries. Thus there is considerable evidence that CaBPs confer resistance as well as vulnerability in CNS injury process, but the mechanism of this involvement and the regulation of the response has still to be worked out.
• a gene familly comprising the functionally unidentified gene (MO25) was recently isolated from a mouse derived cDNA library (Miyamoto et al., Mol. Reprod. Dev., 34, 1-7, 1993).
• the library was constructed from RNA isolated from an early embrionic mouse.
• the predicted amino acid sequence for Mo25 revealed that the MO25 gene may have structural homology with Ca2+ binding proteins and lack membrane spanning domaines, indicating that the protein might be involved in cytosolic development of the unfertilized egg. However the real function of this protein remains unknown.
• Another Mo25 like gene has been cloned from a Drosophila cDNA library (Nozaki et al. DNA Cell Biol., 15, 505-09,
• the present invention relates to ANIC-BP, in particular ANIC-BP polypeptides and ANIC-BP polynucleotides, recombinant materials and methods for their production.
• ANIC-BP in particular ANIC-BP polypeptides and ANIC-BP polynucleotides
• Such polypeptides and polynucleotides are of interest in relation to methods of treatment of certain diseases, including, but not limited to stroke and acute head trauma, multiple sclerosis and spinal cord injury, hereinafter referred to as " diseases of the invention”.
• the invention relates to methods for identifying agonists and antagonists (e.g., inhibitors) using the materials provided by the invention, and treating conditions associated with ANIC-BP imbalance with the identified compounds.
• the invention relates to diagnostic assays for detecting diseases associated with inappropriate ANIC-BP activity or levels.
• the present invention relates to ANIC-BP polypeptides.
• ANIC-BP polypeptides include:
• Polypeptides of the present invention are believed to be members of * Calcium binding protein family of polypeptides. They are therefore of interest because they could serve as an novel drug target.
• ANIC-BP biological activity of ANIC-BP
• ANIC-BP activity biological activity of ANIC-BP
• a polypeptide of the present invention exhibits at least one biological activity of ANIC-BP.
• Polypeptides of the present invention also includes variants of the aforementioned polypeptides, including all allelic forms and splice variants. Such polypeptides vary from the reference polypeptide by insertions, deletions, and substitutions that may be conservative or non-conservative, or any combination thereof. Particularly preferred variants are those in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from
• Preferred fragments of polypeptides of the present 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 an isolated polypeptide comprising an amino acid sequence having at least 30, 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID NO: 2.
• Preferred fragments are biologically active fragments that mediate the biological activity of ANIC-BP, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also preferred are those fragments that are antigenic or immunogenic in an animal, especially in a human. Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
• polypeptides of the present invention may be in the form of the "mature" protein or may be a part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amino acid sequence that contains secretory or leader sequences, pro-sequences, sequences that aid in purification, for instance multiple histidine residues, or an additional sequence for stability during recombinant production.
• Polypeptides of the present invention can be prepared in any suitable manner, for instance by isolation form naturally occurring sources, from genetically engineered host cells comprising expression systems (vide infra) or by chemical synthesis, using for instance automated peptide synthesisers, or a combination of such methods.
• Means for preparing such polypeptides are well understood in the art.
• the present invention relates to ANIC-BP polynucleotides.
• Such polynucleotides include:
• an isolated polynucleotide comprising a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2;
• Preferred fragments of polynucleotides of the present invention include an isolated polynucleotide comprising an nucleotide sequence having at least 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQ ID NO: 1 , or an isolated polynucleotide comprising an sequence having at least 30, 50 or 100 contiguous nucleotides truncated or deleted from the sequence of SEQ ID NO: 1.
• Preferred variants of polynucleotides of the present invention include splice variants, allelic variants, and polymorphisms, including polynucleotides having one or more single nucleotide polymorphisms (SNPs).
• SNPs single nucleotide polymorphisms
• Polynucleotides of the present invention also include polynucleotides encoding polypeptide variants that comprise the amino acid sequence of SEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1 or 1 amino acid residues are substituted, deleted or added, in any combination.
• the present invention provides polynucleotides that are RNA transcripts of the DNA sequences of the present invention. Accordingly, there is provided an RNA polynucleotide that: (a) comprises an RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
• (b) is the RNA transcript of the DNA sequence encoding the polypeptide of SEQ ID NO:2;
• (c) comprises an RNA transcript of the DNA sequence of SEQ ID NO: 1
• (d) is the RNA transcript of the DNA sequence of SEQ ID NO:1 ; and RNA polynucleotides that are complementary thereto.
