WO2000000513A1 - Gene humain krab-np (npaawe05) - Google Patents

Gene humain krab-np (npaawe05) Download PDF

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Publication number
WO2000000513A1
WO2000000513A1 PCT/CN1998/000109 CN9800109W WO0000513A1 WO 2000000513 A1 WO2000000513 A1 WO 2000000513A1 CN 9800109 W CN9800109 W CN 9800109W WO 0000513 A1 WO0000513 A1 WO 0000513A1
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Prior art keywords
polypeptide
identity
seq
subject
sequence
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PCT/CN1998/000109
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English (en)
Inventor
Huaidong Song
Renming Hu
Jialun Chen
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Shanghai Second Medical University
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Priority to PCT/CN1998/000109 priority Critical patent/WO2000000513A1/fr
Publication of WO2000000513A1 publication Critical patent/WO2000000513A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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

  • 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 NPAAWE05, in particular NPAAWE05 polypeptides and NPAAWE05 polynucleotides, recombinant materials and methods for their production.
  • the invention relates to methods for using such polypeptides and polynucleotides, including the treatment of endocrine disorders and diabetes mellitus, hereinafter referred to as "the Diseases", amongst others.
  • the invention relates to methods for identifying agonists and antagonists/inhibitors using the materials provided by the invention, and treating conditions associated with NPAAWE05 imbalance with the identified compounds.
  • the invention relates to-diagnostic assays for detecting diseases associated with inappropriate NPAAWE05 activity or levels.
  • the present invention relates to NPAAWE05 polypeptides.
  • peptides include isolated polypeptides comprising an amino acid 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, most preferably at least 97-99% identity, to that of SEQ ID NO 2 over the entire length of SEQ ID NO 2
  • polypeptides mclude those compnsmg the ammo acid of SEQ ID NO.2
  • peptides of the present invention include isolated polypeptides in which the ammo 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 mclude the polypeptide of SEQ ID NO 2
  • peptides of the present mvention include isolated polypeptides encoded by a polynucleotide compnsmg the sequence contained in SEQ ID NO 1
  • Polypeptides of the present mvention are believed to be members of the gene family including the Kruppel associated box (KRAB) domain family of polypepudes Thev are therefore of interest because the Kruppel-associated box is often found in zinc finger proteins The box is often located in the the non-zmc finger N-termmal regions of the gene, and is thought to act as an activating or suppressing domain of zmc finger protein genes
  • the polypeptide of the present invention has only one KRAB domam, but no zmc finger domains These properties are hereinafter referred to as "NPAAWE05 activity” or "NPAAWE05 polypeptide activity” or "biological activity of NPAAWE05" Also included amongst these activities are antigenic and immunogenic activities of said NPAAWE05 polypeptides, in particular the anugenic and immunogenic activities of the polypeptide of SEQ ID NO 2
  • a polypeptide of the present invention exhibits at least one biological activity of NPAAWE05
  • polypeptides of the present mvention may be in the form of the "mature" protem or may be a part of a larger protem such as a fusion protem It is often advantageous to mclude an additional ammo acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid m punfication such as multiple histidme residues, or an ad ⁇ tionai sequence for stability during recombinant production
  • the present invention also mcludes mclude variants of the aforemen ⁇ oned polypeptides, that is polypeptides that vary from the referents by conservative ammo acid substitutions, whereby a residue is substituted by another with like charactenstics 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 ammo acids are substituted, deleted, or added in any combination
  • Polypepudes of the present mvention can be prepared in any suitable manner
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypepudes, synthetically produced polypepudes, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood m the art
  • the present inven ⁇ on relates to NPAAWE05 polynucleotides
  • polynucleotides mclude isolated polynucleotides compnsmg 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 ammo acid sequence of SEQ ID NO 2, over the enure length of SEQ ID NO 2
  • polypepudes 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 polynucleoudes include a polynucleotide compnsmg the nucleotide sequence contained m SEQ ID NO 1 encoding the polypeptide of SEQ ID NO 2
  • polynucleotides of the present mvention mclude isolated polynucleotides compnsmg 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 codmg 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 mvention mclude isolated polynucleotides compnsmg 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.
