[go: up one dir, main page]

WO1999002664A1 - Human n-arginine dibasic convertase - Google Patents

Human n-arginine dibasic convertase Download PDF

Info

Publication number
WO1999002664A1
WO1999002664A1 PCT/US1998/014022 US9814022W WO9902664A1 WO 1999002664 A1 WO1999002664 A1 WO 1999002664A1 US 9814022 W US9814022 W US 9814022W WO 9902664 A1 WO9902664 A1 WO 9902664A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypφtide
nrd convertase
convertase
nrd
seq
Prior art date
Application number
PCT/US1998/014022
Other languages
French (fr)
Inventor
Sanjay Kumar
Stephanie Van Horn
Michael William Lark
Original Assignee
Smithkline Beecham Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Publication of WO1999002664A1 publication Critical patent/WO1999002664A1/en

Links

Classifications

    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • 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 polynucleotides, polypeptides 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 invention relate to NRD convertase family, hereinafter referred to as h-NRD convertase. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides. BACKGROUND OF THE INVENTION
  • NRDs N-arginine dibasic convertase class
  • metalloendopeptidases which have been suggested to activate numerous proteins including somatostatin-28 and dynorphins A and B.
  • the metallo-NRDs are related to protease m from E. coli and to insulinase.
  • Furin has been proposed to activate this family of membrane type matrix metalloproteinases as well as other proteins including TGF-b [Dubois, et al. J. Biol. Chem. 270(18): 10618-10624, 1995] and endopeptidase-24.18 [Milhiet, et al., Biochem. J. 309: 683- 688, 1995].
  • Furin cleaves C-terminal to the Arg-X-Arg/Lys-Arg basic sequence and has been shown to activate an mtMMP-related enzyme, stromelysin-3, at this site [Pel and Weiss, Nature 375: 244-247, 1995].
  • furin is the enzyme responsible for activation of membrane type matix metalloproteinases in vivo. It is possible that furin, or another endopeptidase such as an NRD could be responsible for this activation in vivo.
  • the present invention describes a novel member of the NRD family of endopeptidases. This indicates that the NRD convertase family has an established, proven history as therapeutic targets.
  • NRD convertase family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease.
  • diseases including, but not limited to, chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoprolife
  • the invention relates to h-NRD convertase polypeptides and recombinant materials and methods for their production.
  • Another aspect of the invention relates to methods for using such h-NRD convertase polypeptides and polynucleotides.
  • Such uses include the treatment of chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg flammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • autoimmune diseases eg flammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AIDS, metabolic
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with h-NRD convertase imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate h-NRD convertase activity or levels.
  • h-NRD convertase refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or an allelic variant thereof.
  • h-NRD convertase activity or h-NRD convertase polypeptide activity or "biological activity of the h-NRD convertase or h-NRD convertase polypeptide” refers to the metabolic or physiologic function of said h-NRD convertase including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said h-NRD convertase.
  • h-NRD convertase gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or allelic variants thereof and/or their complements.
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin 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 original environment, or both.
  • a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polyp ⁇ tide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term 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, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Polyp ⁇ tide refers to any p ⁇ tide or protein comprising two or more amino acids joined to each other by peptide bonds or modified p ⁇ tide bonds, i.e., p ⁇ tide isosteres.
  • Polyp ⁇ tide refers to both short chains, commonly referred to as peptides, oligop ⁇ tides or oligomers, and to longer chains, generally referred to as proteins. Polyp ⁇ tides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in a polyp ⁇ tide, including the p ⁇ tide backbone, the amino acid side- chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polyp ⁇ tide. Also, a given polypeptide may contain many types of modifications.
  • Polyp ⁇ tides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • Variant is a polynucleotide or polyp ⁇ tide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
  • Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polyp ⁇ tide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polyp ⁇ tide. Generally, differences are limited so that the sequences of the reference polyp ⁇ tide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polyp ⁇ tide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polyp ⁇ tide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non- naturally occurring variants of polynucleotides and polyp ⁇ tides may be made by mutagenesis techniques or by direct synthesis.
  • "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. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING:
  • identity is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM 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., SIAM J Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs.
  • Preferred 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 , J Molec 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 exc ⁇ t 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 polyp ⁇ tide having an amino acid sequence having at least, for example, 95 % "identity" to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polyp ⁇ tide is identical to the reference sequence exc ⁇ t that the polyp ⁇ tide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2.
  • up to 5 % of the amino acid residues in the reference sequence may be deleted or substituted with another amino 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 amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the present invention relates to h-NRD convertase polypeptides (or h-NRD convertase proteins) .
  • the h-NRD convertase polypeptides include the polyp ⁇ tide of SEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polyp ⁇ tides comprising 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.
  • h- NRD convertase polyp ⁇ tides having the amino acid sequence which have at least 80% identity to the polyp ⁇ tide 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 LD NO:2. Furthermore, those with at least 97- 99 % are highly preferred.
  • h-NRD convertase polyp ⁇ tide exhibit at least one biological activity of h-NRD convertase.
  • the h-NRD convertase polyp ⁇ tides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
  • Fragments of the h-NRD convertase polyp ⁇ tides are also included in the invention.
  • a fragment is a polyp ⁇ tide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned h-NRD convertase polyp ⁇ tides.
  • fragments may be "free-standing," or comprised within a larger polyp ⁇ tide of which they form a part or region, most preferably as a single continuous region.
  • R ⁇ resentative examples of polyp ⁇ tide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of h-NRD convertase polyp ⁇ tide.
  • “about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
  • Preferred fragments include, for example, truncation polyp ⁇ tides having the amino acid sequence of h-NRD convertase polyp ⁇ tides, exc ⁇ t for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha- helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming 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 preferred fragments are biologically active fragments.
  • Biologically active fragments are those that mediate h-NRD convertase activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
  • polyp ⁇ tide fragments retain the biological activity of the h- NRD convertase, including antigenic activity.
  • fragment is that having the amino acid sequence of SEQ ID NO: 4.
  • Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions — i.e. , those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and lie; 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, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • the h-NRD convertase polyp ⁇ tides of the invention can be prepared in any suitable manner.
  • Such polyp ⁇ tides include isolated naturally occurring polyp ⁇ tides, recombinantly produced polyp ⁇ tides, synthetically produced polyp ⁇ tides, or polyp ⁇ tides produced by a combination of these methods. Means for preparing such polyp ⁇ tides are well understood in the art.
  • h-NRD convertase polynucleotides include isolated polynucleotides which encode the h-NRD convertase polyp ⁇ tides and fragments, and polynucleotides closely related thereto. More specifically, h-NRD convertase polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a h-NRD convertase polyp ⁇ tide of SEQ ED NO: 2, and polynucleotides having the particular sequences of SEQ ID NOS: 1 and 3.
  • h-NRD convertase 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 h-NRD convertase polyp ⁇ tide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to that of SEQ ED NO:l over its entire length.
  • polynucleotides at least 90% identical are particularly preferred, and those with at least 95 % are especially preferred.
  • h-NRD convertase 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 h-NRD convertase polynucleotides.
  • h-NRD convertase of the invention is structurally related to other proteins of the NRD convertase family, as shown by the results of sequencing the cDNA encoding human h-NRD convertase.
  • the cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 162 to 3614) encoding a polyp ⁇ tide of 1151 amino acids of SEQ ID NO:2.
  • the amino acid sequence of Table 2 (SEQ ID NO:2) has about 93.6% identity (using BestFit) in 1151 amino acid residues with rat NRD convertase (GENBANK Accession # L27124) (A.R. Herotti et al. , Proc. Natl. Acad. Sci., USA.
  • amino acid sequence of Table 1 (SEQ ID NO: 2) has about 33.4% identity to rat insulin-degrading enzyme (GENBANK Accession ft X67269) over 981 amino acids residues (H. Baumeister et al., FEES Lett. 317:250-254, 1993).
  • the nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 89.4% identity (using BestFit) in 3684 nucleotide residues with rat NRD convertase (GENBANK Accession # L27124) (A.R. Pierotti et al. , Proc. Natl. Acad. Sci., USA. 91:6078-6082, 1994).
  • nucleotide sequence of Table 1 (SEQ ED NO:l) has about 53.3 % identity to drosophila insulin-degrading enzyme (GENBANK Accession ft M58465) over 843 nucleotide base residues (W. Kuo et al., Mol. Endocrinol. 4:1580-1591, 1990).
  • h-NRD convertase polyp ⁇ tides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polyp ⁇ tides and polynucleotides, and their utility is obvious to anyone skilled in the art.
  • a nucleotide sequence of a human h-NRD convertase (SEQ ID NO: 1).
  • One polynucleotide of the present invention encoding h-NRD convertase may be obtained using standard cloning and screening, from a cDNA hbrary derived from mRNA in cells of human primary dendritic cells, osteoblasts, osteoarthritic cartilage, spleen, helper T cells and retinoic acid-treated NTRA2 cells using the expressed sequence tag (EST) analysis (Adams, M.D., et al Science (1991) 252:1651-1656; Adams, M.D. et al , Nature, (1992) 555:632-634; 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 h-NRD convertase polyp ⁇ tide of SEQ ID NO:2 may be identical to the polyp ⁇ tide encoding sequence contained in Table 1 (nucleotide number 162 to 3614 of SEQ ID NO: 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polyp ⁇ tide of SEQ ID NO:2.
  • the polynucleotide may include the coding sequence for the mature polyp ⁇ tide or a fragment thereof, by itself; the coding sequence for the mature polyp ⁇ tide 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 p ⁇ tide portions.
  • a marker sequence which facilitates purification of the fused polyp ⁇ tide can be encoded.
  • the marker sequence is a hexa-histidine p ⁇ tide, as provided in the pQE vector (Qiagen, Inc.) and described 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 transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • polynucleotides encoding h-NRD convertase variants comprising the amino acid sequence of h-NRD convertase polyp ⁇ tide of Table 2 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination.
  • Table 3 SEQ ID NO: 3 encoding the amino acid sequence of Table 4 (SEQ ID NO: 4).
  • a partial nucleotide sequence of a human h-NRD convertase (SEQ ID NO: 3).
  • a partial amino acid sequence of a human h-NRD convertase (SEQ ID NO: 4).
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization 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 invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ED NO: 1 or a fragment thereof (including that of SEQ ID NO: 3), may be used as hybridization probes for cDNA and genomic DNA, to isolate full- length cDNAs and genomic clones encoding h-NRD convertase polyp ⁇ tide 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 similarity to the h-NRD convertase gene.
  • Such hybridization techniques are known to those of skill in the art.
  • these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent.
  • the probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
  • to obtain a polynucleotide encoding h-NRD convertase polyp ⁇ tide, including homologs and orthologs from species other than human comprises the st ⁇ s of screening an appropriate hbrary under stingent hybridization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment thereof (including that of SEQ ED NO: 3), and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • Such hybridization techniques are well known to those of skill in the art.
  • h- NRD convertase polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO: 3). Also included with h-NRD convertase polyp ⁇ tides are polyp ⁇ tide comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition.
  • Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C.
  • 5xSSC 150mM NaCl, 15mM trisodium citrate
  • 50 mM sodium phosphate pH7.6
  • 5x Denhardt's solution 10 % dextran sulfate
  • 20 microgram/ml denatured, sheared salmon sperm DNA followed by washing the filters in 0. lx SSC at about 65°C.
  • polynucleotides and polyp ⁇ tides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
  • the present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polyp ⁇ tides of the invention by recombinant techniques.
  • Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention.
  • Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al. , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
  • R ⁇ resentative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
  • bacterial cells such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells
  • fungal cells such as yeast cells and Aspergillus cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells
  • plant cells include bacterial cells, such as streptococci, sta
  • Such systems include, among others, chromosomal, ⁇ isomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast ⁇ isomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviiuses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • viruses such as baculoviiuses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such
  • the expression systems may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides to produce a polyp ⁇ tide in a host may be used.
  • the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al. , MOLECULAR CLONING, A LABORATORY MANUAL (supra).
  • secretion signals may be incorporated into the desired polyp ⁇ tide. These signals may be endogenous to the polyp ⁇ tide or they may be heterologous signals.
  • the polyp ⁇ tide be produced at the surface of the cell.
  • the cells may be harvested prior to use in the screening assay. If h-NRD convertase polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polyp ⁇ tide is recovered.
  • h-NRD convertase polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polyp ⁇ tide is denatured during isolation and or purification.
  • This invention also relates to the use of h-NRD convertase polynucleotides for use as diagnostic reagents. Detection of a mutated form of h-NRD convertase gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or susc ⁇ tibility to a disease which results from under-expression, over-expression or altered expression of h-NRD convertase. Individuals carrying mutations in the h-NRD convertase gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
  • RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled h-NRD convertase nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobihty of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing.
  • an array of oligonucleotides probes comprising h-NRD convertase nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g. , genetic mutations.
  • Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.
  • the diagnostic assays offer a process for diagnosing or determining a susc ⁇ tibility to chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease through detection of mutation in the h- NRD convertase gene by the methods described.
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease through detection
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease
  • methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of h-NRD convertase polyp ⁇ tide or h-NRD convertase 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 hybridization methods.
  • Assay techniques that can be used to determine levels of a protein, such as an h- NRD convertase polyp ⁇ tide, in a sample derived from a host are well-known to those of skill in the art.
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and EEISA assays.
  • the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, A DS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, which comprises:
  • a h-NRD convertase polynucleotide preferably the nucleotide sequence of SEQ ED NO: 1, or a fragment thereof ;
  • b a nucleotide sequence complementary to that of (a);
  • a h-NRD convertase polypeptide preferably the polyp ⁇ tide of SEQ ID NO: 2, or a fragment thereof ;
  • an antibody to a h-NRD convertase polypeptide, preferably to the polyp ⁇ tide of SEQ ID NO: 2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
  • the nucleotide sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • the mapping of relevant sequences to chromosomes according to the present invention is an important first st ⁇ in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library).
  • genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
  • linkage analysis coinheritance of physically adjacent genes.
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
  • polyp ⁇ tides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the h-NRD convertase polyp ⁇ tides.
  • immunospecific means that the antibodies have substantial! greater affinity for the polyp ⁇ tides of the invention than their affinity for other related polyp ⁇ tides in the prior art.
  • Antibodies generated against the h-NRD convertase polyp ⁇ tides can be obtained by administering the polyp ⁇ tides or ⁇ itope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al. ,
  • Antibodies against h-NRD convertase polyp ⁇ tides may also be employed to treat chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with h-NRD convertase polyp ⁇ tide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
  • h-NRD convertase polyp ⁇ tide, or a fragment thereof adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering h-NRD convertase polyp ⁇ tide via a vector directing expression of h-NRD convertase polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases. Further aspect of the invention relates to an immunological/vaccine formulation
  • composition which, when introduced into a mammalian host, induces an immunological response in that mammal to a h-NRD convertase polyp ⁇ tide wherein the composition comprises a h-NRD convertase polyp ⁇ tide or h-NRD convertase gene.
  • the vaccine formulation may further comprise a suitable carrier. Since h-NRD convertase polyp ⁇ tide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will d ⁇ end on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the h-NRD convertase polyp ⁇ tide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the h-NRD convertase polyp ⁇ tide of the present invention.
  • polyp ⁇ tides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
  • These agonists or antagonists may be natural or modified substrates, ligands, enzymes, rec ⁇ tors, etc. , as the case may be, of the polyp ⁇ tide of the present invention; or may be structural or functional mimetics of the polyp ⁇ tide of the present invention.
  • h-NRD convertase polyp ⁇ tides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate h-NRD convertase polyp ⁇ tide on the one hand and which can inhibit the function of h-NRD convertase polyp ⁇ tide on the other hand.
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs.
  • Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs.
  • screening procedures may involve using appropriate cells which express the h-NRD convertase polyp ⁇ tide or respond to h-NRD convertase polyp ⁇ tide of the present invention.
  • Such cells include cells from mammals, yeast, Drosophila or E. coli.
  • Cells which express the h-NRD convertase polyp ⁇ tide (or cell membrane containing the expressed polyp ⁇ tide) or respond to h-NRD convertase polyp ⁇ tide are then contacted with a test compound 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 h-NRD convertase activity.
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the h-NRD convertase 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 h-NRD convertase polyp ⁇ tide, using detection systems appropriate to the cells bearing the h-NRD convertase 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 st ⁇ s of mixing a candidate compound with a solution containing a h-NRD convertase polyp ⁇ tide to form a mixture, measuring h-NRD convertase activity in the mixture, and comparing the h-NRD convertase activity of the mixture to a standard.
  • the h-NRD convertase 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 h-NRD convertase mRNA and protein in cells.
  • an ELISA may be constructed for measuring secreted or cell associated levels of h-NRD convertase 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 h-NRD convertase (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
  • the h-NRD convertase protein may be used to identify membrane bound or soluble rec ⁇ tors, if any, through standard rec ⁇ tor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the h-NRD convertase is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a p ⁇ tide sequence suitable for detection or purification, and incubated with a source of the putative rec ⁇ tor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
  • a radioactive isotope eg 1251
  • chemically modified eg biotinylated
  • Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy.
  • binding assays can be used to identify agonists and antagonists of h-NRD convertase which compete with the binding of h-NRD convertase to its receptors, if any. Standard methods for conducting screening assays are well understood in the art.
  • Examples of potential h-NRD convertase polyp ⁇ tide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, enzymes, rec ⁇ tors, etc., as the case may be, of the h-NRD convertase polyp ⁇ tide, e.g., a fragment of the ligands, substrates, enzymes, rec ⁇ tors, etc.; or small molecules which bind to the polyp ⁇ tide of the present invention but do not ehcit a response, so that the activity of the polyp ⁇ tide is prevented.
  • the present invention relates to a screening kit for identifying agonists, antagonists, ligands, rec ⁇ tors, substrates, enzymes, etc. for h-NRD convertase polyp ⁇ tides; or compounds which decrease or enhance the production of h- NRD convertase polypeptides, which comprises: (a) a h-NRD convertase polyp ⁇ tide, preferably that of SEQ ED NO:2;
  • kits may comprise a substantial component.
  • This invention provides methods of treating abnormal conditions such as, chronic and acute inflammation, arthritis, osteoarthritis, s ⁇ ticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AEDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymph ⁇ roliferative disorders), atherosclerosis, and Alzheimers disease, related to both an excess of and insufficient amounts of h-NRD convertase polyp ⁇ tide activity.
  • autoimmune diseases eg inflammatory bowel disease, psoriasis
  • transplant rejection graft vs. host disease
  • infection stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis
  • brain injury AEDS, metabohc and other bone diseases (eg osteoporosis)
  • cancer eg lymph ⁇ roliferative disorders
  • atherosclerosis
  • h-NRD convertase polyp ⁇ tide comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically acc ⁇ table carrier in an amount effective to inhibit the fimction of the h-NRD convertase polyp ⁇ tide, such as, for example, by blocking the binding of ligands, substrates, enzymes, rec ⁇ tors, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition.
  • soluble forms of h-NRD convertase polyp ⁇ tides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous h-NRD convertase polyp ⁇ tide may be administered. Typical embodiments of such competitors comprise fragments of the h-NRD convertase polyp ⁇ tide.
  • soluble forms of h-NRD convertase polyp ⁇ tides still capable of binding the ligand in competition with endogenous h-NRD convertase polyp ⁇ tide may be administered.
  • Typical embodiments of such competitors comprise fragments of the h- NRD convertase polyp ⁇ tide.
  • expression of the gene encoding endogenous h-NRD convertase polyp ⁇ tide can be inhibited using expression blocking techniques.
  • Known such techniques involve the use of antisense sequences, either internally generated or s ⁇ arately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988).
  • oligonucleotides which form triple helices with the gene can be supplied.
  • oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
  • h-NRD convertase For treating abnormal conditions related to an under-expression of h-NRD convertase and its activity, several approaches are also available.
  • One approach comprises administering to a subject a therapeutically effective amount of a compound which activates h-NRD convertase polyp ⁇ tide, i.e., an agonist as described above, in combination with a pharmaceutically acc ⁇ table carrier, to thereby alleviate the abnormal condition.
  • gene therapy may be employed to effect the endogenous production of h-NRD convertase by the relevant cells in the subject.
  • a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above.
  • the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polyp ⁇ tide of the present invention 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 engineering cells in vivo and expression of the polyp ⁇ tide in vivo.
  • gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based
  • P ⁇ tides such as the soluble form of h-NRD convertase polyp ⁇ tides, and agonists and antagonist p ⁇ tides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier include 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 invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Polyp ⁇ tides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection.
  • Other injection routes such as subcutaneous, intramuscular, or intraperitoneal, can be used.
  • Alternative means for systemic administration include transmucosal and transdermal administration using 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, in the form of salves, pastes, gels and the like. The dosage range required depends on the choice of p ⁇ tide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner.
  • Suitable dosages are in the range of 0.1-100 ⁇ g/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
  • Polyp ⁇ tides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above.
  • cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polyp ⁇ tide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
  • a polynucleotide such as a DNA or RNA
  • Example 1 An EST encoding the human homologue of rat NRD convertase was identified through a search of a commercial EST database. The full length sequence of this gene shares 89.4 % identity over 3684 nucleotides and 93.3 % identity over 1151 amino acid residues with rat NRD convertase (A.R. Pierotti et al., Proc. Natl. Acad. Sci., USA. 91:6078-6082, 1994). Therefore, this gene was named h-NRD convertase. The clone encoding h-NRD convertase was found in a human primary dendritic cells cDNA hbrary. It was also found in human osteoblasts, osteoarthritic cartilage, spleen, helper T cells and retinoic acid-treated NTRA2 cells cDNA libraries.
  • Tyr Arg Tyr lie Lys Leu Gin Asn Gly Leu Gin Ala Leu Leu lie Ser 115 120 125
  • Glu Val lie Gly Glu Ala Leu Asn Gin Leu Val Pro Gin Lys Ala Asn 595 600 605 Leu Val Leu Leu Ser Gly Ala Asn Glu Gly Lys Cys Asp Leu Lys Glu 610 615 620
  • Lys Lys lie Glu Glu Phe Leu Ser Ser Phe Glu Glu Lys lie Glu Asn
  • 1045 1050 1055 lie Glu Ala Leu Lys Ser Phe Ser Lys Ser Asp Leu Val Asn Trp Phe
  • ACGAATTCCC CCCCCCGGCG AGAGGGAGAC TGGGTTGGGG GAGGGGTTCA GGCCTGTTCC 60 CCGCGGCTGC GGCAGCACCA GGGCCGGCCG CCACCGCCTC TAGAACGCGG AGGAGGTGGG 120
  • Tyr Arg Tyr lie Lys Leu Gin Asn Gly Leu Gin Ala Leu Leu lie Ser 115 120 125

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

h-NRD convertase polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing h-NRD convertase polypeptides and polynucleotides in the design of protocols for the treatment of chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (e.g. inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (e.g. osteoporosis), cancer (e.g. lymphoproliferative disorders), atherosclerosis, and Alzheimer's disease, among others, and diagnostic assays for such conditions.