• polynucleotide sequence of SEQ ID NO:1 shows homoiogy with Hym A (Nozaki et al. DNA cell Biol., 15, 505-09, 1996) and Mo25 (Karos et al.,
• the polynucleotide sequence of SEQ ID NO:1 is a cDNA sequence that encodes the polypeptide of SEQ ID NO:2.
• the polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence of SEQ ID NO:1 or it may be a sequence other than SEQ ID NO:1 , which, as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
• the polypeptide of the SEQ ID NO:2 is related to other proteins of the * Calcium binding protein family, having homoiogy and/or structural similarity with Hym A and Mo25 proteins.
• Preferred polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides. Furthermore, preferred polypeptides and polynucleotides of the present invention have at least an ANIC-BP activity.
• Polynucleotides of the present invention may be obtained using standard cloning and screening techniques from a cDNA library derived from mRNA cells of the human central nervous system, (see for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Polynucleotides of the invention can also be obtained from natural sources such as genomic
• the polynucleotide may include the coding sequence for the mature polypeptide, by itself, or the coding sequence for the mature polypeptide in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
• a marker sequence that facilitates purification of the fused polypeptide can be encoded.
• the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc Natl Acad Sci USA (1989) 86:821-824, or is an HA tag.
• the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
• Polynucleotides that are identical, or have sufficient identity to a polynucleotide sequence of SEQ ID NO:1 may be used as hybridization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification reaction (for instance, PCR). Such probes and primers may be used to isolate full-length cDNAs and genomic clones encoding polypeptides of the present invention and to isolate cDNA and genomic clones of other genes (including genes encoding paralogs from human sources and orthologs and paralogs from species other than humans) that have a high sequence similarity to SEQ ID NO: 1 , typically at least 95% identity.
• Preferred probes and primers will generally 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 will have between 30 and 50 nucleotides. Particularly preferred primers will have between 20 and 25 nucleotides.
• a polynucleotide encoding a polypeptide of the present invention may be obtained by a process comprising the steps of screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides; and isolating full- length cDNA and genomic clones containing said polynucleotide sequence.
• Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA; followed by washing the filters in 0.1 x SSC at about 65°C.
• the present invention also includes isolated polynucleotides, preferably with a nucleotide sequence of at least 100, obtained by screening a library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides.
• an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide does not extend all the way through to the 5' terminus. This is a consequence of reverse transcriptase, an enzyme with inherently low "processivity" (a measure of the ability of the enzyme to remain attached to the template during the polymerisation reaction), failing to complete a
• PCR reaction is then repeated using 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence).
• primers designed to anneal within the amplified product typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence.
• the products of this reaction can then be analysed by DNA sequencing and a full-length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
• Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems.
• the present invention relates to expression systems comprising a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression sytems and to the production of polypeptides of the invention by recombinant techniques.
• Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
• host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
• Polynucleotides may be introduced into host cells by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al.(ibid).
• Preferred methods of introducing polynucleotides into host cells include, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
• bacterial cells such as Streptococci, Staphylococci, E. 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
• plant cells include bacterial cells, such as Streptococci, Staphylococci, E. 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
• plant cells include bacterial cells, such as Streptococci, Staphylococci, E.
• expression systems can be used, for instance, chromosomal, episomal and virus-derived systems, e.g. , vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
• the expression systems may contain control regions that regulate as well as engender expression.
• any system or vector that is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used.
• the appropriate polynucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, 5 for example, those set forth in Sambrook et al., (ibid).
• Appropriate secretion signals may be incorporated into the desired polypeptide to allow secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals.
• a polypeptide of the present invention is to be expressed for use in screening assays, it is generally preferred that the polypeptide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If the polypeptide is secreted into the medium, the medium can be recovered in order to i s recover and purify the polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
• Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange
• Polynucleotides of the present invention may be used as diagnostic reagents, through detecting mutations in the associated gene. Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NO: 1
• 30 NO:1 in the cDNA or genomic sequence and which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at
• Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
• the genomic DNA may be used directly for detection or it may be amplified enzymatically by using PCR, preferably RT-PCR, or other amplification techniques prior to analysis.
• RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled ANIC-BP nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
• DNA sequence difference may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, for instance, Myers et al., Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and
• An array of oligonucleotides probes comprising ANIC-BP polynucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g., genetic mutations.
• Such arrays are preferably high density arrays or grids.
• Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability, see, for example, M.Chee et al., Science, 274, 610- 613 (1996) and other references cited therein.
• Detection of abnormally decreased or increased levels of polypeptide or mRNA expression may also be used for diagnosing or determining susceptibility of a subject to a disease of the invention. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT- PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Thus in another aspect, the present invention relates to a diagnostic kit comprising:
• a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO: 1 , or a fragment or an RNA transcript thereof;
• polypeptide of the present invention preferably the polypeptide of SEQ ID NO:2 or a fragment thereof; or
• kits may comprise a substantial component.
• Such a kit will be of use in diagnosing a disease or susceptibility to a disease, particularly diseases of the invention, amongst others.
• the polynucleotide sequences of the present invention are valuable for chromosome localisation studies.
• the sequence is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
• the mapping of relevant sequences to chromosomes according to the present invention is an important first step in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
• the polynucleotide sequences of the present invention are also valuable tools for tissue expression studies. Such studies allow the determination of expression patterns of polynucleotides of the present invention which may give an indication as to the expression patterns of the encoded polypeptides in tissues, by detecting the mRNAs that encode them.
• the techniques used are well known in the art and include in situ hydridisation techniques to clones arrayed on a grid, such as cDNA microarray hybridisation (Schena et al, Science, 270, 467-470, 1995 and Shalon et al, Genome Res, 6, 639-645, 1996) and nucleotide amplification techniques such as PCR.
• a preferred method uses the TAQMAN (Trade mark) technology available from Perkin Elmer. Results from these studies can provide an indication of the normal function of the polypeptide in the organism. In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by an alternative form of the same gene (for example, one having an alteration in polypeptide coding potential or a regulatory mutation) can provide valuable insights into the role of the polypeptides of the present invention, or that of inappropriate expression thereof in disease. Such inappropriate expression may be of a temporal, spatial or simply quantitative nature.
• the polypeptides of the present invention are expressed in human brain.
• a further aspect of the present invention relates to antibodies.
• the polypeptides of the invention or their fragments, or cells expressing them, can be used as immunogens to produce antibodies that are immunospecific for polypeptides of the present invention.
• immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
• Antibodies generated against polypeptides of the present invention may be obtained by administering the polypeptides or epitope-bearing fragments, or cells to an animal, preferably a non-human animal, using routine protocols.
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, O, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al. , Monoclonal Antibodies and Cancer Therapy, 77-96, Alan R. Liss, Inc., 1985).
• antibodies may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
• Antibodies against polypeptides of the present invention may also be employed to treat diseases of the invention, amongst others.
• polypeptides and polynucleotides of the present invention may also be used as vaccines. Accordingly, in a further aspect, the present invention relates to a method for inducing an immunological response in a mammal that comprises inoculating the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said animal from disease, whether that disease is already established within the individual or not.
• An immunological response in a mammal may also be induced by a method comprises delivering a polypeptide of the present invention via a vector directing expression of the polynucleotide and coding for the polypeptide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases of the invention.
• One way of administering the vector is by accelerating it into the desired cells as a coating on particles or otherwise.
• Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
• a polypeptide or a nucleic acid vector will be normally provided as a vaccine formulation (composition).
• the formulation may further comprise a suitable carrier.
• a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
• parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents or thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze- dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
• the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
• Polypeptides of the present invention have one or more biological functions that are of relevance in one or more disease states, in particular the diseases of the invention hereinbefore mentioned. It is therefore useful to to identify compounds that stimulate or inhibit the function or level of the polypeptide. Accordingly, in a further aspect, the present invention provides for a method of screening compounds to identify those that stimulate or inhibit the function or level of the polypeptide. Such methods identify agonists or antagonists that may be employed for therapeutic and prophylactic purposes for such diseases of the invention as hereinbefore mentioned. Compounds may be identified from a variety of sources, for example, cells, cell-free preparations, chemical libraries, collections of chemical compounds, and natural product mixtures.
• Such agonists or antagonists so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc., as the case may be, of the polypeptide; a structural or functional mimetic thereof (see Coligan et al. , Current
• the screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearing the polypeptide, or a fusion protein thereof, by means of a label directly or indirectly associated with the candidate compound.
