WO1999051738A1 - Proteine beta 4 humaine a jonction lacunaire - Google Patents

Proteine beta 4 humaine a jonction lacunaire Download PDF

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Publication number
WO1999051738A1
WO1999051738A1 PCT/CN1998/000055 CN9800055W WO9951738A1 WO 1999051738 A1 WO1999051738 A1 WO 1999051738A1 CN 9800055 W CN9800055 W CN 9800055W WO 9951738 A1 WO9951738 A1 WO 9951738A1
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Prior art keywords
polypeptide
leu
seq
identity
val
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PCT/CN1998/000055
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English (en)
Inventor
Jiahui Xia
Qian Pan
Chunyu Liu
Duo Zheng
Wei Xie
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Hunan Medical University
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Priority to PCT/CN1998/000055 priority Critical patent/WO1999051738A1/fr
Publication of WO1999051738A1 publication Critical patent/WO1999051738A1/fr

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    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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 bein ⁇ 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 nno ⁇ t ⁇ date claimed
  • 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
  • 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 mate ⁇ als and methods for their production
  • 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
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the mate ⁇ als provided by the invention, and treating conditions associated with gap junction protein beta-4 imbalance with the identified compounds
  • the invention relates to diagnostic assays for detecting diseases associated with inappropriate gap junction protein beta-4 activity or levels
  • 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
  • polypeptides 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.
  • polypeptides include those comprising the amino acid of SEQ ID NO:2.
  • 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.
  • polypeptides include the polypeptide of SEQ ID NO:2.
  • 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.
  • gap junction protein beta-4 activity or "gap junction protein beta-4 polypeptide activity” or "biological activity of gap junction protein beta-4”.
  • 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.
  • a polypeptide of the present invention exhibits at least one biological activity of gap junction protein beta-4.
  • 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.
  • 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
  • the present invention relates to gap junction protein beta-4 polynucleotides
  • polynucleotides include isolated polynucleotides comp ⁇ sing 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
  • 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 comp ⁇ sing the nucleotide sequence contained in SEQ ID NO 1 encoding the polypeptide of SEQ ID NO 2
  • polynucleotides of the present invention include isolated polynucleotides comp ⁇ sing 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
  • 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
  • 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
  • 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 comp ⁇ sing 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 mu ⁇ ne connexin 31 1
  • the nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and comp ⁇ ses a polypeptide encoding
  • nucleotide 168 to 986 encoding a polypeptide of 273 amino acids
  • 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 simila ⁇ ty 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 de ⁇ ved 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
  • EST expressed sequence tag
  • 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
  • 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
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded
  • 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
  • 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 transc ⁇ ptase, an enzyme with inherently low 'processivity' (a measure of the ability of the enzyme to remam attached to the template du ⁇ ng the polyme ⁇ sation reaction), failing to complete a DNA copy of the mRNA template du ⁇ ng 1st strand cDNA synthesis
  • PCR reaction is then repeated using 'nested' p ⁇ mers, that is, p ⁇ mers designed to anneal within the amplified product (typically an adaptor specific p ⁇ mer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence)
  • 'nested' p ⁇ mers that is, p ⁇ mers designed to anneal within the amplified product (typically an adaptor specific p ⁇ mer 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 comp ⁇ smg expression systems Accordmgly, in a further aspect, the present mvention relates to expression systems which comp ⁇ se 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 de ⁇ ved from the DNA constructs of the present mvention
  • 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
  • appropnate hosts include bacte ⁇ al 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
  • bacte ⁇ al 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
  • a great va ⁇ ety of expression systems can be used, for instance, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasrmds, from bacte ⁇ ophage, 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 de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage 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 approp ⁇ ate 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.
  • 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)
  • an array of oligonucleotides probes comp ⁇ smg 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 va ⁇ ety 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
  • diseases may be diagnosed by methods comp ⁇ sing determining from a sample de ⁇ ved 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 de ⁇ ved from a host are well-known to those of skill the art Such assay methods mclude radiounmunoassays, competitive-binding assays, Western Blot analysis and
  • a polynucleotide of the present invention preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof ,
  • polypeptide of the present invention preferably the polypeptide of SEQ ID NO 2 or a fragment thereof, or
  • any such kit may comp ⁇ se 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 hyb ⁇ dize 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 Inhe ⁇ tance 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 (coinhe ⁇ tance 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
  • 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 p ⁇ or art Antibodies generated against polypeptides of the present mvention may be obtained by administering the polypeptides or epitope-bea ⁇ ng 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 t ⁇ oma technique, the human B-cell hybndoma techmque (Kozbor et al ,
  • 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
  • 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
  • the present invention relates to genetically engineered soluble fusion proteins comp ⁇ sing a polypeptide of the present invention, or a fragment thereof, and va ⁇ ous portions of the constant regions of heavy or light chains of lmmunoglobulms of va ⁇ ous subclasses (IgG, IgM, IgA, IgE) Preferred as an lmmunoglobuhn is the constant part of the heavy chain of
  • the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa
  • this invention relates to processes for the preparation of these fusion proteins by genetic enginee ⁇ ng, 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 comp ⁇ ses 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 comp ⁇ ses a polypeptide or polynucleotide of the present mvention
  • the vaccine formulation may further comp ⁇ se 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, bacte ⁇ ostats 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
  • 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
  • the present mvention provides for a method of screening compounds to identify those which stimulate or which inhibit the function of the polypeptide
  • agonists or antagonists may be employed for therapeutic and prophylactic purposes for such Diseases as herembefore mentioned
  • Compounds may be identified from a va ⁇ ety 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 bea ⁇ ng 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 bea ⁇ ng 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 comp ⁇ se the steps of mixing a candidate compound with a solution containing a polypeptide of the present invention, to form
  • 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
  • an ELISA assay may be constructed for measu ⁇ ng 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
  • 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
  • polypeptide antagonists examples include 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
  • 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 comp ⁇ ses (a) a polypeptide of the present invention,
  • 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,
  • 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
  • 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
  • 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
  • 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, intramus
  • 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 ⁇ g/kg of subject Wide vanations m the needed dosage, however, are to be expected view of the va ⁇ ety of compounds available and the differing efficiencies of va ⁇ ous 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
  • cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex
  • 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 comp ⁇ sing the sequence of SEQ ID NO 1 and/or a polypeptide sequence encoded thereby
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, 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 o ⁇ gmal environment, or both
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated," but the same polynucleotide or polypeptide separated from the coexisting mate ⁇ als of its natural state is “isolated", as the term is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include
  • 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 refe ⁇ ed 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
  • 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 occur ⁇ ng such as an alle c vanant, or it may be a variant that is not known to occur naturally
  • Non-naturally occur ⁇ ng va ⁇ ants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis
  • Identity 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
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between st ⁇ ngs 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,
  • Polynucleotide embodiments further include an isolated polynucleotide comp ⁇ sing 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 nucle
  • 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%
  • • 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 p ⁇ or to
  • 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
  • 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
  • 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 p ⁇ or to subtracting it from x n
  • 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
  • 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%
  • • 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
  • 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 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
  • • 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
  • 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
  • SEQ ID NO:2 polypeptide sequence of human gap junction protein beta-4

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Abstract

L'invention concerne des polypeptides et des polynucléotides de protéine bêta 4 à jonction lacunaire et des procédés permettant d'obtenir ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides de la protéine bêta 4 à jonction lacunaire pour le traitement de pathologies associées, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000055 1998-04-03 1998-04-03 Proteine beta 4 humaine a jonction lacunaire WO1999051738A1 (fr)

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PCT/CN1998/000055 WO1999051738A1 (fr) 1998-04-03 1998-04-03 Proteine beta 4 humaine a jonction lacunaire

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PCT/CN1998/000055 WO1999051738A1 (fr) 1998-04-03 1998-04-03 Proteine beta 4 humaine a jonction lacunaire

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8404644B2 (en) 2007-09-07 2013-03-26 Meat & Livestock Australia Limited Agents with angiogenic and wound healing activity

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4420791C1 (de) * 1994-06-15 1995-05-18 Hinrich Dr Luehring Verfahren zur Expression von genetisch modifizierten Molekülen und dafür geeignete Wirtszellen
US5650317A (en) * 1994-09-16 1997-07-22 Michigan State University Human breast epithelial cell type with stem cell and luminal epithelial cell characteristics
WO1997028179A1 (fr) * 1996-01-31 1997-08-07 The Regents Of The University Of California Procede d'inhibition de la croissance de cellules tumorales

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4420791C1 (de) * 1994-06-15 1995-05-18 Hinrich Dr Luehring Verfahren zur Expression von genetisch modifizierten Molekülen und dafür geeignete Wirtszellen
US5650317A (en) * 1994-09-16 1997-07-22 Michigan State University Human breast epithelial cell type with stem cell and luminal epithelial cell characteristics
WO1997028179A1 (fr) * 1996-01-31 1997-08-07 The Regents Of The University Of California Procede d'inhibition de la croissance de cellules tumorales

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8404644B2 (en) 2007-09-07 2013-03-26 Meat & Livestock Australia Limited Agents with angiogenic and wound healing activity

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