Description

HUMAN N-ARGININE DIBASIC CONVERTASE
This application claims the benefit of U.S. Provisional Application No. 60/051,793, filed July 1, 1997.
FIELD OF INVENTION This invention relates to newly identified polynucleotides, polypeptides 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 invention relate to NRD convertase family, hereinafter referred to as h-NRD convertase. The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides. BACKGROUND OF THE INVENTION
Many bioactive proteins are synthesized as inactive precursors requiring activation through cleavage between two basic amino acids. One class of endopeptidase that has recently been reported to have this activity is the N-arginine dibasic convertase class (NRDs) [Pierotti, et al., Proc. Natl. Acad. Sci. USA 91:6078-6082, (1994)]. These enzymes cleave between two basic amino acids, N-terminal to an arginine. One subclass of the NRDs is the metalloendopeptidases which have been suggested to activate numerous proteins including somatostatin-28 and dynorphins A and B. The metallo-NRDs are related to protease m from E. coli and to insulinase.
Recently, several membrane type matrix metalloproteinases have been described [Sato, et al. , Nature 370: 61-65(1994); Okada, et al. , Proc. Natl. Acad. Sci. , USA 92: 2730-2734 (1995); Will, et al., Eur. J. Biochem. 231: 602-608 (1995); Takino, et al., J. Biol. Chem. 270: 23013-23020 (1995); Puente, et al., Cancer Res. 56: 944-949 (1996)]. These enzymes may be involved in connective tissue remodeling, indirectly, by activation of other matrix metalloproteinases [Sato, et al., Nature 370: 61-65(1994)] or, directly, through matrix degradation in many pathologies including metastatic cancer [Yamamoto, et. al. , Cancer Res. 56: 384-392, 1996] and osteoarthritis [Buttner, et al., Arthrit. and Rheum. 40 (4): 704-709 91997)]. One of the hallmarks of these enzymes is an insertion between the propeptide and catalytic domain which terminates with the basic putative furin activation site, Arg-X- Arg/Lys-Arg. Furin has been proposed to activate this family of membrane type matrix metalloproteinases as well as other proteins including TGF-b [Dubois, et al. J. Biol. Chem. 270(18): 10618-10624, 1995] and endopeptidase-24.18 [Milhiet, et al., Biochem. J. 309: 683- 688, 1995]. Furin cleaves C-terminal to the Arg-X-Arg/Lys-Arg basic sequence and has been shown to activate an mtMMP-related enzyme, stromelysin-3, at this site [Pel and Weiss, Nature 375: 244-247, 1995]. However, to date, no data has been generated to show that furin is the enzyme responsible for activation of membrane type matix metalloproteinases in vivo. It is possible that furin, or another endopeptidase such as an NRD could be responsible for this activation in vivo. The present invention describes a novel member of the NRD family of endopeptidases. This indicates that the NRD convertase family has an established, proven history as therapeutic targets. Clearly there is a need for identification and characterization of further members of NRD convertase family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease.
SUMMARY OF THE INVENTION
In one aspect, the invention relates to h-NRD convertase polypeptides and recombinant materials and methods for their production. Another aspect of the invention relates to methods for using such h-NRD convertase polypeptides and polynucleotides. Such uses include the treatment of chronic and acute inflammation, arthritis, osteoarthritis, septicemia, autoimmune diseases (eg flammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others. In still another aspect, the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with h-NRD convertase imbalance with the identified compounds. Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate h-NRD convertase activity or levels.
DESCRIPnON OF THE INVENTION
Definitions The following definitions are provided to facilitate understanding of certain terms used frequently herein.
"h-NRD convertase" refers, among others, generally to a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or an allelic variant thereof.
"h-NRD convertase activity or h-NRD convertase polypeptide activity" or "biological activity of the h-NRD convertase or h-NRD convertase polypeptide" refers to the metabolic or physiologic function of said h-NRD convertase including similar activities or improved activities or these activities with decreased undesirable side-effects. Also included are antigenic and immunogenic activities of said h-NRD convertase.
"h-NRD convertase gene" refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 1 or allelic variants thereof and/or their complements.
"Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin 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 original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypφtide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. "Polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double- stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term 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, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.
"Polypφtide" refers to any pφtide or protein comprising two or more amino acids joined to each other by peptide bonds or modified pφtide bonds, i.e., pφtide isosteres. "Polypφtide" refers to both short chains, commonly referred to as peptides, oligopφtides or oligomers, and to longer chains, generally referred to as proteins. Polypφtides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino 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 described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypφtide, including the pφtide backbone, the amino acid side- chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypφtide. Also, a given polypeptide may contain many types of modifications.
Polypφtides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993 and Wold, F. , Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLAΗONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, 1983; Seifter et al. , "Analysis for protein modifications and nonprotein cofactors", Meth Enz mol (1990) 182:626-646 and Rattan et al. , "Protein Synthesis: Posttranslational Modifications and Aging", Ann NY Acad Sci (1992) 663:48-62. "Variant" as the term is used herein, is a polynucleotide or polypφtide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypφtide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypφtide. Generally, differences are limited so that the sequences of the reference polypφtide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypφtide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypφtide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non- naturally occurring variants of polynucleotides and polypφtides may be made by mutagenesis techniques or by direct synthesis. "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. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A.M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING:
INFORMATICS AND GENOME PROJECTS, Smith, D.W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G, Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM 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., SIAM J Applied Math (1988) 48:1073. Methods to determine identity and similarity are codified in computer programs. Preferred 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 , J Molec Biol (1990) 215:403).
As an illustration, by 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 excφt 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. In other words, to obtain 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.
Similarly, by a polypφtide having an amino acid sequence having at least, for example, 95 % "identity" to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypφtide is identical to the reference sequence excφt that the polypφtide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2. In other words, to obtain a polypeptide having an amino acid sequence at least 95 % identical to a reference amino acid sequence, up to 5 % of the amino acid residues in the reference sequence may be deleted or substituted with another amino 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 amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
Polypeptides of the Invention
In one aspect, the present invention relates to h-NRD convertase polypeptides (or h-NRD convertase proteins) . The h-NRD convertase polypeptides include the polypφtide of SEQ ID NOS:2 and 4; as well as polypeptides comprising the amino acid sequence of SEQ ID NO: 2; and polypφtides comprising 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 h- NRD convertase polypφtides are polypφtides having the amino acid sequence which have at least 80% identity to the polypφtide 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 LD NO:2. Furthermore, those with at least 97- 99 % are highly preferred. Preferably h-NRD convertase polypφtide exhibit at least one biological activity of h-NRD convertase. The h-NRD convertase polypφtides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production.
Fragments of the h-NRD convertase polypφtides are also included in the invention. A fragment is a polypφtide having an amino acid sequence that entirely is the same as part, but not all, of the amino acid sequence of the aforementioned h-NRD convertase polypφtides. As with h-NRD convertase polypφtides, fragments may be "free-standing," or comprised within a larger polypφtide of which they form a part or region, most preferably as a single continuous region. Rφresentative examples of polypφtide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of h-NRD convertase polypφtide. In this context "about" includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
Preferred fragments include, for example, truncation polypφtides having the amino acid sequence of h-NRD convertase polypφtides, excφt for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Also preferred are fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha- helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming 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 preferred fragments are biologically active fragments. Biologically active fragments are those that mediate h-NRD convertase activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human. Preferably, all of these polypφtide fragments retain the biological activity of the h- NRD convertase, including antigenic activity. Among the most preferred fragment is that having the amino acid sequence of SEQ ID NO: 4. Variants of the defined sequence and fragments also form part of the present invention. Preferred variants are those that vary from the referents by conservative amino acid substitutions — i.e. , those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and lie; 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, or 1-2 amino acids are substituted, deleted, or added in any combination.