• the screening method may involve measuring or detecting (qualitatively or quantitatively) the competitive binding of a candidate compound to the polypeptide against a labeled competitor (e.g. agonist or antagonist). Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropriate to the cells bearing the polypeptide.
• Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuring a ANIC-BP activity in the mixture, and comparing the ANIC-BP activity of the mixture to a control mixture which contains no candidate compound.
• Polypeptides of the present invention may be employed in conventional low capacity screening methods and also in high-throughput screening
• HTS HyperText Transfer Protocol
• HTS formats include not only the well-established use of 96- and, more recently, 384-well micotiter plates but also emerging methods such as the nanowell method described by Schullek et al, Anal Biochem., 246, 20-29, (1997).
• Fusion proteins such as those made from Fc portion and ANIC-BP polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists for the polypeptide of the present invention (see 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 techniques
• polypeptides and antibodies to the polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and polypeptide in cells.
• an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents that may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
• a polypeptide of the present invention may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the polypeptide is labeled with a radioactive isotope (for instance, 'l 25 l), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. These screening methods may also be used to identify agonists and antagonists of the polypeptide that compete with the binding of the polypeptide to its receptors, if any. Standard methods for conducting such assays are well understood in the art.
• antagonists of polypeptides of the present invention include antibodies or, in some cases, oligonucleotides or proteins that are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or a small molecule that bind to the polypeptide of the present invention but do not elicit a response, so that the activity of the polypeptide is prevented.
• transgenic technology may also involve the use of transgenic technology and ANIC-BP gene.
• ANIC-BP gene may be introduced through microinjection into the male pronucleus of fertilized oocytes, retroviral transfer into pre- or post-implantation embryos, or injection of genetically modified, such as by electroporation, embryonic stem cells into host blastocysts.
• Particularly useful transgenic animals are so-called "knock- in” animals in which an animal gene is replaced by the human equivalent within the genome of that animal. Knock-in transgenic animals are useful in the drug discovery process, for target validation, where the compound is specific for the human target.
• transgenic animals are so-called “knock-out” animals in which the expression of the animal ortholog of a polypeptide of the present invention and encoded by an endogenous DNA sequence in a cell is partially or completely annulled.
• the gene knock-out may be targeted to specific cells or tissues, may occur only in certain cells or tissues as a consequence of the limitations of the technology, or may occur in all, or substantially all, cells in the animal.
• Transgenic animal technology also offers a whole animal expression- cloning system in which introduced genes are expressed to give large amounts of polypeptides of the present invention
• Screening kits for use in the above described methods form a further aspect of the present invention.
• Such screening kits comprise:
• polypeptide is preferably that of SEQ ID NO:2.
• kits may comprise a substantial component.
• Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or another immunoglobulin expression library.
• Isolated means altered “by the hand of man” from its natural state, i.e. , if it occurs in nature, it has been changed or removed from its original environment, or both.
• a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
• a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated” even if it is still present in said organism, which organism may be living or non-living.
• Polynucleotide generally refers to any polyribonucleotide (RNA) or polydeoxribonucleotide (DNA), which may be unmodified or modified RNA or DNA.
• Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• the term “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
• Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
• Polypeptide refers to any polypeptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
• Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
• Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
• Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide.
• polypeptides may contain many types of modifications.
• Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Protein
• “Fragment” of a polypeptide sequence refers to a polypeptide sequence that is shorter than the reference sequence but that retains essentially the same biological function or activity as the reference polypeptide.
• “Fragment” of a polynucleotide sequence refers to a polynucloetide sequence that is shorter than the reference sequence of SEQ ID NO: 1..
• Variant refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains the essential properties thereof.
• a typical variant of a polynucleotide differs in nucleotide sequence from the reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
• a typical variant of a polypeptide differs in amino acid sequence from the reference polypeptide.
• a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, insertions, deletions in any combination.
• a substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Typical conservative substitutions include Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe and Tyr.
• a variant of a polynucleotide or polypeptide may be naturally occurring such as an allele, or it may be a variant that is not known to occur naturally.
• Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis. Also included as variants are polypeptides having one or more post-translational modifications, for instance glycosylation, phosphorylation, methylation, ADP ribosylation and the like. Embodiments include methylation of the N-terminal amino acid, phosphorylations of serines and threonines and modification of C- terminal glycines.
