WO1999051738A1 - Human gap junction protein beta-4 - Google Patents

Abstract

Gap junction protein beta-4 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing gap junction protein beta-4 polypeptides and polynucleotides in therapy, and diagnostic assays for such.

Description

International application No

I N TE RN ATIONAL SEA RCH REPORT PCT/CN98/00055

A. CLASSIFIC ΛTION OF SUBJECT MATTER

IPC⁶ C 12N15/12

\ccordinc to iniemauonal Patent C lassification! I PC ) or 10 both national classification and IPC

B. FIELDS SEARCHED

Minimum αocumentation searched! classification s\ stem followed b\ classification s\mools ι

IPC⁶ C12N 15/12

Documentation searcned other than minimum documentation to tne extent that such documents are included in the field searched

Electronic data base consulted during the international search! name ot data base and. where practicable, search terms used)

CNPAT , EPOQUE(WPI)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Cateeorv* Citation of document, with indication, where appropπate. of the relevant passages Relevant claim No

A US. Λ. 5650317 (UNIV. MICHIGAN STATE) 1-12 22 July 1997. See the whole document

WO. A 1. 9728179 (UNIV CALIFORNIA) 1-12 07 August 1997 , See the whole document

DE. C l . 4420791 (LUEHRTNG H) 1-12

18 Mav 1995 . See the whole document

I I Further documents are listed in the continuation of Box C [Xj See patent family annex

Special categories ol cited documents later document published alter the international tiling date or prιoπι\ document dctimne the general state of the art which is not considered date and not in conflict w ith the application but cited to understand the principle or iheon, undcrlv ing the invention to be ol particular relevance document ol particular rcle\ ancc the claimed invention cannol he earlier document but published on or alter the international filing date considered novel or cannot be considered to involv e an inv entiv e document which mas throw doubts on prioπu claimlsl or which is step when the document is taken alone cited lo establish the publication date ol another citation or other document of particular relevance the claimed invention cannot be special reason! as specilicdl considered to involv e an inv entiv e step w nen ihe document is document rcterrin- to an oral disclosure use exhibition or other combined with one or more other u_^h documents such combination means beinε obvious to a person skilled in the an document published prior to the international film- date but later than document member ol the same patent tamiK the nnoπtΛ date claimed

Date ot the actual completion ot the international searcn I Date oi mailing ot the international searcn report

19 Decemr er 1998( 19 12 98) 31 December 1998 ( 31 .12.98)

Name and mailing address ot the ISA- I Authorized officer

The Cninese Patent Office b Xnucnen" Road. Haidian District PAN, aiquπ Bening. 100088. China Facsimile No 86- 10-6201945 ! iTeleDnone No 86-10-62093906

Form PCT ISA 210( second sheet)! Julv 1992) INTERNATIONAL SEARCH REPORT International application No.

Information on patent family members PCT/CN98/00055

Patent document Publication Patent family Publication cited in search report date members date

US-A- 5650317 22.07.97 none

WO-A 1-9728179 07.08.97 22. 08.97

AU-A-1846297

DE-C 1 -4420791 18.05.95 none

Form PCT,'ISA'210(patent family annex)(JuIy 1992) Human Gap Junction Protein Beta-4

Field of the Invention

This invention relates to newly identified polypeptides and polynucleotides encoding such polypeptides, to their use in therapy and in identifying compounds which may be agonists. antagonists and/or inhibitors which are potentially useful in therapy, and to production of such polypeptides and polynucleotides

Background of the Invention The drug discovery process is currently undergoing a fundamental revolution as it embraces

'functional genomics', that is, high throughput genome- or gene-based biology This approach is rapidly superceding earlier approaches based on 'positional cloning' A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available There is a continuing need to identify and characterise further genes and their related polypeptides/proteins, as targets for drug discovery

Summary of the Invention

The present invention relates to gap junction protein beta-4. in particular gap junction protein beta-4 polypeptides and gap junction protein beta-4 polynucleotides, recombinant mateπals and methods for their production In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of neurological diseases, epidermal diseases, deafness, cataracts, and AIDS, hereinafter referred to as "the Diseases", amongst others In a further aspect, the invention relates to methods for identifying agonists and antagonists/inhibitors using the mateπals provided by the invention, and treating conditions associated with gap junction protein beta-4 imbalance with the identified compounds In a still further aspect, the invention relates to diagnostic assays for detecting diseases associated with inappropriate gap junction protein beta-4 activity or levels

Description of the Invention

In a first aspect, the present invention relates to gap junction protein beta-4 polypeptides Such peptides include isolated polypetides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more

1 preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include those comprising the amino acid of SEQ ID NO:2.

Further peptides of the present invention include isolated polypeptides in which the amino acid sequence has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to the ammo acid sequence of SEQ ID NO:2 over the entire length of SEQ ID NO:2. Such polypeptides include the polypeptide of SEQ ID NO:2.

Further peptides of the present invention include isolated polypeptides encoded by a polynucleotide comprising the sequence contained in SEQ ID NO: 1.

Polypeptides of the present invention are believed to be members of the gap junction protein family of polypeptides. They are therefore of interest because gap junctions which were characterized as regionally specialized structures on plasma membranes of contacting adherent cells were shown to consist of cell-to-cell channels. Proteins, called connexins, purified from fractions of enriched gap junctions from different tissues differ. The connexins are designated by their molecular mass. Another system of nomenclature divides gap junction proteins into 2 categories, alpha and beta, according to sequence similarities at the nucleotide and amino acid levels. For example, CX43 is designated alpha- 1 gap junction protein, whereas CX32 and CX26 are called beta-1 and beta-2 gap junction proteins, respectively. This nomenclature emphasizes that CX32 and CX26 are more homologous to each other than either of them is to CX43. Several members of the connexin family are responsible for some diseases. For example, mutation of CX26 causes deafness. These properties are hereinafter referred to as "gap junction protein beta-4 activity" or "gap junction protein beta-4 polypeptide activity" or "biological activity of gap junction protein beta-4". Also included amongst these activities are antigenic and immunogenic activities of said gap junction protein beta-4 polypeptides, in particular the antigenic and immunogenic activities of the polypeptide of SEQ ID NO:2. Preferably, a polypeptide of the present invention exhibits at least one biological activity of gap junction protein beta-4.