The h-NRD convertase polypφtides of the invention can be prepared in any suitable manner. Such polypφtides include isolated naturally occurring polypφtides, recombinantly produced polypφtides, synthetically produced polypφtides, or polypφtides produced by a combination of these methods. Means for preparing such polypφtides are well understood in the art.
Polynucleotides of the Invention
Another aspect of the invention relates to h-NRD convertase polynucleotides. h-NRD convertase polynucleotides include isolated polynucleotides which encode the h-NRD convertase polypφtides and fragments, and polynucleotides closely related thereto. More specifically, h-NRD convertase polynucleotide of the invention include a polynucleotide comprising the nucleotide sequence contained in SEQ ID NO: 1 encoding a h-NRD convertase polypφtide of SEQ ED NO: 2, and polynucleotides having the particular sequences of SEQ ID NOS: 1 and 3. h-NRD convertase 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 h-NRD convertase polypφtide of SEQ ID NO:2, and a polynucleotide comprising a nucleotide sequence that is at least 80% identical to that of SEQ ED NO:l over its entire length. In this regard, polynucleotides at least 90% identical are particularly preferred, and those with at least 95 % are especially preferred. Furthermore, those with at least 97% are highly preferred and those with at least 98-99 % are most highly preferred, with at least 99 % being the most preferred. Also included under h-NRD convertase 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 h-NRD convertase polynucleotides. h-NRD convertase of the invention is structurally related to other proteins of the NRD convertase family, as shown by the results of sequencing the cDNA encoding human h-NRD convertase. The cDNA sequence of SEQ ID NO: 1 contains an open reading frame (nucleotide number 162 to 3614) encoding a polypφtide of 1151 amino acids of SEQ ID NO:2. The amino acid sequence of Table 2 (SEQ ID NO:2) has about 93.6% identity (using BestFit) in 1151 amino acid residues with rat NRD convertase (GENBANK Accession # L27124) (A.R. Herotti et al. , Proc. Natl. Acad. Sci., USA. 91:6078-6082, 1994). Furthermore, the amino acid sequence of Table 1 (SEQ ID NO: 2) has about 33.4% identity to rat insulin-degrading enzyme (GENBANK Accession ft X67269) over 981 amino acids residues (H. Baumeister et al., FEES Lett. 317:250-254, 1993). The nucleotide sequence of Table 1 (SEQ ID NO: 1) has about 89.4% identity (using BestFit) in 3684 nucleotide residues with rat NRD convertase (GENBANK Accession # L27124) (A.R. Pierotti et al. , Proc. Natl. Acad. Sci., USA. 91:6078-6082, 1994). Furthermore, the nucleotide sequence of Table 1 (SEQ ED NO:l) has about 53.3 % identity to drosophila insulin-degrading enzyme (GENBANK Accession ft M58465) over 843 nucleotide base residues (W. Kuo et al., Mol. Endocrinol. 4:1580-1591, 1990). Thus, h-NRD convertase polypφtides and polynucleotides of the present invention are expected to have, inter alia, similar biological functions/properties to their homologous polypφtides and polynucleotides, and their utility is obvious to anyone skilled in the art.
Table Is
1 ACGAATTCCC CCCCCCGGCG AGAGGGAGAC TGGGTTGGGG GAGGGGTTCA
51 GGCCTGTTCC CCGCGGCTGC GGCAGCACCA GGGCCGGCCG CCACCGCCTC
101 TAGAACGCGG AGGAGGTGGG TCCTGGGAAG CGGGATGTCC ATCGCTCCAG
151 CTTGGTGGTG AATGCTGAGG AGAGTCACTG TTGCTGCAGT CTGTGCCACC
201 CGGAGGAAGT TGTGTGAGGC CGGGCGGGAG CTCGCGGCGC TCTGGGGAAT
251 CGAAACGCGG GGTCGGTGCG AAGACTCTGC TGCTGCCAGA CCCTTTCCTA
301 TTCTGGCCAT GCCTGGAAGG AACAAGGCGA AGTCTACCTG CAGCTGCCCT
351 GACCTGCAGC CCAATGGACA GGATCTGGGC GAGAACAGCC GGGTTGCCCG
401 TCTAGGAGCG GATGAATCTG AGGAAGAGGG ACGGAGGGGG TCTCTCAGTA
451 ATGCTGGGGA CCCTGAGATC GTCAAGTCTC CCAGCGACCC CAAGCAATAC
501 CGATACATCA AATTACAGAA TGGCTTGCAG GCACTTCTGA TTTCAGACCT
551 AAGTAATATG GAAGGTAAAA CAGGAAATAC AACAGATGAT GAAGAAGAAG
601 AGGAGGTGGA GGAAGAAGAA GAagaTGATG ATGAAGATtC TGGAgCTGAA
651 ATAGAAGATG ACGATGAAGA GGGTTTTGAT GATGAAGATG AGTTTGATGA
701 TGAACATGAT GATGATCTTG ATACTGAGGA TAATGAATTG GAAGAATTAG
751 AAGAGAGAGC AGAAGCTAGA AAAAAAACTA CTGAAAAACA GTCTGCAGCG
801 GCTCTTTGTG TTGGAGTTGG GAgTTTCGCT GATCCAGATG ACCTGCCGGG
851 GCTGGCACAC TTTTTGGAGC ACATGGTATT CATGGGTAGT TTGAAaTATC
901 CAGATGAGAA TGGATTTGAT GCCTTCCTGA AGAAGCATGG GGGTAGTGAT
951 AATGCCTCAA CTGATTGTGA ACGCACTGTC TTTCAGTTTG ATGTCCAGAG
1001 GAAGTACTTC AAGGAAGCTC TTGATAGATG GGCGCAGTTC TTCATCCACC
1051 CACTAATGAT CAGAGATGCA ATTGACCGTG AAGTTGAAGC TGTTGATAGT
1101 GAATATCAAC TTGCAAGGCC TTCTGATGCA AACAGAAAGG AAATGTTGTT
1151 TGGAAGCCTT GCTAGACCTG GACATCCTAT GGGAAAATTT TTTTGGGGAA
1201 ATGCTGAGAC GCTCAAGCAT GAGCCAAGAA AGAATAATAT TGATACACAT
1251 GCTAGATTGA GAGAATTCTG GATGCGTTAC TACTCTTCTC ATTACATGAC 1301 TTTAGTGGTT CAATCCAAAG AAACACTGGA TACTTTGGAA AAATGGGTGA
1351 CTGAAATCTT CTCTCAGATA CCAAACAATG GGTTAcCCAG ACCAAACTTT
1401 GGCCATTTAA CGGATCCATT TGACACACcA GCATTTAACA AACTTTATAG
1451 AGTTGTTCCA ATCAGAAAAA TTCATGCTCT GACCATCACA TGGGCACTTC
1501 CTCCTCAACA GCAACATTAC AGGGTGAAGC CACTTCATTA TATATCCTGG
1551 CTGGTTGGAC ATGAAGGCAA AGGCAGCATT CTTTCTTTCC TTAGGAAAAA
1601 ATGCTGGGCT CTTGCACTGT TTGGTGGAAA TGGTGAGACA GGATTTGAGC
1651 AAAATTCTAC TTATTCAGTG TTCAGCATTT CTATTACATT GACTGATGAG 1701 GGTTATGAAC ATTTTTATGA GGTTGCTTAC ACTGTCTTTC AGTATTTAAA
1751 AATGCTGCAG AAGCTAGGCC CAGAAAAAAG AATTTTTGAA GAGATTCGGA
1801 AAATTGAGGA TAATGAATTT CATTACCAAG AACAGACAGA TCCAGTTGAG
1851 TATGTGGAAA ACATGTGTGA GAACATGCAG CTGTACCCAT TGCAGGACAT
1901 TCTCACTGGA GATCAGCTTC TTTTTGAATA CAAGCCAGAA GTCATTGGTG
1951 AAGCCTTGAA TCAGCTAGTT CCTCAAAAAG CAAATCTTGT TTTACTGTCT
2001 GGTGCTaATG AAGGAAAATG TGACCTCAAG GAGAAATGGT TTGGAACTCA
2051 ATATAGTATA GAAGATATTG AAAACTCTTG GGCTGAACTG TGGAATAGTA
2101 ATTTCGAATT AAATCCAGaT CTTCaTCTTC CAGCTGAAAA CAAGTACATA
2151 GCCACGGACT TTACGTTGAA GGCTTTCGAT TGCCCGGAAA CAGAATACCC
2201 AGTTAAAATT GTGAATACTC CACAAGGTTG CCTGTGGTAT AAGAAAGACA
2251 ACAAATTCAA AATCCCCAAA GCATATATAC GTTTCCATCT AATTTCACCG
2301 TTGATACAGA AATCTGCAGC AAATGTGGTC CTCTTTGATA TCTTTGTCAA
2351 TATCCTTACG CATAACCTTG CGGAACCAGC TTATGAAGCA GATGTGGCAC
2401 AGCTGGAGTA TAAACTGGTA GCTGGAGAAC ATGGTTTAAT TATTCGAGTG
2451 AAAGGATTTA ACCACAAACT ACCTCTACTG TTTCAGCTCA TTATTGACTA
2501 CTTAGCTGAG TTCAATTCCA CACCAGCTGT CTTTACAATG ATAACTGAGC
2551 AGTTGAAGAA GACCTACTTT AACATCCTCA TCAAGCCCGA GACTTTGGCC
2601 AAAGATGTAC GGCTTTTAAT CTTGGAATAT GCCCGTTGGT CTATGATTGA
2651 CAAGTACCAG GCTTTGATGG ACGGCCTTTC CCTTGAGTCT CTGCTGAGCT 2701 TCGTCAAAGA ATTCAAATCC CAGCTCTTTG TGGAGGGCCT GGTACAAGGG
2751 AATGTCACAA GCACAGAATC TATGGATTTC CTGAAATATG TTGTTGACAA
2801 ACTAAACTTC AAGCCTCTGG AGCAGGAGAT GCCTGTGCAG TTCCAGGTGG
2851 TAGAGCTGCC CAGTGGCCAC CATCTATGCA AAGTGAAAGC TCTGAACAAG
2901 GGTGATGCCA ACTCTGAAGT CACTGTGTAC TACCAGTCAG GTACCAGGAG
2951 TCTAAGAGAA TATACGCTTA TGGAGCTGCT TGTGATGCAC ATGGAAGAAC
3001 CTTGTTTTGA CTTCCTTCGA ACCAAGCAGA CCCTTGGGTA CCATGTCTAC
3051 CCTACCTGTA GGAACACATC CGGGATTCTA GGATTTTCTG TCACTGTGGG
3101 GACTCAGGCA ACCAAATACA ATTCTGAAGT TGTTGATAAG AAGATAGAAG
3151 AGTTTCTTTC TAGCTTTGAG GAGAAGATTG AGAACCTCAC TGAAGAGGCA
3201 TTCAACACCC AGGTCACAGC TCTCATCAAG CTGAAGGAGT GTGAGGATAC
3251 CCACCTTGGG GAGGAGGTGG ATAGGAACTG GAATGAAGTG GTTACACAGC
3301 AGTACCTCTT TGACCGCCTT GCCCACGAGA TTGAAGCACT GAAGTCATTC
3351 TCAAAATCAG ACCTGGTCAA CTGGTTCAAG GCCCATAGAG GGCCAGGAAG
3401 TAAAATGCTC AGCGTTCATG TTGTTGGATA TGGGAAGTAT GAACTGGAAG
3451 AGGATGGTAC CCCTTCTAGT GAGGATTCAA ATTCTTCTTG TGAAGTGATG
3501 CAGCTGACCT ACCTGCCAAC CTCTCCTCTG CTGGCAGATT GTATCATCCC
3551 CATTACTGAT ATCAGGGCTT TCACAACAAC AATCAACCTT TTCCCCTACC
3601 ATAAAATAGT CAAATAAATA AACTGCAGTC AAAAAAAAAA AAAAAAAAAA
3651 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAA
A nucleotide sequence of a human h-NRD convertase (SEQ ID NO: 1).
Table 2-
1 MLRRVTVAAV CATRRKLCEA GRE AAL GI ETRGRCEDSA AARPFPILAM
51 PGRNKAKSTC SCPDLQPNGQ DLGENSRVAR LGADESEEEG RRGSLSNAGD
101 PEIVKSPSDP KQYRYIKLQN GLQALLISDL SNMEGKTG T TDDEEEEEVE
151 EEEEDDDEDS GAEIEDDDEE GFDDEDEFDD EHDDDLDTED NELΞΞLEERA
201 EARK TTEKQ SAAALCVGVG SFADPDDLPG LAHFLEH VF MGSLKYPDEN
Figure imgf000016_0001
One polynucleotide of the present invention encoding h-NRD convertase may be obtained using standard cloning and screening, from a cDNA hbrary derived from mRNA in cells of human primary dendritic cells, osteoblasts, osteoarthritic cartilage, spleen, helper T cells and retinoic acid-treated NTRA2 cells using the expressed sequence tag (EST) analysis (Adams, M.D., et al Science (1991) 252:1651-1656; Adams, M.D. et al , Nature, (1992) 555:632-634; 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 h-NRD convertase polypφtide of SEQ ID NO:2 may be identical to the polypφtide encoding sequence contained in Table 1 (nucleotide number 162 to 3614 of SEQ ID NO: 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypφtide of SEQ ID NO:2.
When the polynucleotides of the invention are used for the recombinant production of h-NRD convertase polypφtide, the polynucleotide may include the coding sequence for the mature polypφtide or a fragment thereof, by itself; the coding sequence for the mature polypφtide 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 pφtide portions. For example, a marker sequence which facilitates purification of the fused polypφtide can be encoded. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine pφtide, as provided in the pQE vector (Qiagen, Inc.) and described 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 transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA. Further preferred embodiments are polynucleotides encoding h-NRD convertase variants comprising the amino acid sequence of h-NRD convertase polypφtide of Table 2 (SEQ ED NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, in any combination. Among the preferred polynucleotides of the present invention is contained in Table 3 (SEQ ID NO: 3) encoding the amino acid sequence of Table 4 (SEQ ID NO: 4). Table 3£
1 ACGAATTCCC CCCCCCGGCG AGAGGGAGAC TGGGTTGGGG GAGGGGTTCA
51 GGCCTGTTCC CCGCGGCTGC GGCAGCACCA GGGCCGGCCG CCACCGCCTC
101 TAGAACGCGG AGGAGGTGGG TCCTGGGAAG CGGGATGTCC ATCGCTCCAG