• Allele refers to one of two or more alternative forms of a gene occuring at a given locus in the genome.
• Polymorphism refers to a variation in nucleotide sequence (and encoded polypeptide sequence, if relevant) at a given position in the genome within a population.
• Single Nucleotide Polymorphism refers to the occurence of nucleotide variability at a single nucleotide position in the genome, within a population.
• An SNP may occur within a gene or within intergenic regions of the genome.
• SNPs can be assayed using Allele Specific Amplification (ASA). For the process at least 3 primers are required. A common primer is used in reverse complement to the polymorphism being assayed. This common primer can be between 50 and 1500 bps from the polymorphic base.
• ASA Allele Specific Amplification
• the other two (or more) primers are identical to each other except that the final 3' base wobbles to match one of the two (or more) alleles that make up the polymorphism.
• Two (or more) PCR reactions are then conducted on sample DNA, each using the common primer and one of the Allele Specific Primers.
• RNA molecules produced from RNA molecules initially transcribed from the same genomic DNA sequence but which have undergone alternative RNA splicing.
• RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of that may encode different amino acid sequences.
• the term splice variant also refers to the proteins encoded by the above cDNA molecules.
• Identity reflects a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing the sequences. In general, identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of the two polynucleotide or two polypeptide sequences, respectively, over the length of the sequences being compared.
• % Identity For sequences where there is not an exact correspondence, a “% identity” may be determined.
• the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment.
• a % identity may be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or very similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
• Similarity is a further, more sophisticated measure of the relationship between two polypeptide sequences.
• similarity means a comparison between the amino acids of two polypeptide chains, on a residue by residue basis, taking into account not only exact correspondences between a between pairs of residues, one from each of the sequences being compared (as for identity) but also, where there is not an exact correspondence, whether, on an evolutionary basis, one residue is a likely substitute for the other. This likelihood has an associated "score” from which the "% similarity" of the two sequences can then be determined.
• BESTFIT is more suited to comparing two polynucleotide or two polypeptide sequences that are dissimilar in length, the program assuming that the shorter sequence represents a portion of the longer.
• GAP aligns two sequences, finding a "maximum similarity", according to the algorithm of Neddleman and Wunsch (J Mol Biol, 48, 443-453, 1970).
• GAP is more suited to comparing sequences that are approximately the same length and an alignment is expected over the entire length.
• the parameters "Gap Weight” and "Length Weight” used in each program are 50 and 3, for polynucleotide sequences and 12 and 4 for polypeptide sequences, respectively.
• % identities and similarities are determined when the two sequences being compared are optimally aligned.
• the BLOSUM62 amino acid substitution matrix (Henikoff S and Henikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915-10919, 1992) is used in polypeptide sequence comparisons including where nucleotide sequences are first translated into amino acid sequences before comparison.
• the program BESTFIT is used to determine the % identity of a query polynucleotide or a polypeptide sequence with respect to a reference polynucleotide or a polypeptide sequence, the query and the reference sequence being optimally aligned and the parameters of the program set at the default value, as hereinbefore desc ⁇ bed.
• Identity Index is a measure of sequence relatedness which may be used to compare a candidate sequence (polynucleotide or polypeptide) and a reference sequence.
• a candidate polynucleotide sequence having, for example, an Identity Index of 0.95 compared to a reference polynucleotide sequence is identical to the reference sequence except that the candidate polynucleotide sequence may include on average up to five differences per each 100 nucleotides of the reference sequence. Such differences are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion.
• a candidate polypeptide sequence having, for example, an Identity Index of 0.95 compared to a reference polypeptide sequence is identical to the reference sequence except that the polypeptide sequence may include an average of up to five differences per each 100 amino acids of the reference sequence. Such differences are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion. These differences may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between these terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
• an average of up to 5 in every 100 of the amino acids in the reference sequence may be deleted, substituted or inserted, or any combination thereof, as hereinbefore described.
• 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
• I is the Identity Index
• • is the symbol for the multiplication operator, and in which any non-integer product of x a and I is rounded down to the nearest integer prior to subtracting it from x a .
• “Homolog” is a generic term used in the art to indicate a polynucleotide or polypeptide sequence possessing a high degree of sequence relatedness to a reference sequence. Such relatedness may be quantified by determining the degree of identity and/or similarity between the two sequences as hereinbefore defined. Falling within this generic term are the terms “ortholog”, and “paralog”. "Ortholog” refers to a polynucleotide or polypeptide that is the functional equivalent of the polynucleotide or polypeptide in another species. "Paralog” refers to a polynucieotideor polypeptide that within the same species which is functionally similar.