  • polynucleotides include a polynucleotide compnsmg 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.l shows homology with M67509, human Kruppel- associated box (KRAB) (C D Constantmou-Deltas, et al Genomics, 1992,12 581-589)
  • the nucleotide sequence of SEQ ID NO 1 is a cDNA sequence and compnses a polypeptide encoding sequence (nucleotide 140 to 421) encoding a polypeptide of 94 ammo 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 encodmg sequence contained m 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 SEQ ID NO 2 is structurally related to other proteins
  • Preferred polypeptides and polynucleotides of the present mvention are expected to have, inter alia, similar biological functions/properties to their homologous polypeptides and polvnucleotides Furthermore, preferred polypeptides and polynucleotides of the present mvention have at least one NPAAWE05 activity
  • Polynucleotides of the present mvention may be obtained, using standard cloning and screening techniques, from a cDNA library de ⁇ ved from mRNA m cells of human normal pituitary, usmg the expressed sequence tag (EST) analysis (Adams, M D , et al Science (1991) 252 1651-1656, Adams.
  • EST expressed sequence tag
  • polynucleotides of the invention can also be obtained from natural sources such as genomic DNA hbranes 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 m reading frame with other codmg 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
  • polypeptide va ⁇ ants which comp ⁇ se the ammo 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, m any combination Polynucleotides which are identical or sufficientiy identical to a nucleotide sequence contained m
  • SEQ ID NO 1 may be used as hyb ⁇ dization probes for cDNA and genomic DNA or as p ⁇ mers for a nucleic acid amplification (PCR) reaction, to isolate full-length cDNAs and genomic clones encoding polypeptides of the present mvention 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 simila ⁇ ty to SEQ ID NO 1 Typically these nucleoude 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 comp ⁇ se at least 15 nucleotides, preferably, at least 30 nucleotides and may have at least 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 comp ⁇ ses the steps of screening an approp ⁇ ate library under strmgent hyb ⁇ dization conditions with a labeled probe havmg the sequence of SEQ ID NO 1 or a fragment thereof, and isolating full-length cDNA and genomic clones containing said polynucleotide sequence
  • strmgent hyb ⁇ dization conditions mclude overnight mcubation at 42°C in a solution compnsmg 50% formamide, 5xSSC (150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt's solution, 10 % dextran sulfate, and 20
  • an isolated cDNA sequence will be mcomplete, m that the region codmg for the po peptide 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 during the polymensation reaction), failing to complete a DNA copy of the mRNA template during 1st strand cDNA synthesis
  • the PCR reaction is then repeated usmg 'nested' primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pnmer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' m the known gene sequence)
  • primers designed to anneal within the amplified product typically an adaptor specific pnmer that anneals further 3' m the adaptor sequence and a gene specific pnmer that anneals further 5' m the known gene sequence
  • the products of this reaction can then be analysed by DNA sequencmg 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 usmg the new sequence information for the design of the 5' pnmer
  • Recombinant polypeptides of the present mvention may be prepared bv processes well known m the art from genetically engmeered host cells compnsmg expression systems Accordmgly, m 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 bv 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 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 m Molecular Biology (1986) and Sambrook et al , Molecular Cloning A Laboratory Manual.
  • 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
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subtihs cells, fungal cells, such as yeast cells and Aspergillus cells, insect cells such as Drosoph ⁇ a 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 coh, Streptomyces and Bacillus subtihs cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosoph ⁇ a 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 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 denved 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 m a host may be used The
  • the cells may be harvested pnor to use m the screening assay If the polypeptide is secreted mto the medium, the medium can be recovered m order to recover and punfy the polypeptide If produced lntracellularly, 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, amon or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinity chromatography, h droxylapa ⁇ te 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 mvention also relates to the use of polynucleotides of the present mvention as diagnostic reagents Detection of a mutated form of the gene characte ⁇ sed by the polynucleotide of SEQ ID NO 1 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
  • 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 pnor 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 NPAAWE05 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 denatu ⁇ ng agents, or by direct DNA sequencing (ee, e g , Myers et al
  • the diagnostic assays offer a process for diagnosing or determinmg a susceptibility to the Diseases through detection of mutation m the NPAAWE05 gene by the methods descnbed
  • diseases may be diagnosed by methods compnsmg determining from a sample denved from a subject an abnormally decreased or increased level of polypeptide or mRNA Decreased or increased expression can be measured at the RNA level usmg any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for mstance PCR, RT-PCR, RNase protection, Northern blottmg and other hybndization methods
  • Assay techniques that can be used to determine levels of a protem, such as a polypeptide of the present mvention, in a sample denved from a host are well-known to those of skill m the art Such assay methods mclude radioimmunoassays, competitive-bmdmg assay
  • 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
  • kits may compnse a substantial component
  • Such a kit will be of use in diagnosing a disease or suspectabihty to a disease, particularly endocrine disorders and diabetes melhtus, 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 mdividual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step in conelating 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 conelated with genetic map data Such data are found in, for example, V McKusick, Mendehan Inhentance m Man (available on-line 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 (coinhentance 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 m 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 immunospecific for polypeptides of the present mvention
  • immunospecrfic means that the antibodies have substantially greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides m the pnor 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, usmg routine protocols For preparation of monoclonal antibodies, any techmque which provides antibodies produced by continuous cell line cultures can be used Examples mclude the hyb ⁇ doma techmque (Kohler, G and Milstem C , Nature (1975) 256 495-497), the t ⁇ oma techmque, the human B-cell hyb ⁇ doma techmque (Kozbor et al , Immunology Today (1983) 4 72) and the EBV- hybndoma techmque (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)
  • 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
  • the present mvention relates to genetically engmeered soluble fusion proteins compnsmg a polypeptide of the present mvention, or a fragment thereof, and va ⁇ ous portions of the constant regions of heavy or light chains of immunoglobulins of vanous subclasses (IgG, IgM, IgA, IgE) Preferred as an immunoglobulm is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hmge region
  • 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 engineenng, and to the use thereof for drug screening, diagnosis and therapy
  • a further aspect of the invention also relates to polynucleotides encoding such fusion proteins Examples of fusion protem technology can be found m International Patent Application Nos W094/29458
  • Another aspect of the mvention relates to a method for mducmg an lmmunological response in a mammal which compnses moculatmg the mammal with a polypeptide of the present mvention, 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 mvention relates to a method of mducmg lmmunological response in a mammal which comp ⁇ ses, delivering a polypeptide of the present mvention via a vector directing expression of the polynucleotide and codmg for the polypeptide in vivo in order to mduce such an unmunological response to produce antibody to protect said animal from diseases
  • a further aspect of the mvention relates to an immunological/vaccine formulation (composition) which, when introduced mto a mammalian host, induces an unmunological response m that mammal to a polypeptide of the present mvention wherem the composition comp ⁇ ses a polypeptide or polynucleotide of the present mvention
  • the vaccine formulation may further compnse a suitable earner Since a polypeptide may be broken down in the stomach, it is preferably administered parenterally (for instance, subcutaneous, intramuscular, mtravenous, or lntradermal 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 mstomc with the blood of the recipient, and aqueous and non- aqueous ste ⁇ le suspensions which may mclude suspending agents or thickening agents
  • 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 polypeptide Accordmgly, m 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
  • 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 libranes, 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.
  • the screenmg method may simply measure the binding of a candidate compound to the polypeptide, or to cells or membranes bearmg 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 mvolve competition with a labeled competitor Further, these screemng methods may test whether the candidate compound results m a signal generated by activation or inhibition of the polypeptide, usmg detection systems approp ⁇ ate to the cells bearing the polypeptide Inhibitors of activation are generally assayed m the presence of a known agomst and the effect on activation by the agomst by the presence of the candidate compound is observed Constitutively active polpypeptides may be employed m screenmg methods for inverse agomsts or inhibitors, m the absence of an agonist or inhibitor,
  • polypeptides and antibodies to the polypeptide of the present mvention may also be used to configure screenmg methods for detectmg the effect of added compounds on the production of mRNA and polypeptide m cells
  • an ELISA assay may be constructed for measunng secreted or cell associated levels of polypeptide usmg 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 agomst, 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, hgand binding and crosslinking assays m which the polypeptide is labeled with a radioactive isotope (for mstance, 2->I), chemically modified (for instance, biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supematants, 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 in the art.
  • polypeptide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, receptors, enzymes, etc., as the case may be, of the polypeptide, e.g., a fragment of the ligands, substrates, receptors, enzymes, etc.; or small molecules which bind to the polypeptide of the present invention 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, ligands, receptors, substrates, enzymes, etc. for polypeptides of the present invention; or compounds which decrease or enhance the production of such polypeptides, which comprises:
  • kits may comprise a substantial component.
  • 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:
  • the present mvention provides methods of treating abnormal conditions such as, for instance, endocrine disorders and diabetes melhtus, related to either an excess of, or an under- expression of, NPAAWE05 polypeptide activity
  • mhibitor compound 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 ligands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of the polypeptides still capable of binding the ligand, substrate, enzymes, receptors, etc m competition with endogenous polypeptide may be administered Typical examples of such competitors mclude fragments of the NPAAWE05 polypeptide
  • NPAAWE05 polypeptide expression of the gene encoding endogenous NPAAWE05 polypeptide can be inhibited usmg expression blocking techniques
  • antisense sequences either internally generated or separately administered
  • ohgonucleotides which form t ⁇ ple 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)
  • ohgomers can be administered /?er se or the relevant ohgomers can be expressed in vivo
  • NPAAWE05 For treating abnormal conditions related to an under-expression of NPAAWE05 and its activity, several approaches are also available One approach compnses administering to a subject a therapeutically effective amount of a compound which activates a polypeptide of the present mvention, l e , an agomst as descnbed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal con ⁇ tion
  • gene therapy may be employed to effect the endogenous production of NPAAWE05 by the relevant cells m the subject
  • 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 mto a packaging cell transduced with a retroviral plasmid vector containing RNA encoding 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 administered to a subject for
  • the present mvention provides for pharmaceutical compositions compnsmg 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, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof
  • the mvention further relates to pharmaceutical packs and kits compnsmg 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
  • composition will be adapted to the route of administration, for instance by a systemic or an oral route
  • Prefened forms of systemic admmistration m include injection, typically by intravenous injection
  • Other injection routes such as subcutaneous, mtramuscular, or mtrapentoneal
  • Alternative means for systemic admmistration m include transmucosal and transdermal admmistration usmg penetrants such as bile salts or fusi c acids or other detergents
  • oral admmistration may also be possible
  • Admmistration 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 admmistration, the nature of the formulation, the nature of
  • Polypeptides used in treatment can also be generated endogenously m the subject, m treatment modalities often refened to as "gene therapy" as descnbed above
  • m treatment modalities often refened 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 vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced mto 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 m a computer readable medium and then usmg the stored data to search a sequence database usmg well known searchmg tools, such as GCC Accordingly, in a further aspect, the present mvention provides for a computer readable medium havmg stored thereon a polynucleotide compnsmg the sequence of SEQ ID NO 1 and/or a polypeptide sequence encoded thereby
  • Antibodies as used herein mcludes polyclonal and monoclonal antibodies, chime ⁇ c, smgle cham, and humamzed antibo ⁇ es, as well as Fab fragments, mcludmg the products of an Fab or other immunoglobulm 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 onginal 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 matenals of its natural state is “isolated", as the term is employed herein
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeoxnbonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides mclude, without limitation, smgle- and double-stranded DNA, DNA that is a mixture of smgle- and double- stranded regions, smgle- and double-stranded RNA, and RNA that is mixture of smgle- and double-stranded regions, hybnd molecules compnsmg DNA and RNA that may be smgle-stranded or, more typically, double-stranded or a mixture of smgle- and double-stranded regions
  • polynucleotide refers to tnple-stranded regions compnsmg RNA or DNA or both RNA and DNA
  • polynucleotide also mcludes DNAs or RNAs containing
  • Polypeptide refers to any peptide or protem comprising 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 chains, commonly refened to as peptides, ohgopeptides or ohgomers, and to longer chains, generally refened to as proteins Polypeptides may contain ammo acids other than the 20 gene-encoded ammo acids
  • Polypeptides include ammo acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known m the art Such modifications are well descnbed m basic texts and m more detailed monographs, as well as m a voluminous research literature Modifications may occur anywhere in a polypeptide, mclu ⁇ ng the peptide backbone, the ammo acid side-chams and the ammo or carboxyl termini It will be appreciated that the same type of modification
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determmed by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determmed by the match between strings of such sequences
  • Identity can be readily calculated by known methods, mcludmg but not limited to those descnbed m ⁇ Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, G ⁇ ffin, A M , and Gnffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology, von Hemje, G , Academic
  • Parameters for polypeptide sequence companson mclude the following 1) Algonthm Needleman and Wunsch, J Mol Biol 48 443-453 (1970) Compa ⁇ son matnx BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl Acad Sci USA 89 10915-10919 (1992) Gap Penalty 12 Gap Length Penalty 4 A program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Ma ⁇ son WI The aforementioned parameters are the default parameters for peptide compansons (along with no penalty for end gaps)
  • Polynucleotide embodiments further include an isolated polynucleotide compnsmg a polynucleotide sequence havmg 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 include up to a certam integer number of nucleotide alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one nucleotide deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, mterspersed either individually among the nucleotides m the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of nucleotide alterations is determmed by multiplying the total number
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides m SEQ LD 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
  • any non-mteger product of x n and y is rounded down to the nearest mteger pnor to subtractmg it from x n
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO 2 may create nonsense, missense or frameshift mutations m this codmg 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 may mclude up to a certam mteger 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 consistmg of at least one nucleic acid deletion, substitution, mcludmg transition and transversion, or insertion, and wherem said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal 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 determmed by multiplying the total number of ammo acids m SEQ ID NO 2 by the mteger defining the percent identity divided by 100 and then subtracting that product from said total number of ammo acids in SEQ
  • n n is the number of ammo acid alterations
  • x n is the total number of ammo acids m SEQ ID NO 2
  • y is, for mstance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger product of x n and y is rounded down to the nearest mteger pnor to subtracting it from x n
  • Polypeptide embodiments further mclude an isolated polypeptide compnsmg a polypeptide havmg at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO 2, wherem said polypeptide sequence may be identical to the reference sequence of SEQ ID NO 2 or may mclude up to a certam integer number of ammo acid alterations as compared to the reference sequence, wherem said alterations are selected from the group consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non- conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the ammo acids m the reference sequence or m one or more contiguous groups within the reference sequence, and wherem said number of ammo acid alterations is determined by multiplying the total number of ammo acids in SEQ ID NO 2 by the
  • 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 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-mteger product of x a and y is rounded down to the nearest mteger pnor to subtractmg it from x a
  • a polypeptide sequence of the present mvention may be identical to the reference sequence of SEQ ID NO 2, that is it may be 100% identical, or it may include up to a certain mteger 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 consistmg of at least one ammo acid deletion, substitution, mcludmg conservative and non-conservative substitution, or insertion, and wherem said alterations may occur at the ammo- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, mterspersed either individually among the ammo acids m the reference sequence or m 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 ammo acids m SEQ ID NO 2 by the integer defining the percent identity divided by 100 and then subtractmg that product from said total number of ammo acids m SEQ ID NO
  • n a is the number of ammo acid alterations
  • x a is the total number of ammo acids m SEQ ID NO 2
  • y is, for mstance 0 70 for 70%, 0 80 for 80%, 0 85 for 85% etc
  • • is the symbol for the multiplication operator, and wherem any non-mteger 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 protem encoded by two, often unrelated, fused genes or fragments thereof
  • EP-A-0 464 ⁇ scloses fusion protems compnsmg va ⁇ ous portions of constant region of immunoglobulm molecules together with another human protem or part thereof
  • employmg an immunoglobulm Fc region as a part of a fusion protem is advantageous for use in therapy and diagnosis resulting m, for example, improved pharmacokinetic properties [see, e g , EP-A 0232 262]
  • it would 00/00513
  • MOLECULE TYPE protein
  • Leu Trp lie Met Thr Thr Ala Thr Arg Arg Gin Gly Asn Leu Asp Thr 65 70 75 80

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Abstract

L'invention concerne des polypeptides et des polynucléotides NPAAWE05 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 NPAAWE05 pour le traitement de pathologies associées, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000109 1998-06-29 1998-06-29 Gene humain krab-np (npaawe05) WO2000000513A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/CN1998/000109 WO2000000513A1 (fr) 1998-06-29 1998-06-29 Gene humain krab-np (npaawe05)

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Application Number Priority Date Filing Date Title
PCT/CN1998/000109 WO2000000513A1 (fr) 1998-06-29 1998-06-29 Gene humain krab-np (npaawe05)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023538A2 (fr) * 1999-09-28 2001-04-05 Incyte Genomics, Inc. Molecules de detection et de traitement de maladies
WO2001083541A1 (fr) * 2000-04-29 2001-11-08 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine 42 de liaison a l'adn associee a kruppel, et polynucleotide codant pour ce polypeptide

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DNA CELL BIOL., 14(2), (1995), ABRINK M. et al., "Isolation of cDNA Clones for 42 Different Kruppel-Related Zinc Finger Proteins Expressed in the Human Monoblast Cell Line U-937", pages 125-136. *
GENBANK, AF027513, (1998), SHANNON M. et al., "Homo Sapiens Zinc Finger Protein (HZF6) mRNA, 5 UTR and Parital Cds". *
GENOMICS, 12, (1992), CONSTANTINOU-DELTAS C.D. et al., "The Identification and Characterization of KRAB-Domain-Containing Zinc Finger Proteins", pages 581-589. *
GENOMICS, 33(1), (1996), SHANNON M. et al., "Comparative Analysis of a Conserved Zinc Finger Gene Cluster on Human Chromosome 19q and Mouse Chromosome 7", pages 112-120. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001023538A2 (fr) * 1999-09-28 2001-04-05 Incyte Genomics, Inc. Molecules de detection et de traitement de maladies
WO2001023538A3 (fr) * 1999-09-28 2002-05-02 Incyte Genomics Inc Molecules de detection et de traitement de maladies
WO2001083541A1 (fr) * 2000-04-29 2001-11-08 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine 42 de liaison a l'adn associee a kruppel, et polynucleotide codant pour ce polypeptide

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