151 CTTGGTGGTG AATGCTGAGG AGAGTCACTG TTGCTGCAGT CTGTGCCACC
201 CGGAGGAAGT TGTGTGAGGC CGGGCGGGAG CTCGCGGCGC TCTGGGGAAT
251 CGAAACGCGG GGTCGGTGCG AAGACTCTGC TGCTGCCAGA CCCTTTCCTA
301 TTCTGGCCAT GCCTGGAAGG AACAAGGCGA AGTCTACCTG CAGCTGCCCT
351 GACCTGCAGC CCAATGGACA GGATCTGGGC GAGAACAGCC GGGTTGCCCG
401 TCTAGGAGCG GATGAATCTG AGGAAGAGGG ACGGAGGGGG TCTCTCAGTA
451 ATGCTGGGGA CCCTGAGATC GTCAAGTCTC CCAGCGACCC CAAGCAATAC
501 CGATACATCA AATTACAGAA TGGCTTGCAG GCACTTCTGA TTTCAGACCT
551 AAGTAATATG GAAGGTAAAA CAGGAAATAC AACC
A partial nucleotide sequence of a human h-NRD convertase (SEQ ID NO: 3).
Table 4-
1 MLRRVTVAAV CATRRK CEA GRE AAL GI ETRGRCEDSA AARPFPI A
51 PGRNKAKSTC SCPDLQPNGQ DLGENSRVAR LGADESEEEG RRGSLSNAGD
101 PEIVKSPSDP KQYRYIK QN G QALLISDL SNMEGKTGNT T
A partial amino acid sequence of a human h-NRD convertase (SEQ ID NO: 4).
The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization 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 invention, which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ED NO: 1 or a fragment thereof (including that of SEQ ID NO: 3), may be used as hybridization probes for cDNA and genomic DNA, to isolate full- length cDNAs and genomic clones encoding h-NRD convertase polypφtide 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 similarity to the h-NRD convertase gene. Such hybridization techniques are known to those of skill in 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 comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
In one embodiment, to obtain a polynucleotide encoding h-NRD convertase polypφtide, including homologs and orthologs from species other than human, comprises the stφs of screening an appropriate hbrary under stingent hybridization conditions with a labeled probe having the SEQ ID NO: 1 or a fragment thereof (including that of SEQ ED NO: 3), and isolating full-length cDNA and genomic clones containing said polynucleotide sequence. Such hybridization techniques are well known to those of skill in the art. Thus in another aspect, h- NRD convertase polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having SEQ ED NO: 1 or a fragment thereof (including that of SEQ ED NO: 3). Also included with h-NRD convertase polypφtides are polypφtide comprising amino acid sequence encoded by nucleotide sequence obtained by the above hybridization condition. Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0. lx SSC at about 65°C.
The polynucleotides and polypφtides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease. Vectors, Host Cells, Expression
The present invention also relates to vectors which comprise a polynucleotide or polynucleotides of the present invention, and host cells which are genetically engineered with vectors of the invention and to the production of polypφtides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof for polynucleotides of the present invention. Introduction of polynucleotides into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al. , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al. , MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, canonic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection.
Rφresentative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.
A great variety of expression systems can be used. Such systems include, among others, chromosomal, φisomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast φisomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviiuses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage 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 polypφtide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al. , MOLECULAR CLONING, A LABORATORY MANUAL (supra).
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the desired polypφtide. These signals may be endogenous to the polypφtide or they may be heterologous signals.
If the h-NRD convertase polypφtide is to be expressed for use in screening assays, generally, it is preferred that the polypφtide be produced at the surface of the cell. In this event, the cells may be harvested prior to use in the screening assay. If h-NRD convertase polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed before the polypφtide is recovered. h-NRD convertase polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypφtide is denatured during isolation and or purification.
Diagnostic Assays
This invention also relates to the use of h-NRD convertase polynucleotides for use as diagnostic reagents. Detection of a mutated form of h-NRD convertase gene associated with a dysfunction will provide a diagnostic tool that can add to or define a diagnosis of a disease or suscφtibility to a disease which results from under-expression, over-expression or altered expression of h-NRD convertase. Individuals carrying mutations in the h-NRD convertase gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis. RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled h-NRD convertase nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in electrophoretic mobihty of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al. , Science (1985) 230:1242. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method. See Cotton et al. , Proc Natl Acad Sci USA (1985) 85: 4397-4401. In another embodiment, an array of oligonucleotides probes comprising h-NRD convertase nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of e.g. , genetic mutations. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability. (See for example: M.Chee et al, Science, Vol 274, pp 610-613 (1996)). The diagnostic assays offer a process for diagnosing or determining a suscφtibility to chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease through detection of mutation in the h- NRD convertase gene by the methods described.
In addition, chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of h-NRD convertase polypφtide or h-NRD convertase 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 hybridization methods. Assay techniques that can be used to determine levels of a protein, such as an h- NRD convertase polypφtide, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and EEISA assays.
Thus in another aspect, the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, A DS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, which comprises:
(a) a h-NRD convertase polynucleotide, preferably the nucleotide sequence of SEQ ED NO: 1, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a);
(c) a h-NRD convertase polypeptide, preferably the polypφtide of SEQ ID NO: 2, or a fragment thereof ; or
(d) an antibody to a h-NRD convertase polypeptide, preferably to the polypφtide of SEQ ID NO: 2. It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
Chromosome Assays
The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes according to the present invention is an important first stφ in correlating those sequences with gene associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in 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 (coinheritance of physically adjacent genes). The differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
Antibodies
The polypφtides of the invention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for the h-NRD convertase polypφtides. The term "immunospecific" means that the antibodies have substantial! greater affinity for the polypφtides of the invention than their affinity for other related polypφtides in the prior art.
Antibodies generated against the h-NRD convertase polypφtides can be obtained by administering the polypφtides or φitope-bearing fragments, analogs or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature (1975) 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , Immunology Today (1983) 4:72) and the EBV-hybridoma technique (Cole et al. ,
MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).
Techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to polypφtides of this invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.
The above-described antibodies may be employed to isolate or to identify clones expressing the polypφtide or to purify the polypφtides by affinity chromatography. Antibodies against h-NRD convertase polypφtides may also be employed to treat chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others.
Vaccines
Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with h-NRD convertase polypφtide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease, among others. Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering h-NRD convertase polypφtide via a vector directing expression of h-NRD convertase polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases. Further aspect of the invention relates to an immunological/vaccine formulation
(composition) which, when introduced into a mammalian host, induces an immunological response in that mammal to a h-NRD convertase polypφtide wherein the composition comprises a h-NRD convertase polypφtide or h-NRD convertase gene. The vaccine formulation may further comprise a suitable carrier. Since h-NRD convertase polypφtide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation instonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will dφend on the specific activity of the vaccine and can be readily determined by routine experimentation.
Screening Assays The h-NRD convertase polypφtide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the h-NRD convertase polypφtide of the present invention. Thus, polypφtides of the invention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, recφtors, etc. , as the case may be, of the polypφtide of the present invention; or may be structural or functional mimetics of the polypφtide of the present invention. See Coligan et al. , Current Protocols in Immunology l(2):Chapter 5 (1991). h-NRD convertase polypφtides are responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which stimulate h-NRD convertase polypφtide on the one hand and which can inhibit the function of h-NRD convertase polypφtide on the other hand. In general, agonists are employed for therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AEDS, metabolic and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease. Antagonists may be employed for a variety of therapeutic and prophylactic purposes for such conditions as chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AIDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphoproliferative disorders), atherosclerosis, and Alzheimers disease. In general, such screening procedures may involve using appropriate cells which express the h-NRD convertase polypφtide or respond to h-NRD convertase polypφtide of the present invention. Such cells include cells from mammals, yeast, Drosophila or E. coli. Cells which express the h-NRD convertase polypφtide (or cell membrane containing the expressed polypφtide) or respond to h-NRD convertase polypφtide are then contacted with a test compound 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 h-NRD convertase activity.
