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WO1999061626A2 - Homologues de l'hydrolase humaine, asparagine amidohydrolase n-terminale, glycosylhydrolase, glucohydrolase, biotinidase, et n-acetylglucosamine 6-p-deacetylase - Google Patents

Homologues de l'hydrolase humaine, asparagine amidohydrolase n-terminale, glycosylhydrolase, glucohydrolase, biotinidase, et n-acetylglucosamine 6-p-deacetylase Download PDF

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Publication number
WO1999061626A2
WO1999061626A2 PCT/US1999/012021 US9912021W WO9961626A2 WO 1999061626 A2 WO1999061626 A2 WO 1999061626A2 US 9912021 W US9912021 W US 9912021W WO 9961626 A2 WO9961626 A2 WO 9961626A2
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hhh
polynucleotide
seq
sequence
polypeptide
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PCT/US1999/012021
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WO1999061626A3 (fr
Inventor
Olga Bandman
Jennifer L. Hillman
Henry Yue
Preeti Lal
Neil C. Corley
Karl J. Guegler
Chandra Patterson
Mariah R. Baughn
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Incyte Pharmaceuticals, Inc.
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Priority to CA002329076A priority Critical patent/CA2329076A1/fr
Priority to AU43222/99A priority patent/AU4322299A/en
Priority to EP99953359A priority patent/EP1080203A2/fr
Priority to JP2000551010A priority patent/JP2003500002A/ja
Publication of WO1999061626A2 publication Critical patent/WO1999061626A2/fr
Publication of WO1999061626A3 publication Critical patent/WO1999061626A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • glycosyl hydrolases are a large group of hydrolase enzymes that cleave the glycosidic bond between two carbohydrates, or between a carbohydrate and a non- carbohydrate moiety. Based on sequence similarity, these enzymes have been classified into at least 54 families that are named according to their substrate specificities. Examples of glycosyl hydrolases include glucosidases. glucanases. xylanases. amylases. galactosidases. sialidases. and mannosidases. Disorders of carbohydrate metabolism are frequently associated with glycosyl hydrolases.
  • the invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO:7 and fragments thereof, as well as a purified agonist and a purified antagonist to the polypeptide.
  • the invention also provides a method for treating or preventing a reproductive disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO:7 and fragments thereof.
  • complementarity refers to the natural binding of polynucleotides by base pairing.
  • sequence A-G-T
  • complementary sequence T-C-A
  • Complementarity between two single-stranded molecules may be "partial,” such that only some of the nucleic acids bind, or it may be “complete.” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • composition comprising a given polynucleotide sequence or a “composition comprising a given amino acid sequence. " as these terms are used herein, refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding HHH or fragments of HHH may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation. pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • oligonucleotide refers to a nucleic acid sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides. and most preferably about 20 to 25 nucleotides. which can be used in PCR amplification or in a hybridization assay or microarray.
  • oligonucleotide is substantially equivalent to the terms "amplimer,” “primer,” “oligomer,” and “probe.” as these terms are commonly defined in the art.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of. for example, a certain population, a disease state, or a propensity for a disease state.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 M NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide. while high stringency hybridization can be obtained in the presence of at least about 35% formamide. and most preferably at least about 50% formamide.
  • Stringent temperature conditions w ill ordinarily include temperatures of at least about 30°C. more preferably of at least about 37°C. and most preferably of at least about 42°C.
  • sequence preparation is automated with machines such as the ABI Catalyst 800 (Perkin Elmer) or a Hamilton Micro Lab 2200 (Hamilton. Reno NV) in combination with thermal cyclers (for PCR).
  • Sequencing is then carried out using either ABI 373 or 377 DNA sequencers (Perkin Elmer) or the MEGABASE capillary electrophoresis (Molecular Dynamics), and the sequences are analyzed using tools (computer programs and algorithms) which are well known in the art. (Ausubel (1997. unit 7.7); Meyers, R.A. (1995: Molecular Biology and Biotechnology. Wiley VCH. Inc. New York NY. p 856-853).
  • primers may be designed using commercially available software, such as OLIGOTM 4.06 Primer Analysis software (National Biosciences Inc., Madison. MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • the nucleotide sequences encoding HHH or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding HHH.
  • Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HHH.
  • Yeast expression systems may be used for production of HHH.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase. and PGH, may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris.
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of HHH. Transcription of sequences encoding HHH may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV. (Takamatsu, N. ( 1987) EMBO J. 3:17-31 1.) Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680: Brogue, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.
  • viral promoters e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters may
  • a number of viral-based expression systems may be utilized.
  • sequences encoding HHH may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses HHH in host cells.
  • dhfr confers resistance to methotrexate: neo confers resistance to the aminoglycosides neomycin and G-418; and als ox pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • neo confers resistance to the aminoglycosides neomycin and G-418; and als ox pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • als ox pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding HHH is inserted within a marker gene sequence
  • transformed cells containing sequences encoding HHH can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding HHH under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding HHH and that express HHH may be identified by a variety of procedures known to those of skill in the art.
  • DNA-DNA or DNA-RNA hybridizations include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
  • Immunological methods for detecting and measuring the expression of HHH using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • natural, modified, or recombinant nucleic acid sequences encoding HHH may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric HHH protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HHH activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to.