• Fusion protein refers to a protein encoded by two, unrelated, fused genes or fragments thereof. Examples have been disclosed in US
• Fc-ANIC-BP employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for performing the functional expression of Fc-ANIC-BP, to improve pharmacokinetic properties of such a fusion protein when used for therapy and to generate a dimeric Fc-ANIC-BP.
• the Fc-ANIC-BP DNA construct comprises in 5' to 3' direction, a secretion cassette, i.e. a signal sequence that triggers export from a mammalian cell, DNA encoding an immunoglobulin Fc region fragment, as a fusion partner, and a DNA encoding Fc-ANIC-BP.
• a secretion cassette i.e. a signal sequence that triggers export from a mammalian cell
• DNA encoding an immunoglobulin Fc region fragment as a fusion partner
• a DNA encoding Fc-ANIC-BP DNA encoding Fc-ANIC-BP.
• L2 cDNA of TBI rat non lesioned side
• R2 cDNA of TBI rat lesioned side.
• TBI traumatic brain injury
• Control rats were anesthetized and the temporal muscle was retracted, but no craniotomy was performed. After a five-day survival, all rats were anesthetized, killed, and the brain was dissected and frozen in liquid nitrogen.
• TBI rats were used for histochemical and immunohistochemical staining.
• TBI rats were anesthetized and perfused with saline followed by 3% paraformaldehyde.
• the brains were cut on a freezing microtome into 30 ⁇ m coronal sections.
• Cerebellar sections were labeled with monoclonal antibodies against the calcium binding protein.
• the immuno complex was visualized using the avidin-biotin/DAB method.
• the Calbindin-D 28kD has been used as a reliable marker of cerebellar Purkinje cells.
• Oligodeoxynucleotides selected from Sequence No.1 have been designed according to standard methods apperent to those skilled in the art. These probes have been used for in situ hybridisation in rat brain tissue sections according to methods known in the art. Autoradiograph have been visualized after 5 day exposure on Kodak BioMax-Film.
• mRNA differential display has been developed as a method to identify and analyze altered gene expression at the mRNA level in any eukaryotic cell (Liang and Pardee, Science 257, 967, 1992).
• this invention we used this method to study genes up or down regulated in response to traumatic brain injury, in order to obtain a better insight into the molecular effects of CNS injury (den Daas et al.; Meeting of the American Neuroscience Society Washington D.C., USA; 1998).
• the animal model for traumatic brain injury is called the lateral fluid percussion model and is performed as follows: a moderate traumatic brain injury centered on the right parietal cortex of the brain was induced with the lateral fluid percussion method in male Sprague-Dawley rats.
• RNA differential display Reverse transcription of mRNAs was performed using oligo-dT primers with two additional nucleotides in all possible combinations (downstream primers; 13 mere), thus anchoring the reaction to the beginning of the poly (A) tail.
• Amplification of the cDNA was conducted with the same 3 ' primer and a second decamer arbitrary 5 ' primer (upstream primer). Amplification products were analyzed on a nondenaturing 10% polyacrylamide gel
• RNA from control and TBI animals were used for the RT-PCR. More gene validation has been performed using the dotblot technique with probes from rat Mo25 and Northern blot analysis performed with probes from human Mo25.
• ANIC-BP Distribution of ANIC-BP in the rat brain after head trauma was examined by the Real Time TaqMan PCR technique in a ABI prism 7700 sequence detection system (PE, Applied Biosystems, Germany). With this technique, absolute concentrations of mRNA can be measured with high sensitivity. Special primers with a length of 25 and 29 bp and a 32 mere
• TaqMan probe reporter dye: FAM/ quencher dye: TAMRA
• Protein separated by SDS-PAGE was blotted onto nitro cellulose membrane and assayed for binding of radiolabled Ca 2+ using the method described by Maruyama, K. et al. (J. Biochem. 95:511-519, 1984). After incubation the surplus of isotope was washed off and the membrane was exposed to Kodak XOMAT.TM film for an appropriate time. A band developed at the position of the binding protein. The presence of the binding protein was confirmed by using antibody specific for the binding protein and applying that antibody by the western blot method that is well known in the art.

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