The assays may simply test binding of a candidate compound wherein adherence to the cells bearing the h-NRD convertase 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 h-NRD convertase polypφtide, using detection systems appropriate to the cells bearing the h-NRD convertase 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.
Further, the assays may simply comprise the stφs of mixing a candidate compound with a solution containing a h-NRD convertase polypφtide to form a mixture, measuring h-NRD convertase activity in the mixture, and comparing the h-NRD convertase activity of the mixture to a standard. The h-NRD convertase 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 h-NRD convertase mRNA and protein in cells. For example, an ELISA may be constructed for measuring secreted or cell associated levels of h-NRD convertase 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 h-NRD convertase (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
The h-NRD convertase protein may be used to identify membrane bound or soluble recφtors, if any, through standard recφtor binding techniques known in the art. These include, but are not limited to, ligand binding and crosslinking assays in which the h-NRD convertase is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a pφtide sequence suitable for detection or purification, and incubated with a source of the putative recφtor (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 h-NRD convertase which compete with the binding of h-NRD convertase to its receptors, if any. Standard methods for conducting screening assays are well understood in the art.
Examples of potential h-NRD convertase polypφtide antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the ligands, substrates, enzymes, recφtors, etc., as the case may be, of the h-NRD convertase polypφtide, e.g., a fragment of the ligands, substrates, enzymes, recφtors, etc.; or small molecules which bind to the polypφtide of the present invention but do not ehcit a response, so that the activity of the polypφtide is prevented.
Thus in another aspect, the present invention relates to a screening kit for identifying agonists, antagonists, ligands, recφtors, substrates, enzymes, etc. for h-NRD convertase polypφtides; or compounds which decrease or enhance the production of h- NRD convertase polypeptides, which comprises: (a) a h-NRD convertase polypφtide, preferably that of SEQ ED NO:2;
(b) a recombinant cell expressing a h-NRD convertase polypφtide, preferably that of SEQ ID NO:2;
(c) a cell membrane expressing a h-NRD convertase polypφtide; preferably that of SEQ ID NO: 2; or
(d) antibody to a h-NRD convertase polypeptide, preferably that of SEQ ID NO: 2.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component.
Prophylactic and Therapeutic Methods This invention provides methods of treating abnormal conditions such as, chronic and acute inflammation, arthritis, osteoarthritis, sφticemia, autoimmune diseases (eg inflammatory bowel disease, psoriasis), transplant rejection, graft vs. host disease, infection, stroke, ischemia, acute respiratory disease syndrome, renal disorders, restenosis, brain injury, AEDS, metabohc and other bone diseases (eg osteoporosis), cancer (eg lymphφroliferative disorders), atherosclerosis, and Alzheimers disease, related to both an excess of and insufficient amounts of h-NRD convertase polypφtide activity.
If the activity of h-NRD convertase polypφtide is in excess, several approaches are available. One approach comprises administering to a subject an inhibitor compound (antagonist) as hereinabove described along with a pharmaceutically accφtable carrier in an amount effective to inhibit the fimction of the h-NRD convertase polypφtide, such as, for example, by blocking the binding of ligands, substrates, enzymes, recφtors, etc., or by inhibiting a second signal, and thereby alleviating the abnormal condition. In another approach, soluble forms of h-NRD convertase polypφtides still capable of binding the ligand, substrate, enzymes, receptors, etc. in competition with endogenous h-NRD convertase polypφtide may be administered. Typical embodiments of such competitors comprise fragments of the h-NRD convertase polypφtide.
In another approach, soluble forms of h-NRD convertase polypφtides still capable of binding the ligand in competition with endogenous h-NRD convertase polypφtide may be administered. Typical embodiments of such competitors comprise fragments of the h- NRD convertase polypφtide.
In still another approach, expression of the gene encoding endogenous h-NRD convertase polypφtide can be inhibited using expression blocking techniques. Known such techniques involve the use of antisense sequences, either internally generated or sφarately administered. See, for example, O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression. CRC Press, Boca Raton, FL (1988). Alternatively, oligonucleotides which form triple 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 oligomers can be administered per se or the relevant oligomers can be expressed in vivo.
For treating abnormal conditions related to an under-expression of h-NRD convertase and its activity, several approaches are also available. One approach comprises administering to a subject a therapeutically effective amount of a compound which activates h-NRD convertase polypφtide, i.e., an agonist as described above, in combination with a pharmaceutically accφtable carrier, to thereby alleviate the abnormal condition. Alternatively, gene therapy may be employed to effect the endogenous production of h-NRD convertase by the relevant cells in the subject. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypφtide of the present invention 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 engineering cells in vivo and expression of the polypφtide in vivo. For overview of gene therapy, see Chapter 20, Gene Therapy and other Molecular Genetic-based
Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers i ά (1996). Another approach is to administer a therapeutic amount of h-NRD convertase polypφtides in combination with a suitable pharmaceutical carrier. Formulation and Administration
Pφtides, such as the soluble form of h-NRD convertase polypφtides, and agonists and antagonist pφtides or small molecules, may be formulated in combination with a suitable pharmaceutical carrier. Such formulations comprise a therapeutically effective amount of the polypφtide or compound, and a pharmaceutically accφtable carrier or excipient. Such carriers include 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 invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypφtides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents. In addition, if properly formulated in enteric or encapsulated formulations, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like. The dosage range required depends on the choice of pφtide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
Polypφtides used in treatment can also be generated endogenously in the subject, in treatment modalities often referred to as "gene therapy" as described above. Thus, for example, cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypφtide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
Example 1 An EST encoding the human homologue of rat NRD convertase was identified through a search of a commercial EST database. The full length sequence of this gene shares 89.4 % identity over 3684 nucleotides and 93.3 % identity over 1151 amino acid residues with rat NRD convertase (A.R. Pierotti et al., Proc. Natl. Acad. Sci., USA. 91:6078-6082, 1994). Therefore, this gene was named h-NRD convertase. The clone encoding h-NRD convertase was found in a human primary dendritic cells cDNA hbrary. It was also found in human osteoblasts, osteoarthritic cartilage, spleen, helper T cells and retinoic acid-treated NTRA2 cells cDNA libraries.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fiilly set forth.
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: KUMAR, SANJAY
VAN HORN, STEPHANIE LARK, MICHAEL
(ii) TITLE OF THE INVENTION: HUMAN N-ARGININE DIBASIC CONVERTASE
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: RATNER & PRESTIA
(B) STREET: P.O. BOX 980
(C) CITY: VALLEY FORGE (D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19482
(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TO BE ASSIGNED
(B) FILING DATE: 07-NOV-1997
(C) CLASSIFICATION: UNKNOWN (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/051,793
(B) FILING DATE: 07-JUL-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: PRESTIA, PAUL F
(B) REGISTRATION NUMBER: 23,031
(C) REFERENCE/DOCKET NUMBER: GH-7013E
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-407-0700
(B) TELEFAX: 610-407-0701
(C) TELEX: 846169
(2) INFORMATION FOR SEQ ID Nθ:l:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3684 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ACGAATTCCC CCCCCCGGCG AGAGGGAGAC TGGGTTGGGG GAGGGGTTCA GGCCTGTTCC 60
CCGCGGCTGC GGCAGCACCA GGGCCGGCCG CCACCGCCTC TAGAACGCGG AGGAGGTGGG 120
TCCTGGGAAG CGGGATGTCC ATCGCTCCAG CTTGGTGGTG AATGCTGAGG AGAGTCACTG 180 TTGCTGCAGT CTGTGCCACC CGGAGGAAGT TGTGTGAGGC CGGGCGGGAG CTCGCGGCGC 240
TCTGGGGAAT CGAAACGCGG GGTCGGTGCG AAGACTCTGC TGCTGCCAGA CCCTTTCCTA 300
TTCTGGCCAT GCCTGGAAGG AACAAGGCGA AGTCTACCTG CAGCTGCCCT GACCTGCAGC 360
CCAATGGACA GGATCTGGGC GAGAACAGCC GGGTTGCCCG TCTAGGAGCG GATGAATCTG 420
AGGAAGAGGG ACGGAGGGGG TCTCTCAGTA ATGCTGGGGA CCCTGAGATC GTCAAGTCTC 480 CCAGCGACCC CAAGCAATAC CGATACATCA AATTACAGAA TGGCTTGCAG GCACTTCTGA 540
TTTCAGACCT AAGTAATATG GAAGGTAAAA CAGGAAATAC AACAGATGAT GAAGAAGAAG 600
AGGAGGTGGA GGAAGAAGAA GAAGATGATG ATGAAGATTC TGGAGCTGAA ATAGAAGATG 660
ACGATGAAGA GGGTTTTGAT GATGAAGATG AGTTTGATGA TGAACATGAT GATGATCTTG 720
ATACTGAGGA TAATGAATTG GAAGAATTAG AAGAGAGAGC AGAAGCTAGA AAAAAAACTA 780 CTGAAAAACA GTCTGCAGCG GCTCTTTGTG TTGGAGTTGG GAGTTTCGCT GATCCAGATG 840
ACCTGCCGGG GCTGGCACAC TTTTTGGAGC ACATGGTATT CATGGGTAGT TTGAAATATC 900