The 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 fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The present invention also includes include variants of the aforementioned polypetides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics Typical such substitutions are among Ala. Val, Leu and He, among Ser and Thr, among the acidic residues Asp and Glu, among Asn and Gin. and among the basic residues Lys and Arg, or aromatic residues Phe and Tyr Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acids are substituted, deleted, or added in any combination Polypeptides of the present invention can be prepared m any suitable manner Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood in the art

In a further aspect, the present invention relates to gap junction protein beta-4 polynucleotides Such polynucleotides include isolated polynucleotides compπsing a nucleotide sequence encoding a polypeptide which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to the amino acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2 In this regard, polypeptides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred Such polynucleotides include a polynucleotide compπsing the nucleotide sequence contained in SEQ ID NO 1 encoding the polypeptide of SEQ ID NO 2

Further polynucleotides of the present invention include isolated polynucleotides compπsing a nucleotide sequence that has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to a nucleotide sequence encoding a polypeptide of SEQ ID NO 2, over the entire coding region In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% identity are more highly preferred, and those with at least 99% identity are most highly preferred

Further polynucleotides of the present invention include isolated polynucleotides comprising a nucleotide sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, to SEQ ID NO 1 over the entire length of SEQ ID NO 1 In this regard, polynucleotides which have at least 97% identity are highly preferred, whilst those with at least 98-99% ldentiy are more highly preferred, and those with at least 99% identity are most highly preferred Such polynucleotides include a polynucleotide compπsing the polynucleotide of SEQ ID NO 1 as well as the polynucleotide of SEQ ID NO 1

The invention also provides polynucleotides which are complementary to all the above descnbed polynucleotides

The nucleotide sequence of SEQ ID NO 1 shows homology with rat or muπne connexin 31 1 The nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and compπses a polypeptide encoding

3 sequence (nucleotide 168 to 986) encoding a polypeptide of 273 amino acids, the polypeptide of SEQ ID NO 2 The nucleotide sequence encoding the polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in SEQ ID NO 1 or it may be a sequence other than the one contained in 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 structurally related to other proteins of the gap junction protein family, having homology and/or structural similaπty with rat or murine connexin 31 1

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 mvention have at least one gap junction protein beta-4 activity

Polynucleotides of the present invention may be obtained, using standard cloning and screening techniques, from a cDNA library deπved from mRNA in cells of human placenta and others, using the expressed sequence tag (EST) analysis (Adams, M O , et al Science ( 1991) 252 1651-1656, Adams, M D et al , Nature, (1992) 355 632-634, Adams, M D , et al , Nature (1995) 377

Supp 3-174) Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques

When polynucleotides of the present invention are used for the recombinant production of polypeptides of the present invention, the polynucleotide may include the codmg sequence for the mature polypeptide, by itself, or the codmg sequence for the mature polypeptide in reading frame with other coding sequences, such as those encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions For example, a marker sequence which facilitates purification of the fused polypeptide can be encoded In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed in Gentz et al , Proc Natl Acad S USA ( 1989) 86 821 -824, or is an HA tag The polynucleotide may also contain non-coding 5' and 3' sequences, such as transcπbed, non-translated sequences, splicing and polyadenylation signals, πbosome binding sites and sequences that stabilize mRNA Further embodiments of the present invention mclude polynucleotides encodmg polypeptide vanants which compπse the amino acid sequence of SEQ ID NO 2 and m which several, for instance from 5 to 10, 1 to 5, 1 to 3, 1 to 2 or 1, ammo acid residues are substituted, deleted or added, in any combination Polynucleotides which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO 1, may be used as hybπdization probes for cDNA and genomic DNA or as primers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encodmg polypeptides of the present mvention and to isolate cDNA and genomic clones of other genes (including genes encodmg homologs and orthologs from species other than human) that have a high sequence similaπty to SEQ ID NO 1 Typically these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to that of the referent The probes or primers will generally compπse at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 50 nucleotides Particularly preferred probes will have between 30 and 50 nucleotides A polynucleotide encoding a polypeptide of the present mvention, including homologs and orthologs from species other than human, may be obtained by a process which compπses the steps of screening an appropπate library under strmgent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence Such hybπdization techniques are well known to the skilled artisan Preferred strmgent hybπdization conditions mclude overnight mcubation at 42°C m a solution compπsing 50% formamide, 5xSSC (150mM NaCl, 15mM tπsodium 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 lx SSC at about 65°C Thus the present mvention also includes polynucleotides obtainable by screening an appropπate library under stingent hybπdization conditions with a labeled probe having the sequence of SEQ ID NO 1 or a fragment thereof

The skilled artisan will appreciate that, in many cases, an isolated cDNA sequence will be incomplete, in that the region codmg for the polypeptide is cut short at the 5' end of the cDNA This is a consequence of reverse transcπptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remam attached to the template duπng the polymeπsation reaction), failing to complete a DNA copy of the mRNA template duπng 1st strand cDNA synthesis

There are several methods available and well known to those skilled in the art to obtain full-length cDNAs, or extend short cDNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman et al , PNAS USA 85, 8998- 9002, 1988) Recent modifications of the technique, exemplified by the Marathon™' technology (Clontech Laboratories Inc ) for example, have significantly simplified the search for longer cDNAs In the Marathon™ technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence hgated onto each end Nucleic acid amplification (PCR) is

5 then carried out to amplify the 'missing' 5' end of the cDNA using a combination of gene specific and adaptor specific o gonucleotide pπmers The PCR reaction is then repeated using 'nested' pπmers, that is, pπmers designed to anneal within the amplified product (typically an adaptor specific pπmer 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 mvention may be prepared by processes well known in the art from genetically engmeered host cells compπsmg expression systems Accordmgly, in a further aspect, the present mvention relates to expression systems which compπse a polynucleotide or polynucleotides of the present mvention, to host cells which are genetically engmeered with such expression sytems and to the production of polypeptides of the mvention by recombinant techniques Cell-free translation systems can also be employed to produce such proteins using RNAs deπved from the DNA constructs of the present mvention

For recombinant production, host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention Introduction of polynucleotides into host cells can be effected by methods descnbed m many standard laboratory manuals, such as Davis et aL, Basic Methods in Molecular Biology (1986) and Sambrook et al , Molecular Cloning A Laboratory Manual, 2nd Ed , Cold Sprmg Harbor Laboratory Press, Cold Sprmg Harbor, N Y (1989) Preferred such methods mclude, for instance, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection

Representative examples of appropnate hosts mclude bacteπal cells, such as streptococci, staphylococci , E colt, Streptomyces and Bacillus subhlis 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 great vaπety of expression systems can be used, for instance, chromosomal, episomal and virus-deπved systems, e g , vectors deπved from bacteπal plasrmds, from bacteπophage, 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 deπved from combinations thereof, such as those deπved from plasmid and bacteπophage genetic elements, such as cosmids and phagemids The expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector which is able to maintain, propagate or express a polynucleotide to produce a polypeptide in a host may be used. The appropπate nucleotide sequence may be inserted into an expression system by any of a vanety of well-known and routme techniques, such as. for example, those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL (supra). 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.

If 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 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 chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the polynucleotide of SEQ ID NOT 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 expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques .

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 may be amplified enzymatically by using 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 gap junction protein beta-4 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (ee, e g , Mvers et al , Science (1985) 230 1242) Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (see Cotton et al , Proc Natl Acad Sci USA (1985) 85 4397-4401) In another embodiment, an array of oligonucleotides probes compπsmg gap junction protem beta-4 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e g , genetic mutations Array technology methods are well known and have general applicability and can be used to address a vaπety of questions m molecular genetics including gene expression, genetic linkage, and genetic variability (see for example M Chee et al , Science, Vol 274, pp 610-613 (1996))

The diagnostic assavs offer a process for diagnosing or determimng a susceptibility to the Diseases through detection of mutation m the gap junction protem beta-4 gene by the methods descnbed In addition, such diseases may be diagnosed by methods compπsing determining from a sample deπved from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known m 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 hybndization methods Assay techniques that can be used to determine levels of a protein, such as a polypeptide of the present mvention, m a sample deπved from a host are well-known to those of skill the art Such assay methods mclude radiounmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays Thus in another aspect, the present invention relates to a diagonostic kit which comprises

(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,

(b) a nucleotide sequence complementary to that of (a),

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

(d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQ ID NO 2

It will be appreciated that m any such kit, (a), (b), (c) or (d) may compπse a substantial component Such a kit will be of use m diagnosing a disease or suspectabi ty to a disease, particularly neurological diseases, epidermal diseases, deafness, cataracts, and AIDS, amongst others

The nucleotide sequences of the present mvention are also valuable for chromosome identification The sequence is specifically targeted to, and can hybπdize with, a particular location on an individual human chromosome The mapping of relevant sequences to chromosomes according to the present mvention 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 m, for example, V McKusick, Mende an Inheπtance in Man (available on-lme through Johns Hopkins University Welch Medical Library) The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheπtance of physically adjacent genes)

The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease

The polypeptides of the mvention or their fragments or analogs thereof, or cells expressmg them, can also be used as lmmunogens to produce antibodies immunospecific for polypeptides of the present mvention The term ''immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pπor art Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-beaπng fragments, analogs or cells to an animal, preferably a non-human animal, using routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used Examples mclude the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the tπoma technique, the human B-cell hybndoma techmque (Kozbor et al , Immunology Today ( 1983) 4 72) and the EBV- hybπdoma techmque (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)

Techniques for the production of smgle chain antibodies, such as those descnbed m U S Patent No 4,946,778, can also be adapted to produce smgle chain antibodies to polypeptides of this mvention Also, transgemc mice, or other organisms, mcludmg other mammals, may be used to express humanized antibodies

The above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to punfy the polypeptides by affinity chromatography

Antibodies against polypeptides of the present mvention may also be employed to treat the Diseases, amongst others

In a further aspect, the present invention relates to genetically engineered soluble fusion proteins compπsing a polypeptide of the present invention, or a fragment thereof, and vaπous portions of the constant regions of heavy or light chains of lmmunoglobulms of vaπous subclasses (IgG, IgM, IgA, IgE) Preferred as an lmmunoglobuhn is the constant part of the heavy chain of

9 human IgG, particularly IgGl, where fusion takes place at the hinge region In a particular embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineeπng, and to the use thereof for drug screening, diagnosis and therapy A further aspect of the invention also relates to polynucleotides encodmg such fusion proteins Examples of fusion protein technology can be found in International Patent Application Nos W094/29458 and W094/22914

Another aspect of the invention relates to a method for inducing an immunological response in a mammal which compπses moculatmg the mammal with a polypeptide of the present invention, adequate to produce antibody and/or T cell immune response to protect said animal from the Diseases herembefore mentioned, amongst others Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which 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

A further aspect of the invention relates to an lmmunological/vacc e formulation (composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a polypeptide of the present invention wherein the composition compπses a polypeptide or polynucleotide of the present mvention The vaccine formulation may further compπse a suitable earner Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or lntradermal injection) Formulations suitable for parenteral administration mclude aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteπostats and solutes which render the formulation mstonic with the blood of the recipient, and aqueous and non- aqueous sterile suspensions which 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-dπed condition requiring only the addition of the steπle liquid earner immediately pπor to use The vaccine formulation may also include adjuvant systems for enhancing the lmmunogemcity of the formulation, such as oil-m water systems and other systems known m 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 mvention are responsible for many biological functions, mcludmg many disease states, m particular the Diseases herembefore mentioned It is therefore desirous to devise screening methods to identify compounds which stimulate or which inhibit the function of the

10 polypeptide Accordingly, in a further aspect, the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide In general, agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned Compounds may be identified from a vaπety of sources, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures Such agonists, antagonists or inhibitors so-identified may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide, or may be structural or functional mimetics thereof (see Cohgan et al , Current Protocols m Immunology 1(2) Chapter 5 (1991))

The screening method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes beaπng the polypeptide, or a fusion protem thereof by means of a label directly or indirectly associated with the candidate compound Alternatively, the screening method may involve competition with a labeled competitor Further, these screening methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems appropnate to the cells beaπng the polypeptide Inhibitors of activation are generally assayed m the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed Constitutively active polpypeptides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide Further, the screening methods may simply compπse the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form a mixture, measuπng gap junction protem beta-4 activity in the mixture, and comparing the gap junction protem beta-4 activity of the mixture to a standard Fusion proteins, such as those made from Fc portion and gap junction protem beta-4 polypeptide, as hereinbefore descnbed, 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 Recogmtion, 8 52-58 (1995), and K Johanson et al , J Bi Chem, 270(16) 9459-9471 (1995))

The polynucleotides, polypeptides and antibodies to the polypeptide of the present mvention may also be used to configure screenmg methods for detect g the effect of added compounds on the production of mRNA and polypeptide in cells For example, an ELISA assay may be constructed for measuπng secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known m the art This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues

1 1 The polypeptide 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, gand binding and cross nkmg assays in which the polypeptide is labeled with a radioactive isotope (for instance, ^1), chemically modified (for instance, biotmylated), 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 which compete with the binding of the polypeptide to its receptors, if any Standard methods for conducting such assays are well understood m the art

Examples of potential polypeptide antagonists mclude antibodies or, m some cases, oligonucleotides or proteins which are closely related to the gands, substrates, receptors, enzymes, etc , as the case may be, of the polypeptide, e g , a fragment of the hgands, substrates, receptors, enzymes, etc , or small molecules which bmd to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented

Thus, in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc for polypeptides of the present mvention, or compounds which decrease or enhance the production of such polypeptides, which compπses (a) a polypeptide of the present invention,

(b) a recombinant cell expressing a polypeptide of the present invention,

(c) a cell membrane expressing a polypeptide of the present invention, or

(d) antibody to a polypeptide of the present invention, which polypeptide is preferably that of SEQ ID NO 2 It will be appreciated that in any such kit, (a), (b), (c) or (d) may compπse a substantial component

It will be readily appreciated by the skilled artisan that a polypeptide of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the polypeptide, by (a) determining in the first instance the three-dimensional structure of the polypeptide,

(b) deducing the three-dimensional structure for the likely reactive or bindmg sιte(s) of an agonist, antagonist or inhibitor,

(c) synthesmg candidate compounds that are predicted to bind to or react with the deduced binding or reactive site, and

12 (d) testing whether the candidate compounds are indeed agonists antagonists or inhibitors It will be further appreciated that this will normally be an interative process

In a further aspect, the present mvention provides methods of treating abnormal conditions such as, for instance, neurological diseases, epidermal diseases, deafness, cataracts, and AIDS, related to either an excess of, or an under-expression of, gap junction protem beta-4 polypeptide activity

If the activity of the polypeptide is excess, several approaches are available One approach compπses admmistenng to a subject m need thereof an inhibitor compound (antagonist) as hereinabove descnbed, optionally m combination with a pharmaceutically acceptable earner, m an amount effective to inhibit the function of the polypeptide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of the polypeptides still capable of binding the gand, substrate, enzymes, receptors, etc in competition with endogenous polypeptide may be administered Typical examples of such competitors include fragments of the gap junction protem beta-4 polypeptide In still another approach, expression of the gene encoding endogenous gap junction protem beta-4 polypeptide can be inhibited using expression blocking techniques Known such techniques involve the use of antisense sequences, either internally generated or separately administered (see, for example, O'Connor, J Neurochem (1991) 56 560 in Ohgodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)) Alternatively, oligonucleotides which form tπple helices with the gene can be supplied (see, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360) These o gomers can be administered per se or the relevant ohgomers can be expressed in vivo

For treating abnormal conditions related to an under-expression of gap junction protem beta-4 and its activity, several approaches are also available One approach compπses admmistenng to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present mvention, l e , an agonist as descnbed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition Alternatively, gene therapy may be employed to effect the endogenous production of gap junction protem beta-4 by the relevant cells in the subject For example, a polynucleotide of the mvention may be engmeered for expression m a replication defective retroviral vector, as discussed above The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containing the gene of interest These producer cells may be admmistered to a subject for engineering cells in vivo and

13 expression of the polypeptide in vivo For an overview of gene therapy, see Chapter 20 Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996) Another approach is to administer a therapeutic amount of a polypeptide of the present mvention in combination with a suitable pharmaceutical earner

In a further aspect, the present mvention provides for pharmaceutical compositions compnsing a therapeutically effective amount of a polypeptide, such as the soluble form of a polypeptide of the present mvention, agonist/antagonist peptide or small molecule compound, m combination with a pharmaceutically acceptable earner or excipient Such earners mclude, but are not limited to, salme, buffered salme, dextrose, water, glycerol, ethanol, and combinations thereof The mvention further relates to pharmaceutical packs and kits compnsing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds The composition will be adapted to the route of administration, for instance by a systemic or an oral route Preferred forms of systemic administration mclude injection, typically by intravenous injection Other injection routes, such as subcutaneous, intramuscular, or lntrapentoneal, can be used Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents In addition, if a polypeptide or other compounds of the present mvention can be formulated in an enteπc or an encapsulated formulation, oral administration may also be possible Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels, and the like

The dosage range required depends on the choice of peptide or other compounds of the present mvention, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attendmg practitioner Suitable dosages, however, are m the range of 0 1 - 100 μg/kg of subject Wide vanations m the needed dosage, however, are to be expected view of the vaπety of compounds available and the differing efficiencies of vaπous routes of administration For example, oral administration would be expected to require higher dosages than administration by intravenous injection Vanations m these dosage levels can be adjusted usmg standard empincal routines for optimization, as is well understood in the art

Polypeptides used in treatment can also be generated endogenously m the subject, in treatment modalities often referred to as "gene therapy" as descnbed above Thus, for example, cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex

14 v7vo, and for example, b\ the use of a retroviral plasmid vector The cells are then introduced into the " subject

Polynucleotide and polypeptide sequences form a valuable information resource with which to identify further sequences of similar homology This is most easily facilitated by storing the sequence in a computer readable medium and then usmg the stored data to search a sequence database usmg well known searchmg tools, such as GCC Accordmgly, m a further aspect, the present mvention provides for a computer readable medium having stored thereon a polynucleotide compπsing the sequence of SEQ ID NO 1 and/or a polypeptide sequence encoded thereby

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

"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeπc, single chain, and humamzed antibodies, as well as Fab fragments, mcludmg the products of an Fab or other lmmunoglobulin expression library "Isolated" means altered "by the hand of man" from the natural state If an "isolated" composition or substance occurs in nature, it has been changed or removed from its oπgmal environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting mateπals of its natural state is "isolated", as the term is employed herein "Polynucleotide" generally refers to any polyπbonucleotide or polydeoxπbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation, single- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybπd molecules compπsmg DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions In addition, "polynucleotide" refers to tπple-stranded regions compπsing RNA or DNA or both RNA and DNA The term "polynucleotide" also mcludes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases mclude, for example, tπtylated bases and unusual bases such as mosme A vaπety of modifications may be made to DNA and RNA, thus, "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 characteπstic of viruses and cells "Polynucleotide" also embraces relatively short polynucleotides, often refeπed to as oligonucleotides

15 "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined^' to each other by peptide bonds or modified peptide bonds, 1 e , peptide isosteres "Polypeptide" refers to both short chains, commonly referred to as peptides, o gopeptides or ohgomers and to longer chains, generally refeπed to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded ammo acids "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art Such modifications are well descnbed 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 ammo acid side-chains and the ammo 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 Also, a given polypeptide 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-nbosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a hpid or hpid derivative, covalent attachment of phosphotidylinositol, cross-linking, cychzation, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, ga ma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, lodination, methylation, myπstoylation, oxidation, proteolytic processmg, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of ammo acids to proteins such as argmylation, and ubiquitination (see, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed , T E Creighton, W H Freeman and Company, New York, 1993, Wold, F , Post-translational Protein Modifications Perspectives and Prospects, pgs 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B C Johnson, Ed , Academic Press, New York, 1983, Seifter et al , "Analysis for protein modifications and nonprotein cofactors", Meth Enzymol (1990) 182 626-646 and Rattan et al , "Protein Synthesis Post-translational Modifications and Aging", Ann NYAcadSci (1992) 663 48-62) "Vanant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties A typical vanant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the vanant may or may not alter the ammo acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result m amino acid

16 substitutions, additions, deletions, fusions and truncations m the polypeptide encoded by the reference sequence, as discussed below A typical vanant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions m any combination A substituted or inserted amino acid residue may or may not be one encoded by the genetic code A vanant of a polynucleotide or polypeptide may be a naturally occurπng such as an alle c vanant, or it may be a variant that is not known to occur naturally Non-naturally occurπng vaπants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis

"Identity," as known m the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between stπngs of such sequences "Identity" can be readily calculated by known methods, mcludmg but not limited to those descnbed in (Computational olecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputmg Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis m Molecular Biology, von Hemje, G , Academic Press, 1987, and Sequence Analysis Primer, Gπbskov, M and Devereux, J , eds , M Stockton Press, New York, 1991, and Canllo, H , and Lipman, D , SIAM J Applied Math , 48 1073 (1988) Methods to determine identity are designed to give the largest match between the sequences tested Moreover, methods to determine identity are codified m publicly available computer programs Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S F et al , J Molec Bwl 215 403-410 (1990) The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Bwl 215 403-410 (1990) The well known Smith Waterman algorithm may also be used to determine identity Parameters for polypeptide sequence compaπson include the following

1) Algorithm Needleman and Wunsch J Mol Biol 48 443-453 (1970) Compaπson matπx BLOSSUM62 from Hentikoff and Hentikoff, Proc Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12

17 Gap Length Penalty 4

A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI The aforementioned parameters are the default parameters for peptide compaπsons (along witii no penalty for end gaps) Parameters for polynucleotide compaπson include the following

1) Algorithm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Companson matπx matches = +10, mismatch = 0 Gap Penalty 50 Gap Length Penalty 3 Available as The "gap" program from Genetics Computer Group, Madison WI These are the default parameters for nucleic acid compaπsons

A prefeπed meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided m (1) and (2) below

(1) Polynucleotide embodiments further include an isolated polynucleotide compπsing a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO 1 , wherem said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO 1 or may mclude up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or msertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those termmal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides m SEQ ID NO 1, or

"n ≤ ^xn " (*n * y)>

wherem n_n is the number of nucleotide alterations, x_n is the total number of nucleotides in SEQ ID NO 1, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherem any non-integer product of x_n and y is rounded down to the nearest mteger pπor to

18 subtracting it from x_n Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations

By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it mav include up to a certain integer number of ammo acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, mcludmg transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' termmal positions of the reference polynucleotide sequence or anywhere between those termmal positions, interspersed either individually among the nucleic acids m the reference sequence or m one or more contiguous groups within the reference sequence The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or

ⁿn ≤ ^xn " (^xn ^# y)>

wherein n_n is the number of amino acid alterations, x_n is the total number of ammo acids in SEQ ID NO 2, y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , • is the symbol for the multiplication operator, and wherein any non-integer product of x_n and y is rounded down to the nearest integer pπor to subtracting it from x_n

(2) Polypeptide embodiments further mclude an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one ammo acid deletion, substitution, including conservative and non- conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those termmal positions, interspersed either individually among the ammo acids m the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the

19 mteger defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID NO 2, or

ⁿa ^{≤ x}a " (^xa • y).

wherem n_a is the number of amino acid alterations, x_a is the total number of ammo acids in SEQ ID NO 2, y is 0 50 for 50%, 0 60 for 60%, 0 70 for 70%, 0 80 for 80%, 0 85 for 85%, 0 90 for 90%, 0 95 for 95%, 0 97 for 97% or 1 00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x_a and y is rounded down to the nearest integer pnor to subtracting it from x_a

By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may mclude up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity Such alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the ammo- or carboxy-termmal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids in the reference sequence or in one or more contiguous groups within the reference sequence The number of ammo acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids m SEQ ID NO 2, or

n_a < x_a ( _a • y).

wherein n_a is the number of ammo acid alterations, x_a is the total number of ammo acids in SEQ ID NO 2, y is, for instance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc , and • is the symbol for the multiplication operator, and wherein any non-integer product of x_a and y is rounded down to the nearest mteger pnor to subtracting it from x_a "Fusion protem" refers to a protein encoded by two, often unrelated, fused genes or fragments thereof In one example, EP-A-0 464 discloses fusion proteins compnsing vanous portions of constant region of immunoglobulin molecules together with another human protem or part thereof In many cases, employing an immunoglobulin Fc region as a part of a fusion protein

20 is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262] On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and puπfied

All publications, mcludmg but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth

21 SEQUENCE INFORMATION

SEQ ID NO:l Polynucleotide sequence of human gap junction protein beta-4

TAAAAAGCAAAAGAATTCGCGGCCGCGTCGACACGGGCTTCCCCGAAAACCTTCCCCGCTTCTGGATATGAAATTCAAGC TGCTTGCTGAGTCCTATTGCCGGCTGCTGGGAGCCAGGAGAGCCCTGAGGAGTAGTCACTCAGTAGCAGCTGACGCGTGG GTCCACCATGAACTGGAGTATCTTTGAGGGACTCCTGAGTGGGGTCAACAAGTACTCCACAGCCTTTGGGCGCATCTGGC TGTCTCTGGTCTTCATCTTCCGCGTGCTGGTGTACCTGGTGACGGCCGAGCGTGTGTGGAGTGATGACCACAAGGACTTC GACTGCAATACTCGCCAGCCCGGCTGCTCCAACGTCTGCTTTGATGAGTTCTTCCCTGTGTCCCATGTGCGCCTCTGGGC CCTGCAGCTTATCCTGGTGACATGCCCCTCACTGCTCGTGGTCATGCACGTGGCCTACCGGGAGGTTCAGGAGAAGAGGC ACCGAGAAGCCCATGGGGAGAACAGTGGGCGCCTCTACCTGAACCCCGGCAAGAAGCGGGGTGGGCTCTGGTGGACATAT GTCTGCAGCCTAGTGTTCAAGGCGAGCGTGGACATCGCCTTTCTCTATGTGTTCCACTCATTCTACCCCAAATATATCCT CCCTCCTGTGGTCAAGTGCCACGCAGATCCATGTCCCAATATAGTGGACTGCTTCATCTCCAAGCCCTCAGAGAAGAACA TTTTCACCCTCTTCATGGTGGCCACAGCTGCCATCTGCATCCTGCTCAACCTCGTGGAGCTCATCTACCTGGTGAGCAAG AGATGCCACGAGTGCCTGGCAGCAAGGAAAGCTCAAGCCATGTGCACAGGTCATCACCCCCACGGTACCACCTCTTCCTG CAAACAAGACGACCTCCTTTCGGGTGACCTCATCTTTCTGGGCTCAGACAGTCATCCTCCTCTCTTACCAGACCGCCCCC GAGACCATGTGAAGAAAACCATCTTGTGAGGGGCTGCCTGGACTGGTCTGGCAGGTTGGGCCTGGATGGGGAGGCTCTAG CATCTCTCATAGGTGCAACCTGAGAGTGGGGGAGCTAAGCCATGAGGTAGGGGCAGGCAAGAGAGAGGATTCAGACGCTC TGGGAGCCAGTTCCTAGTCCTCAACTCCAGCCACCTGCCCCAGCTCGACGGCACTGGGCCAGTTCCCCCTCTGCTCTGCA GCTCGGTTTCCTTTTCTAGAATGGAAATAGTGAGGGCCAATGC

SEQ ID NO:2 polypeptide sequence of human gap junction protein beta-4

MNWSI FEGLLSGVN YSTAFGRIWLSLVFI FRVLVYLVTAERVWSDDH DFDCNTRCPGCSNVCFDE FFPVSHVRLWALQ LILVTCPS LWMHVAYREVQEKRHREAHGENSGRLYLNPGKKRGG WWTYVCSLVFKASVDIAF YVFHSFYPKYI PP W CHADPCPNIVDCFISKPSEKNI FTLFMVATAAICI LNLVELIYLVSKRCHECLAARKAQAMCTGHH PHGTTSSC Q DDL SGDLI FLGSDSHPPLLPDRPRDHVKKTIL .

22 SEQUENCE LISTING

;i) GENERAL INFORMATION

(i) APPLICANT: Hunan Medical University

(ii) TITLE OF THE INVENTION: HUMAN GAP JUNCTION PROTEIN

BETA-4

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: RATNER & PRESTIA

(B) STREET: P.O. BOX 980

(C) CITY: VALLEY FORGE

(D) STATE: PA (E) COUNTRY: USA

(F) ZIP: 19482

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: TO BE ASSIGNED

(B) FILING DATE:

(C) CLASSIFICATION: UNKNOWN

( vii ) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER:

( B) FILING DATE :

( viii ) ATTORNEY/AGENT INFORMATION :

(A) NAME: PRESTIA, PAUL F

(B) REGISTRATION NUMBER: 23,031

(C) REFERENCE/DOCKET NUMBER: GP-70422

23

Claims

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE : 610- 407 - 0700

( B ) TELEFAX : 610- 4 07 - 0701

( C ) TELEX : 846169

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1243 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TAAAAAGCAA AAGAATTCGC GGCCGCGTCG ACACGGGCTT CCCCGAAAAC CTTCCCCGCT 60 TCTGGATATG AAATTCAAGC TGCTTGCTGA GTCCTATTGC CGGCTGCTGG GAGCCAGGAG 120

AGCCCTGAGG AGTAGTCACT CAGTAGCAGC TGACGCGTGG GTCCACCATG AACTGGAGTA 180

TCTTTGAGGG ACTCCTGAGT GGGGTCAACA AGTACTCCAC AGCCTTTGGG CGCATCTGGC 240

TGTCTCTGGT CTTCATCTTC CGCGTGCTGG TGTACCTGGT GACGGCCGAG CGTGTGTGGA 300

GTGATGACCA CAAGGACTTC GACTGCAATA CTCGCCAGCC CGGCTGCTCC AACGTCTGCT 360 TTGATGAGTT CTTCCCTGTG TCCCATGTGC GCCTCTGGGC CCTGCAGCTT ATCCTGGTGA 420

CATGCCCCTC ACTGCTCGTG GTCATGCACG TGGCCTACCG GGAGGTTCAG GAGAAGAGGC 480

ACCGAGAAGC CCATGGGGAG AACAGTGGGC GCCTCTACCT GAACCCCGGC AAGAAGCGGG 540

GTGGGCTCTG GTGGACATAT GTCTGCAGCC TAGTGTTCAA GGCGAGCGTG GACATCGCCT 600

TTCTCTATGT GTTCCACTCA TTCTACCCCA AATATATCCT CCCTCCTGTG GTCAAGTGCC 660 ACGCAGATCC ATGTCCCAAT ATAGTGGACT GCTTCATCTC CAAGCCCTCA GAGAAGAACA 720

TTTTCACCCT CTTCATGGTG GCCACAGCTG CCATCTGCAT CCTGCTCAAC CTCGTGGAGC 780

TCATCTACCT GGTGAGCAAG AGATGCCACG AGTGCCTGGC AGCAAGGAAA GCTCAAGCCA 840

TGTGCACAGG TCATCACCCC CACGGTACCA CCTCTTCCTG CAAACAAGAC GACCTCCTTT 900

CGGGTGACCT CATCTTTCTG GGCTCAGACA GTCATCCTCC TCTCTTACCA GACCGCCCCC 960 GAGACCATGT GAAGAAAACC ATCTTGTGAG GGGCTGCCTG GACTGGTCTG GCAGGTTGGG 1020

CCTGGATGGG GAGGCTCTAG CATCTCTCAT AGGTGCAACC TGAGAGTGGG GGAGCTAAGC 1080

CATGAGGTAG GGGCAGGCAA GAGAGAGGAT TCAGACGCTC TGGGAGCCAG TTCCTAGTCC 1140

TCAACTCCAG CCACCTGCCC CAGCTCGACG GCACTGGGCC AGTTCCCCCT CTGCTCTGCA 1200

GCTCGGTTTC CTTTTCTAGA ATGGAAATAG TGAGGGCCAA TGC 1243

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

24 (A) LENGTH: 273 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Met Asn Trp Ser lie Phe Glu Gly Leu Leu Ser Gly Val Asn Lys Tyr 1 5 10 15

Ser Thr Ala Phe Gly Arg lie Trp Leu Ser Leu Val Phe lie Phe Arg

20 25 30

Val Leu Val Tyr Leu Val Thr Ala Glu Arg Val Trp Ser Asp Asp His 35 40 45 Lys Asp Phe Asp Cys Asn Thr Arg Gin Pro Gly Cys Ser Asn Val Cys 50 55 60

Phe Asp Glu Phe Phe Pro Val Ser His Val Arg Leu Trp Ala Leu Gin 65 70 75 80

Leu lie Leu Val Thr Cys Pro Ser Leu Leu Val Val Met His Val Ala 85 90 95

Tyr Arg Glu Val Gin Glu Lys Arg His Arg Glu Ala His Gly Glu Asn

100 105 110

Ser Gly Arg Leu Tyr Leu Asn Pro Gly Lys Lys Arg Gly Gly Leu Trp 115 120 125 Trp Thr Tyr Val Cys Ser Leu Val Phe Lys Ala Ser Val Asp lie Ala 130 135 140

Phe Leu Tyr Val Phe His Ser Phe Tyr Pro Lys Tyr lie Leu Pro Pro 145 150 155 160

Val Val Lys Cys His Ala Asp Pro Cys Pro Asn lie Val Asp Cys Phe 165 170 175 lie Ser Lys Pro Ser Glu Lys Asn lie Phe Thr Leu Phe Met Val Ala

180 185 190

Thr Ala Ala lie Cys lie Leu Leu Asn Leu Val Glu Leu lie Tyr Leu 195 200 205 Val Ser Lys Arg Cys His Glu Cys Leu Ala Ala Arg Lys Ala Gin Ala 210 215 220

Met Cys Thr Gly His His Pro His Gly Thr Thr Ser Ser Cys Lys Gin 225 230 235 240

Asp Asp Leu Leu Ser Gly Asp Leu lie Phe Leu Gly Ser Asp Ser His 245 250 255

Pro Pro Leu Leu Pro Asp Arg Pro Arg Asp His Val Lys Lys Thr lie

260 265 270

Leu

25 What is claimed is:

1 An isolated polypeptide selected from the group consisting of

(1) an isolated polypeptide comprising an amino acid sequence selected from the group having at least

(a) 70% identity,

(b) 80% identity,

(c) 90% identity, or

(d) 95% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID

NO 2, (n) an isolated polypeptide compπsing the ammo acid sequence of SEQ ID NO 2 or (in) an isolated polypeptide which is the amino acid sequence of SEQ ID NO 2

2 An isolated polynucleotide selected from the group consisting of

(I) an isolated polynucleotide compπsmg a nucleotide sequence encodmg a polypeptide that has at least

(a) 70% identity,

(b) 80% identity, (c) 90% identity, or

(d) 95% identity, to the ammo acid sequence of SEQ ID NO 2, over the entire length of SEQ ID NO 2, (n) an isolated polynucleotide compnsing a nucleotide sequence that has at least (a) 70% identity (b) 80% identity,

(c) 90% identity, or

(d) 95% identity, over its entire length to a nucleotide sequence encodmg the polypeptide of SEQ ID NO 2, (in) an isolated polynucleotide compnsing a nucleotide sequence which has at least

(a) 70% identity,

(b) 80% identity,

(c) 90% identity, or

(d) 95% identity,

26 to that of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1; (iv) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO:2;

(vi) an isolated polynucleotide which is the polynucleotide of SEQ ID NO: 1 ; or (vi) an isolated polynucleotide obtainable by screenmg an appropπate library under strmgent hybridization conditions with a labeled probe having the sequence of SEQ ID NO: 1 or a fragment thereof; or a nucleotide sequence complementary to said isolated polynucleotide.

3. An antibody immunospecific for the polypeptide of claim 1.

4. A method for the treatment of a subject:

(i) in need of enhanced activity or expression of the polypeptide of claim 1 comprising:

(a) administering to the subject a therapeutically effective amount of an agonist to said polypeptide; and/or

(b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence encoding said polypetide in a form so as to effect production of said polypeptide activity in vivo.; or

(ii) having need to inhibit activity or expression of the polypeptide of claim 1 comprising:

(a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or

(b) administering to the subject a nucleic acid molecule that inhibits the expression of a nucleotide sequence encoding said polypeptide; and/or (c) administering to the subject a therapeutically effective amount of a polypeptide that competes with said polypeptide for its ligand, substrate , or receptor.

5. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of the polypeptide of claim 1 in a subject comprising:

(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said polypeptide in the genome of said subject; and/or

(b) analyzing for the presence or amount of said polypeptide expression in a sample derived from said subject.

27 6 A method for screening to identify compounds which stimulate or which inhibit the function of the polypeptide of claim 1 which compπses a method selected from the group consisting of

(a) measuπng the binding of a candidate compound to the polypeptide (or to die cells or membranes beanng the polypeptide) or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound,

(b) measuπng the binding of a candidate compound to the polypeptide (or to the cells or membranes beaπng the polypeptide) or a fusion protein thereof in the presense of a labeled competitior, (c) testmg whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide, using detection systems appropπate to the cells or cell membranes beaπng the polypeptide,

(d) mixing a candidate compound with a solution containing a polypeptide of claim 1 , to form a mixture, measuring activity of the polypeptide in the mixture, and comparing the activity of the mixture to a standard, or

(e) detectmg the effect of a candidate compound on the production of mRNA encodmg said polypeptide and said polypeptide in cells, using for instance, an ELISA assay

7 An agonist or an antagonist of the polypeptide of claim 1

8 An expression system compnsing a polynucleotide capable of producing a polypeptide of claim 1 when said expression system is present in a compatible host cell

9 A process for producing a recombinant host cell comprising transforming or transfectmg a cell with an expression system of claim 8 such the the host cell, under appropπate culture conditions, produces a polypeptide compπsing an amino acid sequence having at least 70% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2

10 A recombinant host cell produced by the process of claim 9

1 1 A membrane of a recombinant host cell of claim 10 expressing a polypeptide compπsing an ammo acid sequence having at least 70% identity to the ammo acid sequence of SEQ ID NO 2 over the entire length of SEQ ID NO 2

28 12 A process for producing a polypeptide compnsing culturing a recombinant host cell of claim 10 under conditions sufficient for the production of said polypeptide and recovering the polpeptide form the culture

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