  • an antagonist of HHH may be administered to a subject to treat or prevent a reproductive disorder.
  • a reproductive disorder may include, but is not limited to, disorders of prolactin production; infertility, including tubal disease, ovulatory defects, and endometriosis; disruptions of the estrous cycle, disruptions of the menstrual cycle, polycystic ovary syndrome, ovarian hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis; cancer of the breast, fibrocystic breast disease, and galactorrhea; disruptions of spermatogenesis.
  • An antagonist of HHH may be produced using methods which are generally known in the art.
  • purified HHH may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HHH.
  • Antibodies to HHH may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal. monoclonal, chimeric, and single chain antibodies.
  • Neutralizing antibodies i.e., those which inhibit dimer formation are especially preferred for therapeutic use.
  • the oligopeptides. peptides, or fragments used to induce antibodies to HHH have an amino acid sequence consisting of at least about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HHH amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Expression vectors derived from retroviruses. adenoviruses. or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding HHH (See, e g , SambrooL supra, and Ausubel. supra )
  • Genes encoding HHH can be turned off b ⁇ transforming a cell or tissue with expression vectors which express high levels of a polynucleotide. or fragment thereof, encoding HHH
  • Such constructs mav be used to introduce untranslatable sense or antisense sequences into a cell Even in the absence ot integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system
  • RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ⁇ bonuclease protection assays
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HHH Such DNA sequences may be incorporated into a wide va ⁇ et> of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly. can be introduced into cell lines, cells, or tissues RNA molecules mav be modified to increase intracellular stability and half-life.
  • flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine. queosine. and wvbutosine. as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine. cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to. hydrochloric, sulfuric. acetic, lactic, tartaric. malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol. at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HHH or closely related molecules may be used to identify nucleic acid sequences which encode HHH
  • the specificity of the probe whether it is made from a highly specific region, e g , the 5' regulatory region, or from a less specific region, e g , a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding HHH. allelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the HHH encoding sequences
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:8 through SEQ ID NO 14 or from genomic sequences including promoters, enhancers, and introns of the HHH gene
  • Means for producing specific hybridization probes for DNAs encoding HHH include the cloning of polynucleotide sequences encoding HHH or HHH derivatives into vectors for the production of mRNA probes Such vectors are known in the art.
  • disruptions of the estrous cycle disruptions of the menstrual cycle, polycvstic ovary syndrome, ovarian hyperstimulation syndrome, endomet ⁇ al and ovarian tumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis, cancer of the breast, fibrocystic breast disease and galactorrhea.
  • disruptions ot spermatogenesis abnormal sperm physiology, cancer ot the testis. cancer of the prostate, benign prostatic hyperplasia, prostatitis.
  • melanoma myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus. thyroid, and uterus.
  • Hybridization probes derived from SEQ ID NO:8 through SEQ ID NO: 14 are employed to screen cDNAs, genomic DNAs. or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGOTM 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham. Chicago. IL), and T4 polynucleotide kinase (DuPont NEN*.
  • Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENETM. Full-length cDNAs. ESTs. or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g.. UV cross- linking followed by thermal and chemical treatments and subsequent drying. (See. e.g., Schena, M. et al.
  • Fluorescent probes are prepared and used for hybridization to the elements on the substrate.
  • the substrate is analyzed by procedures described above.
  • HHH in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica californica nuclear polyhedrosis virus
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding HHH by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus.
  • HHH is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST glutathione S-transferase
  • a peptide epitope tag such as FLAG or 6-His
  • an assay measuring the ⁇ -glucosidase activity of an HHH molecule is described. Varying amounts of HHH are incubated with 1 M 4-nitrophenyl ⁇ -D- glycopyranoside (a substrate) in 50 mM sodium acetate buffer. pH 5.0. for various times (typically 1-5 minutes) at 37°C. The reaction is halted by heating to 100°C for 2 minutes. The absorbance is measured spectrophotometrically at 410 nm. and is proportional to the activity of HHH in the sample. (Hrmova, M. et al. ( 1998) J. Biol. Chem. 273:1 1 134-1 1 143.)
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP) (Clontech, Palo Alto, CA), CD64, or a CD64-GFP fusion protein.
  • Flow cytometry an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for example, their apoptotic state.
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down- regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake: alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. ( 1994) Flow Cytometry, Oxford, New York. NY.
  • HHH substantially purified using polyacryiamide gel electrophoresis (PAGE)(see. e.g., Harrington. M.G. ( 1990) Methods Enzymol. 182:488-495). or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
  • PAGE polyacryiamide gel electrophoresis
  • LASERGENETM software is used to analyze the HHH amino acid sequence.
  • oligopeptides 15 residues in length are synthesized using an Applied Bioelectron sequence.
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • Naturally occurring or recombinant HHH is substantially purified by immunoaffinity chromatography using antibodies specific for HHH.
  • An immunoaffinity column is constructed by covalently coupling anti-HHH antibody to an activated chromatographic resin, such as CNBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • HHH. or biologically active fragments thereof are labeled with 125 I Bolton-Hunter reagent. (See. e.g.. Bolton et al. ( 1973) Biochem. J. 133:529.)
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HHH. washed, and any wells with labeled HHH complex are assayed. Data obtained using different concentrations of HHH are used to calculate values for the number, affinity, and association of HHH with the candidate molecules.

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Abstract

L'invention porte sur des homologues de l'hydrolase humaine (HHH) et des polynucléotides identifiant la HHH et codant pour elle. L'invention porte également sur des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes associés ainsi que sur des méthodes de diagnostic, traitement et prévention de troubles associés à l'expression du HHH.
PCT/US1999/012021 1998-05-29 1999-05-28 Homologues de l'hydrolase humaine, asparagine amidohydrolase n-terminale, glycosylhydrolase, glucohydrolase, biotinidase, et n-acetylglucosamine 6-p-deacetylase WO1999061626A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002329076A CA2329076A1 (fr) 1998-05-29 1999-05-28 Homologues de l'hydrolase humaine, asparagine amidohydrolase n-terminale, glycosylhydrolase, glucohydrolase, biotinidase, et n-acetylglucosamine 6-p-deacetylase
AU43222/99A AU4322299A (en) 1998-05-29 1999-05-28 Human hydrolase homologs: n-terminal asparagine amidohydrolase, glycosyl hydrolase, glucohydrolase, biotinidase, and n-acetylglucosamine 6-p deacetylase
EP99953359A EP1080203A2 (fr) 1998-05-29 1999-05-28 Homologues de l'hydrolase humaine, asparagine amidohydrolase n-terminale, glycosylhydrolase, glucohydrolase, biotinidase, et n-acetylglucosamine 6-p-deacetylase
JP2000551010A JP2003500002A (ja) 1998-05-29 1999-05-28 ヒトヒドロラーゼ相同体：ｎ−末端アスパラギンアミドヒドロラーゼ、グリコシルヒドロラーゼ、グルコヒドロラーゼ、ビオチニダーゼ及びｎ−アセチルグルコサミン６−ｐデアセチラーゼ

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US8723698P 1998-05-29 1998-05-29
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EP (1) EP1080203A2 (fr)
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AU (1) AU4322299A (fr)
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WO (1) WO1999061626A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1092032A2 (fr) * 1998-06-30 2001-04-18 Incyte Pharmaceuticals, Inc. Proteines oxydoreductase humaines
WO2001049733A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, glycosyl hydrolase 12, et polynucleotide codant pour ce polypeptide
WO2000039160A3 (fr) * 1998-12-24 2001-08-23 Yeda Res & Dev
WO2003054153A2 (fr) * 2001-12-12 2003-07-03 University Of Cincinnati Polypeptides de l'apyrase et acides nucleiques codant pour ces polypeptides
WO2003093427A2 (fr) * 2002-04-30 2003-11-13 Incyte Corporation Molecules utilisees dans la detection et le traitement de maladies

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6537547B1 (en) * 1996-06-07 2003-03-25 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Polypeptides having L-asparaginase activity
US5958750A (en) * 1996-07-03 1999-09-28 Inctye Pharmaceuticals, Inc. Human hyaluronidase

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1092032A2 (fr) * 1998-06-30 2001-04-18 Incyte Pharmaceuticals, Inc. Proteines oxydoreductase humaines
WO2000039160A3 (fr) * 1998-12-24 2001-08-23 Yeda Res & Dev
US6762283B1 (en) 1998-12-24 2004-07-13 Yeda Research And Development Co. Ltd. Caspase-8 interacting proteins
US7339047B2 (en) 1998-12-24 2008-03-04 Yeda Research And Development Company Ltd. Caspase-8 interacting proteins
WO2001049733A1 (fr) * 1999-12-29 2001-07-12 Fudan University Nouveau polypeptide, glycosyl hydrolase 12, et polynucleotide codant pour ce polypeptide
WO2003054153A2 (fr) * 2001-12-12 2003-07-03 University Of Cincinnati Polypeptides de l'apyrase et acides nucleiques codant pour ces polypeptides
WO2003054153A3 (fr) * 2001-12-12 2003-11-06 Univ Cincinnati Polypeptides de l'apyrase et acides nucleiques codant pour ces polypeptides
WO2003093427A2 (fr) * 2002-04-30 2003-11-13 Incyte Corporation Molecules utilisees dans la detection et le traitement de maladies
WO2003093427A3 (fr) * 2002-04-30 2005-09-01 Incyte Corp Molecules utilisees dans la detection et le traitement de maladies

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WO1999061626A3 (fr) 2000-04-06
CA2329076A1 (fr) 1999-12-02
EP1080203A2 (fr) 2001-03-07
AU4322299A (en) 1999-12-13
JP2003500002A (ja) 2003-01-07

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