CAGATGAGAA TGGATTTGAT GCCTTCCTGA AGAAGCATGG GGGTAGTGAT AATGCCTCAA 960
CTGATTGTGA ACGCACTGTC TTTCAGTTTG ATGTCCAGAG GAAGTACTTC AAGGAAGCTC 1020
TTGATAGATG GGCGCAGTTC TTCATCCACC CACTAATGAT CAGAGATGCA ATTGACCGTG 1080 AAGTTGAAGC TGTTGATAGT GAATATCAAC TTGCAAGGCC TTCTGATGCA AACAGAAAGG 1140
AAATGTTGTT TGGAAGCCTT GCTAGACCTG GACATCCTAT GGGAAAATTT TTTTGGGGAA 1200
ATGCTGAGAC GCTCAAGCAT GAGCCAAGAA AGAATAATAT TGATACACAT GCTAGATTGA 1260
GAGAATTCTG GATGCGTTAC TACTCTTCTC ATTACATGAC TTTAGTGGTT CAATCCAAAG 1320
AAACACTGGA TACTTTGGAA AAATGGGTGA CTGAAATCTT CTCTCAGATA CCAAACAATG 1380 GGTTACCCAG ACCAAACTTT GGCCATTTAA CGGATCCATT TGACACACCA GCATTTAACA 1440
AACTTTATAG AGTTGTTCCA ATCAGAAAAA TTCATGCTCT GACCATCACA TGGGCACTTC 1500
CTCCTCAACA GCAACATTAC AGGGTGAAGC CACTTCATTA TATATCCTGG CTGGTTGGAC 1560
ATGAAGGCAA AGGCAGCATT CTTTCTTTCC TTAGGAAAAA ATGCTGGGCT CTTGCACTGT 1620
TTGGTGGAAA TGGTGAGACA GGATTTGAGC AAAATTCTAC TTATTCAGTG TTCAGCATTT 1680 CTATTACATT GACTGATGAG GGTTATGAAC ATTTTTATGA GGTTGCTTAC ACTGTCTTTC 1740
AGTATTTAAA AATGCTGCAG AAGCTAGGCC CAGAAAAAAG AATTTTTGAA GAGATTCGGA 1800
AAATTGAGGA TAATGAATTT CATTACCAAG AACAGACAGA TCCAGTTGAG TATGTGGAAA 1860
ACATGTGTGA GAACATGCAG CTGTACCCAT TGCAGGACAT TCTCACTGGA GATCAGCTTC 1920
TTTTTGAATA CAAGCCAGAA GTCATTGGTG AAGCCTTGAA TCAGCTAGTT CCTCAAAAAG 1980 CAAATCTTGT TTTACTGTCT GGTGCTAATG AAGGAAAATG TGACCTCAAG GAGAAATGGT 2040
TTGGAACTCA ATATAGTATA GAAGATATTG AAAACTCTTG GGCTGAACTG TGGAATAGTA 2100
ATTTCGAATT AAATCCAGAT CTTCATCTTC CAGCTGAAAA CAAGTACATA GCCACGGACT 2160
TTACGTTGAA GGCTTTCGAT TGCCCGGAAA CAGAATACCC AGTTAAAATT GTGAATACTC 2220
CACAAGGTTG CCTGTGGTAT AAGAAAGACA ACAAATTCAA AATCCCCAAA GCATATATAC 2280 GTTTCCATCT AATTTCACCG TTGATACAGA AATCTGCAGC AAATGTGGTC CTCTTTGATA 2340
TCTTTGTCAA TATCCTTACG CATAACCTTG CGGAACCAGC TTATGAAGCA GATGTGGCAC 2400
AGCTGGAGTA TAAACTGGTA GCTGGAGAAC ATGGTTTAAT TATTCGAGTG AAAGGATTTA 2460
ACCACAAACT ACCTCTACTG TTTCAGCTCA TTATTGACTA CTTAGCTGAG TTCAATTCCA 2520
CACCAGCTGT CTTTACAATG ATAACTGAGC AGTTGAAGAA GACCTACTTT AACATCCTCA 2580 TCAAGCCCGA GACTTTGGCC AAAGATGTAC GGCTTTTAAT CTTGGAATAT GCCCGTTGGT 2640
CTATGATTGA CAAGTACCAG GCTTTGATGG ACGGCCTTTC CCTTGAGTCT CTGCTGAGCT 2700
TCGTCAAAGA ATTCAAATCC CAGCTCTTTG TGGAGGGCCT GGTACAAGGG AATGTCACAA 2760
GCACAGAATC TATGGATTTC CTGAAATATG TTGTTGACAA ACTAAACTTC AAGCCTCTGG 2820
AGCAGGAGAT GCCTGTGCAG TTCCAGGTGG TAGAGCTGCC CAGTGGCCAC CATCTATGCA 2880 AAGTGAAAGC TCTGAACAAG GGTGATGCCA ACTCTGAAGT CACTGTGTAC TACCAGTCAG 2940
GTACCAGGAG TCTAAGAGAA TATACGCTTA TGGAGCTGCT TGTGATGCAC ATGGAAGAAC 3000
CTTGTTTTGA CTTCCTTCGA ACCAAGCAGA CCCTTGGGTA CCATGTCTAC CCTACCTGTA 3060
GGAACACATC CGGGATTCTA GGATTTTCTG TCACTGTGGG GACTCAGGCA ACCAAATACA 3120
ATTCTGAAGT TGTTGATAAG AAGATAGAAG AGTTTCTTTC TAGCTTTGAG GAGAAGATTG 3180 AGAACCTCAC TGAAGAGGCA TTCAACACCC AGGTCACAGC TCTCATCAAG CTGAAGGAGT 3240
GTGAGGATAC CCACCTTGGG GAGGAGGTGG ATAGGAACTG GAATGAAGTG GTTACACAGC 3300
AGTACCTCTT TGACCGCCTT GCCCACGAGA TTGAAGCACT GAAGTCATTC TCAAAATCAG 3360
ACCTGGTCAA CTGGTTCAAG GCCCATAGAG GGCCAGGAAG TAAAATGCTC AGCGTTCATG 3420
TTGTTGGATA TGGGAAGTAT GAACTGGAAG AGGATGGTAC CCCTTCTAGT GAGGATTCAA 3480 ATTCTTCTTG TGAAGTGATG CAGCTGACCT ACCTGCCAAC CTCTCCTCTG CTGGCAGATT 3540
GTATCATCCC CATTACTGAT ATCAGGGCTT TCACAACAAC AATCAACCTT TTCCCCTACC 3600 ATAAAATAGT CAAATAAATA AACTGCAGTC AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 3660 AAAAAAAAAA AAAAAAAAAA AAAA 3684
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1151 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Leu Arg Arg Val Thr Val Ala Ala Val Cys Ala Thr Arg Arg Lys 1 5 10 15
Leu Cys Glu Ala Gly Arg Glu Leu Ala Ala Leu Trp Gly lie Glu Thr
20 25 30
Arg Gly Arg Cys Glu Asp Ser Ala Ala Ala Arg Pro Phe Pro lie Leu 35 40 45
Ala Met Pro Gly Arg Asn Lys Ala Lys Ser Thr Cys Ser Cys Pro Asp
50 55 60
Leu Gin Pro Asn Gly Gin Asp Leu Gly Glu Asn Ser Arg Val Ala Arg 65 70 75 80 Leu Gly Ala Asp Glu Ser Glu Glu Glu Gly Arg Arg Gly Ser Leu Ser
85 90 95
Asn Ala Gly Asp Pro Glu lie Val Lys Ser Pro Ser Asp Pro Lys Gin
100 105 110
Tyr Arg Tyr lie Lys Leu Gin Asn Gly Leu Gin Ala Leu Leu lie Ser 115 120 125
Asp Leu Ser Asn Met Glu Gly Lys Thr Gly Asn Thr Thr Asp Asp Glu
130 135 140
Glu Glu Glu Glu Val Glu Glu Glu Glu Glu Asp Asp Asp Glu Asp Ser 145 150 155 160 Gly Ala Glu lie Glu Asp Asp Asp Glu Glu Gly Phe Asp Asp Glu Asp
165 170 175
Glu Phe Asp Asp Glu His Asp Asp Asp Leu Asp Thr Glu Asp Asn Glu
180 185 190
Leu Glu Glu Leu Glu Glu Arg Ala Glu Ala Arg Lys Lys Thr Thr Glu 195 200 205
Lys Gin Ser Ala Ala Ala Leu Cys Val Gly Val Gly Ser Phe Ala Asp
210 215 220
Pro Asp Asp Leu Pro Gly Leu Ala His Phe Leu Glu His Met Val Phe 225 230 235 240 Met Gly Ser Leu Lys Tyr Pro Asp Glu Asn Gly Phe Asp Ala Phe Leu
245 250 255
Lys Lys His Gly Gly Ser Asp Asn Ala Ser Thr Asp Cys Glu Arg Thr
260 265 270
Val Phe Gin Phe Asp Val Gin Arg Lys Tyr Phe Lys Glu Ala Leu Asp 275 280 285
Arg Trp Ala Gin Phe Phe lie His Pro Leu Met lie Arg Asp Ala lie
290 295 300
Asp Arg Glu Val Glu Ala Val Asp Ser Glu Tyr Gin Leu Ala Arg Pro 305 310 315 320 Ser Asp Ala Asn Arg Lys Glu Met Leu Phe Gly Ser Leu Ala Arg Pro
325 330 335
Gly His Pro Met Gly Lys Phe Phe Trp Gly Asn Ala Glu Thr Leu Lys
340 345 350
His Glu Pro Arg Lys Asn Asn lie Asp Thr His Ala Arg Leu Arg Glu 355 360 365
Phe Trp Met Arg Tyr Tyr Ser Ser His Tyr Met Thr Leu Val Val Gin 370 375 380
Ser Lys Glu Thr Leu Asp Thr Leu Glu Lys Trp Val Thr Glu lie Phe 385 390 395 400
Ser Gin lie Pro Asn Asn Gly Leu Pro Arg Pro Asn Phe Gly His Leu 405 410 415
Thr Asp Pro Phe Asp Thr Pro Ala Phe Asn Lys Leu Tyr Arg Val Val
420 425 430
Pro lie Arg Lys lie His Ala Leu Thr lie Thr Trp Ala Leu Pro Pro 435 440 445 Gin Gin Gin His Tyr Arg Val Lys Pro Leu His Tyr lie Ser Trp Leu 450 455 460
Val Gly His Glu Gly Lys Gly Ser lie Leu Ser Phe Leu Arg Lys Lys 465 470 475 480
Cys Trp Ala Leu Ala Leu Phe Gly Gly Asn Gly Glu Thr Gly Phe Glu 485 490 495
Gin Asn Ser Thr Tyr Ser Val Phe Ser lie Ser lie Thr Leu Thr Asp
500 505 510
Glu Gly Tyr Glu His Phe Tyr Glu Val Ala Tyr Thr Val Phe Gin Tyr 515 520 525 Leu Lys Met Leu Gin Lys Leu Gly Pro Glu Lys Arg lie Phe Glu Glu 530 535 540 lie Arg Lys lie Glu Asp Asn Glu Phe His Tyr Gin Glu Gin Thr Asp 545 550 555 560
Pro Val Glu Tyr Val Glu Asn Met Cys Glu Asn Met Gin Leu Tyr Pro 565 570 575
Leu Gin Asp lie Leu Thr Gly Asp Gin Leu Leu Phe Glu Tyr Lys Pro
580 585 590
Glu Val lie Gly Glu Ala Leu Asn Gin Leu Val Pro Gin Lys Ala Asn 595 600 605 Leu Val Leu Leu Ser Gly Ala Asn Glu Gly Lys Cys Asp Leu Lys Glu 610 615 620
Lys Trp Phe Gly Thr Gin Tyr Ser lie Glu Asp lie Glu Asn Ser Trp 625 630 635 640
Ala Glu Leu Trp Asn Ser Asn Phe Glu Leu Asn Pro Asp Leu His Leu 645 650 655
Pro Ala Glu Asn Lys Tyr lie Ala Thr Asp Phe Thr Leu Lys Ala Phe
660 665 670
Asp Cys Pro Glu Thr Glu Tyr Pro Val Lys lie Val Asn Thr Pro Gin 675 680 685 Gly Cys Leu Trp Tyr Lys Lys Asp Asn Lys Phe Lys lie Pro Lys Ala 690 695 700
Tyr lie Arg Phe His Leu lie Ser Pro Leu lie Gin Lys Ser Ala Ala 705 710 715 720
Asn Val Val Leu Phe Asp lie Phe Val Asn lie Leu Thr His Asn Leu 725 730 735
Ala Glu Pro Ala Tyr Glu Ala Asp Val Ala Gin Leu Glu Tyr Lys Leu
740 745 750
Val Ala Gly Glu His Gly Leu lie lie Arg Val Lys Gly Phe Asn His 755 760 765 Lys Leu Pro Leu Leu Phe Gin Leu lie lie Asp Tyr Leu Ala Glu Phe 770 775 780
Asn Ser Thr Pro Ala Val Phe Thr Met lie Thr Glu Gin Leu Lys Lys 785 790 795 800
Thr Tyr Phe Asn lie Leu lie Lys Pro Glu Thr Leu Ala Lys Asp Val 805 810 815
Arg Leu Leu lie Leu Glu Tyr Ala Arg Trp Ser Met lie Asp Lys Tyr
820 825 830
Gin Ala Leu Met Asp Gly Leu Ser Leu Glu Ser Leu Leu Ser Phe Val 835 840 845 Lys Glu Phe Lys Ser Gin Leu Phe Val Glu Gly Leu Val Gin Gly Asn 850 855 860 Val Thr Ser Thr Glu Ser Met Asp Phe Leu Lys Tyr Val Val Asp Lys
865 870 875 880
Leu Asn Phe Lys Pro Leu Glu Gin Glu Met Pro Val Gin Phe Gin Val
885 890 895 Val Glu Leu Pro Ser Gly His His Leu Cys Lys Val Lys Ala Leu Asn
900 905 910
Lys Gly Asp Ala Asn Ser Glu Val Thr Val Tyr Tyr Gin Ser Gly Thr
915 920 925
Arg Ser Leu Arg Glu Tyr Thr Leu Met Glu Leu Leu Val Met His Met 930 935 940
Glu Glu Pro Cys Phe Asp Phe Leu Arg Thr Lys Gin Thr Leu Gly Tyr
945 950 955 960
His Val Tyr Pro Thr Cys Arg Asn Thr Ser Gly lie Leu Gly Phe Ser
965 970 975 Val Thr Val Gly Thr Gin Ala Thr Lys Tyr Asn Ser Glu Val Val Asp
980 985 990
Lys Lys lie Glu Glu Phe Leu Ser Ser Phe Glu Glu Lys lie Glu Asn
995 1000 1005
Leu Thr Glu Glu Ala Phe Asn Thr Gin Val Thr Ala Leu lie Lys Leu 1010 1015 1020
Lys Glu Cys Glu Asp Thr His Leu Gly Glu Glu Val Asp Arg Asn Trp
025 1030 1035 1040
Asn Glu Val Val Thr Gin Gin Tyr Leu Phe Asp Arg Leu Ala His Glu
1045 1050 1055 lie Glu Ala Leu Lys Ser Phe Ser Lys Ser Asp Leu Val Asn Trp Phe
1060 1065 1070
Lys Ala His Arg Gly Pro Gly Ser Lys Met Leu Ser Val His Val Val
1075 1080 1085
Gly Tyr Gly Lys Tyr Glu Leu Glu Glu Asp Gly Thr Pro Ser Ser Glu 1090 1095 1100
Asp Ser Asn Ser Ser Cys Glu Val Met Gin Leu Thr Tyr Leu Pro Thr 105 1110 1115 1120
Ser Pro Leu Leu Ala Asp Cys lie lie Pro lie Thr Asp lie Arg Ala 1125 1130 1135 Phe Thr Thr Thr lie Asn Leu Phe Pro Tyr His Lys lie Val Lys 1140 1145 1150
(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 584 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
ACGAATTCCC CCCCCCGGCG AGAGGGAGAC TGGGTTGGGG GAGGGGTTCA GGCCTGTTCC 60 CCGCGGCTGC GGCAGCACCA GGGCCGGCCG CCACCGCCTC TAGAACGCGG AGGAGGTGGG 120
TCCTGGGAAG CGGGATGTCC ATCGCTCCAG CTTGGTGGTG AATGCTGAGG AGAGTCACTG 180
TTGCTGCAGT CTGTGCCACC CGGAGGAAGT TGTGTGAGGC CGGGCGGGAG CTCGCGGCGC 240
TCTGGGGAAT CGAAACGCGG GGTCGGTGCG AAGACTCTGC TGCTGCCAGA CCCTTTCCTA 300
TTCTGGCCAT GCCTGGAAGG AACAAGGCGA AGTCTACCTG CAGCTGCCCT GACCTGCAGC 360 CCAATGGACA GGATCTGGGC GAGAACAGCC GGGTTGCCCG TCTAGGAGCG GATGAATCTG 420
AGGAAGAGGG ACGGAGGGGG TCTCTCAGTA ATGCTGGGGA CCCTGAGATC GTCAAGTCTC 480
CCAGCGACCC CAAGCAATAC CGATACATCA AATTACAGAA TGGCTTGCAG GCACTTCTGA 540
TTTCAGACCT AAGTAATATG GAAGGTAAAA CAGGAAATAC AACC 584 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Leu Arg Arg Val Thr Val Ala Ala Val Cys Ala Thr Arg Arg Lys 1 5 10 15
Leu Cys Glu Ala Gly Arg Glu Leu Ala Ala Leu Trp Gly lie Glu Thr
20 25 30
Arg Gly Arg Cys Glu Asp Ser Ala Ala Ala Arg Pro Phe Pro lie Leu 35 40 45
Ala Met Pro Gly Arg Asn Lys Ala Lys Ser Thr Cys Ser Cys Pro Asp
50 55 60
Leu Gin Pro Asn Gly Gin Asp Leu Gly Glu Asn Ser Arg Val Ala Arg 65 70 75 80 Leu Gly Ala Asp Glu Ser Glu Glu Glu Gly Arg Arg Gly Ser Leu Ser
85 90 95
Asn Ala Gly Asp Pro Glu lie Val Lys Ser Pro Ser Asp Pro Lys Gin
100 105 110
Tyr Arg Tyr lie Lys Leu Gin Asn Gly Leu Gin Ala Leu Leu lie Ser 115 120 125
Asp Leu Ser Asn Met Glu Gly Lys Thr Gly Asn Thr Thr 130 135 140

