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WO1999036524A1 - Cbmaae03: gene semblable a la proteine ribosomique l33 - Google Patents

Cbmaae03: gene semblable a la proteine ribosomique l33 Download PDF

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Publication number
WO1999036524A1
WO1999036524A1 PCT/CN1998/000005 CN9800005W WO9936524A1 WO 1999036524 A1 WO1999036524 A1 WO 1999036524A1 CN 9800005 W CN9800005 W CN 9800005W WO 9936524 A1 WO9936524 A1 WO 9936524A1
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Prior art keywords
polypeptide
cbmaae03
seq
nucleotide sequence
polynucleotide
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PCT/CN1998/000005
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English (en)
Inventor
Qiu-Hua Huang
Jian Gu
Ya-Xin Wang
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Shanghai Second Medical University
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Application filed by Shanghai Second Medical University filed Critical Shanghai Second Medical University
Priority to PCT/CN1998/000005 priority Critical patent/WO1999036524A1/fr
Publication of WO1999036524A1 publication Critical patent/WO1999036524A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • CBMAAE03 A Gene Similar to Ribosomal Protein L33
  • This invention relates to newly identified polynucleotides, polypepUdes encoded by them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present mvention relate to the nbosomal protein family, hereinafter referred to as CBMAAE03 The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides
  • the E cob and yeast ⁇ bosomal L33 proteins are 54 and 69 a mo acids in length, respectively No human L33 gene was previously known
  • the ⁇ bosomal protein family has an established, proven history as therapeutic targets Clearly there is a need for identification and characterization of further members of the ⁇ bosomal protein family which can play a role in preventing, amehoratmg or correcting dysfunctions or diseases, including, but not limited to, infertility, miscamage, kidney disease, and liver disease
  • the mvention relates to CBMAAE03 polypeptides and recombinant mate ⁇ als and methods for their production
  • Another aspect of the invention relates to methods for using such CBMAAE03 polypeptides and polynucleotides
  • Such uses include the treatment of infertility, miscar ⁇ age, kidney disease, and liver disease, among others
  • the invention relates to methods to identify agonists and antagonists usmg the mate ⁇ als provided by the invention, and treating conditions associated with CBMAAE03 imbalance with the identified compounds
  • Yet another aspect of the mvention relates to diagnostic assays for detecting diseases associated with inapprop ⁇ ate CBMAAE03 activity or levels
  • CBMAAE03 refers, among others, generally to a polypeptide havmg the amino acid sequence set forth in SEQ ID NO 2 or an alle c va ⁇ ant thereof
  • CBMAAE03 activity or CBMAAE03 polypeptide activity or "biological activity of the CBMAAE03 or CBMAAE03 polypeptide” refers to the metabolic or physiologic function of said CBMAAE03 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogemc activities of said CBMAAE03 "CBMAAE03 gene” refers to a polynucleotide having the nucleotide sequence set forth in SEQ
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulm expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated" composition or substance occurs m nature, it has been changed or removed from its o ⁇ gmal environment, or both
  • a polynucleotide or a polypeptide naturally present m a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated", as the term is employed herem
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation single- and double-strand
  • polynucleotide refers to t ⁇ ple-stranded regions comp ⁇ smg RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, t ⁇ tylated bases and unusual bases such as inosine
  • Modified bases include, for example, t ⁇ tylated bases and unusual bases such as inosine
  • polynucleotide embraces chemically, enzymatically or metabo cally modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as o gonucleotides
  • Polypeptide refers to any peptide or protein compnsmg two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, I e , peptide isosteres "Polypeptide” refers to both short chams, commonly referred to as peptides, o gopeptides or oligomers, and to longer chams, generally referred to as proteins Polypeptides may contain amino acids other than the 20 gene-encoded amino acids "Polypeptides” include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art Such modifications are well desc ⁇ bed in basic texts and in more detailed monographs, as well as m a voluminous research literature Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the ammo or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees
  • Va ⁇ ant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical va ⁇ ant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide Changes m the nucleotide sequence of the va ⁇ ant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in ammo acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below
  • a typical va ⁇ ant 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 va ⁇ ant are closely similar overall and, m many regions, identical
  • a variant and reference polypeptide may differ m ammo acid sequence by one
  • Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques.
  • identity is well known to skilled artisans (Carillo, H., and Lipton, D., SLAM J Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SLAM J Applied Math (1988) 48: 1073. Methods to determine identity and similarity are codified in computer programs.
  • Prefe ⁇ ed computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al, JMolec Biol (1990) 215:403).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having an ammo acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO 2 is intended that the ammo acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five ammo acid alterations per each 100 ammo acids of the reference ammo acid of SEQ ID NO 2
  • the reference sequence may be deleted or substituted with another ammo acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence
  • These alterations of the reference sequence may occur at the ammo or carboxy terminal positions of the reference ammo acid sequence or anywhere between those termmal positions, mterspersed either individually among residues m the reference sequence or in one or more contiguous
  • the present mvention relates to CBMAAE03 polypeptides (or CBMAAE03 proteins)
  • the CBMAAE03 polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comp ⁇ sing the ammo acid sequence of SEQ ID NO 2, and polypeptides comp ⁇ sing the amino acid sequence which have at least 80% identity to that of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Also included within CBMAAE03 polypeptides are polypeptides having the ammo acid sequence which have at least 80% identity to the polypeptide having the amino acid sequence of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly prefe ⁇ ed Preferably C
  • the CBMAAE03 polypeptides may be in the form of the "mature" protem or may be a part of a larger protein such as a fusion protem 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 histid e residues, or an additional sequence for stability du ⁇ ng recombinant production
  • a fragment is a polypeptide having an ammo acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned CBMAAE03 polypeptides
  • fragments may be "free-standing," or comp ⁇ sed within a larger polypeptide of which they form a part or region, most preferably as a smgle continuous region
  • Representative examples of polypeptide fragments of the mvention, m clude, for example, fragments from about ammo acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of CBMAAE03 polypeptide
  • “about” m cludes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 ammo acid at either extreme or at both extremes
  • Preferred fragments m clude, for example, truncation polypeptides having the ammo acid sequence of
  • CBMAAE03 polypeptides except for deletion of a continuous se ⁇ es of residues that mcludes the ammo terminus, or a continuous se ⁇ es of residues that mcludes the carboxyl terminus or deletion of two continuous se ⁇ es of residues, one including the ammo terminus and one including the carboxyl terminus
  • fragments characte ⁇ zed by structural or functional attributes such as fragments that comp ⁇ se alpha-helix and alpha-helix formmg regions, beta-sheet and beta-sheet-formmg regions, turn and tum-formmg regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions
  • Other prefe ⁇ ed fragments are biologically active fragments
  • Biologically active fragments are those that mediate CBMAAE03 activity, including those with a similar activity or an improved activity, or with
  • va ⁇ ants of the defined sequence and fragments also form part of the present mvention
  • va ⁇ ants are those that vary from the referents by conservative ammo acid substitutions i e , those that substitute a residue with another of like characte ⁇ stics Typical such substitutions are among Ala, Val, Leu and Be, 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
  • va ⁇ ants m which several, 5-10, 1-5, or 1-2 ammo acids are substituted, deleted, or added m any combination
  • the CBMAAE03 polypeptides of the mvention can be prepared in any suitable manner Such polypeptides mclude 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 m the art
  • CBMAAE03 polynucleotides mclude isolated polynucleotides which encode the CBMAAE03 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBMAAE03 polynucleotide of the mvention mclude a polynucleotide compnsing the nucleotide sequence contained m SEQ ID NO 1 encoding a CBMAAE03 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO: 1.
  • CBMAAE03 polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBMAAE03 polypeptide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to of SEQ ID NO: 1 over its entire length.
  • polynucleotides at least 90% identical are particularly prefe ⁇ ed, and those with at least 95% are especially prefe ⁇ ed.
  • CBMAAE03 polynucleotides are a nucleotide sequence which has sufficient identity to a nucleotide sequence contained in SEQ ID NO: 1 to hybridize under conditions useable for amplification or for use as a probe or marker.
  • the invention also provides polynucleotides which are complementary to such CBMAAE03 polynucleotides.
  • CBMAAE03 of the invention is structurally related to other proteins of the ribosomal protein family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO:l) encoding human CBMAAE03.
  • the cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 36 to 230) encoding a polypeptide of 65 amino acids of SEQ ID NO:2.
  • the amino acid sequence of Table 2 (SEQ ID NO:2) has about 44.4% identity (using FASTA) in 45 amino acid residues with E. coli ribosomal protein L33 (J.S. Lee, et al. Mol. Gen. Genet.l84:218-223,1981).
  • CBMAAE03 (SEQ ID NO:2) is 40.4% identical over 52 amino acid residues to yeast ribosomal protein L33 (Z49810) (Lgrohmann et al,FEBS Lett, 284:51-56,1991).
  • the nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 60.9% identity (using FASTA) in 138 nucleotide residues with E. coli ribosomal protein L33 (J.S. Lee, et al. Mol. Gen. Genet.l84:218-223,1981).
  • CBMAAE03 polypeptides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled in the art.
  • AAAAACTGAC TCTTTTGCAT TATGATCCAG TTGTGAAACA AAGAGTCCTC
  • a nucleotide sequence of a human CBMAAE03 (SEQ ID NO: 1).
  • One polynucleotide of the present invention encoding CBMAAE03 may be obtained using standard cloning and screening, from a cDNA library derived from mRNA in cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M.D., et al. Science (1991) 252:1651-1656; Adams, M.D. et l., Nature, (1992) 155:632-634; 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.
  • the nucleotide sequence encoding CBMAAE03 polypeptide of SEQ ID NO:2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 36 to 230 of SEQ ID NO:l), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO:2.
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and desc ⁇ bed in Gentz et al , Proc Natl Acad Sci USA ( 1989) 86 821 -824, or is an HA tag
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA
  • polynucleotides encoding CBMAAE03 va ⁇ ants comp ⁇ se the ammo acid sequence CBMAAE03 polypeptide of Table 2 (SEQ ID NO 2) m which several, 5-10, 1-5, 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, in any combination
  • the present mvention further relates to polynucleotides that hyb ⁇ dize to the herein above-desc ⁇ bed sequences
  • the present mvention especially relates to polynucleotides which hyb ⁇ dize under stringent conditions to the herem above-desc ⁇ bed polynucleotides
  • stringent conditions means hyb ⁇ dization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences
  • Polynucleotides of the mvention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO 1 or a fragment thereof, may be used as hyb ⁇ dization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding CBMAAE03 polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence similanty to the CBMAAE03 gene
  • Such hyb ⁇ dization techniques are known to those of skill m the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent
  • the probes generally will comp ⁇ se at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly prefe ⁇ ed probes will range between 30 and 50
  • CBMAAE03 polynucleotides of the present mvention further mclude a nucleotide sequence comp ⁇ smg a nucleotide sequence that hyb ⁇ dize under stringent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof
  • polypeptide compnsmg ammo acid sequence encoded by nucleotide sequence obtained by the above hyb ⁇ dization condition Such hyb ⁇ dization
  • the present mvention also relates to vectors which comp ⁇ se a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engineered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techniques
  • Cell-free translation systems can also be employed to produce such proteins usmg RNAs de ⁇ ved from the DNA constructs of the present mvention
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides mto host cells can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis et al , BASIC METHODS LN MOLECULAR BIOLOGY (1986) and Sambrook et al , MOLECULAR CLONLNG A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E col , Streptomyces and Bacillus subtilis cells, fi gal 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, C 127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • bacte ⁇ al cells such as streptococci, staphylococci, E col , Streptomyces and Bacillus subtilis cells
  • fi gal 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, C 127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • a great va ⁇ ety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-de ⁇ ved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasmids, 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 suitable to maintain, propagate or express polynucleotides to produce a polypeptide m a host may be used The
  • approp ⁇ ate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • the cells may be harvested prior to use m the screenmg assay If CBMAAE03 polypeptide is secreted into the medium, the medium can be recovered m order to recover and pu ⁇ fy the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered
  • CBMAAE03 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobe 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 dunng isolation and or punfication
  • This mvention also relates to the use of CBMAAE03 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBMAAE03 gene 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 CBMAAE03 Individuals carrying mutations m the CBMAAE03 gene may be detected at the DNA level by a va ⁇ ety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, u ⁇ ne, saliva, tissue biopsy or autopsy mate ⁇ al
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification techniques p ⁇ or to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions can be detected by a change m size of the amplified product m compa ⁇ son to the normal genotype
  • Pomt mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled CBMAAE03 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures
  • DNA sequence differences may also be detected by alterations m electrophoretic mobility of DNA fragments m gels, with or without denaturmg agents, or by direct DNA sequencmg See, e g , Myers e
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to infertility, miscamage, kidney disease, and liver disease through detection of mutation in the CBMAAE03 gene by the methods desc ⁇ bed
  • infertility, miscamage, kidney disease, and hver disease can be diagnosed by methods comp ⁇ sing determining from a sample de ⁇ ved from a subject an abnormally decreased or increased level of CBMAAE03 polypeptide or CBMAAE03 mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hyb ⁇ dization methods Assay techniques that can be used to determine levels of a protem, such as an CBMAAE03 polypeptide. m a sample de ⁇ ved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present mvention relates to a diagonostic kit for a disease or suspectability to a disease, particularly infertility, miscamage, kidney disease, and liver disease, which comp ⁇ ses
  • a CBMAAE03 polynucleotide preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof,
  • a CBMAAE03 polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • any such kit, (a), (b), (c) or (d) may comp ⁇ se a substantial component
  • 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 mappmg of relevant sequences to chromosomes according to the present mvention is an important first step m co ⁇ elating 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 co ⁇ elated with genetic map data
  • genetic map data are found, for example, in V McKusick, Mendehan 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 m 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 expressing them can also be used as lmmunogens to produce antibodies -mmunospecific for the CBMAAE03 polypeptides
  • immunospecific means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the p ⁇ or art
  • Antibodies generated against the CBMAAE03 polypeptides can be obtained by administering the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routine protocols For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell lme cultures can be used Examples mclude the hyb ⁇ doma techmque (Kohler, G and Milstein, C , Nature ( 1975) 256 495-497), the t ⁇ oma technique, the human B-cell hyb ⁇ doma techmque (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hyb ⁇ doma techmque (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Lie , 1985)
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to purify the polypeptides by affimty chromatography
  • Antibodies against CBMAAE03 polypeptides may also be employed to treat infertility, miscamage, kidney disease, and liver disease, among others
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comp ⁇ ses inoculating the mammal with CBMAAE03 polypeptide, or a fragment thereof, adequate to produce antibody and or T cell immune response to protect said animal from infertility, miscamage, kidney disease, and liver disease, among others
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering CBMAAE03 polypeptide via a vector directing expression of CBMAAE03 polynucleotide in vivo m order to induce such an immunological response to produce antibody to protect said animal from diseases
  • composition which, when introduced mto a mammalian host, mduces an immunological response m that mammal to a CBMAAE03 polypeptide wherein the composition comp ⁇ ses a CBMAAE03 polypeptide or CBMAAE03 gene
  • the vaccine formulation may further comp ⁇ se a suitable earner Since CBMAAE03 polypeptide may be broken down m the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc injection)
  • parenterally including subcutaneous, intramuscular, intravenous, intradermal etc injection
  • Formulations suitable for parenteral administration mclude aqueous and non-aqueous ste ⁇ le injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation lnstonic with the blood of the recipient, and aqueous and non-aqueous ste ⁇ le suspensions which may include suspending agents or thickening agents
  • the formulations may be presented in unit
  • the CBMAAE03 polypeptide of the present mvention may be employed m a screenmg process for compounds which activate (agomsts) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBMAAE03 polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical hbra ⁇ es, and natural product mixtures
  • agomsts or antagonists may be natural or modified substrates, gands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Coligan et al , Current Protocols in Lmmunology 1(2) Chapter 5 (1991)
  • CBMAAE03 polypeptides are responsible for many biological functions, including many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBMAAE03 polypeptide on the one hand and which can inhibit the function of CBMAAE03 polypeptide on the other hand
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as infertility, miscarriage, kidney disease, and liver disease
  • Antagonists may be employed for a va ⁇ ety of therapeutic and prophylactic purposes for such conditions as infertility, miscamage, kidney disease, and liver disease
  • screening procedures may involve using appropriate cells which express the CBMAAE03 polypeptide or respond to CBMAAE03 polypeptide of the present invention.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • Cells which express the CBMAAE03 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBMAAE03 polypeptide are then contacted with a test compoimd to observe binding, or stimulation or inhibition of a functional response.
  • the ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for CBMAAE03 activity.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the CBMAAE03 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor. Further, these assays may test whether the candidate compound results in a signal generated by activation of the CBMAAE03 polypeptide, using detection systems appropriate to the cells bearing the CBMAAE03 polypeptide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing a CBMAAE03 polypeptide to form a mixture, measuring CBMAAE03 activity in the mixture, and comparing the CBMAAE03 activity of the mixture to a standard.
  • the CBMAAE03 cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBMAAE03 mRNA and protein in cells.
  • an ELISA may be constructed for measuring secreted or cell associated levels of CBMAAE03 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of CBMAAE03 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the CBMAAE03 protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art.
  • ligand binding and crosslinking assays include, but are not limited to, ligand binding and crosslinking assays in which the CBMAAE03 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. In addition to being used for purification and cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBMAAE03 which compete with the binding of CBMAAE03 to its receptors, if any.
  • a radioactive isotope eg 1251
  • chemically modified eg biotinylated
  • a peptide sequence suitable for detection or purification and incubated with a source of the
  • CBMAAE03 polypeptide antagonists mclude antibodies or, in some cases, oligonucleotides or proteins which are closely related to the gands, substrates, receptors, enzymes, etc , as the case may be, of the CBMAAE03 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 agomsts, antagonists, hgands, receptors, substrates, enzymes, etc for CBMAAE03 polypeptides, or compounds which decrease or enhance the production of CBMAAE03 polypeptides, which compnses (a) a CBMAAE03 polypeptide, preferably that of SEQ ID NO 2, (b) a recombinant cell expressing a CBMAAE03 polypeptide, preferably that of SEQ ID NO 2,
  • This mvention provides methods of treating abnormal conditions such as, infertility, miscamage, kidney disease, and liver disease, related to both an excess of and insufficient amounts of CBMAAE03 polypeptide activity
  • CBMAAE03 polypeptide If the activity of CBMAAE03 polypeptide is excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagomst) as hereinabove descnbed along with a pharmaceutically acceptable earner m an amount effective to inhibit the function of the CBMAAE03 polypeptide, such as, for example, by blocking the binding of gands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of CBMAAE03 polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc m competition with endogenous CBMAAE03 polypeptide may be admimstered Typical embodiments of such competitors comprise fragments of the CBMAAE03 polypeptide
  • expression of the gene encoding endogenous CBMAAE03 polypeptide can be inhibited using expression blocking techniques
  • antisense sequences either internally generated or separately administered See, for example, O'Connor, JNeurochem (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression.
  • oligonucleotides which form tnple 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 ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • gene therapy may be employed to effect the endogenous production of CBMAAE03 by the relevant cells in the subject
  • Peptides such as the soluble form of CBMAAE03 polypeptides, and agomsts and antagomst peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner
  • a suitable pharmaceutical earner Such formulations comp ⁇ se a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient
  • earners mclude but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art
  • the mvention further relates to pharmaceutical packs and kits comp ⁇ smg 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 in conjunction with other compounds, such as therapeutic compounds
  • Prefe ⁇ ed forms of systemic administration of the pharmaceutical compositions 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.
  • oral administration may also be possible Administration of these compounds may also be topical and or localized, m the form of salves, pastes, gels
  • the dosage range required depends on the choice of peptide, 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 in the range of 0 1-100 ⁇ g/kg of subject Wide vanations in the needed dosage, however, are to be expected m view of the va ⁇ ety of compounds available and the differing efficiencies of va ⁇ ous
  • Polypeptides used m treatment can also be generated endogenously m the subject, m treatment modalities often refe ⁇ ed to as "gene therapy" as descnbed above
  • m treatment modalities often refe ⁇ ed to as "gene therapy" as descnbed above
  • cells from a sub j ect may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasrmd vector The cells are then introduced mto the subject
  • TELECOMMUNICATION INFORMATION (A) TELEPHONE: 610-407-0700 (B) TELEFAX: 510-407-0701
  • GCTTCAACAC CAAGAGAAAC CGACTGCGGG AAAAACTGAC TCTTTTGCAT TATGATCCAG 180 TTGTGAAACA AAGAGTCCTC TTCGTGGAAA AGAAAAAAAT ACGCTCCCTT TAAACGGTGG 240

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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des polypeptides et des polynucléotides CBMAAE03 et des procédés d'obtention de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides CBMAAE03 dans la conception de protocoles pour le traitement de la stérilité, de fausses couches, de maladies du rein, et de maladies du foie, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000005 1998-01-18 1998-01-18 Cbmaae03: gene semblable a la proteine ribosomique l33 WO1999036524A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000005 WO1999036524A1 (fr) 1998-01-18 1998-01-18 Cbmaae03: gene semblable a la proteine ribosomique l33

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN1998/000005 WO1999036524A1 (fr) 1998-01-18 1998-01-18 Cbmaae03: gene semblable a la proteine ribosomique l33

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WO1999036524A1 true WO1999036524A1 (fr) 1999-07-22

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FEBS LETT., 68(1), (1976), WITTMANN-LIEBOLD B. et al., "Primary Structure of Protein L33 from the Large Subunit of the Escherichia Coli Ribosome", pp. 115-118. *
GENOMICS, 16(3), (1993), BURLAND V. et al., "DNA Sequence and Analysis of 136 Kilobases of the Escherichia Coli Genome: Organizational Symmetry Around the Origin of Replication", pp. 551-561. *
MOL. GEN. GENET., 184(2), (1981), LEE J.S. et al., "Cloning and the Nucleotide Sequence of the Genes for Escherichia Coli Ribosomal Proteins L28 (rpmB) and L33 (rpmG)", pp. 218-223. *
SCIENCE, 269(5223), (1995), FLEISCHMANN R.D. et al., "Whole-Genome Random Sequencing and Assembly of Haemophilus Influenza Rd", pp. 496-512. *

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