Claims

What is claimed is:
1. An isolated polynucleotide comprising a nucleotide sequence that has at least 90% identity over its entire length to a nucleotide sequence encoding the h-NRD convertase polypφtide of SEQ ID NO:2; or a nucleotide sequence complementary to said isolated polynucleotide.
2. The polynucleotide of claim 1 wherein said polynucleotide comprises the nucleotide sequence contained in SEQ ED NO:l encoding the h-NRD convertase polypφtide of SEQ ID NO:2.
3. The polynucleotide of claim 1 wherein said polynucleotide comprises a nucleotide sequence that is at least 90% identical to that of SEQ ID NO: 1 over its entire length.
4. The polynucleotide of claim 3 which is the polynucleotide of SEQ ID NO:
1.
5. The polynucleotide of claim 1 which is DNA or RNA.
6. A DNA or RNA molecule comprising an expression system, wherein said expression system is capable of producing a h-NRD convertase polypφtide comprising an amino acid sequence, which has at least 94% identity with the polypφtide of SEQ ID NO: 2 when said expression system is present in a compatible host cell.
7. A host cell comprising the expression system of claim 6.
8. A process for producing a h-NRD convertase polypφtide comprising culturing a host of claim 7 under conditions sufficient for the production of said polypeptide and recovering the polypφtide from the culture.
9. A process for producing a cell which produces a h-NRD convertase polypeptide thereof comprising transforming or transfecting a host cell with the expression system of claim 6 such that the host cell, under appropriate culture conditions, produces a h-NRD convertase polypeptide.
10. A h-NRD convertase polypeptide comprising an amino acid sequence which is at least 94% identical to the amino acid sequence of SEQ ID NO: 2 over its entire length.
11. The polypφtide of claim 10 which comprises the amino acid sequence of SEQ ID NO:2.
12. An antibody immunospecific for the h-NRD convertase polypφtide of claim 10.
13. A method for the treatment of a subject in need of enhanced activity or expression of h-NRD convertase polypeptide of claim 10 comprising:
(a) administering to the subject a therapeutically effective amount of an agonist to said polypeptide; and/or
(b) providing to the subject an isolated polynucleotide comprising a nucleotide sequence that has at least 90% identity to a nucleotide sequence encoding the h-NRD convertase polypφtide of SEQ ED NO: 2 over its entire length; or a nucleotide sequence complementary to said nucleotide sequence in a form so as to effect production of said polypφtide activity in vivo.
14. A method for the treatment of a subject having need to inhibit activity or expression of h-NRD convertase polypeptide of claim 10 comprising:
(a) administering to the subject a therapeutically effective amount of an antagonist to said polypeptide; and/or
(b) administering to the subject a nucleic acid molecule that inhibits the expression of the nucleotide sequence encoding said polypeptide; and/or
(c) administering to the subject a therapeutically effective amount of a polypφtide that competes with said polypeptide for its ligand, substrate , or recφtor.
15. A process for diagnosing a disease or a susceptibility to a disease in a subject related to expression or activity of h-NRD convertase polypφtide of claim 10 in a subject comprising:
(a) determining the presence or absence of a mutation in the nucleotide sequence encoding said h-NRD convertase polypφtide in the genome of said subject; and/or
(b) analyzing for the presence or amount of the h-NRD convertase polypφtide expression in a sample derived from said subject.
16. A method for identifying compounds which inhibit (antagonize) or agonize the h-NRD convertase polypφtide of claim 10 which comprises:
(a) contacting a candidate compound with cells which express the h-NRD convertase polypφtide (or cell membrane expressing h-NRD convertase polypφtide) or respond to h-NRD convertase polypφtide; and
(b) observing the binding, or stimulation or inhibition of a functional response; or comparing the abihty of the cells (or cell membrane) which were contacted with the candidate compounds with the same cells which were not contacted for h-NRD convertase polypφtide activity.
17. An agonist identified by the method of claim 16.
18. An antagonist identified by the method of claim 16.
19. A recombinant host cell produced by a method of Claim 9 or a membrane thereof expressing a h-NRD convertase polypeptide.
PCT/US1998/014022 1997-07-07 1998-07-07 Human n-arginine dibasic convertase WO1999002664A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US5179397P 1997-07-07 1997-07-07
US60/051,793 1997-07-07
US96590597A 1997-11-07 1997-11-07
US08/965,905 1997-11-07

Publications (1)

Publication Number Publication Date
WO1999002664A1 true WO1999002664A1 (en) 1999-01-21

Family

ID=26729830

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/014022 WO1999002664A1 (en) 1997-07-07 1998-07-07 Human n-arginine dibasic convertase

Country Status (1)

Country Link
WO (1) WO1999002664A1 (en)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DRAOUI M., ET AL.: "EXPRESSION AND RETINOID MODULATION OF N-ARGININE DIBASIC CONVERTASE AND AN AMINOPEPTIDASE-B IN HUMAN NEUROBLASTOMA CELL LINES.", JOURNAL OF NEURO-ONCOLOGY., KLUWER, BOSTON., US, vol. 31., no. 01/02., 1 January 1997 (1997-01-01), US, pages 99 - 106., XP002912137, ISSN: 0167-594X, DOI: 10.1023/A:1005745717231 *
GEORGIOU G.: "OPTIMIZING THE PRODUCTION OF RECOMBINANT PROTEINS IN MICROORGANISMS", HEWLETT-PACKARD JOURNAL., HEWLETT-PACKARD CO. PALO ALTO., US, vol. 34., no. 08., 1 August 1988 (1988-08-01), US, pages 1233 - 1248., XP002912141 *
JOULIE C., ET AL.: "HUMAN AND RAT TESTIS EXPRESS TWO MRNA SPECIES ENCODING VARIANTS OF NRD CONVERTASE, A METALLOENDOPEPTIDASE OF THE INSULINASE FAMILY.", BIOCHEMICAL JOURNAL, PORTLAND PRESS LTD., GB, vol. 327., 1 November 1997 (1997-11-01), GB, pages 773 - 779., XP002912139, ISSN: 0264-6021 *
SAMBROOK J., FRITSCH E. F., MANIATIS T.: "MOLECULAR CLONING. LABORATORY MANUAL.", 1 January 1987, NEW YORK, COLD SPRING HARBOUR PRESS., US, article "CONSTRUCTION AND ANALYSIS OF CDNA LIBRARIES.", pages: 8.01 - 8.86 + 17.01, XP002912138, 016613 *

Similar Documents

Publication Publication Date Title
US6870030B2 (en) Asp2
US6319688B1 (en) Polynucleotide encoding human sodium dependent phosphate transporter (IPT-1)
WO1999021991A1 (en) Bmzf12: a zinc finger gene cloned from bone marrow
EP0848062A2 (en) Aspartic protease ASP1
EP0887414A2 (en) Human serine proteases HGBAB90
US5922546A (en) Human disintegrin metalloprotease KUZ gene
US6365715B1 (en) Human cardiac/brain tolloid-like protein
US5948669A (en) Rat cathepsin K polynucleotide and polypeptide sequence
US20080318835A1 (en) Novel Integrin Ligand ITGL-TSP
US20020103354A1 (en) Splicing variant of human membrane-type matrix metalloproteinase-5 (MT-MMP5-L)
US6214600B1 (en) Membrane-type matrix metalloproteinase-5 gene
EP0874050A2 (en) Integrin ligand ITGL-TSP
EP1002865A1 (en) Adipose specific protein
US5932446A (en) Hmvab41
EP0881294A2 (en) HOEFCC11, a HAS2 splicing variant
WO1999002664A1 (en) Human n-arginine dibasic convertase
EP0894856A1 (en) A human sMAD3 splice variant
EP0887406A2 (en) A member of the FRZB family, franzzled
WO1999022006A1 (en) CBLAFC02: A SUBUNIT OF VACUOLAR H(+)-ATPase
WO1999021988A1 (en) THE HUMAN VESICLE TRAFFICKING PROTEIN SEC22b GENE OF CBFBBA01
WO1999021885A1 (en) A human abc transporter-7 (habc7) gene
EP0892050A2 (en) Human HFIZG53
WO1999021982A1 (en) Human m6b1 gene
WO2001023587A2 (en) Serine protease
WO1999025822A1 (en) The exostosin-4 gene

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1999508832

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase