WO2002033087A2 - Proteins and nucleic acids encoding same - Google Patents

Proteins and nucleic acids encoding same Download PDF

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Publication number
WO2002033087A2
WO2002033087A2 PCT/US2001/032496 US0132496W WO0233087A2 WO 2002033087 A2 WO2002033087 A2 WO 2002033087A2 US 0132496 W US0132496 W US 0132496W WO 0233087 A2 WO0233087 A2 WO 0233087A2
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WIPO (PCT)
Prior art keywords
nucleic acid
polypeptide
amino acid
protein
seq
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Application number
PCT/US2001/032496
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French (fr)
Other versions
WO2002033087A3 (en
Inventor
Shlomit Edinger
Valerie Gerlach
John R. Macdougall
Uriel M. Malyankar
Glennda Smithson
Isabelle Millet
John A. Peyman
David J. Stone
Erik Gunther
Karen Ellerman
Richard A. Shimkets
Muralidhara Padigaru
Xiaojia Guo
Meera Patturajan
Raymond J. Taupier
Catherine E. Burgess
Bryan D. Zerhusen
Ramesh Kekuda
Kimberly A. Spytek
Esha A. Gangolli
Elma R. Fernandes
Linda Gorman
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Curagen Corporation
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Publication date
Priority to AU1663702A priority Critical patent/AU1663702A/en
Priority claimed from US09/981,151 external-priority patent/US20030212256A1/en
Application filed by Curagen Corporation filed Critical Curagen Corporation
Priority to AU2002216637A priority patent/AU2002216637A1/en
Publication of WO2002033087A2 publication Critical patent/WO2002033087A2/en
Publication of WO2002033087A3 publication Critical patent/WO2002033087A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention generally relates to nucleic acids and polypeptides encoded thereby.
  • the invention generally relates to nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleiG acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
  • the invention is based in part upon the discovery of nucleiG acid sequences encoding novel polypeptides.
  • novel nucleic acids and polypeptides are referred to herein as NOVX, or NOVl , NOV2, NOV3, NOV4, NOV5, NOV6, NOV7, NOV8, NOV9, and NOV10 nucleic acids and polypeptides.
  • NOVX nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as ⁇ NOVX" nucleic acid or polypeptide sequences.
  • the invention provides an isolated NOVX nucleic acid molecule encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ DD NOS.l, 3, 5, 1, 9, 11 , 13, 15, 11, 19, 21, 23, 25, and 28.
  • the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof
  • the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS. l , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, and 28.
  • an oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic aGid (e.g., SEQ ID NOS.l , 3, 5, 7, 9, 1 1 , 13, 1 5, 17, 19, 21 , 23, 25, and 28) or a complement o said oligonucleotide.
  • substantially purified NOVX polypeptides SEQ ID NOS;2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29.
  • the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide.
  • the invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier.
  • the therapeutic Gan be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
  • the invention includes a method of detecting the presence of a NOVX polypeptide in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
  • the invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.
  • Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
  • the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g.
  • adrenoleukodystrophy congenital adrenal hyperplasia, leukodystrophies, breast cancer, Small- cell cancer of lung, squamous cell carcinomas, Colorectal cancer, Malignant melanoma, cutaneous, Neuroblastoma, Prostate cancer-brain cancer susceptibility, Alzheimer's disease, epilepsy, Huntington's disease, anxiety, ataxia-telangiectasia, behavioral disorders, multiple sclerosis, muscular dystrophy, myasthenia gravis, neurodegeneration, neuroprotection, Parkinson's disease, pain, stroke, Stroke, Aneurysm, Embolism, autoimmune disease, allergies, addiction, asthma, ARDS, allergy, endometriosis, endocrine dysfunctions, graft versus host disease, graft versus host disease (GVHD), idiopathic thrombocytopenic purpura, immunodeficiencies, IgA nephropathy, lymphaedema, systemiG lup
  • Lymphaedema atherosclerosis, aortic stenosis, atrial septal defect (ASD), atrioventricular (A- V) canal defect, ductus arteriosus, cerebral palsy, cirrhosis, cardiomyopathy, congenital heart defects, hypertension, hemophilia, hypercoagulation, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, bacterial and viral infections, cerebral vascular disease, osteoarthritis, rheumatoid arthritis, Heart block, nonprogressive, Heart blo ⁇ k, progressive, 2, Ventricular fibrillation, idiopathic, entricular tachycardia, idiopathic, Thrombocytopenia, congenital amegakaryocytic, Bypass surgery, Bleeding disorders, Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, hypercoagulation, diabetes, obesity, metabolic disorders su ⁇ h as familial
  • Hype ⁇ rohnemia type II, erythrokeratodermia va ⁇ abilis, palmoplantar keratoderma, diseases and disorders involving inteicellular metabolic and electrical communication, diseases and disordeis involving coordination, proliferation and differentiation, diseases and disorders involving maintainance of tissue homeostasis, growth control, development, and synchronized lesponse of cells to stimuli, Wislcott-Alduch syndrome, cytoskeletal abnormalities, trauma, tissue regeneration (in vitro and in vivo), respiratory disease, gastro-intestinal diseases, muscle, bone, joint and skeletal disorders, hematopoietiG disorders, urinary system disorders, Tissue and organ transplantation, Fibromuscular dysplasia, Hyperparathyroidism, Hypoparathyroidism, Hyperthyroidism and Hypothyroidism, SIDS, Xerostomia, Tonsihtis, Osteoporosis, Ankylosmg spondylitis, Scohosis, Tendinitis, Dental disease and infection, growth
  • the therapeutic can be, e g , a NOVX nudeic acid, a NOVX polypeptide, or a NOVX- specific antibody, or biologically-active derivatives or fragments thereof
  • the compositions of the present invention will have efficacy for treatment of patients suffe ⁇ ng from the diseases and disorders disclosed above and/or other pathologies and disorders of the like
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines They can also be used to screen for potential agonist and antagonist compounds
  • a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof
  • the compositions of the present invention will have efficacy for treatment of patients suffei g fiom the diseases and disorders disclosed above and/or other pathologies and disorders of the like
  • the invention further includes a method for screening for a modulator of disorders or syndiomes including, e g , the diseases and disorders disclosed above and/or other pathologies and disorders of the like
  • the method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said NOVX polypeptide Binding of the lest compound to the NOVX polypeptide indicates the test compound is a modulatoi of activity, or of latency or predisposition to the aforementioned disoiders or syndromes
  • a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly- expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency o the disorder or syndrome.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide, a NOVX nucleic acid, or both, in a subject (e.g., a human subject).
  • the method includes measuring the amount of the NOVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the NOVX polypeptide present in a control sample.
  • An alteration in the level of the NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders o the like.
  • the expression levels o the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
  • the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition.
  • the disorder includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art.
  • the present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their encoded polypeptides. The sequences are collectively referred to herein as “NOVX nucleiG acids” or “NOVX polynucleotides” and the corresponding encoded polypeptides are referred to as “NOVX polypeptides” or “NOVX proteins.” Unless indicated otherwise, “NOVX” is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary ofthe NOVX nucleic acids and their encoded polypeptides.
  • NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts.
  • the various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
  • NOVl is homologous to a Zinc Metalloprotease/ADAM-TS 7-like family of proteins.
  • the NOVl nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; adrenoleukodystrophy , Alzheimer's disease, autoimmune disease, allergies, addiction, anxiety, ataxia-telangiectasia, asthma, ARDS, atherosclerosis, behavioral disorders, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, allergy, cerebral palsy, congenital adrenal hyperplasia, cirrhosis, cardiomyopathy, congenital heart defects, diabetes, diverticular disease, epilepsy, emphysema, endometriosis, endocrine dysfunctions, graft versus host disease, glomerulonephritis, graft versus host disease (GVHD),
  • NOV2 is homologous to the Alpha-2-macroglobulin precursor-like family of proteins.
  • NOV2 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; asthma, allergy and psoriasis, Alzheimer disease, Emphysema, pulmonary disease, immune disorders and Cancer and/or other pathologies and disorders.
  • NOV3 is homologous to a family of Heal Sodium/Bile Acid Cotransporter-like proteins.
  • the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: Primary bile acid malabsorption (PBAM, an idiopathic intestinal disorder), congenital diarrhea, steatorrhea, refractory infantile diarrhea, interruption of the enterohepatic circulation of bile acids, reduced plasma cholesterol levels, croh-n's disease, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Cirrhosis, Transplantation, Hypercalceimia, Ulcers, growth failure and/or other pathologies.
  • PBAM Primary bile acid malabsorption
  • congenital diarrhea congenital diarrhea
  • steatorrhea refractory infantile diarrhea
  • interruption of the enterohepatic circulation of bile acids reduced plasma cholesterol levels
  • croh-n's disease Inflammatory
  • NOV4 is homologous to the Prohibitin-like family of proteins.
  • NOV4 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: breast cancer (In a sporadic breast cancer, Sato et al. (1992) found a missense mutation from valine (GTC) to alanine (GCC) at codon 88 of the PHB gene), and/or other pathologies.
  • GTC valine
  • GCC alanine
  • NOV5 is homologous to the Macrophage Stimulating Protein Precursor-like family of proteins.
  • NOV5 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: Aicardi-Goutieres syndrome 1, Brugada syndrome, Deafness, autosomal recessive 6, Heart block, nonprogressive, Heart block, progressive, 2, Ichthyosiforme erythroderma, congenital, nonbullous, Long QT syndrome-3, Night blindness, congenital stationary, Pituitary ACTH-secreting adenoma, Small-cell cancer of lung, Ventricular fibrillation, idiopathic, entricular tachycardia, idiopathic, HIV infection, susceptibility/resistance to, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, and/or other pathologies/disorders.
  • VHL Von Hippel-L
  • NOV6 is homologous to the Fatty Acid-Binding Protein-like family of proteins.
  • nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Transplantation, Endometriosis, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Crohn's Disease, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis, Von Hippel-Lindau (V
  • NOV7 is homologous to members of the Gap junction beta-5 protein-like family of proteins.
  • the NOV7 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Deafness, autosomal dominant 2, Elliptocytosis-1 , Fucosidosis, Hypophosphatasia (adult, childhood, infantile), Muscle-eye-brain disease, Neuropathy, paraneoplastic sensory, Porphyria cutanea tarda, Porphyria, hepatoerythropoietic, Schwartz- Jampel syndrome, Thrombocytopenia, congenital amegakaryocytic, Charcot-Marie-Tooth neuropathy-2A, Galactose epimerase deficiency, Glucose transport defect, blood-brain barrier, Kostmann neutropenia, Muscular dystrophy, congenital, with early spine rigidity, Myopathy due to succinate dehydrogenase deficiency,
  • NOV8 is homologous to the Metallothionein-like family of proteins.
  • NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Gitelman syndrome, Menkes disease, Wilson's disease, acrodermatitis enteropathica, myelomonocytic leukemia, eosinophil disorders, hepatic disorders such as hepatic copper toxicity, and/or other pathologies/disorders.
  • NOV9 is homologous to the CDC-42 interacting protein 4-like family of proteins.
  • NOV9 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Wiskott-Aldrich syndrome, immunodeficiency, thrombocytopenia, eczema, lymphoid malignancy cytoskeletal abnormalities, impaired monocyte motility, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Fertility, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart
  • NOVl 0 is homologous to the hepsin/plasma transmembrane serine protease/spinesin- like family of proteins.
  • NOV 10 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; cancer, trauma, tissue regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, immunological disease, respiratory disease, gastro-intestinal diseases, reproductive health, neurological and neurodegenerative diseases, bone marrow transplantation, metabolic and endocrine diseases, allergy and inflammation, nephrological disorders, cardiovascular diseases, muscle, bone, joint and skeletal disorders, hematopoietic disorders, urinary system disorders, Tissue and organ transplantation, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis ,
  • Tuberous sclerosis hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy,Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Endocrine dysfunctions, Growth and reproductive disorders, Myasthenia gravis, Leukodystrophies, Pain, Neuroprotection, Systemi ⁇ lupus erythematosus, Autoimmune disease, Emphysema, Scleroderma, ARDS, Pharyngitis, Laryngitis, Asthma, Hearing loss, Tinnitus, Psoriasis, Actinic keratosis, Tuberous sclerosis, Acne, Hair growth, allopecia, pigmentation disorders, endocrine disorders, cystitis, incontinence, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerul
  • the NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function.
  • the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
  • NOVl NOV I includes three novel zinc metal loprotease/ADAM-TS 7-Iilce proteins disclosed below.
  • the disclosed sequences have been named NOVl a, NOVl b, NOV l c, and NOV ld.
  • NOVl a A disclosed NOVl a nucleic acid of 2997 nucleotides (also referred to as SC138213196 _A) encoding a novel Zinc Metalloprotease-like protein is shown in Table 1 A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 10-12 and ending with a TAA codon at nucleotides 2968-2970.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table I A. The start and stop codons are in bold letters.
  • Table 1A NOVla nucleotide sequence (SEQ ID NO:l).
  • the NOV l a nudeic acid sequence, located on chromsome 5 has 250 of 375 bases (66%) identical to a zinc metalloprotease ADAMTS6 (ADAMTS6) mRNA from Homo sapiens (GENBANK.-ID: AF140674).
  • Public nucleotide databases include all GenBank databases and the GeneScq patent database.
  • the "E-value” or "Expect” value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched.
  • the probability that the subject (“Sbjct”) retrieved from the NOVl BLAST analysis, e.g., thioredoxin mRNA from Ovis aries, matched the Query NOVl sequence purely by chance is 9.4e ' .
  • the Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
  • the Expect value is used as a convenient way to create a significance threshold for reporting results.
  • the default value used for blasting is typically set to 0.0001.
  • the Expect value is also used instead of the P value (probability) to report the significance of matches.
  • an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance.
  • An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/, Occasionally, a string of X's or N's will result from a BLAST search.
  • NOVl a polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:l has 986 amino acid residues and is presented in Table IB using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOVla has a signal peptide and is likely to be localized in extraceliularly with a certainty of 0.5469.
  • NOVl a may also be localized to the lysosome (lumen) with acertainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1297 or in the endoplasmic reticulum (membrane) with a certainty of , 1000.
  • the most likely cleavage site for a NOVla peptide is between amino acids 26 and 27, at: VAE-QV. Table IB. Encoded NOVla protein sequence (SEQ ID NO:2).
  • NOVl a is expressed in the lung.
  • NOVlb A disclosed NOVlb nucleic acid of 2433 nucleotides (also referred to as
  • Table 1 C 137043926_EXT_1) encoding a novel zinc metalloprotease-1-like protein is shown in Table 1 C.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TAA codon at nucleotides 2404-2406.
  • a putative untranslated regions upstream from the initiation codon and downstream of the termination codon are underlined in Table IC. The start and stop codons are in bold letters.
  • Table IC NOVlb nucleotide sequence (SEQ ID NO:3).
  • the NOVlb nucleic add sequence, located on chromsome 5 has 101 of 126 bases (80%) identical to agb:GENBANK- ID:HSA400877
  • acc:AJ400877.1 mRNA from Homo sapiens (Homo sapiens ASCL3 gene, CEGPl gene, CI l orfl 4 gene, Cl l orfl 5 gene, Cl l orfl ⁇ gene and CI lorfl 7 gene) (E 2.3e "7 ).
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • NOVlb polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 791 amino acid residues and is presented in Table ID using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOVlb has a signal peptide and is likely to be localized in extracellularlye with a certainty of 0.5469.
  • NOVlb may also be localized to the lysosome (lumen) with acertainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1 144 or in the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • the most likely cleavage site for a NOVlb peptide is between amino acids 23 and 24, at: VAE-QV.
  • Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
  • NOVlb is expressed in at least the following tissues: brain, liver, spleen, uterus, colon, tonsil, lung, germ cells. This information was derived by detennining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Genomic Clone sources, Literature sources, and/or RACE sources.
  • the target sequence identified previously, NOVl a was subjected to the exon linking process to confirm the sequence.
  • PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached.
  • Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species.
  • primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus.
  • a disclosed NOVlc nucleic acid of 2902 nucleotides (also referred to as CG52952-03) encoding a novel ADAM-TS 7-like protein is shown in Table I E.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 182-184 and ending with a TAA codon at nucleotides 2750-2752.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table IE. The start and stop codons are in bold letters.
  • Table I E. NOVlc nucleotide sequence (SEQ ID NO:5).
  • the NOVl c nucleic acid sequence, located on chromsome 5 has 646 of 1089 bases (59%) identical to a gb:GENBAN - !D:AFl40675
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • NOVl c polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 856 amino acid residues and is presented in Table I F using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOV l c has no signal peptide and is likely to be localized in the nucleus with a certainty of 0.7000.
  • NOVl c may also be localized to the microbody (peroxisome) with a certainty of 0.3813, the mitochondrial matrix space with a certainty of 0, 1000, or in the lysosome (lumen) with a certainty of 0.1000.
  • Table I F Encoded NOVlc protein sequence (SEQ ID NO:6).
  • NOVlc amino acid sequence has 256 of 579 amino acid residues (44%) identical to, and 352 of 579 amino acid residues (60%) similar to, the 997 amino acid residue ptnr;SWISSNEW-ACC:Q9UKP4 protein from Homo sapiens (Human) (ADAM-TS 7 precursor (EC 3.4.24.-) (A Disintegrin And
  • ADAMTS-7 Metalloproteinase With Thrombospondin Motifs 7
  • ADAM-TS7 Metalloproteinase With Thrombospondin Motifs 7
  • E 1.9 ⁇ " 1 ).
  • Public amino acid databases include the GenBank databases, SwissProt, PDB and PER..
  • NOVl c is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus.
  • Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of NOVlc.
  • a disclosed NOVl d nucleic acid of 2895 nucleotides (also referred to as CG52952-04) encoding a novel ADAM-TS 7-like protein is shown in Table I G.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 10-12 and ending with a TAA codon at nucleotides 2866-2868.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table I G. The start and stop codons are in bold letters.
  • Table IG NOVl d nucleotide sequence (SEQ ID NO:7).
  • the NOVl d nucleic acid sequence, located on chromsome 5 has 380 of 614 bases (61%) identical to a gb:GENBANK- ID:AF 140675(acc:AF140675.1 mRNA from Homo sapiens (Homo sapiens zinc metalloprotease ADAMTS7 (ADAMTS7) mRNA, complete eds) (E - 7.0e "16 ).
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • NOV l d polypeptide (SEQ ID NO'8) encoded by SEQ ID NO:7 has 952 amino acid residues and is presented in Table 1 H using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOVl d has a signal peptide and is likely to be localized extracellualrly with a certainty of 0.3700.
  • NOVl d may also be localized to the lysosome (lumen) with a certainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1270, or in the endoplasmic reticulum (membrane) with a certainty of Ori OOO.
  • the most likely cleavage site for a NOVl a peptide is between amino acids 23 and 24, at: VAE-QV.
  • Table I H Encoded NOVld protein sequence (SEQ ID NO:8).
  • Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
  • NOVld is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus.
  • Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOVld and the expression pattern of (GENBANK-ID: gb:GENBAN -ID:AF140675
  • the proteins encoded by the NOVl a, lb, lc, and Id nucleotides are very closely homologous as is shown in the alignment in Table 1 1.
  • TaqMan data for NOVl can be found below in Example 2.
  • SNP data for NOVl a can be found below in Example 3.
  • NOVl Homologies to any ofthe above NOVl proteins will be shared by the other two NOVl proteins insofar as they are homologous to each other as shown above. Any reference to NOVl is assumed to refer to all three of the NOVl proteins in general, unless otherwise noted.
  • the disclosed NOV l a polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table U.
  • Novel NOVla SEQ ID NO : 2
  • Novel NOVlb SEQ ID NO : 4
  • Novel NOVlc ⁇ SEQ ID NO . 6
  • Novel NOVld SEQ ID NO ; 8
  • RNLRPFHCQFLAGIPPPLSM ⁇ CNPEPCEAWQVEPWSQCSR go. I 11493589 I - PPTTQQCEA g-L j 10645199 I WLQLPA g ⁇ jl53099 3 l
  • Tables 1 L-l O lists the domain description from DOMAIN analysis results against NOVl . This indicates that the NOVl sequence has properties similar to those of other proteins known to contain this domain.
  • the members of this family are enzymes that cleave peptides. These proteases require zinc for catalysis. Members of this family are also known as adamalysins . Most members of this family are snake venom endopeptidases, but there are also some mammalian proteins, and fertilin. Fertilin and closely related proteins appear to not have some active site residues and may not be active enzymes. (SEQ ID NO:76)
  • CD-Length 48 residues, 100.0% aligned
  • Thrombospondin- 1 associates with the extracellular matrix and inhibits angiogenesis in vivo.
  • THBSl blocks capillary-like tube formation and endothelial cell proliferation.
  • the antiangiogenic activity is mediated by a region that contains 3 type 1 (properdin or thrombospondin) repeats.
  • ADAMTS8 is a secreted protein that has an N- tc ⁇ ninal signal peptide, a zinc metalloprotease domain containing a zinc-binding site, and a cysteine-rich region containing 2 putative disintegrin loops.
  • the C terminus of ADAMTS8 has 2 heparin-binding thrombospondin repeats with 6 cys and 3 tip residues.
  • Southern blot analysis showed that ADAMTS8 is a single-copy gene distinct from that encoding ADAMTS 1.
  • ADAMTS8 is expressed as a 98-kD protein, a 79-kD protein after cleavage at the subtihsin site, or as a 64-kD protein, which is most abundant, generated by an additional processing event.
  • Functional analysis determined that ADAMTS 8 disrupts angiogenesis in vivo and in vitro more efficiently than THBSl or endostatin but somewhat less efficiently than ADAMTSl .
  • the novel protein described here contains thrombospondin type I domains and Reprolysin domain. It is homologous to AD AM genes. Thrombospondin type I domain [1PR000884; (TSP 1)] was found in the thrombospondin protein where it is repeated 3 times. Now a number of proteins involved in the complement pathway (properdin, C6, C7, C8A, C8B, C9) as well as extracellular matrix protein like indin, F-spondin, SCO-spondin and even the circumsporozoite surface protein 2 and TRAP proteins of Plasmodium contain one or more instance of this repeat. It has been involved in cell-cell interraction, inhibition of angiogenesis, apoptosis. The intron-exon organisation of the properdin gene confirms the hypothesis that the repeat might have evolved by a process involving exon shuffling. A study of properdin structure provides some information about the structure of the thrombospondin type I repeat.
  • Reprolysin family propeptide [IPR002870; (Pep_M12B_propep)] domain is contained in the propeptide for members of peptidase family M12B.
  • the propeptide contains a sequence motif similar to the 'cysteine switch 1 o the matrixins. This motif is found at the C terminus of the alignment but is not well aligned.
  • the mouse homologue of member o the ADAM (a disintegrin and metalloprotease) family of proteins was identified. This rapidly growing gene family encodes cell surface proteins that possess putative adhesion and protease domains.
  • Northern blot analyses demonstrated that the mouse ADAM gene, te ⁇ ned ADAM7, is expressed in the caput region of the epididymis and in the anterior pituitary gonadotropes with no detectable expression in the twenty-six other tissues examined.
  • ADAM7 messenger RNA exhibited an apical localization within the proximal caput epididymal epithelium that may correlate with an unusual sparsely granulated endoplasmic reticulum uniquely present in the proximal region of the epididymidis and to which no known function has been ascribed.
  • Hormonal, surgical, and genetiG strategies demonstrated that ADAM7 gene expression requires, in a region-dependent manner, androgens as well as testicular factors for expression.
  • ADAM7 mRNA maps to the central region of mouse
  • Chromosome 14 approximately 4-5 cM distal from the fertilin beta locus, whi ⁇ h encodes another reproductive-specific ADAM protein (1).
  • the disclosed NOVl nucleic acid ofthe invention encoding a ADAM-TS 7 -like protein includes the nucleic acid whose sequence is provided in Table 1A, IC, IE, and IG or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 1A, I C, IE and IG while still encoding a protein that maintains its A AM-TS 7-like activities and physiological functions, or a fragment of such a nucleic acid,
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
  • the disclosed NOVl protein of the invention includes the ADAM-TS 7-like protein whose sequence is provided in Table I B, ID, IF, or I H.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table I B, I D, I F, or I H while still encoding a protein that maintains its ADAM-TS 7 -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 62% percent of the residues may be so changed.
  • the invention further encompasses antibodies and antibody fragments, such as F a b or (Fa b ) 2 , that bind immunospecifically to any of the proteins ofthe invention.
  • NOVl ADAM-TS 7-like protein
  • ADAM-TS 7 family ADAM-TS 7 family
  • the NOV l nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • NOVl nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to various pathologies and disorders as indicated below.
  • a cDNA encoding the ADAM-TS 7-like protein (NOVl) may be useful in gene therapy, and the ADAM-TS 7 -like protein (NOVl) may be useful when administered to a subject in need thereof.
  • compositions o the present invention will have efficacy for treatment of patients suffering from adrenoleukodystrophy , Alzheimer's disease, autoimmune disease, allergies, addiction, anxiety, ataxia-telangiectasia, asthma, ARDS, atherosclerosis, behavioral disorders, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, allergy, cerebral palsy, congenital adrenal hyperplasia, cirrhosis, cardiomyopathy, congenital heart defects, diabetes, diverticular disease, epilepsy, emphysema, endometriosis, endocrine dysfunctions, graft versus host disease, glomerulonephritis, graft versus host disease (GVHD), growth and reproductive disorders, hemophilia, hypercoagulation, hypercalceimia, Huntington's disease, hypertension, hypogonadism, fertility, idi
  • NOV lc protein has multiple hydrophilic regions, each of which can be used as an immunogen
  • a contemplated NOVl c epitope is from about ammo acids 1 to 50
  • a NOVlc epitope is from about amino acids 80 to 100
  • NOVl c epitopes are from about amino acids 1 10 to 260, from about ammo acids 280 to 320, from about amino acid 350 to 400, from about ammo acid 420 to 470, from about amino acid 480 to 620, from about ammo acid 700 to 750, and from about amino acids 770 to 810
  • the disclosed NOV l d protein has multiple hydrophilic regions, each of w hich can be used as an immunogen
  • a contemplated NOVld epitope is from about amino acids 20 to 180
  • a NOVl d epitope is from about amino acids 190 to 280.
  • NOVld epitopes sre from about amino acids 300 to 360, from about amino acids 400 to 530, from about amino acid 540 to 550, from about ammo acid 580 to 610, from about amino acid 630 to 680, and from about amino acids 710 to 750.
  • novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed NOV2 nucleic acid of 4488 nucleotides (also referred to as SC_78316254_A) encoding a novel alpha-2-macroglobulin precursor-like protein is shown in Table 2A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1 -3 and ending with a TGA codon at nucleotides 4477-4479.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters.
  • Table 2A NOV2 nucleotide sequence (SEQ ID NO:9).
  • a NOV2 polypeptide (SEQ ID NO: 10) encoded by SEQ ID NO:9 has 1492 amino acid residues and is presented using the one-letter code in Table 2B. Signal P, Psort and/or Hydropathy results predict that NOV2 does contain a signal peptide and is likely to be localized extraceliularly with a certainty of 0.3703. In other embodiments, NOV2 may also be localized to the lysosome (lumen) with a certainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1585, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The most likely cleavage site for a NOV2 peptide is between amino acids 17 and 18, at: AlA- EE.
  • NOV2 is expressed in at least the following tissues: Hematopoietic tissues, blood plasma, fetal lung, and the coronary artery.
  • NOV2 also has homology to the ammo acid sequences shown in the BLASTP data listed in Table 2C.
  • Tables 2E-F list the domain description from DOMAIN analysis results against NOV2.
  • NOV2 sequence has properties similar to those of other proteins known to contain this domain.
  • A2M-binding alpha-macroglobulins [1] are large glycoproteins found in the plasma of vertebrates, in the hemolymph of some invertebrates and in reptilian and avian egg white. A2M-like proteins are able to inhibit all four classes of proteinases by a 'trapping' mechanism. They have a peptide stretch, called the 'bait region', which contains specific cleavage sites for different proteinases. When a proteinase cleaves the bait region, a conformational change is induced in the protein, thus trapping the proteinase.
  • the entrapped enzyme remains active against low molecular weight substrates, whilst its activity toward larger substrates is greatly reduced, due to steric hindrance.
  • a thiol ester bond formed between the side chains of a cysteine and a glutamine, is cleaved and mediates the covalent binding ofthe A2M-like protein to the proteinase.
  • Alpha2 -Macroglobulin is a proteinase inhibitor found in association with senile plaques (SP) in Alzheimer's disease (AD).
  • A2M has been implicated biochemically in binding and degradation of the amyloid beta (Abeta) protein which accumulates in SP.
  • VallOOO GTCyilelOOO (ATC)
  • ATC amyloid beta
  • the G/G genotype is over-represented in Alzheimer's disease in an additional independent data set: a group of 359 controls and 566 Alzheimer's disease patients.
  • the G/G genotype increased from 0.07 in controls to 0.12 in Alzheimer's disease (P ⁇ 0.05, Fisher's exact test).
  • the odds ratio for Alzheimer's disease associated with the G/G genotype was 1.77 (1.16-2.70, P ⁇ 0.01) and in combination with APOE4 was 9.68 (95% CI 3.91 -24.0, P ⁇ 0 001).
  • the presence of the G allele was associated with an increase in Abeta burden in a small series.
  • the A2M receptor is a multifunctional receptor whose ligands include apolipoprotein E and the amyloid precursor protein. These four proteins have each been genetically linked to Alzheimer's disease, suggesting that they may participate in a common disease pathway.
  • Six alpha 2-macroglobuli ⁇ cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation.
  • the coding sequence for rat alpha 2- macroglobulin including its 27-residue signal peptide and the 3' - and part of he 5' nontranslated regions were detemiined.
  • the mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA.
  • rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 1 8 h. By 24 h the levels had decreased to less than 30% o the maximum value.
  • alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein.
  • 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) BMBO I. 7,4119-4127).
  • alpha 2M receptor-associated protein The primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning.
  • the deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein.
  • HBP-44 mouse heparin-binding protein
  • alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain.
  • Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.
  • Alpha-2-macroglobulin is a serum pan-protease inhibitor.
  • a possible correlation to this gene is the following reference on Rhinovirus infections causing exacerbations of eosinophilic airway disease.
  • the acute effects of allergen-challenge on nasal interleukin-8 (IL- 8), eosinophil cationic protein (ECP), and alpha2-macroglobulin were examined in atopic subjects with common cold symptoms. Twenty-three patients with seasonal allergic rhinitis were inoculated with human rhinovirus 16 outside the pollen season. Diluent and allergen challenges, followed by nasal lavages, were carried out about 3 months before and 4 days after virus inoculation.
  • Alpha-2-macroglobulin has been implicated in Alzheimer disease (AD) based on its ability to mediate the clearance and degradation of A-beta, the major component of amyloid beta deposits.
  • AD Alzheimer disease
  • Blacker et al. (1998) analyzed a deletion in the A2M gene at the 5-prime splice site of 'exon II' o the bait region (exon 18) and found that inheritance of the deletion, designated A2M-2, conferred increased risk for AD.
  • the disclosed NOV2 nucleic acid of the invention encoding a Alpha-2-macroglobulin precursor-like protein includes the nucleic acid whose sequence is provided in Table 2A or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 2A while still encoding a protein that maintains its Alpha-2-macroglobulin precursor -like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability o the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 40% percent of the bases may be so ⁇ hanged.
  • the disclosed NOV2 protein of the invention includes the Alpha-2-macroglobulin precursor -like protein whose sequence is provided in Table 2B.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2B while still encoding a protein that maintains its Alpha-2- macroglobulin precursor -like activities and physiological functions, or a functional fragment thereof.
  • a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2B while still encoding a protein that maintains its Alpha-2- macroglobulin precursor -like activities and physiological functions, or a functional fragment thereof.
  • up to about 62% percent of the residues may be so changed.
  • the NOV2 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in asthma, allergy and psoriasis, Alzheimer disease, Emphysema, pulmonary disease, immune disorders and Cancer and/or other pathologies and disorders.
  • the NOV2 nucleic acid encoding Alpha-2-macroglobulin precursor-like protein, and the Alpha-2-macroglobulin precursor-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
  • NOV2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV2 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV2 epitope is from about amino acids 80 to 100.
  • a NOV2 epitope is from about amino acids 110 to 200.
  • NOV2 epitopes are from about amino acids 290 to 340, from about 380 to 400, from about amino acids 410 to 580, from about amino acids 620 to 730, from about amino acids 810 to 900, from about amino acids 990 to 1 100, from about ammo acids 1 190 to 1210, from about amino acids 1240 to 1320, and from about amino acids 1400 to 1450.
  • novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed NOV3 nucleic acid of 987 nucleotides (also referred to as GMAC079237_A) encoding a novel Heal Sodium/Bile Acid Cotransporter-hke protein is shown in Table 3 A.
  • An open reading frame was identified beginning with a ATG initiation codon at nucleotides 1 -3 and ending with a TGA codon at nucleotides 979-981. The start and stop codons are in bold letters, and the 3' untranslated region is underlined.
  • a disclosed NOV3 protein (SEQ ID NO: 12) encoded by SEQ ID NO: 1 1 has 326 amino acid residues, and is presented using the one-letter code in Table 3B.
  • Signal P, Psort and/or Hydropathy results predict that NOV3 does not have a signal peptide, and is likely to be localized to the plasma membrane with a certainty of 0.6000.
  • NOV3 is also likely to be localized to Golgi body with a certainty of 0.4000, to the mitochondrial inner membrane with a certainty of 0.3815, or to the endoplasmic reticulum (membrane) with a certainty of 0.3000.
  • NOV3 protein sequence (SEQ ID NO:12).
  • the disclosed NOV3 amino acid has 129 of 331 amino acid residues (38%) identical to, and 198 of 331 amino acid residues (59%) similar to, the 348 amino acid residue ileal sodium/bile acid cotransporter protein from Cricetul
  • NOV3 sequence is predicted to be expressed in ileum because of the expression pattern of (GENBANK-ID: NTCI_HUMAN) a closely related ILEAL SODIUM/BILE ACID COTRANSPORTER homolog in species Homo sapien.
  • TaqMan data for NOV3 can be found below in Example 2.
  • NOV3 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 3E.
  • Table 3G list the domain description from DOMAl-N analysis results against NOV3. This indicates that the NOV3 sequence has properties similar to those of other proteins known to contain this domain.
  • Bile acids are synthesized from cholesterol in the liver and secreted into the small intestine, where they facilitate absorption of fat-soluble vitamins and cholesterol. Wong et al. (1996) noted that, rather than being excreted, the majority of bile acids are reabsorbed from the intestine and returned to the liver via the portal circulation. In the liver, bile acids are quantitatively extracted and resecreted into bile, thereby eliminating the need for substantial de novo hepatic bile acid synthesis.
  • the ileum is the major site of active uptake of bile acids from Ihe intestine, where the sodium-gradient-driven transporter has been identified in the ileal enterocyte.
  • Mutations in the transporter or other ileal genes that participate in the transepithelial transport of bile acids are predicted to affect bile acid and cholesterol metabolism significantly.
  • Heubi et l. (1982) described an apparent familial defect in active ileal bile acid transport.
  • cloned a cDNA encoding an ileal sodium/bile acid cotransporter gene (designated ISBT by them). They also isolated a genomic clone for human ISBT.
  • the gene encodes a 348-amino acid polypeptide with 7 predicted transmembrane domains and a predicted molecular mass of 38 kD.
  • the native human protein has a relative molecular mass of 40 kD on SDS gel electrophoresis due to N- linked glycosylation.
  • Wong et al. (1 95) demonstrated a dysfunctional mutation (P290S) in the ileal sodium-bile acid cotransporter gene in the course of cloning the human cDNA.
  • PBAM Primary bile acid malabsorption
  • the L243P, T262M, and double mutant did not affect transporter protein expression or trafficking to the plasma membrane; however, transport of taurocholate and other bile acids was abolished.
  • the A171S mutation had no effect on taurocholate uptake.
  • the dysfunctional mutations were not detected in 104 unaffected control subjects, whereas the A171S was present in 28% of that population.
  • EHC enterohepatic circulation
  • the polar ileal enterocytes and the hepatocytes each have 3 unique molecules: a receptor that binds bile salts on one surface and translocates them into the cell, a cellular bile salt binding protein that moves them across the cell, and an exit molecule that moves bile salts out of the other side ofthe cell.
  • ileal sodium/bile salt transporter ISBT
  • I BP ileal lipid-binding protein
  • ILBP allows the bile salts to move through the cytoplasm to the basolateral membrane o the ileal intestinal epithelial cell, where a sodium-independent organic ion exchange system secretes bile salts into portal capillaries.
  • Bile salts in portal capillaries bind to albumin and flow to the liver. There they are recognized by a transporter with high homology to ISBT, the sodium taurocholate cotransporting polypeptide (SLClOAl). Small et al.
  • the disclosed NOV3 nucleic acid of the invention encoding a Ileal Sodium/Bile Acid Cotransporter-like protein includes the nucleic acid whose sequence is provided in Table 3A or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 3A while still encoding a protein that maintains its Ileal Sodium/Bile Acid Cotransporter-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
  • the disclosed NOV3 protein ofthe invention includes the Ileal Sodium/Bile Acid
  • Cotransporter-like protein whose sequence is provided in Table 3B or 3D.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 3B or 3D while still encoding a protein that maintains its Ileal Sodium/Bile Acid Cotransporter-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 61% percent of the residues may be so changed.
  • NOV3 Heal Sodium/Bile Acid Cotransporter-like protein and nucleic acid
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount o the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • compositions of the present invention will have efficacy for treatment of patients suffering from Primary bile acid malabsorption (PBAM, an idiopathic intestinal disorder), congenital diarrhea, steatorrhea, refractory infantile diarrhea, interruption of the enterohepatic circulation of bile acids, reduced plasma cholesterol levels, crohn's disease, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Cirrhosis, Transplantation,
  • PBAM Primary bile acid malabsorption
  • congenital diarrhea steatorrhea
  • refractory infantile diarrhea interruption of the enterohepatic circulation of bile acids
  • reduced plasma cholesterol levels crohn's disease
  • Inflammatory bowel disease Diverticular disease
  • Hirschsprung's disease Hirschsprung's disease
  • Cirrhosis Transplantation
  • NOV3 nucleic acid, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic cid or the protein are to be assessed.
  • NOV3 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV3 protein have multiple hydrophilic regions, each of which can be used as an immunogen.
  • contemplated NOV3 epitope is from about amino acids 5 to 30.
  • a NOV3 epitope is from about amino acids 55 to 60.
  • NOV3 epitopes are from about amino acids 140 to 150, from about amino acids 180 to 190, and from about amino acids 280 to 330.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • NOV4 nucleic acid of 850 nucleotides (designated CuraGen Ace. No. AL161453_A) encoding a novel Prohibitin -like protein is shown in Table 4A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 6-8 and ending with a TGA codon at nucleotides 822-824.
  • a putative untranslated regions upstream of the initiation codon and downstream from the termination codon are underlined in Table 4A, and the start and stop codons are in bold letters.
  • Table 4A NOV4 Nucleotide Sequence (SEQ ID NO:13)
  • a NOV4 polypeptide (SEQ ID NO: 14) encoded by SEQ ID NO: 13 is 272 amino acid residues and is presented using the one letter code in Table 4B.
  • Signal P, Psort and/or Hydropathy results predict that NOV4 has a signal peptide and is likely to be localized at the endoplasmic reticulum (membrane) with a certainty of 0 5500.
  • NOV4 may also be localized to the lysosome (lumen) with a certainty of 0.2631, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or extraceliularly with a certainty of 0.1000.
  • the most likely cleavage site for a NOV4 peptide is between amino acids 25 and 26, at: VNS-AL.
  • NOV4 protein is widely expressed in a variety of tissues TaqMan data for NOV4 can be found below in Example 2.
  • NOV4 also has homology to the ammo acid sequences shown in the BLASTP data listed in Table 4C.
  • N0V4 (SEQ ID NO: 14)
  • Tables 4E-F list the domain description from DOMAIN analysis results against NOV4. This indicates that the NOV4 sequence has properties similar to those of other proteins known to contain this domain.
  • CD-Length 191 residues, 91.6% aligned
  • CD-Length 160 residues, 98.8% aligned
  • Prohibitin a novel cytoplasmic anti- proliferative protein widely expressed in a variety of tissues, inhibits DNA synthesis.
  • Prohibitin may be a suppressor gene and is associated with tumor development and/or progression of at least some breast cancers. Sequence comparisons suggest that the Prohibitin gene is an analogue of Cc, a Drosophila gene that is vital for normal development.
  • Prohibitin is a 30-kD intracellular, antiproliferative protein.
  • White et al. (1991) mapped the gene to chromosome 17 by analysis of human-mouse somatic cell hybrid cell lines using a genomic fragment of human Prohibitin DNA isolated from a library using the rat Prohibitin cDNA clone. By a study of cell lines containing portions of human chromosome 17, they determined that the PHB gene was located in the 17qll.2-q23 region. By in situ hybridization, they localized the gene to 17q21.
  • Sato et al. (1992) isolated the human homolog of the rat Prohibitin gene and mapped it to 17ql2-q21 by in situ hybridization. Sato et al.
  • the human Prohibitin gene family consists of 1 functional PHB gene on 7q21 and 4 processed pseudogenes, each on a different chromosome: PHBP1 on 6q25, PHBP2 on 1 l pl l.2, PHBP3 on l p3 .3, and PHBP4 on 2q21.
  • the disclosed NOV4 nucleic acid ofthe invention encoding a Prohibitin -like protein includes the nucleic acid whose sequence is provided in Table 4A or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 4A while still encoding a protein that maintains its Prohibitin -like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications indude, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability o the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
  • the disclosed NOV4 protein of the invention includes the Prohibitin -like protein whose sequence is provided in Table 4B.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 4B while still encoding a protein that maintains its Prohibitin -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 43% percent of the residues may be so changed.
  • the protein similarity information, expression pattern, and map location for the Prohibitin-like protein and nucleic acid (NOV4) disclosed herein suggest that this NOV4 protein may have important structural and/or physiological functions characteristic ofthe Prohibitin family. Therefore, the NOV4 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • the NOV4 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below.
  • compositions of the present invention will have efficacy for treatment of patients suffering from breast cancer (In a sporadic breast cancer, Sato et al. (1992) found a missense mutation from valine (GTC) to alanine (GCC) at codon 88 of the PHB gene), and/or other pathologies.
  • the NOV4 nucleic acids, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • NOV4 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV4 protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOV4 epitope is from about amino acids 40 to 45. In another embodiment, a NOV4 epitope is from about amino acids 60 to 75.
  • NON4 epitopes are from about amino acids 100 to 130, from about amino acids 140 to 160,, from about amino acids 180 to 220, and from about amino acids 240 to 260. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed NOV5 nudeic acid of 2011 nucleotides also referred to as dj l 182al4__dal
  • Table 5A An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TAG codon at nucleotides 1999-2001.
  • a putative untranslated region downstream from the termination codon is underlined in Table 5A, and the start and stop codons are in bold letters.
  • the NOV5 nucleic acid was identified on the q21 region of chromosome 3 and has 1508 of 1524 bases (98%) identical to a gb: GENBANK-ID :HUMMST1 A
  • acc:L11924 mRNA from Homo sapiens (Homo sapiens macrophage-stimulating protein (MSTl) mRNA, complete eds (E 0.0).
  • a disclosed NOV5 polypeptide (SEQ ID NO: 16) encoded by SEQ ID NO: 15 is 666 amino acid residues and is presented using the one-letter code in Table 5B.
  • Signal P, Psort and/or Hydropathy results predict that NOV5 has a signal peptide and is likely to be localized in the lysosome (lumen) with a certainty of 0.5493.
  • NOV5 may also be localized to extraceliularly with acertainty of 0.3700, the microbody (peroxisome) with a certainty of 0.1588, or the endoplasmic reticulum (membrane) with a certainty of 0.1000.
  • the most likely cleavage site for a NOV5 peptide is between amino acids 18 and 19, at: VPG-QR.
  • NOV5 is expressed in at least HepG2 (liver)
  • sequence is predicted to be expressed in the following tissues because o the expression pattern of (GENBANK-ID: HUMMSTlA
  • TaqMan data for NOV5 can be found below in Example 2.
  • NOV5 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 5C.
  • Tables 5E-N list the domain description from DOMAIN analysis results against NOV5. This indicates that the NOV5 sequence has properties similar to those of other proteins known to contain this domain.
  • CD-Length 79 residues, 100.0% aligned
  • Kringle domains have been found in plasminogen, hepatocyte growth factors, prothrombin, and apolipoprotein A. Structure is disulfide-rich, nearly all-beta.
  • CD-Length 79 residues, 100.0% aligned
  • CD-Length 79 residues, 100.0% aligned
  • Table 5H Domain Analysis of NOV5 gnl (Smar [smart00130, KR, Kringle domain; Named after a Danish pastry. Found in several serine proteases and in ROR-Iike receptors. Can occur in up to 38 copies (in apolipoprotein (a) ) . Plasminogen-like kringles possess affinity for free lysine and lysine- containing peptides. (SEQ ID NO: 86)
  • CD-Length 83 residues, 97.6% aligned
  • Table 5J Domain Analysis of NOV5 gnl (Smart [ smart00130, KR, Krmgle domain; Named after a Danish pastry. Found in several serine proteases and in ROR-like receptors. Can occur in up to 38 copies (in apolipoprotein (a) ) . Plasminogen-like kringles possess affinity for free lysine and lysine- containing peptides. (SEQ ID NO; 86)
  • Table 5L Domain Analysis of NOV5 gnlj Pfam
  • ORFs and the extracellular portion of plant receptor-like protein kinases Predicted to possess protein- and/or carbohydrate-binding functions. (SEQ ID NO: 89)
  • CD-Length 79 residues, 94.9% aligned
  • MSP MaGrophage-stimulating protein
  • HGF hepatocyte growth factor
  • RON tyrosine kinase a new member of he HGF receptor family.
  • the MSP- RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated.
  • HGF receptor which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct.
  • Macrophage stimulating protein (MSP; 142408), also known as hepatocyte growth factor-like protein (HGFL), is structurally related to hepatocyte growth factor.
  • MSP Macrophage stimulating protein
  • HGFL hepatocyte growth factor-like protein
  • Ronsin et al. (Oncogene 8: 1 195-1202, 1993) mapped the RON gene to 3p21 , with the most probable location being 3p21.3.
  • the gene encoding MSP is also located on 3p21 , a region of frequent deletion or mutation in small cell lung and renal carcinoma.
  • HGF hepatocyte growth factor
  • MET receptor hepatocyte growth factor
  • HepG2 human hepatoma (HepG2) cell line library was screened with an oligonucleotide probe for macrophage stimulating protein (MSP) to clone an MSP cDNA(Yoshimura T, et.al.; J Biol Chem 1993 Jul 25;268(21): 15461-8). Deduced sequences of isolated clones were compared with peptide fragment sequences of MSP. MSP9 cDNA encoded most of the known sequence of MSP except for a small segment of the 5' end o the open reading frame. Consequently, a hybrid 2300-base pair cDNA that encoded the complete MSP amino acid sequence was constructed from 2 clones.
  • MSP macrophage stimulating protein
  • MSP hepatocyte growth factor
  • the MSP cDNA hybridized strongly to mR A from liver, and to a lesser extent to mRNA from kidney and pancreas, suggesting that a cell type in the liver is the source of MSP.
  • MSP cDNAs had insertions or deletions, suggesting that alternatively spliced MSP mRNAs may occur. This was reflected in Northern blots probed with an MSP cDNA, which showed more than one mRNA species. Furthermore, although the gene coding for MSP is on chromosome 3, the sequence of one o the cDNAs was identical with a unique sequence in chromosome 1, indicating that there may be a family of MSP genes, located on chromosomes 3 and 1.
  • HLP hepatocyte growth factor-like protein
  • the purified protein showed M(r) of 85 kDa on SDS-PAGE, and this M(r) is in agreement with that of macrophage-stimulating protein (MSP) previously purified from human serum, as well as with the predicted M(r) of HLP.
  • Amino acid composition of the purified protein coincided with the compositions of human HLP and MSP.
  • Protein Precursor -like protein includes the nucleic acid whose sequence is provided in Table 5A or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 5A while still encoding a protein that maintains its Macrophage Stimulating Protein Precursor -like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any o the nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability o the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
  • the disclosed NOV5 protein of the invention includes the Macrophage Stimulating Protein Precursor -like protein whose sequence is provided in Table 5B.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 5B while still encoding a protein that maintains its Macrophage Stimulating Protein Precursor -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 39% percent o the residues may be so changed.
  • NOV5 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in Aicardi-Goutieres syndrome 1 ; Brugada syndrome; Deafness, autosomal recessive 6; Heart block, nonprogressive; Heart block, progressive, 2;
  • the NOV5 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
  • NOV5 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV5 protein have multiple hydrophilic regions, each of which can be used as an immunogen.
  • contemplated NOV5 epitope is from about amino acids 20 to 80.
  • NOV5 epitope is from about amino acids 90 to 120, from about amino acids 140 to 180, from about amino acids 190 to 340, from about amino acids 350 to 460, from about amino acids 500 to 530, from about amino acids 570 to 590, and from about amino acids 600 to 620, or from about amino acids 420 to 460.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed NOV6 nucleic acid of 634 nucleotides (also referred to as GM382a20_A) encoding a novel Fatty Acid-Binding Protein-like protein is shown in Table 6A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 58-60 and ending with a TAA codon at nucleotides 460-462.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
  • a disclosed NOV6 polypeptide (SEQ ID NO: 18) encoded by SEQ ID NO: 17 is 134 amino acid residues and is presented using the one-letter amino acid code in Table 6B.
  • Signal P, Psort and/or Hydropathy results predict that NOV6 contains no signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.4500.
  • NOV6 is also likely to be localized to the mitochondrial matrix space with a certainty of 0.3600, or to the lysosome (lumen) with a certainty of 0.1000.
  • Table 6B Encoded NOV6 protein sequence (SEQ ID NO.18). ATVQQLGGRWRLVDSKRFDEYMKEGGVGTALRKMDAMAKPDCIITCDGKNLTIKTESTLKTQFSCTLGEKFEET TADGRKTQTVCSFADGALVQHQE DGKENTITRKLKDGKLWYCVMNNVACTRIYEKVE
  • TaqMan data for NOV6 can be found below in Example 2.
  • NOV6 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 6C.
  • Table 6E lists the domain description from DOMAIN analysis results against NOV6. This indicates that the NOV6 sequence has properties similar to those of other proteins known to contain this domain.
  • Table 6E Domain Analysis of NOV6 gnl [ Pfa ⁇ [pfam00061, lipocalin, Lipocalin / cytosolic fatty-acid binding protein family.
  • Lipocalins are transporters for small hydrophobic molecules, such as lipids, steroid hormones, bilins, and retinoids. Alignment subsumes both the lipocalin and fatty acid binding protein signatures from PROSITE. This is supported on structural and functional grounds. Structure is an eight-stranded beta barrel. (SEQ ID NO: 90)
  • Fatty acid metabolism in mammalian cells depends on a flux of fatty acids, between the plasma membrane and mitochondria or peroxisomes for beta-oxidation, and between other cellular organelles for lipid synthesis.
  • the fatty acid-binding protein (FABP) family consists of small, cytosolic proteins believed to be involved in the uptake, transport, and solubilization of their hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structures. Fatty acid-binding proteins were first isolated in the intestine (FABP2;
  • OMIM- 134640 and later found in liver (FABP1 ; OMIM- 134650), striated muscle (FABP3;
  • E-FABP Epidermal fatty acid binding protein
  • PA-FABP psoriasis-associated fatty acid-binding protein
  • E-FABP Immunohistochemical localization of the expression of E-FABP in psoriasis, basal and squamous cell carcinomas has been carried out in order to obtain indirect information, at the cellular level, on the transport of the fatty acidss. (Masouye et al, 1996, PMID: 8726632).
  • E- FABP was localized in the upper stratum spinosum and stratum granulosum in normal and non-lesional psoriatic skin, hi contrast, lesional psoriatiG epidermis strongly expressed E- FABP in all suprabasal layers, like nonkeratinized oral mucosa.
  • the basal layer did not express E-FABP reactivity in any of these samples.
  • the disclosed NOV6 nucleic acid of the invention encoding a Fatty acid binding protein-like protein includes the nucleic acid whose sequence is provided in Table 6A or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 6A while still encoding a protein that maintains its Fatty acid binding protein-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nuGleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized.
  • modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
  • the mutant or variant nucleic acids, and their complements up to about 10% percent of the bases may be so changed.
  • the disclosed NOV6 protein of the invention includes the Fatty acid binding proteinlike protein whose sequence is provided in Table 6B.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 6B while still encoding a protein that maintains its Fatty acid binding protein-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 14% percent o the residues may be so changed.
  • NOV6 Fatty acid binding protein-like proteins
  • the above defined information for this invention suggests that these Fatty acid binding protein-like proteins (NOV6) may function as a member of a "Fatty acid binding proteinfamily". Therefore, the NOV6 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/Gytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • the nucleic acids and proteins of NOV6 are useful in Cardiomyopathy, AtherosclerosiSjHypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Transplantation, Endometriosis, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Crohn's Disease, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral pal
  • novel NOV6 nucleic acid encoding NOV6 protein, or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
  • NOV6 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV6 protein have multiple hydrophilic regions, each of which can be used as an immunogen.
  • contemplated NOV6 epitope is from about amino acids 10 to 35.
  • NON6 epitope is from about amino acids 40 to 45, from about amino acids 50 to 85, or from about amino acids 90 to 1 10.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed NOV7 nucleic acid of 822 nucleotides (also referred to sggc_draft_dj895c5_20000819) encoding a novel Gap junction beta-5 protein-like protein is shown in Table 7 A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1 -3 and ending with a TAA codon at nucleotides 800-802.
  • Table 7 A the 5' and 3 1 untranslated regions are underlined and the start and stop codons are in bold letters.
  • the disclosed NOV7 nucleic acid sequence localized to the p34.3-36.1 1 region of chromosome 1 , has 682 of 807 bases (84%) identical to a gb:GENBAN -
  • a disclosed NOV7 polypeptide (SEQ ID NO:20) encoded by SEQ ID NO: 19 is 266 amino acid residues and is presented using the one-letter amino acid code in Table 7B.
  • Signal P, Psort and/or Hydropathy results predict that NOV7 has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000.
  • NOV7 is also likely to be localized to the Golgi body with a certainty of 0.4000, to the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the nucleus with a certainty of 0.2400.
  • the most likely cleavage site for a NOV7 peptide is between amino acids 40 and 41, at: VAA- EE.
  • Table 7B Encoded NOV7 protein sequence (SEQ ID NO:20).
  • the disclosed NOV7 amino acid sequence has 230 of 266 amino acid residues (86%) identical to, and 244 of 266 amino acid residues (91%) similar to, the 266 amino acid residue ptnr:SWISSPROT-ACC:Q02738 protein from Mus musculus (Mouse) (Gap Junction Beta-5 Protein (Connexin 30.3) (CX30.3) (4.4e " ⁇ z9 ).
  • NOV7 is expressed in at least kidney tissue.
  • the sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBATNOK-.-ID: RNCON303
  • NOV7 also has homology to the amino acid sequence shown in the BLASTP data listed in Table 7C.
  • Table 7E-F lists the domain description from DOMAIN analysis results against NOV7. This indicates that the NOV7 sequence has properties similar to those of other proteins known to contain this domain.
  • Gap junctions are conduits that allow the direct cell-to-cell passage of small cytoplasmic molecules, including ions, metabolic intermediates, and second messengers, and thereby mediate intercellular metabolic and electrical communication.
  • Gap junction channels consist of connexin protein subunits, which are encoded by a multigene family.
  • Richard et al. identified 2 expressed sequence tags (ESTs) from the human EST database by their similarity to mouse Gjb3 (see 603324) and Gjb5. By radiation hybrid mapping, they placed them in proximity to a sequence tagged site (STS) that is linked to GJA4 (121012) at lp35.1.
  • ESTs 2 expressed sequence tags
  • GJIC gap junctional intercellular communication
  • extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances.
  • GJIC gap junctional intercellular communication
  • the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control.
  • the intercellular signaling system mediated by connexin channels is crucial for maintaining tissue homeostasis, growth control, development, and synchronized response of cells to stimuli (Richard G; Exp Dermatol 2000 Apr;9(2):77-96).
  • This review summarizes the structure, assembly, and properties of the components of the complex and diverse connexin system, and their biological functions in skin.
  • the importance of gap junctional intercellular communication for normal development and differentiation of human epidermis as well as the hearing function of the inner ear is illustrated by the examples of erythrokeratodermia variabilis and palmoplantar keratoderma associated with hearing loss.
  • connexins are sites of cytoplasmic communication between contacting cells.
  • Cxs protein subunits denoted connexins (Cxs) which are encoded by a gene family (Saez JC et.al; Braz J Med Biol Res 2000 Apr;33(4):447-55).
  • gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses.
  • Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps o the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently.
  • Antitumor suicide gene therapy is one of the emerging strategies against cancer (Mesnil et. al.; Cancer Res 2000 Aug l ;60(15):3989-99) . It consists of the introduction into cancer cells of a gene capable of converting a nontoxic prodrug into a cytotoxic drug.
  • this therapeutic gene cannot be easily introduced into the whole cell population of a tumor, the successful eradication of tumors depends on a phenomenon called the "bystander effect," by which the introduced gene can affect even cells in which it is not itself present, From a therapeutic point of view, it may be crucial to enhance this phenomenon through various means to achieve tumor eradication.
  • One such suicide gene the thymidine kinase gene from the herpes simplex virus, in combination with the prodrug ganciclovir, has been extensively and successfully used in some animal models exhibiting a strong bystander effect.
  • GJIC gap junctional intercellular communication
  • Synapses are classically defined as close connections between two nerve cells or between a neuronal cell and a muscle or gland cell across which a chemical signal (i.e., a neurotransmitter) and/or an electrical signal (i.e., current-carrying ions) can pass (Rozental R et.al; Brain Res Brain Res Rev 2000 Apr;32(l);l 1-5).
  • a chemical signal i.e., a neurotransmitter
  • an electrical signal i.e., current-carrying ions
  • GJIC gap junctional intercellular communication
  • mechanotransduction is defined as transduction of a load-induced biophysical signal, such as fluid flow, substrate deformation, or electrokinetic effects, to a cell and ultimately throughout a cellular network.
  • mechanotransduction would include interactions of extracellular signals with cellular membranes, generation of intracellular second messengers, and the propagation of these messengers, or signals they induce, through a cellular network.
  • gap junctions contribute largely to the propagation of intracellular signals.
  • the disclosed NOV7 nucleic acid of the invention encoding a Gap Junction Beta-5 Protein-like protein includes the nucleic acid whose sequence is provided in Table 7A or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 7A while still encoding a protein that maintains its Gap Junction Beta-5 Protein-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
  • modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 16% percent of the bases may be so changed.
  • the disclosed NOV7 protein of the invention includes the Gap Junction Beta-5 Protein-like protein whose sequence is provided in Table 7B.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 7B while still encoding a protein that maintains its Gap Junction Beta- 5 Protein-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 33% percent of the residues may be so changed.
  • the protein similarity information, expression pattern, and map location for the Gap Junction Beta-5 Protein-like protein and nucleic acid (NOV7) disclosed herein suggest that NOV7 may have important structural and/or physiological functions characteristic of the Gap Junction Beta-5 Protein-like family. Therefore, the NOV7 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications.
  • nucleic acid or protein diagnostic and/or prognostic marker serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nuclei ⁇ acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
  • compositions of the present invention will have efficacy for treatment of patients suffering from Deafness, autosomal dominant 2; Elliptocytosis-1 ; Fucosidosis; Hypophosphatasia (adult, childhood, infantile); Muscle-eye- brain disease; Neuropathy, paraneoplasti ⁇ sensory; Porphyria Gutanea tarda; Porphyria, hepatoerythropoietic; Schwartz-Jampel syndrome; Thrombocytopenia, congenital amegakaryocytic; Charcot-Marie-Tooth neuropathy-2A; GalaGtose epimerase deficiency; Glucose transport defect, blood-brain barrier; Kostmann neutropenia; Muscular dystrophy, congenital, with early spine rigidity; Myopathy due to succinate dehydrogenase deficiency; SC
  • NOV7 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below.
  • the disclosed NOV7 protein have multiple hydrophilic regions, each of which can be used as an immunogen.
  • contemplated NOV7 epitope is from about amino acids 40 to 70.
  • NOV7 epitope is from about amino acids 90 to 140, from about amino acids 170 to 180, or from about amino acids 220 to 255.
  • This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
  • a disclosed NOV8 nucleic acid of 546 nucleotides (also referred to as 56072181_dal) encoding a novel MT-like protein is shown in Table 8A.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 214-216 and ending with a TGA codon at nucleotides 397-399.
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 8A. The start and stop codons are in bold letters.
  • Table 8A NOV8 nucleotide sequence (SEQ ID NO:21).
  • the NOV8 nucleic acid sequence is located on the ql 3 region of chromsome 16.
  • the disclosed NOV8 polypeptide (SEQ ID NO:22) encoded by SEQ ID NO:21 has 61 amino acid residues and is presented in Table SB using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOV8 has no signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.6500.
  • NOV8 may also be localized to the mitochondrial matrix space with a certainty of 0.3100, or the lysosome (lumen) with a certainty of 0.1000.
  • Table 8B Encoded NOV8 protein sequence (SEQ ID NO:22).
  • MTs as a family of low molecular weight, heavy metal-binding proteins characterized by a high cysteine content and lack of aromatic amino acids. MTs bind 7 to 12 heavy metal atoms per molecule of protein. They are ubiquitous in the animal and plant kingdoms and are found in prokaryotes. In mammals, the cysteine residues are absolutely conserved and serve to coordinate heavy metal atoms such as zinc, cadmium, and copper via mercaptide linkages. In human liver, MTs occur in 2 major forms, MT-I and MT-II (156360). In HeLa cells, MT synthesis is induced by either ionized zinc or ionized cadmium and by glucocorticoid hormones.
  • metallothioneins are encoded by at least 10 to 12 genes separated into 2 groups designated MT-I and MT-II.
  • Masters et al. (1994) noted that, unlike MT-I and MT-II, which are expressed in most organs, MT-III (139255) expression appears to be restricted to the brain, and MT-IV is only expressed in certain stratified squamous epithelia.
  • MTs have been postulated to detoxify metals; to play a role in zinc and copper homeostasis during development; to regulate synthesis, assembly, or activity of zinc metalloproteins; and to protect against reactive oxygen species.
  • MTs may also protect against copper toxicity in the Menkes disease (309400) and murine Mottled' phenotypes (X-linked diseases resulting in copper deficiency) as well as in Wilson disease (277900); see also the Animal Models section.
  • Karin and Richards (Nature 299: 797-802, 1982) described the molecular cloning and sequence analysis of human metallothionein transcripts.
  • Karin et al. (1984) characterized DNA sequences that are involved in the induction of MT gene expression by cadmium and glucocorticoids. MAPPING
  • Karin et al. (Proc. Nat. Acad. Sci. 81 : 5494-5498, 1984) used several different hybridization probes derived from cloned and functional human MTl and MT2 genes to map the genes in somatic cell hybridization studies. They concluded that most of the human genes are clustered on chromosome 16. Analysis of RNA from somatic cell hybrids indicated that all hybrids that contain human chromosome 16 express both MTl and MT2 mRNA and that expression is regulated by both heavy metal ions and glucocorticoid hormones.
  • the metallothionein genes are on chromosome 8, which has other homology to human chromosome 16; by somatic cell hybridization, Cox and Palmiter (Hum. Genet. 64: 61-64, 1983) assigned the MT-1 structural gene to mouse chromosome 8, which also carries glutathione reductase in the mouse. (By chance the human 8 also carries glutathione reductase.)
  • chromosome 16 contains a cluster of metallothionein sequences, including 2 functional metallothionein I genes (156351 and 156352) and a functional metallothionein II gene.
  • the remaining sequences, including a processed pseudogene are dispersed to at least 4 other autosomes.
  • the absence of metallothionein sequences from the X chromosome indicates that the Menkes disease mutation affects metallothionein expression by a 'trans-acting' mechanism.
  • the processed pseudogene is on chromosome 4 and shows allelic variation (Karin and Richards, Nucleic Acids Res.
  • Two MT genes are on chromosome 1 but not close together: one is on the distal two-thirds of the short arm and the second probably on the long arm.
  • One metallothionein gene is on chromosome 20 and another is on chromosome 18.
  • genes were flanked by the single MT2A gene at one end and a gene labeled MT1X (156359) at the other.
  • mice with targeted disruption of the metallothionein-I and metallothionein-II genes were more sensitive to toxic metal and oxidative stress. In addition they were larger than most strains of mice, becoming significantly heavier at age 5 to 6 weeks. At age 14 weeks, the body weight and food intake of MT-null mice was 16 and 30% higher, respectively, compared with control mice. Most 22- to 39-week-old male MT-null mice were obese.
  • MT-null Seven-week-old MT-null also had significantly higher levels of plasma leptin (601694) and elevated expression of OB (164160), lipoprotein lipase (238600), and CCAAT enhancer binding protein alpha (189965) genes as compared with age-matched control mice. Abnormal accretion of body fat and adipocyte maturation was initiated at 5 to 7 weeks of age, possibly coincident with sexual maturation. Beattie et al. ( 1998) concluded that a link between MT and the regulation of energy balance is implied by these observations. They noted the possibility that obesity and the associated biochemical changes in the MT-null mice may be caused by factors other than lack of MT.
  • the disclosed NOV8 nucleic acid of the invention encoding a MT-like protein includes the nucleic acid whose sequence is provided in Table 8A, or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 8A while still encoding a protein that maintains its MT-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic a ⁇ id fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic add, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
  • the disclosed NOV8 protein of the invention includes the MT-like protein whose sequence is provided in Table 8B.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2 while still encoding a protein that maintains its MT-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 61% percent of the residues may be so changed.
  • the invention further encompasses antibodies and antibody fragments, such as F ab or (Fab) 2 , that bind immunospecifically to any of the proteins o the invention.
  • antibodies and antibody fragments such as F ab or (Fab) 2 , that bind immunospecifically to any of the proteins o the invention.
  • NOV8 MT-like protein
  • the above defined information for this invention suggests that this MT-like protein (NOV8) may function as a member of a "MT family". Therefore, the NOV8 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below.
  • the potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug targel, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
  • the NOV8 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to Inflamation, Autoimmune disorders, Aging and Cancer
  • a cDNA encoding the MT like protein (NOV8) may be useful in gene therapy, and the MT-like protein (NOV8) may be useful when admmisteied to a subject in need thereof
  • the compositions of the piesent invention will have efficacy for treatment of patients suffering fiom Gitelman syndiome, Menkes disease, Wilson's disease, aciodermatitis enteropathica, myelomonocytic leukemia, eosinophil disorders, hepatic disorders such as hepatic copper toxicity and other such conditions
  • the NOV8 nucleic acid encoding MT-like protein, and the MT-hke protein of the invention, or fiagments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be
  • NOV8 nucleic acids and polypeptides are further useful in the generation of antibodies that bind lmmuno-specifically to the novel NOV8 substances for use in therapeutic or diagnostic methods
  • These antibodies may be generated according to methods known in the art, using prediction from hydrophobidty charts, as described in the "Anti-NOVX Antibodies" section below
  • the disclosed NOV8 protein has multiple hydrophilic regions, each of which can be used as an immunogen
  • a contemplated NOV8 epitope is from about amino acids 25 to 40
  • a NOV8 epitope is from about ammo acids 45 to 55
  • NOV9 nucleic acid of 2309 nucleotides also referred to as 2855519_0_19_dal
  • Table 9A An open reading frame was identified beginning with an ATG initiation codon at nucleotides 78-80 and ending with a TGA codon at nucleotides 1719-1721
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 9A
  • the start and stop codons are in bold letters Table 9A.
  • NOV9 nucleotide sequence SEQ ID NO:23).
  • the disclosed NOV9 nucleic acid sequence localized to the p21.2-22.2 region of cliromosome 1, has 916 of 1460 bases (62%.) identical to a 2001 bp cdc42-interactmg protein 4 (CfP4 mRNA from Homo sapiens (GENBANK-ID HSCrP4
  • acc:AJ000414) (E 1.3e "97 ).
  • NOV9 polypeptide (SEQ ID NO:24) encoded by SEQ ID NO:23 has 547 amino acid residues and is presented in Table 9B using the one-letter amino add code.
  • Signal P, Psort and/or Hydropathy results predict that NOV9 has no signal peptide and is likely to be localized in the nucleus with a certainty of 0.7000
  • NOV9 may also be localized to the microbosy (peroxisome) with a certainty of 0.3000, the mitochondrial matrix space with a certainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000.
  • TaqMan data for NOV9 can be found below in Example 2.
  • the disclosed NOV9 polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table 9C.
  • Table 9E-H lists the domain description from DOMAIN analysis results against NOV9. This indicates that the NOV9 sequence has properties similar to those of other proteins known to contain this domain.
  • SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids. Pro-containing polypeptides may bind to SH3 domains in 2 different binding orientations.
  • CD-Length 59 residues, 88.1% aligned
  • CD-Length 57 residues, 91.2% aligned
  • Table 9G Domain Analysis of NOV9 gnl
  • TRs The thyroid hormone receptors
  • RXRs retinoid X receptors
  • TRIP 10 interacted with rat Thrb only in the presence of thyroid hormone. It also showed a ligand- dependent interaction with RXR-alpha (RXRA; 180245), but did not interact with the glucocorticoid receptor (NR3C1 ; 138040) under any condition.
  • TRIP10 contains a Src homology-3 (SH3) domain and shows sequence similarity to a Xenopus fyn homolog (see 137025) and chicken src (see 190090).
  • Northern blot analysis detected a 2.6-kb TRIP 10 transcript in several tissues, with highest expression in skeletal muscle.
  • CTP4 human B-cell cDNA encoding CDC42-interacting protein-4
  • the predicted 545-amino acid CIP4 protein contains an N-terminal domain that bears resemblance to the nonkinase domain of the FER (176942) and Fes/Fps family of tyrosine kinases, and a C-terminal SH3 domain.
  • CIP4 shares sequence similarity with a number of proteins that have roles in regulating the actin cytoskeleton. Aspenstrom (Curr. Biol.
  • CIP4 can bind to activated CDC42 in vitro and in vivo.
  • Overexpression of CIP 4 in fibroblasts reduced the amount of stress fibers in these cells.
  • Recombinant CfP4 protein accumulated at the cell periphery, particularly in areas that exhibited membrane ruffling.
  • Coexpression of activated CDC42 and CIP4 led to clustering of CUM to a large number of foci at the dorsal side of the cells.
  • Northern blot analysis showed a major 2.2-kb CIP4 transcript that was abundant in skeletal muscle, heart, and placenta, present at lower levels in pancreas, lung, liver, and kidney, and barely detectable in brain. Minor transcripts of 3.5 and 5 kb were also detected.
  • CIP4 is a downstream target of activated GTP- bound CDC42, and is similar in sequence to proteins involved in signaling and cytoskeletal control. The author suggested that CIP4 may act as a link between CDC42 signaling and regulation of the actin cytoskeleton.
  • the Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency characterized by thrombocytopenia, eczema, and a tendency toward lymphoid malignancy (Tian L, et.al.; J Biol Chem 2000 Mar 17;275(11 ):7854-61). Lymphocytes from affected individuals have cytoskeletal abnormalities, and monocytes show impaired motility.
  • the Wiskott-Aldrich syndrome protein (WASP) is a multi-domain protein involved in cytoskeletal organization. In a two-hybrid screen, we identified the protein Cdc42-interacting protein 4 (CIP4) as a WASP interactor.
  • CIP4 like WASP, is a Cdc42 effector protein involved in cytoskeletal organization.
  • WASP-CIP4 interaction is mediated by the binding ofthe Src homology 3 domain of CIP4 to the proline-rich segment of WASP.
  • Cdc42 was not required for this interaction.
  • Co-expression of CIP4 and green fluorescent protein- WASP in COS-7 cells led to the association of WASP with microtubules.
  • CIP4 binds to microtubules via its NH(2) terminus.
  • the region of CTP4 responsible for binding to active Cdc42 was localized to amino acids 383-417, and the mutation I398S abrogated binding.
  • the disclosed NOV9 nucleic acid of the invention encoding a CfP4-like protein includes the nucleic acid whose sequence is provided in Table 9 A, or a fragment thereof.
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 9A while still encoding a protein that maintains its CIP4-like activities and physiological functions, or a fragment of such a nucleic acid.
  • the invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described.
  • the invention additionally includes nucleiG acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic adds whose sugar phosphate backbones are modified or derivatized.
  • the disclosed NOV9 protein of the invention includes the CIP4-like protein whose sequence is provided in Table 9B.
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2 while still encoding a protein that maintains its CIP4-like activities and physiological functions, or a functional fragment thereof In the mutant or variant protein, up to about 51% percent ofthe residues may be so changed
  • the invention further encompasses antibodies and antibody fragments, such as F ab or (F J ) 2 that bind immunospecifically to any of the proteins of the invention.
  • NOV9 may function as a member of a "MT family" Therefore, the NO V9 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below
  • the potential therapeutic applications for this invention include, but are not limited to protein therapeutic, small molecule drug target, antibody target (therapeutiG, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in xitro of all tissues and cell types composing (but not limited to) those defined here
  • NOV9 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to Inflamation,
  • compositions of the present invention will have efficacy for treatment of patients suffering from Wiskott-Ald ⁇ ch syndrome, immunodeficiency, thrombocytopenia, eczema, lymphoid malignancy cytoskeletal abnormalities, impaired monocyte motihty, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders,
  • NOV9 nucleic acids and polypeptides are further useful in the generation of antibodies that bind lmmuno-specifically to the novel NOV9 substances for use in therapeutic or diagnostic methods
  • These antibodies may be generated according to methods known in the art, using prediction from hydiophobicity charts, as described in the "Anti-NOVX Antibodies" section below
  • the disclosed NOV9 piotein has multiple hydrophilic regions, each of which can be used as an immunogen
  • a contemplated NOV9 epitope is from about ammo acids 25 to 40
  • a NOV9 epitope is from about amino acids 45 to 55
  • NOV10 NOV10 includes three novel hepsin/plasma transmembrane serine protease-like proteins disclosed below. The disclosed sequences have been named NOV 10a and NOV 10b NOVlOa
  • a disclosed NOVl 0a nucleic acid of 1787 nucleotides (also referred to as 129297354JEXT) encoding a novel hepsin/plasma transmembrane serine protease-hke protein is shown in Table 10A
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 54-56 and ending with a TAA codon at nucleotides 1470-1472
  • a putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 10A The start and stop codons are in bold letters
  • Table 10A NOVlOa nucleotide sequence (SEQ ID NO:25).
  • the NOV l Oa nucleic acid sequence has 424 ases (60%) identical to a gb:GENBAN -ID:AF243500
  • acc:AF243500 transmembrane protein X mRNA, complete eds, 1735 bp. mRNA from mouse/ Mus musculus (E 4.9e "19 ).
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • the disclosed NOV 10a polypeptide (SEQ ID NO:27) encoded by SEQ ID NO:25 has 472 amino acid residues and is presented in Table 10B using the one-letter amino acid code.
  • NOVlOa has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.7900.
  • NOVlOa may also be localized to the microbody (peroxisome) with acertainty of 0.5425, the Golgi body with a certainty of 0.3000, or in the endoplasmic reticulum (membrane) with a certainty of 0.2000.
  • the most likely cleavage site for a NOVlOa peptide is between amino acids 13and 14, at: MEA-QY.
  • Table 10B Encoded NOVlOa protein sequence (SEQ ID NO:27).
  • Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
  • NOVlOa is expressed in at least the adrenal gland.
  • NOVlOb nucleic acid of 2148 nucleotides also referred to as CGI 06783- 02 encoding a novel Spinesin-like protein is shown in Table IOC.
  • An open reading frame was identified beginning with an ATG initiation codon at nucleotides 156-158 and ending with a TGA codon at nucleotides 1410-1412.
  • a putative untranslated region upstream from the initiation codon is underlined in Table IOC. The start and stop codons are in bold letters.
  • Table IOC. NOV] Ob nucleotide sequence (SEQ ID NO:28).
  • the NOVlOb nucleic acid sequence, located on chromosome 7 has 1343 of 1446 bases (92%) identical to a gb:GENBANK- ID:AB028140
  • acc:AB028140.1 mRNA from Homo sapiens (Homo sapiens mRNA for spinesin, complete eds) (E 3.2e "268 ).
  • Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
  • NOVlOb polypeptide (SEQ ID NO:29) encoded by SEQ ID NO:28 has 418 amino acid residues and is presented in Table 10D using the one-letter amino acid code.
  • Signal P, Psort and/or Hydropathy results predict that NOV 10a has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.7900.
  • NOVlOa may also be localized to the Golgi body with acertainty of 0.3000, the microbody (peroxisome) with a certainty of 0.2036 or in the endoplasmic reticulum (membrane) with a certainty of 0.2000.
  • the most likely cleavage site for a NOVl Oa peptide is between amino acids 13 and 14, at: MEA-QY.
  • Table 10D Encoded NOVlOa protein sequence (SEQ ID NO:29).
  • Public amino acid databases include the GenBank databases, SwissProt, PDB and P1R.
  • NOVlOb is expressed in at least the following tissues: Colon, Brain, Placenta, Testis, Adrenal Gland/Suprarenal gland, Retina. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOVlOb. The sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBA-NK-ID:AB028140
  • TaqMan data for NOVlOa can be found below in Example 2.
  • the proteins encoded by the NOVlOa and 10b nucleotides are very closely homologous as is shown in the alignment in Table 10E.
  • NOVIO is assumed to refer to both ofthe NOV10 proteins in general, unless otherwise noted.
  • the disclosed NOVlOa polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table 10F.
  • Proteins recognized include all proteins xn families SI, S2A, S2B, S2C, and S5 in the classification of peptidases. Also included are proteins that are clearly members, but that lack peptidase activity, such as haptoglobin and protein 3
  • pancreatin represents a therapeutically successful form of enzyme replacement (Townes, 1972).
  • Trypsin EC 3.4.21.4
  • elastase 130120
  • MacDonald et al. (1982) reported nucleotide sequences of cDNAs representing 2 pancreatic rat trypsinogens. Using a rat cDNA probe, Honey et al. (1984, 1984) found that a 3.8-kb DNA fragment containing human trypsin- 1 gene sequences cosegregated with chromosome 1 , and assigned the gene further to 7q22- 7qter by study of hybrids with a deletion of this segment.
  • the trypsin gene is on mouse chromosome 6 (Honey et al., 1984). Carboxypeptidase A (114850) and trypsin are a syntenic pair conserved in mouse and man. Emi et al. (1986) isolated cDNA clones for 2 major human trypsinogen isozymes from a pancreatic cDNA library. The deduced amino acid sequences had 89% homology and the same number of amino acids (247), including a 15-amino acid signal peptide and an 8-amino acid activation peptide. Southern blot analysis of human genomic DNA
  • DNA with the cloned cDNA as a probe showed that the human trypsinogen genes constitute a family of more than 10, some of which may be pseudogenes or may be expressed in other stages of development.
  • Rowen et al. (1996) mapped the gene corresponding to the third pancreatic trypsinogen cDNA by fluorescence in situ hybridization. They used a cosmid clone containing 3 trypsinogen genes. Strong hybridization to chromosome 7 and weaker hybridization to chromosome 9 were observed. They isolated and partially sequenced 4 cosmid clones from the chromosome 9 region. They found that the region represents a duplication and translocation of a DNA segment from the 3-prime end of the TCRB locus that includes at least 7 V(beta) elements and a functional trypsinogen gene denoted T9.
  • the assignment ofthe PRSS1 gene to 7q35 is established by the demonstration of its sequence within the sequence ofthe 'locus' for the T-cell receptor beta-chain (Rowen et al., 1996). Since hereditary pancreatitis (167800) has been mapped rather precisely to 7q35 and since a defect in the trypsinogen gene has been identified in hereditary pancreatitis, the assignment of the trypsinogen gene can be refined from 7q32-qter to 7q35.
  • Whitcomb et al. stated that the high degree of DNA sequence homology (more than 91%) present among this cluster of 5 trypsinogen genes identified by Rowen et al. (1996) demanded that highly specific sequence analysis strategies be developed for mutational screening in families with hereditary pancreatitis. This was necessary to ensure that each sequencing run contained only the 2 alleles corresponding to a single gene, thereby permitting detection of heterozygotes in this autosomal dominant disorder, and not a dozen or more alleles from multiple related trypsinogen-like genes, which would make detection of heterozygotes nearly impossible. In a family with hereditary pancreatitis, Whitcomb et al.
  • the disclosed NOVIO nucleic acid o the invention encoding a spinesin like protein includes the nucleic acid whose sequence is provided in Table 10A and 10D or a fragment theieof
  • the invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 10A and 10D while still encoding a protein that maintains its spinesm like activities and physiological functions, or a fragment of such a nudeic acid
  • the invention further includes nucleiG acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described
  • the invention additionally includes nucleiG adds or nucleic acid fragments, or complements thereto, whose structures include chemical modifications Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be
  • the disclosed NOVIO protein of the invention includes the spinesm-hke protein whose sequence is provided in Table 10B or 10E
  • the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 10B or 10E while still encoding a protein that maintains its spinesin like activities and physiological functions, or a functional fragment thereof In the mutant or vanant protein, up to about 22% percent of the residues may be so changed
  • the invention further encompasses antibodies and antibody fragments, such as F ab or (F a b) 2 that bind immunospecifically to any of the proteins ofthe invention
  • NOV10 spmesin- ke protein
  • the above defined information for this invention suggests that this spmesin- ke protein (NOV10) may function as a member of a "spmesin family" Therefore, the NOV10 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below
  • the potential therapeutic applications for this invention include, but are not limited to protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vit/o of all tissues and cell types composing (but not limited to) those defined here
  • NOV10 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to various pathologies and disorders as indicated below.
  • a cDNA encoding the spinesin-like protein (NOVIO) may be useful in gene therapy, and the spinesin -like protein (NOVIO) may be useful when administered to a subject in need thereof.
  • compositions of the present invention will have efficacy for treatment of patients suffering from cancer, trauma, tissue regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, immunological disease, respiratory disease, gastro-intestinal diseases, reproductive health, neurological and neurodegenerative diseases, bone marrow transplantation, metabolic and endocrine diseases, allergy and inflammation, nephrological disorders, cardiovascular diseases, muscle, bone, joint and skeletal disorders, hematopoietic disorders, urinary system disorders, Tissue and organ transplantation, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis , Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Scleroderma, Obesity, Hypertension, Fibromuscular dysplasia, Stroke, Aneurysm
  • NOVIO nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVIO substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti- NOVX Antibodies" section below.
  • the disclosed NOVIO protein has multiple hydrophilic regions, each of which can be used as an immunogen.
  • a contemplated NOVIO epitope is from about amino acids 5 to 50.
  • a NOVIO epitope is from about amino acids 60 to 70.
  • NOV10 epitope is from about amino acids 100 to 130, from about amino acids 140 to 210, from about amino acid 270 to 320, from about amino acid 340 to 360, from about amino acid 390 to 410, and from about amino acids 430 to 450.
  • novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
  • nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
  • an NOVX nucleic acid can encode a mature NOVX polypeptide.
  • a "mature" form of a polypeptide or protein disclosed in the present invention is the product of naturally occurring polypeptide or precursor form or proprotein.
  • the naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein.
  • the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
  • Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence.
  • a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal of the N-terminal methionine.
  • a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • probes refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe invention e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS: 1 , 3, 5, 1, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, and 28, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, lohn Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.

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Abstract

Disclosed herein are nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies, which immunospecifically-bind to the polypeptide, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The invention further discloses therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these novel human nucleic acids and proteins.

Description

PROTEINS AND NUCLEIC ACIDS ENCODING SAME
FIELD OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded thereby.
BACKGROUND OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded therefrom. More specifically, the invention relates to nucleiG acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides, as well as vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.
SUMMARY OF THE INVENTION The invention is based in part upon the discovery of nucleiG acid sequences encoding novel polypeptides. The novel nucleic acids and polypeptides are referred to herein as NOVX, or NOVl , NOV2, NOV3, NOV4, NOV5, NOV6, NOV7, NOV8, NOV9, and NOV10 nucleic acids and polypeptides. These nucleic acids and polypeptides, as well as derivatives, homologs, analogs and fragments thereof, will hereinafter be collectively designated as αNOVX" nucleic acid or polypeptide sequences.
In one aspect, the invention provides an isolated NOVX nucleic acid molecule encoding a NOVX polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ DD NOS.l, 3, 5, 1, 9, 11 , 13, 15, 11, 19, 21, 23, 25, and 28. In some embodiments, the NOVX nucleic acid molecule will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of a NOVX nucleic acid sequence. The invention also includes an isolated nucleic acid that encodes a NOVX polypeptide, or a fragment, homolog, analog or derivative thereof For example, the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS. l , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, and 28. Also included in the invention is an oligonucleotide, e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of a NOVX nucleic aGid (e.g., SEQ ID NOS.l , 3, 5, 7, 9, 1 1 , 13, 1 5, 17, 19, 21 , 23, 25, and 28) or a complement o said oligonucleotide. Also included in the invention are substantially purified NOVX polypeptides (SEQ ID NOS;2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29). In certain embodiments, the NOVX polypeptides include an amino acid sequence that is substantially identical to the amino acid sequence of a human NOVX polypeptide. The invention also features antibodies that immunoselectively bind to NOVX polypeptides, or fragments, homologs, analogs or derivatives thereof.
In another aspect, the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically- acceptable carrier. The therapeutic Gan be, e.g., a NOVX nucleic acid, a NOVX polypeptide, or an antibody specific for a NOVX polypeptide. In a further aspect, the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
In a further aspect, the invention includes a method of producing a polypeptide by culturing a cell that includes a NOVX nucleic acid, under conditions allowing for expression of the NOVX polypeptide encoded by the DNA. If desired, the NOVX polypeptide can then be recovered.
In another aspect, the invention includes a method of detecting the presence of a NOVX polypeptide in a sample. In the method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound. The complex is detected, if present, thereby identifying the NOVX polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types based on their expression of a NOVX.
Also included in the invention is a method of detecting the presence of a NOVX nucleic acid molecule in a sample by contacting the sample with a NOVX nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a NOVX nucleic acid molecule in the sample.
In a further aspect, the invention provides a method for modulating the activity of a NOVX polypeptide by contacting a cell sample that includes the NOVX polypeptide with a compound that binds to the NOVX polypeptide in an amount sufficient to modulate the activity of said polypeptide. The compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein. Also within the scope of the invention is the use of a therapeutic in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g. , adrenoleukodystrophy, congenital adrenal hyperplasia, leukodystrophies, breast cancer, Small- cell cancer of lung, squamous cell carcinomas, Colorectal cancer, Malignant melanoma, cutaneous, Neuroblastoma, Prostate cancer-brain cancer susceptibility, Alzheimer's disease, epilepsy, Huntington's disease, anxiety, ataxia-telangiectasia, behavioral disorders, multiple sclerosis, muscular dystrophy, myasthenia gravis, neurodegeneration, neuroprotection, Parkinson's disease, pain, stroke, Stroke, Aneurysm, Embolism, autoimmune disease, allergies, addiction, asthma, ARDS, allergy, endometriosis, endocrine dysfunctions, graft versus host disease, graft versus host disease (GVHD), idiopathic thrombocytopenic purpura, immunodeficiencies, IgA nephropathy, lymphaedema, systemiG lupus erythematosus, scleroderma, transplantation, psoriasis, crohn's disease, HIV infection, Muscle-eye-brain disease, Neuropathy, paraneoplastic sensory, Charcot-Marie-Tooth neuropathy-2A, SCID due to LCK deficiency, bone marrow transplantation, Kostmann neutropenia, immunodeficiency, thrombocytopenia, eczema, lymphoid malignancy, impaired monocyte motility,
Lymphaedema, atherosclerosis, aortic stenosis, atrial septal defect (ASD), atrioventricular (A- V) canal defect, ductus arteriosus, cerebral palsy, cirrhosis, cardiomyopathy, congenital heart defects, hypertension, hemophilia, hypercoagulation, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, bacterial and viral infections, cerebral vascular disease, osteoarthritis, rheumatoid arthritis, Heart block, nonprogressive, Heart bloϋk, progressive, 2, Ventricular fibrillation, idiopathic, entricular tachycardia, idiopathic, Thrombocytopenia, congenital amegakaryocytic, Bypass surgery, Bleeding disorders, Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, hypercoagulation, diabetes, obesity, metabolic disorders suσh as familial amyloidotic polyneuropathy, hyperkinetic diseases, Galactose epimerase deficiency, Glucose transport defect, blood-brain barrier, diverticular disease, emphysema, glomerulonephritis, hypercalceimia, interstitial nephritis, inflammatory bowel disease, Lesch-Nyhan syndrome, polycystic kidney disease, pancreatitis, renal artery stenosis, renal tubular acidosis, tuberous sclerosis, Von Hippel-Lindau (VHL) syndrome, ulcers, neonatal apnea, eagle's syndrome, renal fibrogenesis, Meckel syndrome, skin disorders, connective tissue disorders such as type VIIC Ehlers-Danros syndrome, Primary bile acid malabsorption (PBAM, an idiopathic intestinal disorder), congenital diarrhea, steatorrhea, refractory infantile diarrhea, interruption of the enterohepatic circulation of bile acids, reduced plasma cholesterol levels, Hirschsprung's disease, Cirrhosis, growth failure, Aicardi-Goutieres syndrome 1 , Brugada syndrome. Deafness, autosomal recessive 6, Ichthyosi forme erythioderma, congenital, nonbullous, Long QT syndrome-3, Night blindness, congenital stationary, Pituitai y ACTH-secreting adenoma, Elhptocytosιs-1 , Fucosidosis, Hypophosphatasia (adult, childhood, infantile), Poiphyiia cutanea tarda, Po hyria, hepatoerythropoietic, Schwartz- Fainpel syndiome, Myopathy due to succinate dehydrogenase deficiency, Bartter syndiome, type 3, Comeal dystiophy, crystalline, Schnyder,
Hypeφrohnemia, type II, erythrokeratodermia vaπabilis, palmoplantar keratoderma, diseases and disorders involving inteicellular metabolic and electrical communication, diseases and disordeis involving coordination, proliferation and differentiation, diseases and disorders involving maintainance of tissue homeostasis, growth control, development, and synchronized lesponse of cells to stimuli, Wislcott-Alduch syndrome, cytoskeletal abnormalities, trauma, tissue regeneration (in vitro and in vivo), respiratory disease, gastro-intestinal diseases, muscle, bone, joint and skeletal disorders, hematopoietiG disorders, urinary system disorders, Tissue and organ transplantation, Fibromuscular dysplasia, Hyperparathyroidism, Hypoparathyroidism, Hyperthyroidism and Hypothyroidism, SIDS, Xerostomia, Tonsihtis, Osteoporosis, Ankylosmg spondylitis, Scohosis, Tendinitis, Dental disease and infection, growth and reproductive disorders, hypogonadism, fertility, and/or other pathologies and disorders of the like
The therapeutic can be, e g , a NOVX nudeic acid, a NOVX polypeptide, or a NOVX- specific antibody, or biologically-active derivatives or fragments thereof For example, the compositions of the present invention will have efficacy for treatment of patients suffeπng from the diseases and disorders disclosed above and/or other pathologies and disorders of the like The polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines They can also be used to screen for potential agonist and antagonist compounds For example, a cDNA encoding NOVX may be useful in gene therapy, and NOVX may be useful when administered to a subject in need thereof By w ay of non limiting example, the compositions of the present invention will have efficacy for treatment of patients suffei g fiom the diseases and disorders disclosed above and/or other pathologies and disorders of the like
The invention further includes a method for screening for a modulator of disorders or syndiomes including, e g , the diseases and disorders disclosed above and/or other pathologies and disorders of the like The method includes contacting a test compound with a NOVX polypeptide and determining if the test compound binds to said NOVX polypeptide Binding of the lest compound to the NOVX polypeptide indicates the test compound is a modulatoi of activity, or of latency or predisposition to the aforementioned disoiders or syndromes Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes. The test animal expresses a recombinant polypeptide encoded by a NOVX nucleic acid. Expression or activity of NOVX polypeptide is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly- expresses NOVX polypeptide and is not at increased risk for the disorder or syndrome. Next, the expression of NOVX polypeptide in both the test animal and the control animal is compared. A change in the activity of NOVX polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency o the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of a NOVX polypeptide, a NOVX nucleic acid, or both, in a subject (e.g., a human subject). The method includes measuring the amount of the NOVX polypeptide in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the NOVX polypeptide present in a control sample. An alteration in the level of the NOVX polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject. Preferably, the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders o the like. Also, the expression levels o the new polypeptides of the invention can be used in a method to screen for various cancers as well as to determine the stage of cancers.
In a further aspect, the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject a NOVX polypeptide, a NOVX nucleic acid, or a NOVX-specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition. In preferred embodiments, the disorder, includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like. In yet another aspect, the invention can be used in a method to identity the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with antibodies or other specific-interacting molecules. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing o the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel nucleotides and polypeptides encoded thereby. Included in the invention are the novel nucleic acid sequences and their encoded polypeptides. The sequences are collectively referred to herein as "NOVX nucleiG acids" or "NOVX polynucleotides" and the corresponding encoded polypeptides are referred to as "NOVX polypeptides" or "NOVX proteins." Unless indicated otherwise, "NOVX" is meant to refer to any of the novel sequences disclosed herein. Table A provides a summary ofthe NOVX nucleic acids and their encoded polypeptides.
TABLE A. Sequences and Corresponding SEQ ID Numbers
Figure imgf000008_0001
Figure imgf000009_0001
NOVX nucleic acids and their encoded polypeptides are useful in a variety of applications and contexts. The various NOVX nucleic acids and polypeptides according to the invention are useful as novel members of the protein families according to the presence of domains and sequence relatedness to previously described proteins. Additionally, NOVX nucleic acids and polypeptides can also be used to identify proteins that are members of the family to which the NOVX polypeptides belong.
NOVl is homologous to a Zinc Metalloprotease/ADAM-TS 7-like family of proteins. Thus, the NOVl nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; adrenoleukodystrophy , Alzheimer's disease, autoimmune disease, allergies, addiction, anxiety, ataxia-telangiectasia, asthma, ARDS, atherosclerosis, behavioral disorders, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, allergy, cerebral palsy, congenital adrenal hyperplasia, cirrhosis, cardiomyopathy, congenital heart defects, diabetes, diverticular disease, epilepsy, emphysema, endometriosis, endocrine dysfunctions, graft versus host disease, glomerulonephritis, graft versus host disease (GVHD), growth and reproductive disorders, hemophilia, hypercoagulation, hypercalceimia, Huntington's disease, hypertension, hypogonadism, fertility, idiopathic thrombocytopenic purpura, immunodeficiencies, interstitial nephritis, IgA nephropathy, lymphaedema, inflammatory bowel disease, Lesch-Nyhan syndrome, leukodystrophies, multiple sclerosis, muscular dystrophy, myasthenia gravis, neurodegeneration, neuroprotection,obesity, Parkinson's disease, pain, polycystic kidney disease, pulmonary stenosis, pancreatitis, renal artery stenosis, renal tubular acidosis, stroke, systemic lupus erythematosus, scleroderma, subaortic stenosis, transplantation, tuberous sclerosis, Von Hippel-Lindau (VHL) syndrome, ventricular septal defect (VSD), valve diseases, Von Hippel-Lindau (VHL) syndrome, ulcers, bacterial and viral infections, neonatal apnea, eagle's syndrome, atherosclerosis, metabolic disorders such as familial amyloidotic polyneuropathy, hyperkinetic diseases, muscular dystrophy, cerebral vascular disease, hypertension, cardiovascular diseseases, renal fibrogenesis, inflammatory bowel disease, Meckel syndrome, colorectal cancer, papillomavirus infection and cervical carcinoma, liver malignancies, skin disorders, connective tissue disorders such as type VIIC Ehlers-Danros syndrome, osteoarthrifis, rheumatoid arthritis, or other pathologie or conditions. NOV2 is homologous to the Alpha-2-macroglobulin precursor-like family of proteins. Thus NOV2 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; asthma, allergy and psoriasis, Alzheimer disease, Emphysema, pulmonary disease, immune disorders and Cancer and/or other pathologies and disorders.
NOV3 is homologous to a family of Heal Sodium/Bile Acid Cotransporter-like proteins. Thus, the NOV3 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: Primary bile acid malabsorption (PBAM, an idiopathic intestinal disorder), congenital diarrhea, steatorrhea, refractory infantile diarrhea, interruption of the enterohepatic circulation of bile acids, reduced plasma cholesterol levels, croh-n's disease, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Cirrhosis, Transplantation, Hypercalceimia, Ulcers, growth failure and/or other pathologies.
NOV4 is homologous to the Prohibitin-like family of proteins. Thus, NOV4 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: breast cancer (In a sporadic breast cancer, Sato et al. (1992) found a missense mutation from valine (GTC) to alanine (GCC) at codon 88 of the PHB gene), and/or other pathologies.
NOV5 is homologous to the Macrophage Stimulating Protein Precursor-like family of proteins. Thus NOV5 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: Aicardi-Goutieres syndrome 1, Brugada syndrome, Deafness, autosomal recessive 6, Heart block, nonprogressive, Heart block, progressive, 2, Ichthyosiforme erythroderma, congenital, nonbullous, Long QT syndrome-3, Night blindness, congenital stationary, Pituitary ACTH-secreting adenoma, Small-cell cancer of lung, Ventricular fibrillation, idiopathic, entricular tachycardia, idiopathic, HIV infection, susceptibility/resistance to, Von Hippel-Lindau (VHL) syndrome, Cirrhosis, Transplantation, and/or other pathologies/disorders.
NOV6 is homologous to the Fatty Acid-Binding Protein-like family of proteins. Thus NOV6 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example: Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Transplantation, Endometriosis, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Crohn's Disease, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Asthma, allergy, ARDS, Lesch-Nyhan syndrome, Multiple sclerosis, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection Fertility, psoriasis, cancer including but not limited to basal and squamous cell carcinomas, obesity, diabetis, and/or other pathologies and disorders involving fatty acid transport of skin, oral mucosa as well as other organs, and/or other pathologies/disorders.
NOV7 is homologous to members of the Gap junction beta-5 protein-like family of proteins. Thus, the NOV7 nucleic acids, polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Deafness, autosomal dominant 2, Elliptocytosis-1 , Fucosidosis, Hypophosphatasia (adult, childhood, infantile), Muscle-eye-brain disease, Neuropathy, paraneoplastic sensory, Porphyria cutanea tarda, Porphyria, hepatoerythropoietic, Schwartz- Jampel syndrome, Thrombocytopenia, congenital amegakaryocytic, Charcot-Marie-Tooth neuropathy-2A, Galactose epimerase deficiency, Glucose transport defect, blood-brain barrier, Kostmann neutropenia, Muscular dystrophy, congenital, with early spine rigidity, Myopathy due to succinate dehydrogenase deficiency, SCID due to LCK deficiency, Colorectal cancer, resistance to, Bartter syndrome, type 3, Breast cancer, ductal, Corneal dystrophy, crystalline, Schnyder, Hyperprolinemia, type II, Inflammatory bowel disease 7, Malignant melanoma, cutaneous, Neuroblastoma, Prostate cancer-brain cancer susceptibility, erythrokeratodermia variabilis, palmoplantar keratoderma, diseases and disorders involving intercellular metabolic and electrical communication, diseases and disorders involving coordination, proliferation and differentiation, diseases and disorders involving maintainance of tissue homeostasis, growth control, development, and synchronized response of cells to stimuli, diseases and disorders involving the the immune system, diseases and disorders involving regulation of bone cell differentiation, and/or other pathologies/disorders. NOV8 is homologous to the Metallothionein-like family of proteins. Thus, NOV8 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Gitelman syndrome, Menkes disease, Wilson's disease, acrodermatitis enteropathica, myelomonocytic leukemia, eosinophil disorders, hepatic disorders such as hepatic copper toxicity, and/or other pathologies/disorders.
NOV9 is homologous to the CDC-42 interacting protein 4-like family of proteins. Thus, NOV9 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; Wiskott-Aldrich syndrome, immunodeficiency, thrombocytopenia, eczema, lymphoid malignancy cytoskeletal abnormalities, impaired monocyte motility, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Fertility, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis , Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes, Pancreatitis, Obesity, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, ARDS, Cirrhosis, Transplantation, Diabetes, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, and/or other pathologies/disorders. NOVl 0 is homologous to the hepsin/plasma transmembrane serine protease/spinesin- like family of proteins. Thus, NOV 10 nucleic acids and polypeptides, antibodies and related compounds according to the invention will be useful in therapeutic and diagnostic applications implicated in, for example; cancer, trauma, tissue regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, immunological disease, respiratory disease, gastro-intestinal diseases, reproductive health, neurological and neurodegenerative diseases, bone marrow transplantation, metabolic and endocrine diseases, allergy and inflammation, nephrological disorders, cardiovascular diseases, muscle, bone, joint and skeletal disorders, hematopoietic disorders, urinary system disorders, Tissue and organ transplantation, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis , Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Scleroderma, Obesity, Hypertension, Fibromuscular dysplasia, Stroke, Aneurysm, Myocardial infarction, Embolism, Bypass surgery, Anemia , Bleeding disorders, Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Diabetes, Von Hippel-Lindau (VHL) syndrome, Pancreatitis, Hyperparathyroidism, Hypoparathyroidism, Hyperthyroidism and Hypothyroidism, S-IDS, Endometriosis, infertility, Xerostomia, Hypercalceimia, Ulcers, Cirrhosis, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Crohn's Disease, Appendicitis, Hemophilia, hypercoagulation, autoimmume disease,allergies, immunodeficiencies, transplantation, Graft vesus host disease (GVHD), Ataxia-telangiectasia, Autoimmume disease, Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic purpura, Immunodeficiencies, Lymphedema, Allergies, Hemophilia, hypercoagulation, idiopathic thrombocytopenic purpura, Lymphaedema, Tonsilitis, Osteoporosis, Hypercalceimia, Arthritis, Ankylosing spondylitis, Scoliosis, Tendinitis, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis, Dental disease and infection, Alzheimer's disease, Stroke,
Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy,Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Endocrine dysfunctions, Growth and reproductive disorders, Myasthenia gravis, Leukodystrophies, Pain, Neuroprotection, Systemiϋ lupus erythematosus, Autoimmune disease, Emphysema, Scleroderma, ARDS, Pharyngitis, Laryngitis, Asthma, Hearing loss, Tinnitus, Psoriasis, Actinic keratosis, Tuberous sclerosis, Acne, Hair growth, allopecia, pigmentation disorders, endocrine disorders, cystitis, incontinence, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Vesicoureteral reflux, and/or other pathologies/disorders.
The NOVX nucleic acids and polypeptides can also be used to screen for molecules, which inhibit or enhance NOVX activity or function. Specifically, the nucleic acids and polypeptides according to the invention may be used as targets for the identification of small molecules that modulate or inhibit, e.g., neurogenesis, cell differentiation, cell proliferation, hematopoiesis, wound healing and angiogenesis.
Additional utilities for the NOVX nucleic acids and polypeptides according to the invention are disclosed herein.
NOVl NOV I includes three novel zinc metal loprotease/ADAM-TS 7-Iilce proteins disclosed below. The disclosed sequences have been named NOVl a, NOVl b, NOV l c, and NOV ld. NOVl a A disclosed NOVl a nucleic acid of 2997 nucleotides (also referred to as SC138213196 _A) encoding a novel Zinc Metalloprotease-like protein is shown in Table 1 A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 10-12 and ending with a TAA codon at nucleotides 2968-2970. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table I A. The start and stop codons are in bold letters.
Table 1A. NOVla nucleotide sequence (SEQ ID NO:l).
CGCTCCTGGATGAAGCCCCGCGCGCGCGGATGGCGGGGCTTGGCGGCGCTGTGGATGCTGTTGGCGCAGGTG
GCCGAGCAGGTGAGTCCCGGGCGCTCCCACCAGCGCGGAAACCGCGGGTCCGGACAGCTGGAGGCGAGTCCC
CCGCGGCTCCTCTCCCGCGGACCCCGCCGTCTCACCGCGATGTCGCCGCTGTTTTCCGCAGGCACCTGCGTG
CGCCATGGGACCCGCAGCGGCAGCGCCTGGGAGCCCGAGCGTCCCGCGTCCTCCTCCACCCGCGGAGCGGCC
GGGCTGGATGGAAAAGGGCGGGACATGGATGAAGCTGGAAACCATCGTTCTCAGCAAACTAACACAGGAACA
GA-AAACCAAACACTGCATGTTCTCACTCAATATGACCTGGTCTCTGCCTACGAGGTTGACCACAGGGGCGAT
TACGTGTCCCATGAAATCATGCACCATCAGCGGCGGAGAAGAGCAGTGGCCGTGTCCGAGGTTGAGTCTCTT
CACCTTCGGCTGAAAGGCCCCAGGCACGACTTCCACATGGATCTGAGGACTTCCAGCAGCCTAGTGGCTCCT
GGCTTTATTGTGCAGACGTTGGGAAAGACAGGCACTAAGTCTGTGCAGACTTTACCGCCAGAGGACTTCTGT
TTCTATCAAGGCTCTTTGCGATCACACAGAAACTCGCCATCGCATGGAGGGAAGTTCTGTGAGGGCTCCACT
CGCACTCTGAAGCTCIGCAACAGTCAGAAATGTCCCCGGGACAGTGTTGACTTCCGTGCTGCTCAGTGTGCC
GAGCACAACAGCAGACGATTCAGAGGGCGGCACTACAAGTGGAAGCCTTACACTCAAGTAGAAGNNGACTTA
TGCAAACTCTACTGTATCGCAGAAGGATTTGATTTCTTCTTTTCTTTGTCAAATAAAGTCAAAGATGGGACT
CCATGCTCGGAGGATAGCCGTAATGTTTGTATAGATGGGATATGTGAGCTCAGTGTGGTGTCCACATCTGCG
CACATGCCCCAGCCTCCCAAGGAAGACCTCTTCATCTTGCCAGATGAGTATAAGTCTTGCTTACGGCATAAG
CGCTCTCTTCTGAGGTCCCATAGAAATGAAGAACTGAACGTGGAGACCTTGGTGGTGGTCGACAAAAAGATG
ATGCAAAACCATGGCGATGAAAATATCACCACCTACGTGCTCACGATACTCAACATGGTATCTGCTTTATTC
AAΛGATGGAACAATAGGAGGAAACATCAACATTGCAATTGTAGGTCTGATTCTTCTAGAAGATGAACAGCCA
GGACTGGTGATAAGTCACCACGCAGACCACACCTTAAGTAGCTTCTGCCAGTGGCAGTCTGGATTGATGGGG
AAAGATGGGACTCGTCATGACCACGCCATCTTACTGACTGGTCTGGATATATGTTCCTGGAAGAATGAGCCC
TGTGACACTTTGGGATTTGCACCCATAAGTGGAATGTGTAGTAAATATCGCAGCTGCACGATTAATGAAGAT
ACAGGTCTTGGACTGGCCTTCACCATTGCCCAIGAGTCTGGACACAACTTTGGCATGATTCATGATGGAGAA
GGGAACATGTGTAAAAAGTCCGAGGGCAACATCATGTCCCCTACATTGGCAGGACGCAATGGAGTCTTCTCC
TGGTCACCCTGCAGCCGCCAGTATCTACACAAATTTCTAAGCACCGCTCAAGCTATCTGCCTTGCTGATCAG
CCAAAGCCTGTGAAGGAATACAAGTATCCTGAGAAATTGCCAGGAGAATTATATGATGCAAACACACAGTGC
AAGTGGCAGTTCGGAGAGAAAGCCAAGCTCTGCATGCTGGACTTTAAAAAGGACATCTGTAAAGCCCTGTGG
TGCCATCGTATTGGAAGGAAATGTGAGACTAAATTTATGCCAGGAGCAGAAGGCACAATTTGTGGGCATGAC
ATGTGGTGCCGGGGAGGACAGTGTGTGAAATATGGTGATGAAGGCCCCAAGCCCACCCATGGCCACTGGTCG
GACTGGTCTTCTTGGTCCCCAΪGCTCCAGGACCTGCGGAGGGGGAGTATCTCATAGGAGTCGCCTCTGCACC
AACCCCAATCCATCGCATGGAGGGAAGTTCTGTGAGGGCTCCACTCGCACTCTGAAGCTCTGCAACAGTCAG
AAATGTCCCCGGGACAGTGTTGACTTCCGTGCTGCTCAGTGTGCCGAGCACAACAGCAGACGATTCAGAGGG
CGGCACTACAAGTGGAAGCCTCAGGACTTATGCAAACTCTACTGTATCGCAGAAGGATTTGATTTCTTCTTT
TCTTTGTCAAATAAAGTCAAAGATGGGACTCCATGCTCGGAGGATAGCCGTAATGTTTGTATAGATGGGATA
TGTGAGNTTGGATGTGACAATGTCCTTGGATCTGATGCTGTTGAAGACGTCTGTGGGGTGTGTAACGGGAAT
AACTCAGCCTGCACGATTCACAGGGGTCTCTACACCAAGCACCACCACACCAACCATTATCACATGGTCACC
ATTCCTTCTGGAGCCCGGAGTATCCGCATCTATGAAATGAACGTCTCTACCTCCTACATTTCTGTGCGCAAT
GCCCTCAGAAGGTACTACCTGAATGGGCACTGGACCGTGGACTGGCCCGGCCGGTACAAATTTTCGGGCACT
ACTTTCGACTACAGACGGTCCTATAATGAGCCCGAGAACTTAATCGCTACTGGACCAACCAACGAGACACTG
ATTGTGGAGCTGCTGTTTCAGGGAAGGAACCCGGGTGTTGCCTGGGAATACTCCATGCCTCGCTTGGGGACC
GAGAAGCAGCCCCCTGCCCAGCCCAGCTACACTTGGGCCATCGTGCGCTCTGAGTGCTCCGTGTCCTGCGGA
GGGGGTAGGTGCCTTCCAGTGCTGCTCCTGGAGGCAGCATGTCAGCCTTCAGCCACTGCGTACATTGCACTG GCCTTTCTTGAATCCTAATGAGCAGCCCGGNGCTTCTCCCTGCCA
In a search of public sequence databases, the NOV l a nudeic acid sequence, located on chromsome 5 has 250 of 375 bases (66%) identical to a zinc metalloprotease ADAMTS6 (ADAMTS6) mRNA from Homo sapiens (GENBANK.-ID: AF140674). Public nucleotide databases include all GenBank databases and the GeneScq patent database. In all BLAST alignments herein, the "E-value" or "Expect" value is a numeric indication of the probability that the aligned sequences could have achieved their similarity to the BLAST query sequence by chance alone, within the database that was searched. For example, the probability that the subject ("Sbjct") retrieved from the NOVl BLAST analysis, e.g., thioredoxin mRNA from Ovis aries, matched the Query NOVl sequence purely by chance is 9.4e ' . The Expect value (E) is a parameter that describes the number of hits one can "expect" to see just by chance when searching a database of a particular size. It decreases exponentially with the Score (S) that is assigned to a match between two sequences. Essentially, the E value describes the random background noise that exists for matches between sequences.
The Expect value is used as a convenient way to create a significance threshold for reporting results. The default value used for blasting is typically set to 0.0001. In BLAST 2.0, the Expect value is also used instead of the P value (probability) to report the significance of matches. For example, an E value of one assigned to a hit can be interpreted as meaning that in a database ofthe current size one might expect to see one match with a similar score simply by chance. An E value of zero means that one would not expect to see any matches with a similar score simply by chance. See, e.g., http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/, Occasionally, a string of X's or N's will result from a BLAST search. This is a result of automatic filtering o the query for low- complexity sequence that is performed to prevent artifactual hits. The filter substitutes any low-complexity sequence that it finds with the letter "N" in nucleotide sequence (e.g., ''NNNNNNNNNNNNN*') or the letter "X" in protein sequences (e.g., "XXXXXXXXX"). Low-complexity regions can result in high scores that reflect compositional bias rather than significant position-by-position alignment. (Wootton and Federhen, Methods Enzymol 266:554-571 , 1996).
The disclosed NOVl a polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:l has 986 amino acid residues and is presented in Table IB using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOVla has a signal peptide and is likely to be localized in extraceliularly with a certainty of 0.5469. In other embodiments, NOVl a may also be localized to the lysosome (lumen) with acertainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1297 or in the endoplasmic reticulum (membrane) with a certainty of , 1000. The most likely cleavage site for a NOVla peptide is between amino acids 26 and 27, at: VAE-QV. Table IB. Encoded NOVla protein sequence (SEQ ID NO:2).
MKPI-ARG RGLAAIiW IiLAQVAEQVSPGRSHQRGNRGSGQ EASPPRL SRGPRRIjTAMS PLFSAGTCVRHG TRSGSAWEPERPASSSTRGAAG DGKGRDMDEAGNHRSQQTNTGTENQTLHV-3TQYDLVSAYEVDHRGDYVΞ HEIMHHQRRRRAVAVSEVESLHbRLKGPRHDFHMDLRTSSSI VAPGFIVQT GKTGT SVQT PPEDFCFYQ GSLRSHRNSPSHGGKFCEGSTRTIiKLCNSQKCPRDSVDFRAAQCAEHNSRRFRGRHYKWKPYTQVEXDLCKb YCIAEGFDFFFSLSN VKDGTPCSEDSRNVCIDGlCELSWSTSAHMPQPPKEDLFILPDEYKSCLRHKRSli RSHRNEELNVET λπrv/DKKMMQNHGHENITTYVLTILNMVSArjFKDGTIGGNINXAIVGLiILLEDEQPG V ISHHADHTDSS FCQ QSGLMGKDGTRHDHAIL TGBDICS KNE PCDTIJGFAPI SGMCSKYRS CTINEDTGD
GLAFTIAHESGHNFGMIHDGEGNMC KSEGNIMSPTLAGR GVFS SPCSRQYLHKFLSTAQAICLADQPKP VKEYKYPEKLPGELYDANTQCK QFGEKAKLCMLDFKKDICKA WCHRIGRKCETKFMPAAEGTICGHDM C RGGQCVKYGDEGPKPTHGHWSDWSSWSPCSRTCGGGVSHRSRLCTNPNPSHGGKFCEGSTRTLKLCNSQKCP RDSVDFRAAQCAEHNSRRFRGRHYKWKPQD CKI-YCΪAEGFDFFFSLSNKVKDGTPCSEDSRNVCIDGICEX GGDNVLGSDAVEDVCG\/CNGNNSACTIHRGLYTKHHHTT.HYHMVTLPSGARSIRIYEMNVSTSYISVRNABR RYYLNGH TVDWPGRYKFSGTTFDYRRSYNEPEN IATGPTNETIJIVE LIFQGRNPGVA EYSMPR GTEKQ
PPAQPSYTWAIVRSECSVSCGGGRCLPVLIJ EAACQPSATAYIAI AFLES
A search of sequence databases reveals that the NOVl amino acid sequence has 257 of 579 amino acid residues (44%) identical to, and 356 of 579 amino acid residues (61%) similar to, the 997 amino acid residue Zinc Metalloprotease Adamts7 protein from Homo sapiens (Human) (Q9U P4) (E = 6.1 e"149), Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
NOVl a is expressed in the lung.
NOVlb A disclosed NOVlb nucleic acid of 2433 nucleotides (also referred to as
137043926_EXT_1) encoding a novel zinc metalloprotease-1-like protein is shown in Table 1 C. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 31-33 and ending with a TAA codon at nucleotides 2404-2406. A putative untranslated regions upstream from the initiation codon and downstream of the termination codon are underlined in Table IC. The start and stop codons are in bold letters.
Table IC. NOVlb nucleotide sequence (SEQ ID NO:3).
GTGGCCCCTAGCCCCTCGGAGCGCTCCTGGATGAAGCCCCGCGCGCGCGGATGGCGGGGCTTGGCGGCGCTG TGGATGCTGCTGGCGCAGGTGGCCGAGCAGGTGAGTCCCGGGCGCTCCCACCAGCGCGGAAACCGCGGGTCC GGACAGCTGGAGGCGAGTCCCCCGCGGCTCCTCTCCCGCGGACCCCGCCGTCTCACCGCGATGTCGCCGCTG
TTTTCCGCAGGCACCTGCGTGCGCCATGGGACCCGCAGCGGCAGCGCCTGGGAGCCCGAGCGTCCCGCGTCC
TCCTCCACCCGCGGAGCGGCCGGGCTGGATGGAAAAGGGCGGGACATGGATGAAGCTGGAAACCATCGTTCT
CAGCAAACTAACACAGGAACAGAAAACCAAACACTGCATGTTCTCACTCGTGAATATGACCTGGTCTCTGCC ACGAGGTTGACCACAGGGGCGATTACGTGTCCCATGAAATCATGCACCATCAGCGGCGGAGAAGAGCAGTG
GCCGTGTCCGAGGTTGAGTCTCTTCACCTTCGGCTGAAAGGCCCCAGGCACGACTTCCACATGGATCTGAGG ACTTCCAGCAGCCTAGTGGCTCCTGGCTTTATTGTGCAGACGTTGGGAAAGACAGGCACTAAGTCTGTGCAG
ACTTTACCGCCAGAGGACTTCTGTTTCTATCAAGGCTCTTTGCGATCACACAGAAACTCCCCATCGCATGGA GGGAAGTTCTGTGAGGGCTCCACTCGCACTCTGAAGCTCTGCAACAGTCAGAAATGTCCCCGGGACAGTGTT GACTTCCGTGCTGCTCAGTGTGCCGAGCACAACAGCAGACGATTCAGAGGGCGGCACTACAAGTGGAAGCCT TACACTCAAGTAGAACAGGACTTATGCAAACTCTACTGTATCGCAGAAGGATTTGATTTCTTCTTTTCTTTG CAAATAAAGTCAAAGATGGGACTCCATGCTCGGAGGATAGCCGTAATGTTTGTATAGATGGGATATGTGAG ATGCCCCAGCCTCCCAAGGAAGACCTCTTCATCTTGCCAGATGAGTATAAGTCTTGCTTACGGCATAAGCGC TCTCTTCTGAGGTCCCATAGAAATGAAGAACTGAACGTGGAGACCTTGGTGGTGGTCGACAAAAAGATGATG CAAAACCATGGCCATGAAAATAΪCACCACCTACGTGCTCACGATACTCAACATGGTATCTGCTTTATTCAAA GATGGAACAATAGGAGGAAACATCAACATTGCAATTGTAGGTCTGATTCTTCTAGAAGATGAACAGGACATC TGTAAAGCCCTGTGGTGCCATCGTATTGGAAGGAAATGTGAGACTAAATTTATGCCAGCAGCAGAAGGCACA ATTTGTGGGCATGACATGTGGTGCCGGGGAGGACAGTGTGTGAAATATGGTGATGAAGGCCCCAAGCCCACC CATGGCCACTGGTCGGACTGGTCTTCTTGGTCCCCATGCTCCAGGACCTGCGGAGGGGGAGTATCTCATAGG AGTCGCCTCTGCACCAACCCCAGGCCATGGCATGGAGGGAAGTTCTGTGAGGGCTCCACTCGCACTCTGAAG CTCTGCAACAGTCAGAAATGTCCCCGGGACAGTGTTGACTTCCGTGCTGCTCAGTGTGCCGAGCACAACAGC AGACGATTCAGAGGGCGGCACTACAAGTGGAAGCCTCAGGACTTATGCAAACTCTACTGTATCGCAGAAGGA TTTGATTTCTTCTTTTCTTTGTCAAATAAAGTCAAAGATGGGACTCCATGCTCGGAGGATAGCCGTAATGTT TGTATAGATGGGATATGTGAGGGATGTGACAATGTCCTTGGATCTGATGCTGTTGAAGACGTCTGTGGGGTG TGTAACGGGAATAACTCAGCCTGCACGATTCAGAGGGGTCTCTACACCAAGCACCACCACACCAACTATTAT CACATGGTCACCATTCCTTCTGGAGCCCGGAGTATCCGCATCTATGAAATGAACGTCTCTACCTCCTACATT CTGTGCGCAATGCCCTCAGAAGGTACTACCTGAATGGGCACTGGACCGTGGACTGGCCCGGCCGGTACAAA TTTTCGGGCACTACTTTCGACTACAGACGGTCCTATAATGAGCCCGAGAACTTAATCGCTACTGGACCAACC AACGAGACACTGATTGTGGAGCTGCIGTTTCAGGGAAGGAACCCGGGTGTTGCCTGGGAATACTCCATGCCI* CGCTTGGGGACCGAGAAGCAGCCCCCTGCCCAGCCCAGCTACACTTGGGCCATCGTGCGCTCTGAGTGCTCC GTGTCCTGCGGAGGGGGTAGGTGCCTTCCAGTGCTGCTCCTGGAGGCAGCATGTCAGCCTTCAGCCACTGCG TACATTGCACTGGCCTTTCTTGAATCCTAATGAGCAGCCCGGGGCTTCTCCCTGGCA
In a search of public sequence databases, the NOVlb nucleic add sequence, located on chromsome 5 has 101 of 126 bases (80%) identical to agb:GENBANK- ID:HSA400877|acc:AJ400877.1 mRNA from Homo sapiens (Homo sapiens ASCL3 gene, CEGPl gene, CI l orfl 4 gene, Cl l orfl 5 gene, Cl l orflό gene and CI lorfl 7 gene) (E = 2.3e"7). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
The disclosed NOVlb polypeptide (SEQ ID NO:4) encoded by SEQ ID NO:3 has 791 amino acid residues and is presented in Table ID using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOVlb has a signal peptide and is likely to be localized in extracellularlye with a certainty of 0.5469. In other embodiments, NOVlb may also be localized to the lysosome (lumen) with acertainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1 144 or in the endoplasmic reticulum (membrane) with a certainty of 0.1000. The most likely cleavage site for a NOVlb peptide is between amino acids 23 and 24, at: VAE-QV.
Table ID. Encoded NOVlb protein sequence (SEQ ID NO:4).
MKPRARGWRGLAA M--j AQVAEQVSPGRSHQRGNRGSGQLEASPPRLLSRGPRR] TAMSPLFSAGTCVRHG TRSGSAWEPERPASSSTRGAAGLDGKGRDMDEAGNHRSQQTNTGTENQT HV TREYD-aVSAYEVDHRGDYV SHEI HHQRRRRAVAVSEVESLHLRLKGPRHDFHMDIJRTSSSLVAPGFIVQT G TGTKSVQTLIPPEDFCFY
QGS RSHRNSPSHGGKFCEGSTRTLK CNSQKCPRDSVDFRAAQCAEHNSRRFRGRHYK KPYTQVEQDLCK LYCIAEGFDFFFSLSNKVKDGTPCSEDSRNVCIDGICEMPQPP EDLFILPDEYKSC RHKRSIJL-JRSHRNEE I-WETLVVVDKKMMQNHGHENITTYV TI NMVSA-IJFKDGTIGGNLNIAIVG-LJI IJEDEQDICKAL CHRIG RKCETKFMPAAEGTICGHDMWCRGGQCVKYGDEGPKPTHGHWΞDWSSWSPCSRTCGGGVSHRSRLCTNPRPS HGGKFCEGSTRTIJKX.CNSQKCPRDSVDFRAAQCAEHI-LSRRFRGRHYK KPQD CKIJYCIAEGFDFFFS SNK VKDGTPCSEDSR VCIDGICEGCDNV GSDAV-EDVCGVCNGNNSACTIHRGLYTKHHHTNYYHMVTIPSGAR SIRIYEMNVSTSYISVRNAL--TRRYYIJNGH TVD PGRYKFSGTTFDYRRSYNEPEN IATGPTNET IVE--AI-AF QGRNPGVAWEYSMPRLGTEKQPPAQPSYTWAIVRSECSVSCGGGRCLPVLLLEAACQPSATAYIALAFLES
A search of sequence databases reveals that the NOVlb amino acid sequence has 152 of 357 amino acid residues(42%) identical to, and 216 of357 amino acid residues (60%) similar to, the 860 amino acid residue ptnr'SWISSNEW-ACC:Q9UKP5 protein from Homo sapiens (Human) (ADAM-TS 6 precursoi (EC 3.4.24,-) (A Disintegrin And Metalloprotcinasc With Thrombospondin Motifs 6) (ADAMTS-6) (ADAM-TS6) (E = 4.8e'101). Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
NOVlb is expressed in at least the following tissues: brain, liver, spleen, uterus, colon, tonsil, lung, germ cells. This information was derived by detennining the tissue sources of the sequences that were included in the invention including but not limited to SeqCalling sources, Public EST sources, Genomic Clone sources, Literature sources, and/or RACE sources.
NOVlc
In the present invention, the target sequence identified previously, NOVl a, was subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case of the reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Corporation's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for corrections if appropriate. These procedures provide the sequence reported below, which is designated NOVl c. This differs from the previously identified sequence NOVla in having a different N-terminus
A disclosed NOVlc nucleic acid of 2902 nucleotides (also referred to as CG52952-03) encoding a novel ADAM-TS 7-like protein is shown in Table I E. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 182-184 and ending with a TAA codon at nucleotides 2750-2752. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table IE. The start and stop codons are in bold letters.
Table I E. NOVlc nucleotide sequence (SEQ ID NO:5).
TTGGCGGCGCTGTGGATGCTGCTGGCGCAGGTGGCCGAGCAGGTGAGTCCCGGGCGCTCCCACCAGCGCGGA AACCGCGGGTCCGGACAGCTGGAGGCGAGTCCCCCGCGGCTCCTCTCCCGCGGACCCCGCCGTCTCACCGCG ATGTCGCCGCTGTTTTCCGCAGGCACCTGCGTGCGCCATGGGACCCGCAGCGGCAGCGCCTGGGAGCCCGAG CGTCCCGCGTCCTCCTCCAGACCTGGTCTCTGCCTACGAGGTTGACCACAGGGGCGATTACGTGTCCCATGA AATCATGCACCATCAGCGGCGGAGAAGAGCAGTGGCCGΪGTCCGAGGTTGAGCCAGCCTTTCTCCAGGTATG CAGAGCCAGAGAGCTCAGACTGTGTGTGGAGGCCTTTCCCATTGCTAATTCTCAGCCGGGGTTTTTGAACCT TTCCAATGTTCGCTCTCACTGGAGGGAACAGCATGCTTCCAAGAGAATAATAACAAATGCAATGCTTGGAGA ATCGGCCCTGGCTTCAACCAGAAAGTCTAATTGTGTTTTCTTTCTTTCCTTTTATTTTTTCCAGTCAGGCAT GATACGAACAGAAGAGGCAGATTACTTCCTAAGGCCACTTCCTTCACACCTCTCATGGAAACTCGGCAGAGC TGCCCAAGGCAGCTCGCCATCCCACGTACTGTACAAGAGAGAGGTCCTGGTGACCTCAAGGACATGGGAGCT GGCACATCAACCCCTGCACAGCAGCGACCTTCGCCTGGGACTGCCACAAAAGCAGCATTTCTGTGGAAGACG CAAGAAATACATGCCCCAGCCTCCCAAGGAAGACCTCTTCATCTTGCCAGATGAGTATAAGTCTTGCTTACG GCATAAGCGCTCTCTTCTGAGGTCCCATAGAAATGAAGAACTGAACGTGGAGACCTTGGTGGTGGTCGACAA AAAGATGATGCAAAACCATGGCCATGAAAATATCACCACCTACGTGCTCACGATACTCAACATGGTATCTGC TTTATTCAAAGATGGAACAATAGGAGGAAACATCAACATTGCAATTGTAGGTCTGATTCTTCTAGAAGATGA ACAGCCAGGACTGGTGATAAGTCACCACGCAGACCACACCTTAAGTAGTTTCTGCCAGTGGCAGTGTGGATT GATGGGGAAAGATGGGACTCGTCATGACCACGCCATCTTACTGACTGGTCTGGATATATGTTCCTGGAAGAA TGAGCCCTGTGACACTTTGGGATTTGCACCCATAAGTGGAATGTGTAGTAAATATCGCAGCTGCACGATTAA TGAAGATACAGGTCTTGGACTGGCCTTCACCATTGCCCATGAGTCTGGACACAACTTTGGCATGATTCATGA TGGAGAAGGGAACATGTGCAAAAAGTCCGAGGGCAACATCATGTCCCCTACATTGGCAGGACGCAATGGAGT CTTCTCCTGGTCACCCTGCAGCCGCCAGTATCTACACAAATTTCTAAGCACCGCTCAAGCTATCTGCCTTGC TGATCAGCCAAAGCCTGTGAAGGAATACAAGTATCCTGAGAAATTGCCAGGAGAATTATATGGTGCAAACAC ACAGTGCAAGTGGCAGTTCGGAGAGAAAGCCAAGCTCTGCATGCTGGACTTTAAAAAGGACATCTGTAAAGC CCTGTGGTGCCATCGTATTGGAAGGAAATGTGAGACTAAATTTATGCCAGGAGCAGAAGGCACAATTTGTGG GCATGAACATGGTGCCGGAGGACAGTGTGTGAAATATGGTGATGAAGGCCCCAAGCCCACCCATGGCCACTG GTCGGACTGGTCTTCTTGGTGCCCATGCTCCAGGACCTGCGGAGGGGGAGTATCTCATAGGAGTCGCTCTCA AAATACACATTCCAGGCCATCGCATGGAGGGAAGTTCTGTGAGGGCTCCACTCGCACTCTGAAGCTCTGCAA CAGTCAGAAATGTCCCCGGGACAGTGTTGACTTCCGTGCTGCTCAGTGTGCCGAGCACAACAGCAGACGATT CAGAGGGCGGCACTACAAGTGGAAGCCTGATCAGGACTTATGCAAACTCTACTGTATCGCAGAAGGATTTGA TTTCTTCTTTTCTTTGTCAAATAAAGTCAAAGATGGGACTCCATGCTCGGAGGATAGCCGTAATGTTTGTAT AGATGGGATATGTGAGAGAGTTGGATGTGACAATGTCCTTGGATCTGATGCTGTTGAAGACGTCTGTGGGGT GTGTAACGGGAATAACTCAGCCTGCACGATTCACAGGGGTCTCTACCTAGAGTATTATCACATGGTCACCAT TCCTTCTGGAGCGCGGAGTATCCGCATCTATGAAATGAACGTCTCTACCTCCTACATTTCTGTGCGCAATGC CCTCAGAAGGTACTACCTGAATGGGCACTGGACCGTGGACTGGCCCGGCGGGTACAAATTTTCGGGCACTAC TTTCGACTACAGACGGTCCTATAATGAGCCCGAGAACTTAATCGCTACTGGACCAACCAAGGAGACACTGAT TGTGGAGCTGCTGTTTCAGGGAAGGAACCCGGGTGTTGCCTGGGAATACTCCATGCCTCGCTTGGGGACCGA GAAGCAGCCCCCTGCCCAGCCCAGGTACACTTGGGCCATCGTGCGCTCTGAGTGCTCCGTGTCCTGCGGAGG GGGTAGGTGCCTTCCAGTGCTGCTCCTGGAGGCAGCATGTCAGCCTTTAGCCACTGCGTACATTGCACTGGC CTTTCTTGAATCCTAATGAGCAGCCCGGGGCTTCTCCCTGCCAGTAGCAGTGACATTCCCAAGGTGGGGAGT GGTGGTCCTGAGTGTCACTTGTCGGCCCGAGCTGCCT'TC'rCCAGTCTATCTGCTTCAGTGTGTGACTCTGAG GAAGTCAGTAGATGCATTGCTT
In a search of public sequence databases, the NOVl c nucleic acid sequence, located on chromsome 5 has 646 of 1089 bases (59%) identical to a gb:GENBAN - !D:AFl40675|acc:AF140675 1 mRNA from Homo sapiens (Homo sapiens zinc metalloprotease ADAMTS7 (ADAMTS7) mRNA, complete eds) (E - 1.0e"23). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
The disclosed NOVl c polypeptide (SEQ ID NO:6) encoded by SEQ ID NO:5 has 856 amino acid residues and is presented in Table I F using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOV l c has no signal peptide and is likely to be localized in the nucleus with a certainty of 0.7000. In other embodiments, NOVl c may also be localized to the microbody (peroxisome) with a certainty of 0.3813, the mitochondrial matrix space with a certainty of 0, 1000, or in the lysosome (lumen) with a certainty of 0.1000.
Table I F. Encoded NOVlc protein sequence (SEQ ID NO:6).
MGPAAAAPGSPSVPRPPPDIjVSAYEVDHRGDYVSHEIMHHQRRRRAVAVSEVEPAFLQVCRARELR CVEAF PIA SQPGFLNbSNVRSHWREQHASKRIITNAMLGESArjASTRKSNCVFFLSFYFFQSGMIRTEEADYF RP PSHLSWKLGRAAQGSSPSHVLYKREVLVTSRTWE AHQP--jHSSD RL)G--jPQKQHFCGRRKKYMPQPPKEDL. FILPDEYKSCLRHKRS--J RSHRNEELNVETLVVVDKKMMQNHGHENITTYVLTIIJNMVSAI1F DGTIGGNII. IAIVGLILLEDEQPGriVISHHADHT SSFCQWQSG MGKDGTRHDHAIIjIiTG DrCS KNEPCDTIiGFAPIS GMCSKYRSCTI--.EDTGLG AFTIAHESGHMFG IHDGEGNMC KSEGNIMSPTLAGR GVFSWSPCSRQYI1H KF STAQAICLADQPKPVKEYKYPEKljPGELYGANTQCK QFGEKAKLCM DFKK-DICKAIi CHRlGRKCET KFMPAAEGTICGHEHGAGGQCVKYGDEGPKPTHGH SD SSWSPCSRTCGGGVSHRSRSQNTHSRPSHGGKF CEGSTRTLK CNSQKCPRDSVDFRAAQCAEHNSRRFRGRHYKWKPDQDLCKLYCIAEGFDFFFSLΞNKVKDG TPGSEDSRNVCIDGICERVGCD VI GSDAVEDVCGVCNGNNSACTIHRGLY EYYH VTIPSGARSIRIYEM VSTSYrSVRNALRRYYLNGH TVDWPGRYKFSGTTFDYRRSYNEPENl- lATGPTWETrjIVEI- LFQGRNPGV A EYSMPRbGTEKQPPAQPSYT AIVRSECSVSCGGGRCLPV- LEAACQPI-ATAYIAI-AFI-ES
A search of public sequence databases reveals that the NOVlc amino acid sequence has 256 of 579 amino acid residues (44%) identical to, and 352 of 579 amino acid residues (60%) similar to, the 997 amino acid residue ptnr;SWISSNEW-ACC:Q9UKP4 protein from Homo sapiens (Human) (ADAM-TS 7 precursor (EC 3.4.24.-) (A Disintegrin And
Metalloproteinase With Thrombospondin Motifs 7) (ADAMTS-7) (ADAM-TS7) (E = 1.9β" 1 ). Public amino acid databases include the GenBank databases, SwissProt, PDB and PER..
NOVl c is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. Expression information was derived from the tissue sources of the sequences that were included in the derivation of the sequence of NOVlc.
NOVl d
A disclosed NOVl d nucleic acid of 2895 nucleotides (also referred to as CG52952-04) encoding a novel ADAM-TS 7-like protein is shown in Table I G. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 10-12 and ending with a TAA codon at nucleotides 2866-2868. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table I G. The start and stop codons are in bold letters. Table IG. NOVl d nucleotide sequence (SEQ ID NO:7).
CGCTCCTGGATGAAGCCCCGCGCGCGCGGATGGCGGGGCTTGGCGGCGCTGTGGATGCTGTTGGCGCAGGTG
GCCGAGCAGGTGAGTCCCGGGCGCTCCCACCAGCGCGGAAACCGCGGGTCCGGACAGCTGGAGGCGAGTCCC
CCGCGGCTCCTCTCCCGCGGACCCCGCCGTCTCACCGCGATGTCGCCGCTGTTTTCCGCAGGCACCTGCGTG
CGCCATGGGACCCGCAGCGGCAGCGCCTGGGAGCCCGAGCGTCCCGCGTCGTCCTCCACCCGCGGAGCGGCC
GGGCTGGATGGAAAAGGGCGGGACATGGATGAAGCTGGAAACCATCGTTCTCAGCAAACTAACACAGGAACA
GAAAACCAAACACTGCATGTTCTCACTCAATATGACCTGGTCTCTGCCTAGGAGGTTGACCACAGGGGCGAT
TACGTGTCCCATGAAATCATGCACCATCAGCGGCGGAGAAGAGCAGTGGCCGTGTCCGAGGTTGAGTGTCTT
CACCTTCGGCTGAAAGGCCCCAGGCACGACTTCCACATGGATCTGAGGACTTCCAGCAGCCTAGXGGCTCCT
GGCTTTATTGTGCAGACGTTGGGAAAGACAGGCACTAAGTCTGTGCAGACTTTACCGCCAGAGGACTTCTGT TTCTATCAAGGCTCTTTGCGATCACACAGAAACTCGCCATCGCATGGAGGGAAGTTCTGTGAGGGCTCCACT
CGCACTCTGAAGCTCTGCAACAGTCAGAAATGTCCCCGGGACAGTGTTGACTTCCGTGCTGCTCAGTGTGCC GAGCACAACAGCAGACGATTCAGAGGGCGGCACTACAAGTGGAAGCCTTACACTCAAGTAGAAGCCGACTTA TGCAAACTCTACTGTATCGCAGAAGGATTTGATTTCTTCTTTTCTTTGTCAAATAAAGTCAAAGATGGGACT CCATGCTCGGAGGATAGCCGTAATGTTTGTATAGATGGGATATGTGAGCTCAGTGTGGTGTCCACATCTGCG CACATGCCCCAGCCTCCCAAGGAAGACCTCTTCATCTTGCCAGATGAGTATAAGTCTTGCTTACGGCATAAG CGCTCTCTTCTGAGGTCCCATAGAAATGAAGAACTGAACGTGGAGACCTTGGTGGTGGTCGACAAAAAGATG ATGCAAAACCATGGCCATGAAAATATCACCACCTACGTGCTCACGATACTCAAGATGGTATCTGCTTTATTC AAAGATGGATTGATGGGGAAAGATGGGACTCGTCATGACCACGCCATCTTACTGACTGGTCTGGATATATGT TCCTGGAAGAATGAGCCCTGTGACACTTTGGGATTTGCACCCATAAGTGGAATGTGTAGTAAATATCGCAGC TGCACGATTAATGAAGATACAGGTCTTGGACTGGCCTTCACCATTGCCCATGAGTCTGGACACAACTTTGGC ATGATTCATGATGGAGAAGGGAACATGTGTAAAAAGTCCGAGGGCAACATCATGTCCCCTACATTGGCAGGA CGCAATGGAGTCTTCTCCTGGTCACCCTGCAGCCGCCAGTATCTACACAAATTTCTAAGCACCGCTCAAGCT ATCTGCCTTGCTGATCAGCCAAAGCCTGTGΛAGGAATACAAGTATCCTGAGAAATTGCCAGGAGAATTATAT GATGCAAACACACAGTGCAAGTGGCAGTTCGGAGAGAAAGCCAAGCTCTGCATGCTGGACTTTAAAAAGGAC ATCTGTAAAGCCCTGTGGTGCCATCGTATTGGAAGGAAATGTGAGACTAAATTTATGCCAGCAGCAGAAGGC ACAATTTGTGGGCATGACATGTGGTGCCGGGGAGGACAGTGTGTGAAATATGGTGATGAAGGCCCCAAGCCC ACCGATGGCCACTGGTCGGACTGGTCTTCTTGGTCCCCATGCTCCAGGACCTGCGGAGGGGGAGTATCTCAT AGGAGTCGCCTCTGCACCAACCCCAAGCCATCGCATGGAGGGAAGTTCTGTGAGGGCTCCACTCGCACTCTG AAGCTCTGCAACAGTCAGAAATGTCCCCGGGACAGTGTTGACTTCCGTGCTGCTCAGTGTGCCGAGCACAAC AGCAGACGATTCAGAGGGCGGCACTACAAGTGGAAGCCTTACACTCAAGTAGAAGATCAGGACTTATGCAAA CTCTACTGTATCGCAGAAGGATTTGATTTCTTCTTTTCTTTGTCAAATAAAGTCAAAGATGGGACTCCATGC TCGGAGGATAGCCGTAATGTTTGTATAGATGGGATATGTGAGAGAGTTGGATGTGAC-AATGTCCTTGGATCT GATGCTGTTGAAGACGTCTGTGGGGTGTGTAACGGGAATAACTCAGCCTGCACGATTCACAGGGGTCTCTAC ACCAAGCACCACCACACCAACCAGTATTATCACATGGTCACCATTCCTTCTGGAGCCCGGAGTATCCGCATC TATGAAATGAACGTCTCTACCTCCTACATTTCTGTGCGCAATGCCCTCAGAAGGTACTACCTGAATGGGCAC TGGACCGTGGACTGGCCCGGCCGGTACAAATTTTCGGGCACTACTTTCGACTACAGACGGTCCTATAATGAG CCCGAGAACTTAATCGCTACTGGACCAACCAACGAGACACTGATTGTGGAGCTGCTGTTTCAGGGAAGGAAC CCGGGTGTTGCCTGGGAATACTCCATGCCTCGCTTGGGGACCGAGAAGCAGCCCCCTGCCCAGCCCAGCTAC ACTTGGGCCATCGTGCGCTCTGAGTGCTCCGTGTCCTGCGGAGGGGGTAGGTGCCTTCCAGTGCTGCTCCTG GAGGCAGCATGTCAGCCTTCAGCCACTGCGTACATTGCACTGGCCTTTCTTGAATCCTAATGAGCAGCCCGG GGCTTGTCCCTGCCA
In a search of public sequence databases, the NOVl d nucleic acid sequence, located on chromsome 5 has 380 of 614 bases (61%) identical to a gb:GENBANK- ID:AF 140675(acc:AF140675.1 mRNA from Homo sapiens (Homo sapiens zinc metalloprotease ADAMTS7 (ADAMTS7) mRNA, complete eds) (E - 7.0e"16). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
The disclosed NOV l d polypeptide (SEQ ID NO'8) encoded by SEQ ID NO:7 has 952 amino acid residues and is presented in Table 1 H using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOVl d has a signal peptide and is likely to be localized extracellualrly with a certainty of 0.3700. In other embodiments, NOVl d may also be localized to the lysosome (lumen) with a certainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1270, or in the endoplasmic reticulum (membrane) with a certainty of Ori OOO. The most likely cleavage site for a NOVl a peptide is between amino acids 23 and 24, at: VAE-QV.
Table I H. Encoded NOVld protein sequence (SEQ ID NO:8).
MKPRARGWRGLAALWMLLAQVAEQVSPGRSHQRGNRGSGQLEASPPRLLSRGPRRLTAMSP FSAGTCVRHG TRSGSA EPERPASSSTRGAAGLDGKGRDMDEAGNHRSQQTNTGTENQT HVbTQYD VSAYEVDHRGDYVS HEIMHHQRRRRAVAVSEVESLHLRI KGPRHDFHMD RTSSSΓIVAPGFIVQT GKTGTKSVQTLPPEDFCFYQ GSLRSHRNSPSHGGKFCEGSTRT KIJCNSQKCPRDSVDFRAAQCAEHNSRRFRGRHYK KPYTQVEAD CKIJ YCIAEGFDFFFSLΞNKVKDGTPCSEDSRNVCIDGICELSWSTSAHMPQPPKEDDFI PDEYKSCLRHKRSI) RSH NEE ^VETj- VVDKKMMQNHGHENrTTY X)TI^JNMVSA FKD l-JMGKDGTRHDHAIlJLTGEDICSWK NEPCDTI-GFAPISGMCSKYRSCTINEDTG GLAFTIAHESGHNFGMIHDGEGNMCKKSEGNIMSPTI-AGRNG VFS SPCSRQY HKFLSTAQAICLADQP PVKEYKYPE --JPGE YDANTQGK QFGEKAKLCM1-JDFKKIIICK AL CHRIGRKCETKFMPAAEGTICGHDMWCRGGQCVKYGDEGPKPTHGH SDWSSWSPCSRTCGGGVSHRSR IjCTNP PSHGGKFCEGSTRT KLCNSQKCPRDSVDFRAAQCAEHNSRRFRGRHYK KPYTQVEDQD CKLYC IAEGFDFFFSLSNKVKDGTPCSEDSRNVCIDGICERVGCDNV GSDAVEDVCGVCNGNNSACTIHRGLYTKH HHTNQYYHMVTIPSGARSIRIYEMNVSTSYISVRNAIjRRYY NGH TVDWPGRYKFSG FTFDyRRSYNEPEN LIATGPTNETMVEJ-j FQGRNPGVAWEYSMPRLGTEKQPPAQPSYT AIVRSECSVSCGGGRCLPVLL EAA CQPSATAYIAI-AFI-ES
A search of public sequence databases reveals that the NOVld amino acid sequence has 207 of 483 amino acid residues (42%) identical to, and 287 of 483 amino acid residues (59%) similar to, the 997 amino acid residue ptnr:SWISSNEW-ACC:Q9UKP4 protein from Homo sapiens (Human) (ADAM-TS 7 precursor (EC 3.4.24.-) (A Disintegrin And Metalloproteinase With Thrombospondin Motifs 7) (ADAMTS-7) (ADAM-TS7)) (E = 7.0e" lb). Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
NOVld is expressed in at least the following tissues: adrenal gland, bone marrow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea and uterus. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOVld and the expression pattern of (GENBANK-ID: gb:GENBAN -ID:AF140675|acc:AF140675.1) a closely related Homo sapiens zinc metalloprotease ADAMTS7 (ADAMTS7) mRNA, complete eds homolog in species Homo sapiens.
The proteins encoded by the NOVl a, lb, lc, and Id nucleotides are very closely homologous as is shown in the alignment in Table 1 1. TaqMan data for NOVl can be found below in Example 2. SNP data for NOVl a can be found below in Example 3.
Table 11 Alignment of NOVl a, 1 b, 1 c, and 1 d.
10 20 30 40 50
I • • • ■ I
NOVla J 1KPRARG RG AALWML AQVAEQVSPGRSHQRGNRGSGQLEASPPR LS NOVlb IKPRARGWRGLAALWMLLAQVAEQVSPGRSHQRGNRGSGQLEASPPRL
NOVld IKPRARGWRGLAALWMLLAQVAEQVSPGRSHQRGNRGSGQLEASPPRLL!.
NOVlc - MgPAAgAg-
60 70 90 100
NOVla OPRRLTAMSPLFSAGTCVRHGTRSGSAWEPERPASS- NOVlb JGPRRLTAMSPLFSAGTCVRHGTRSGSAWEPERPASSSTRGAAGLDGKGR NOVld GPRRLTAMSPLFSAGTCVRHGTRSGSAWEPERPASSSTRGAAGLDGKGR NOVlc (jjgPSVgRPjgP-
110 120 130 140 150
NOVla MDEAGNHRSQQTNTGTENQTLHVLTTOYD VSAYEVDHRGDYVSHEIM NOVlb DEAGNHRSQQTNTGTENQTLHVL-fflYDIiVSAYEVDHRGpYySHEIiyil NOVld MPEAGNHRSQQT TGTENQTLHV TBΫDLVSAYEVDHRGDYVSHEIMI NOVlc Miy-cfty-----! i/DHRGDYVSHE IMI
Figure imgf000023_0001
210 220 230 240 250
NOVla GTKSVQT PβPEDFCFYQGSLRSHRNSPSHGGKECEGSTRT KL'CNSQI NOVlb GTKSVQ LpBPEDFCFYQGS RSH-^SPSHGGKFCEGSTRtlJRLCNSQ NOVld wriemnms- iψ .v^ tXK S; RSHRNSPSHGGKFCEGSTR LKLCNSQI NOVlc HWREQHAS KRI ITNAMLGES. L0SgTgK§ NCVgFL-S FYFFQgjGM
260 270 280 290 300
NOVla .{M-aawwiB-MwiTtTgwa-Hiiiia;-^ ι:«Λ't«»iiaoa»- NOVlb PRDSyDFRAAQCAEHNSRRFRGRHΫ WKPYTQVEB X CKLYCIAEGF NOVld PRDSVDFRAAQCAEHNSRRFRGRHY HKPYTQVEg .LCKLYCIAEGFD NOVlc IRTEEAgYF RPIjPSgLg KLGRAAQGSSgSHVLYKREV VTSRTWE AH 310 320 330 340 350
I I
NOVla FFSIJSN VKDGTPCSEDSRNVCΓDGICE SWSTS,? MPQPPKEDLFILP NOVlb FFSLSNKV D'GTPGSEDSR VCIDGICEL MPQPPKEDLFILP NOVld FFS SNKVKDGTPCSEDSR VCIDGICEI θWSTS; MPQPPKEDLFILP' NOVlc QPIjHgSDLRL|§LJ§QKQHFCGRRKK - ~ - MPQPPKEDI-iFILP
360 370 380 390 400
NOVla DEYKSGLRHKRSLLRSHRNEELNVETLVVVDKKMMQ HGHENITTYVLTI NOVlb pEYksCLRH RSLLRSHRNEELOTETLVV^ NOVld DEYKSCLRHKRSLLRSHRiJEELNVET VVVDKK IMQNH.GHENITTYVLT:i NOVlc DEYKSCLRH RSELRSHRNEELNVETLVVVDKKMMQNHGHENITT.YVLTI
410 420 430 440 450
1 1 1 1 ( 1 1
NOVla LN VSAI-FKDGTr< NINrAIVGLILLEDEQPGLVISHHADHTLSSFCQW
NOVlb LNMVSALFKDGTK NOVld LNMVSALFKDGgjJj( NOVlc LNMVSALFKDGTII ININIAIVGLILLEDEQPGLVΪSHHADHTIiSSFCQW 460 470 480 490 500
NOVla QSGLMGKg GTRHDHAILLTGLDICSWKNEPCDTLGFAPISGMCSKYRSCT
NOVlb
NOVld DGT----D--AILLTGLDICSWKNEPCDTLGFAPISGMCSKYRSCT MO VIC QSGLMG. DGTRHDHAIL'LTGLDΪCS KNEPCDTLGFAPISGMCSKYRSCT
510 520 530 540 550
NOVla I I
INEDTGLGLAFTIAHESGHMFGMIHDGEGNMCKKSEGNIMSPTLAGRN NOVlb NOVld NOVlc
Figure imgf000024_0001
550 570 580 590 600
NOVla ""SDEEJIS3! NOVlb -UAIVG- NOVld SWSPCSRQYLHKFLSTAQAICLADQPKPVKEYKYPEKLPGELYFFLA T NOVlc SWSPCSRQYLHKFLSTAQAICLADQPKPVKEYKYPEKLPGELY@ANTQ<
610 620 630 640 650
NOVla CWOFGEKAKLCMED KKDICKAL eHRIGRKGETKFMPAAEGTrCGHDMK NOVlb _ ^ ^'!)tϊ!lDICKA CH lGR:KCΕT,KFMP EGTr-CGHDMW NOVld - QFGEIAKLeMLDFKKDIGKALWCHRrGRKC TKF EAAEGTICGHDMg NOVlc OTOFGEKAKLCMEDF tαDICϊ-ALWGHRIGRKeETKFMPAAEGTΪCGH' ΉG
660 670 680 690 700
.. II ..
NOVia :RGGQCVKYGDEGPKPTHGHWSDWSSWSPCSRTCGGGVSHRSR LCTNP
NOVlb RGGQCVKYGDEGPKPTHGH SDWSS SPCSRTCGGGVSHRSRLI-JCTNP NOVld RGGQCVKYGDEGPKPTHGH SD SS SPCSRTCGGGVSHRSRJLQTNP NOVlc A- SGOCVKYGDEGPKPTHGHWSDWSSWSPCSRTCGGGVSHRSL SQNgjHSR 710 720 730 740 750
NOVla PSHGGKFCEGS RT KLCNSQKCPRDSVDFR-AAQeAEH SRRFRGRHYK! NOVlb PSHGGKFCEGSTRTL LC SQKCPRDSVDERAAQCAEHNSRRER:GRHYKi; NOVld PSHGGKFCEGSTRTLKLCNSQKCPRDSVDFRAAQCAEHNSRRFRGRHYK-W NOVlc PSHGGKFCEGSTRTLKLCMSQKCPRDSVDFRAAQCAEHNSRRFRGRHYK-W
760 770 780 790 300 ..I.. ..I..
JDLCKLYCIAEGFDFFFSLSNKVKD-GTPCSEDSR VeieGIC DLCKLYCIAEGFbFFFSLSNKVKDGTPCSED-SRNVCIDGIC DLCKLYCIAEGFDFFESLSNKVKDG PC'SEDSRNVCΪDGIG
Figure imgf000024_0003
DLCKLYCIAEGFbFFFSLSNKVKDG PGSEbsRNVCIDGIG
NOVla NOVlb
NOVld NOVlc
Figure imgf000024_0002
860 370 890 900
NOVla PSGARSIRIYEMNVSTSYISVRNALRRYYLNGHWTVDWPGRYKFSGTTFD NOVlb PSG-ARSΪRIYEMNVSTSYISVrøALRRYYLNGH TVD PGRYKFSGTTFD NOVld PSGARSIRI ElNVSTSYISVI^NA ;RR GH T E)WEGRYKFSGT F NOVlc PSGARSIRIYEiy--^S SYI;SVRNALRRYYLNGHWTVD PGRYKFSGTTFB
910 920 930 940 950
NOVla YRRSYNEPE LIATGPTNETLIVELLFQGRNPGVAWEYSMPREGTEKQPP NOVlb YRRSYNEPENLIATGPTNETLIVELLFQGRNPGVAWEYSMPRLGTEKQPP NOVld RRSYNEPENLIATGPTNETLIVELLFQGRNPGVAWEYSMPRLGTEKQPP NOVlC YRRSYNEPENLIATGPTNETLIVELLFQGRNPGVAWEΫSMPRLGTEKQP'P
960 970 980 990
NOVla iQPSYTWAIVRSECSVSCGGGRCLPVLLLEAAeQPSATAYIALAFLES (SEQ ID NO: 2) NOVlb ^QPSYT AIVRSECSVSCGGGRCLPVLLLEAACQPSATAYISLAFLES (SEQ ID NO: ) NOVld ^QPSYTWAIVRSECSVSCGGGRCLPVLLLEAACQPSATAYIALAFLES (SEQ ID NO:6) NOVlc iQPSYT AIVRi3ECSVSCG(3GRCLPVLLLEAACQP|lATAYIALAFLES (SEQ ID NO:8)
?? Homologies to any ofthe above NOVl proteins will be shared by the other two NOVl proteins insofar as they are homologous to each other as shown above. Any reference to NOVl is assumed to refer to all three of the NOVl proteins in general, unless otherwise noted.
The disclosed NOV l a polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table U.
Figure imgf000025_0001
The homology between these and other sequences is shown graphically in the
ClustalW analysis shown in Table I . In the ClustalW alignment of the NOV l proteins, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function.
Table IK. ClustalW Analysis of NOVl
1) Novel NOVla ( SEQ ID NO : 2 ) 2) Novel NOVlb (SEQ ID NO : 4 ) 3) Novel NOVlc {SEQ ID NO . 6 ) 4) Novel NOVld ( SEQ ID NO ; 8 )
4) gi|l356992 I ref l NP 112217 . 1 a disintegrj-n-like and metalloprotease with thrombospondin type 1 motif, 12 [Homo sapiens] (SEQ ID NO.30)
5) gi 111493589 (gb |AAG35563.1 | AF163762 _1 (AF163762) zinc metalloendopeptidase [Homo sapiens) (SEQ ID NO-31)
6) gi|l0645199|ref |NP_055087 1 | a drsintegrxn and metalloprotease with thrombospondin motifs- 7 preproprotein; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 7 [Homo sapiens] (SEQ ID N0.32)
7) gi|l530993l|ref |XP_054419.1 |l a disintegrin-like and metalloprotease domain with thrombospondin type I repeats 10 [Homo sapiens] (SEQ ID NO: 33)
8) gi[ 7656869 I ef |NP_055088.1 | a disintegrin and metalloprotease with thrombospondin moti s-6 preproprotein; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 6 [Homo sapiens) (SEQ ID NO:34)
10 20 30 4 0
NOVla MKPRARGWRGLAALWMLLAQVAEQVSPGRSHQRGNRGΞGQ
NOVlb MKPRARG RG AA W LLAQVAEQVSPGRSHQRGNRGSGQ
NOVlc
NOVld MPRARGWRGLAA WM LAQVAEQVSPGRSHQRGNRGSGQ
Figure imgf000026_0001
SO 60 70 30
NOVla LEASPPRL SRGPRRLTAMSPLFΞAGTCVRHGTRSGSAWE NOVlb I-EASPPR SRGPRRLTAMSP FSAGTCVRHGTRSGSAWE NOVlc MGP AAAAP GSPSV NOVld LEASPPR SRGPRRLTAMSPIiFSAGTCVRHGTRSGSAWE gi[l3569928| MPCAQRSW A-N SWAQL NFGAI-CYGRQPQPGPVRF gi|ll493589 j gi 1,10645199] MPGGPSPRSPAPLLRP LLLLCA APG - APGPAPGRA gι)l530993l) gi [7656869 | MEILWKTLTWILS lMASSEFHSDHRIi
90 1 00 110 120
.... I .. . . I .. . . |, .... I . ... i .... I .... ( .... (
NOVla PERPASSSTRGAAGLDGKGRDMDEAGNHRSQQTN GTENQ
NOVlb PERPASSSTRGAAGLDGKGRDMDEAGNHRSQQTNTGTENQ
NOVlc PRPPP --
NOVld PERPASSSTRGAAGLDGKGRDMDEAGNHRSQQTNTGTENQ gi (13569928 j PDRRQEHFIKG gijll493539| - - SQDEF SS gi I 10645199 | TEGR gi]l530993l| g± ||7656869 | SYSSQEEFBTYL-
Figure imgf000027_0001
170 180 190 200
NOVla VS EVES-LHLRLKGPRHDFHMpBRTSSSLVAPG
NOVlb s EVES-ΪHLRLKGPRHDFHM SRTSSΞLVAPG
NOVlc S EVEP-AFLQVCRARELRLCVEAFPIANSQPG
NOVld VS Ev S-LHLR KGPRHDFHMDgRTSSSLViiPG gi 113569928 |, LD GSED -VYYRISHEEKDLFFN TVHQGFI,SNSYI gi|ll493589| AT AESRLFYKVASPSTHFLLN|τRSSRLIιiGHVS gi|l0645199 I VS VRRDAPAFYEI»QYRGRELRF1^TANQHLIJAPG|V gi (l530993l| gi 1,7656869 I, DPIDPQQAVS LFFKIiSAYGKHFHLN JTLNTDFySKH T
Figure imgf000027_0002
330 340 350 360
NOVla PCSEDSRNVCIDGICELSWSTSAHMPQPPKEDLFILPDE
NOVlb PCSEDSRNVCIDGICE MPQPPKEDLFILPDE
NOVlc PQKQHFCGRRK YMPQPPKEDLFILPDE
NOVld PCSEDSRNVCIDGICELSWSTSAHMPQPPKEDLFILPDE g± 1 3569928 | NISQ KQELWREK gi |11493589| LRT - -LKPPPARP gi|l0645199l ELEΞ - -RRERWEQR gi|l530993l| gi 7656869 WLN DTSTVSYS
Figure imgf000028_0001
450 460 470 480
NOVla EI)EffipGJ2vgsi^DHX0S^gQggSGt1MGKDG-
NOVlb EDE|--- "
NOVlc EDEWPGg gSiSBDHTgs ^QggUSGiaiGKDG-
NOVld SWKN-
Figure imgf000028_0002
NOVla
Figure imgf000028_0003
NOVlb
Figure imgf000028_0004
Figure imgf000029_0001
610 620 630 640
NOVla DQPKPVKEYKYgEKL[^EIι gJA ^KWi- FGEKAK CMLD NOVlb NOVlc JMFGEKAKLCMLD NOVld DQPKPVKEYKYgEKL JMFGEKAKLCMLD gi|l3569928 | DIPKK-KG KSKVIA! |QL§YG- -PNATFCQ gi|ll493589 ( NRP PR - QDFVT TVA 3 JFHH GVKSRQC gi|l0645199| DPPAX-DIIDFjisVP RLWYG- -AYSAFCE g± 115309931 |ι NRPPR-QDFVX§TV, JF§H GVKSRQC gi|7656869| SRX HHQ
Figure imgf000029_0003
Figure imgf000029_0002
Figure imgf000030_0001
1010 1020 1030 1040
NOVla NOVlb NOVlc NOVld gi)l3569928] GTGIRRQTAHCIKKGRGMVKATFCDPETQPNGRQKKCHEK gi|H493589 ( -APIAR DS gi (10645199 j VP gi |15309931|| -APIAR DS gi)7656869] -VPITRTGSG- DN
NOVla NOVlb
Figure imgf000030_0002
Figure imgf000031_0001
1130 1140 1150 1160
NOVla -YTWAX|R-
NOVlb -YTWAI|R-
NOVlc -YTWAX R-
NOVld - ywAXøR- gi|l3569928| CGGGVRIRSVTCAKNHDEPCDVTRKPNSRALCGLQQCPSS gi (11493589 | CDAGVRSR'SVjgCQR gi|l0645199| - -GGVSPGSVT E-PGS gi(l530993l| CDAGVRSRSVgCQR -- - gi (7656869 ( - -ARWREKH1I.SY
1170 1180 1190 1200
NOVla NOVlb NOVlc NOVld gi|l3569928] RRVLKPNKGTISNGKNPPTLKPVPPPTSRPRMLTTPTGPE gi j 11493589 ) -- RVSAAEEKALDDSACPQ gi|l0645199| -- EPGPPAAAS gi 115309931) RVSAAEEKALDDSACPQ- - gi (7656869 [ ALCLLKKLIGNISCR
Figure imgf000031_0002
1290 1300 1310 1320
.. I ....).... I .... ( .... I ... NOVla
NOVlb
NOVlc
NOVld gi [13569928) GLVATTTSGSGLSSSRNPITWPVTPFYNTLTKGPEMEIHS gi j 11493589 j - ■ PAT gα (10645199 | G-- VHYEYT - IHR g-.|l530993l| PAT gx[7656869 [
1330 1340 1350 1360
NOVla
NOVlb
NOVlc
NOVld gi 113569928 I GSGEEREQPEDKDESNPVIWTKIRVPGNDAPVESTEMPLA gι|ll493589 | RCNLRRCPP gι|l0645199 [ EAGGHDEVPP gι| 15309931) MRCNLRRCPP gi ||7656869 |
1370 1380 1390 1400
NOVla NOVlb NOVlc NOVld gι|l3569928] PPLTPDLSRESWWPPFSTVMEGLLPSQRPTTSETGTPRVE gi j 11493589) gi (10645199 j gi j 15309931 j gi (7656869 [
1410 1420 1430 1440
NOVla
NOVlb
NOVlc
GMVTEKPANTLLPLGGDHQPEPSGKTANRNHLKLPNNMNQ
Figure imgf000032_0001
1450 1460 1470 1480
NOVla
NOVlb
NOVlc
NOVld gi |13569928 | TKSSEPVLTEEDATSLITEGFLLNASNYKQLTNGHGSAHW gι|ll493589 | ARW gι|l0645199 | PVFS - W gι|l530993l| ARW gi|7656869 |
1490 1500 1510 1520
NOVla NOVlb NOVlc NOVld
Si 13569928 IVGNWSECSTTCGLGAYWKRVECTTQMDSDCAAIQRPDPA gα (11493589 | VAGEWGECSAQCGVGQRQRSVRCTS gαjl0645199 j HYGPWTKCTVTCGRGE gι|1530993l| VAGEWGECSAQCGVGQRQRSVRCTS-- gα[7656869 [ 1530 1540 1550 1560
NOVla NOVlb NOVlc NOVld g-i- 113569928 [I KRCHLRPCAGWKVGNWSKCSRNCSGGFKIREIQCVDSRDH gil 11493589 I HTGQAΞHECTEALR gx [10645199 ( KWGRHΞPTCRG - -LVSGQGH gi ( 15309931 j HTGQASHECTEALR - - gι|7656869|
1570 1580 1590 1600
NOVla NOVlb NOVlc NOVld gι|l3569928| RNLRPFHCQFLAGIPPPLSMΞCNPEPCEAWQVEPWSQCSR go. I 11493589 I - PPTTQQCEA g-L j 10645199 I WLQLPA gαjl530993l| PPTTQQCEA gι|7656869 |
1610 1620 1630 1640
NOVla
NOVlb
NOVlc
NOVld gι| 13569928 | SCGGGVQERGVFCPGGLCDWTKRPTSTMSCNEHLCCHWAT gα) 11493589 j KCDS PTPG gα j 10645199 I HC -WAT gι(l530993lj KCDS -- -- - PTPG gα)7656869 (
1650 1660 1670 1680
NOVla
NOVlb
NOVlc
NOVld g (13569928 GNWDLCSTSCGGGFQKRIVQCVPSEGNKTEDQDQCLCDHK gα|H493589 DGPEECKDVN gι|l0645199 TGLEVCFS- - E g j 15309931 DGPEECKDVN gα |7656869 |
1690 1700 1710 1720
NOVla NOVlb NOVlc NOVld gα|l3569928 | PRPPEFKKCNQQACKKSADLLCTKDKLSASFCQTLKAMKK gι|H493589 | _- __ KVAYCPLVLKFQF gα (10545199 j1 p QF S-ICE RLAIAL gα | 15309931] __ _ -KVAYCPLVLKFQF gι|7656869 |
1730 17.0 1750
NOVla NOVlb NOVlc NOVld gα|l3569928 | CSVPTVRAECCFSCPQTHITHTQRQRRQRLLQKSKEL gα [11493589 I CSRAYFRQ CCKTCQGH gι|l0645199| CPRPAGRVHG - g (15309931| CSRAYFRQMCCKTCHGH- - gα [ 7656869
The presence of identifiable domains in NOV l , as well as all other NOVX proteins, was determined by searches using software algorithms such as PROSITB, DOMAIN, Blocks, Pfa , ProDo ain, and Prints, and then determining the Interpro number by crossing the domain match (or numbers) using the Interpro website (http:www.ebi.ac.uk/ interpro). DOMAIN results for NOVl as disclosed in Tables IL-IO, were collected from the Conserved Domain Database (CDD) with Reverse Position Specific BLAST analyses. This BLAST analysis software samples domains found in the Smart and Pfam collections. For Table I E and all successive DOMAIN sequence alignments, fully conserved single residues are indicated by black shading or by the sign (|) and "strong" semi-conserved residues are indicated by grey shading or by the sign (+). The "strong" group of conserved amino acid residues may be any one of the following groups of amino acids: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MTLF, HY, FYW.
Tables 1 L-l O lists the domain description from DOMAIN analysis results against NOVl . This indicates that the NOVl sequence has properties similar to those of other proteins known to contain this domain.
Table IL. Domain Analysis of NOVl qnl 1 Pf m| p am0142 , Reprolysin, Reprolysin (M12B) family zinc metalloprotease. The members of this family are enzymes that cleave peptides. These proteases require zinc for catalysis. Members of this family are also known as adamalysins . Most members of this family are snake venom endopeptidases, but there are also some mammalian proteins, and fertilin. Fertilin and closely related proteins appear to not have some active site residues and may not be active enzymes. (SEQ ID NO:76)
CD-Length = 199 residues, 95.0% aligned Score = 122 bits (307), Expect = 7e-29
Query : 378 DKKMMQNHG~-HENITTYVMI.ONMVSALFKDGTIGGNINIAIVGbILbEDEQPGI-VlSH 435 t I + | i ! μ μ μ +++ | | + + m + | . + Sb^ct i 10 DHGMFTRYGSDLNKIRQRVHQIVB-- VNEIYRPL NIRWLVGLEIWSDGDK- ITVQG 64- Query 436 HADHTLSSFCQ QSGLMGKDGTRHDHAUjLTGJ-jDICSWKNEPCDTLGFAPXSGMCSKyRS 495 μ 1 1 1 + μ + 1 | μ| | | | μ| + μι | + 1 1 1 1 I I Sbj ct : 55 DA DTLHRFBEWRETDbLKR-KSHDNAQ TGlDF DGNT1GAAXVGGMCSPKRS 117 Query . 496 CTINED TGLGLAFTIAHESGHNFG IHDGEGN-MCKKSEGNIMSPTI-AGRNGVFSWS 551
+ + ι I + ι ki n n i I I I I μ i ii μ i + i ø Sbj ct : 118 VGWQDHΞPIV LVAVTMAHELGHHLGMTHDDINKCTCEGGGGCIMNPVASSSPGKK-FS 176 Query 552 PCSRQYLHKFLSTAQAICLADQ 573
I I ++ Sb] ct ' 177 HCSMDDYQQF TKGKPQCLbNK 198 Table I M. Domain Analysis of NOVl gnl I Smart | smarb00209 , TSP1, Thrombospondin type 1 repeats; Type 1 repeats in thrombospondin-1 bind and activate TGF-beta.
(SEQ ID NO: 77) CD-Length = 51 residues, 100.0% aligned Score = 63.5 bits (153), Expect = 5e-ll
Query: 668 WSDWSSWSPCSRTCGGGVSHRSRLCTNPNPSHGGKFCEGSTRTLKL-CNSQKCP 720
( + 1 1 I I Mi I μi I I I I I I + I I I i l
Sbj ct : 1 WGEWSEWSPCSVTCGGGVQTRTRCCNPPPN--GGGPCTGPDΪETRACNEQPCP 51
Table IN. Domain Analysis of NOVl gnl 1 Pfam | pfamO Q090 , tsρ_l , Thrombospondin type 1 domain . ( SEQ ID
NO : 78 )
CD-Length = 48 residues, 100.0% aligned
Score = 54.7 bits (130), Expect = 2e-08
Query : 669 SDWSSWSPCSRTCGGGVSHRSRbCTNPNPSHGGKFCEGSTRTbKLCNSQKC 719 i i i i n n n i i+ i i i + ι m i i + + i i
Sbj ct : 1 SPWSE SPCSVTCGKGIRTRQRTCNSP- - -AGGKPCTGDAQETEACMMDPC 48
Table lO. Domain Analysis of NOVl gnl I Pfam | pfam01562 , Pep_M12B_propep, Reprolysin family propeptide. This region is the propeptide for members of peptidase family M12B . The propeptide contains a sequence motif similar to the "cysteine switch" of the matrixins . This motif is found at the C terminus of the alignment but is not well aligned, repeats. (SEQ ID NO: 79) CD-Length = 117 residues, only 39.3% aligned Score = 38.5 bits (88), Expect = 0.002
Query: 180 LRTSSSLVAPGFIVQTLGKTGTKSVQTLPPEDFCFYQGSLRSHRNS 225 i + i n i | | | | + + | μ n i + + I I
Sbj ct : 2 LBKNRSLLAPDFTVTTYDDDGT-.JVTEHPLIQDHCYYQGYVEGYPNS 47
Thrombospondin- 1 (THBSl) associates with the extracellular matrix and inhibits angiogenesis in vivo. In vitro, THBSl blocks capillary-like tube formation and endothelial cell proliferation. The antiangiogenic activity is mediated by a region that contains 3 type 1 (properdin or thrombospondin) repeats. By searching an EST database for sequences containing the antiangiogenic motif of THBSl , Vazquez et al. (1999) identified heart and lung cDNAs encoding ADAMTSl and ADAMTS8, which they called METH1 and METH2, respectively. Sequence analysis predicted that the 890-amino acid ADAMTS8 protein shares 52% amino acid identity with ADAMTS l . ADAMTS8 is a secreted protein that has an N- tcπninal signal peptide, a zinc metalloprotease domain containing a zinc-binding site, and a cysteine-rich region containing 2 putative disintegrin loops. The C terminus of ADAMTS8 has 2 heparin-binding thrombospondin repeats with 6 cys and 3 tip residues. Southern blot analysis showed that ADAMTS8 is a single-copy gene distinct from that encoding ADAMTS 1. Northern blot analysis detected highest expression of a 3.7-kb ADAMTS8 transcript in adult and fetal lung, with lower expression in brain, placenta, heart, and stomach, as well as fetal brain and kidney. Expression was also detected in a colon carcinoma cell line. SDS-PAGE analysis demonstrated that ADAMTS8 is expressed as a 98-kD protein, a 79-kD protein after cleavage at the subtihsin site, or as a 64-kD protein, which is most abundant, generated by an additional processing event. Functional analysis determined that ADAMTS 8 disrupts angiogenesis in vivo and in vitro more efficiently than THBSl or endostatin but somewhat less efficiently than ADAMTSl .
By interspecific backcross analysis, Georgiadis et al. (1999) mapped the mouse AdamtsS gene to chromosome 9 in a region showing homology of synteny with human 1 lq23- qter. They mapped the human AD AMTS8 gene to l l q25 by PCR analysis of a radiation hybrid mapping panel. The authors noted that a number of disorders have been mapped in the vicinity of the ADAMTS8 gene in mice and humans, most notably, given the expression and functional analyses, lung neoplasms.
The novel protein described here contains thrombospondin type I domains and Reprolysin domain. It is homologous to AD AM genes. Thrombospondin type I domain [1PR000884; (TSP 1)] was found in the thrombospondin protein where it is repeated 3 times. Now a number of proteins involved in the complement pathway (properdin, C6, C7, C8A, C8B, C9) as well as extracellular matrix protein like indin, F-spondin, SCO-spondin and even the circumsporozoite surface protein 2 and TRAP proteins of Plasmodium contain one or more instance of this repeat. It has been involved in cell-cell interraction, inhibition of angiogenesis, apoptosis. The intron-exon organisation of the properdin gene confirms the hypothesis that the repeat might have evolved by a process involving exon shuffling. A study of properdin structure provides some information about the structure of the thrombospondin type I repeat.
Reprolysin family propeptide [IPR002870; (Pep_M12B_propep)] domain is contained in the propeptide for members of peptidase family M12B. The propeptide contains a sequence motif similar to the 'cysteine switch1 o the matrixins. This motif is found at the C terminus of the alignment but is not well aligned.
Through a subtractive hybridization approach to identify genes specifically expressed in the caput epididy idis, the mouse homologue of member o the ADAM (a disintegrin and metalloprotease) family of proteins was identified. This rapidly growing gene family encodes cell surface proteins that possess putative adhesion and protease domains. Northern blot analyses demonstrated that the mouse ADAM gene, teπned ADAM7, is expressed in the caput region of the epididymis and in the anterior pituitary gonadotropes with no detectable expression in the twenty-six other tissues examined. Furthermore, in situ hybridization experiments revealed that the ADAM7 messenger RNA (mRNA) exhibited an apical localization within the proximal caput epididymal epithelium that may correlate with an unusual sparsely granulated endoplasmic reticulum uniquely present in the proximal region of the epididymidis and to which no known function has been ascribed. Hormonal, surgical, and genetiG strategies demonstrated that ADAM7 gene expression requires, in a region-dependent manner, androgens as well as testicular factors for expression. Interestingly, the apical localization of ADAM7 mRNA is dependent upon an intact testis, because in situ hybridization analyses of the proximal caput epididymidis from a testosterone maintained Gastrate mouse did not show the apical localization of ADAM7 mRNA. Finally, chromosomal mapping demonstrated that the ADAM7 gene maps to the central region of mouse
Chromosome 14, approximately 4-5 cM distal from the fertilin beta locus, whiϋh encodes another reproductive-specific ADAM protein (1).
Because o the presence ofthe domain and the homology to the , we anticipate that the novel sequence described here will have useful properties and functions similar to these genes. The disclosed NOVl nucleic acid ofthe invention encoding a ADAM-TS 7 -like protein includes the nucleic acid whose sequence is provided in Table 1A, IC, IE, and IG or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 1A, I C, IE and IG while still encoding a protein that maintains its A AM-TS 7-like activities and physiological functions, or a fragment of such a nucleic acid, The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject, In the mutant or variant nucleic acids, and their complements, up to about 33% percent of the bases may be so changed. The disclosed NOVl protein of the invention includes the ADAM-TS 7-like protein whose sequence is provided in Table I B, ID, IF, or I H. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table I B, I D, I F, or I H while still encoding a protein that maintains its ADAM-TS 7 -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 62% percent of the residues may be so changed.
The invention further encompasses antibodies and antibody fragments, such as Fab or (Fab)2, that bind immunospecifically to any of the proteins ofthe invention.
The above defined information for this invention suggests that this ADAM-TS 7-like protein (NOVl) may function as a member of a "ADAM-TS 7 family". Therefore, the NOV l nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
The NOVl nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to various pathologies and disorders as indicated below. For example, a cDNA encoding the ADAM-TS 7-like protein (NOVl) may be useful in gene therapy, and the ADAM-TS 7 -like protein (NOVl) may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions o the present invention will have efficacy for treatment of patients suffering from adrenoleukodystrophy , Alzheimer's disease, autoimmune disease, allergies, addiction, anxiety, ataxia-telangiectasia, asthma, ARDS, atherosclerosis, behavioral disorders, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, allergy, cerebral palsy, congenital adrenal hyperplasia, cirrhosis, cardiomyopathy, congenital heart defects, diabetes, diverticular disease, epilepsy, emphysema, endometriosis, endocrine dysfunctions, graft versus host disease, glomerulonephritis, graft versus host disease (GVHD), growth and reproductive disorders, hemophilia, hypercoagulation, hypercalceimia, Huntington's disease, hypertension, hypogonadism, fertility, idiopathic thrombocytopenic purpura, immunodeficiencies, interstitial nephritis, IgA nephropathy, lymphaedema, inflammatory bowel disease, Lesch-Nyhan syndrome, leukodystrophies, multiple sclerosis, muscular dystrophy, myasthenia gravis, neurodegeneration, neuroprotection, obesity, Parkinson's disease, pain, polycystic kidney disease, pulmonary stenosis, pancreatitis, renal artery stenosis, renal tubulai acidosis, stroke, systemic lupus erythematosus, scleroderma, subaortic stenosis, transplantation, tuberous scleiosis, Von Hippel-Lindau (VHL) syndrome, ventricular septal defect (VSD), valve diseases, Von Hippel-Lindau (VHL) syndrome, ulceis, bacterial and viral infections, neonatal apnea, eagle's syndrome, atherosclerosis, metabolic disorders such as familial amyloidotic polyneuropathy, hyperk etic diseases, muscular dystrophy, cerebral vascular disease, hypertension, cardiovascular diseseases, renal fibrogenesis, inflammatory bowel disease, Mec el syndrome, colorectal cancer, papillomavnus infection and cervical carcinoma, hvei malignancies, skm disorders, connective tissue disorders such as type VIIC Ehlers-Danros syndrome, osteoarthπtis, rheumatoid arthritis, or other pathologie or conditions The NOVl nucleic acid encoding the ADAM-TS 7-hke protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed NOVl nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVl substances for use in therapeutic or diagnostic methods These antibodies may be generated according to methods known in the ait, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below The disclosed NOVla and b proteins have multiple hydrophilic regions, each of whiGh oan be used as an immunogen In one embodiment, a contemplated NOVla and b epitope is from about amino acids 20 to 70 In another embodiment, a NOVla and b epitope is from about am o acids 80 to 180 In additional embodiments, NOVla and b epitopes are from about ammo acids 200 to 280, from about ammo acids 300 to 360, from about amino acid 400 to 530, from about ammo acid 540 to 550, from about ammo acid 580 to 610, from about ammo acid 630 to 680, and from about ammo acids 710 to 750 Also, the disclosed
NOV lc protein has multiple hydrophilic regions, each of which can be used as an immunogen In one embodiment, a contemplated NOVl c epitope is from about ammo acids 1 to 50 In another embodiment, a NOVlc epitope is from about amino acids 80 to 100 In additional embodiments, NOVl c epitopes are from about amino acids 1 10 to 260, from about ammo acids 280 to 320, from about amino acid 350 to 400, from about ammo acid 420 to 470, from about amino acid 480 to 620, from about ammo acid 700 to 750, and from about amino acids 770 to 810 Also, the disclosed NOV l d protein has multiple hydrophilic regions, each of w hich can be used as an immunogen In one embodiment, a contemplated NOVld epitope is from about amino acids 20 to 180 In another embodiment, a NOVl d epitope is from about amino acids 190 to 280. In additional embodiments, NOVld epitopes sre from about amino acids 300 to 360, from about amino acids 400 to 530, from about amino acid 540 to 550, from about ammo acid 580 to 610, from about amino acid 630 to 680, and from about amino acids 710 to 750. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV2
A disclosed NOV2 nucleic acid of 4488 nucleotides (also referred to as SC_78316254_A) encoding a novel alpha-2-macroglobulin precursor-like protein is shown in Table 2A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1 -3 and ending with a TGA codon at nucleotides 4477-4479. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 2A, and the start and stop codons are in bold letters.
Table 2A. NOV2 nucleotide sequence (SEQ ID NO:9).
ATGTGGGCTCAGCTCCTTCTAGGAATGTTGGCCCTATCACCAGCCATTGCAGAAGAACTTCCAAACTACCTG GTGACATTACCAGCCCGGCTAAATTTCCCCTCCGTTCAGAAGGTTTGTTTGGACCTGAGCCCTGGGTACAGT GATGTTAAATTCACGGTTACTCTGGAGACCAAGGACAAGACCCAGAAGTTGCTAGAATACTCTGGACTGAAG AAGAGGCACTTACATTGTATCTCCTTTCTTGTACCACCTCCTGCTGGTGGCACAGAAGAAGTGGCCACAATC CGGGTGTCGGGAGTTGGAAATAACATCAGCTTTGAGGAGAAGAAAAAGGTTCTAATTCAGAGGCAGGGGAAC GGCACCTTTGTACAGACTGACAAACCTCTCTACACCCCAGGGCAGCAAGTGTATTTCCGCATTGTCACCATG GATAGCAACTTCGTTCCAGTGAATGACAAGTACTCCATGGTGGAACTACAGGATCCAAATAGCAACAGGATT GCACAGTGGCTGGAAGTGGTACCTGAGCAAGGCATTGTAGACCTGTCCTTCCAACTGGCACCAGAGGCAATG CTGGGCACCTACACTGTGGCAGTGGCTGAGGGCAAGACCTTTGGTACTTTCAGTGTGGAGGAATATGTGCTT TCTCCATTTCTCCTTTTACTCTCTTCAGTGCTGCCGAAGTTTAAGGTGGAAGTGGTGGAACCCAAGGAGTTA TCAACGGTGCAGGAATCTTTCTTAGTAAAAATTTGTTGTAGGTACACCTATGGAAAGCCCATGCTAGGGGCA GTGCAGGTATCTGTGTGTCAGAAGGCAAATACTTACTGGTATCGAGAGGTGGAACGGGAACAGCTTCCTGAC AAATGCAGGAACCTCTCTGGACAGACTGACAAAACAGGATGTTTCTCAGCACCTGTGGACATGGCCACCTTT GACCTCATTGGATATGCGTACAGCCATCAAATCAATATTGTGGCTACTGTTGTGGAGGAAGGGACAGGTGTG GAGGCCAATGCCACTCAGAATATCTACATTTCTCCACAAATGGGATCAATGACCTTTGAAGACACCAGCAAT TTTTACCATCCAAATTTCCCCTTCAGTGGGAAGATGCTGCTCAAGTTTCCGCAAGGCGGTGTGCTCCCTTGC AAGAACCATCTAGTGTTTCTGGTGATTTATGGCACAAATGGAACCTTCAACCAGACCCTGGTTACTGATAAC AATGGCCTAGCTCCCTTTACCTTGGAGACATCCGGTTGGAATGGGACAGACGTTTCTCTGGAGGGAAAGTTΪ CAAATGGAAGACTTAGTATATAATCCGGAACAAGTGCCACGTTACTACCAAAATGCCTACCTGCACCTGCGA CCCTTCTACAGCACAACCCGCAGCTTCCTTGGCATCCACCGGCTAAACGGCCCCTTGAAATGTGGCCAGCCC CAGGAAGTGCTGGTGGATTATTACATCGACCCGGCCGATGCAAGCCCTGACCAAGAGATCAGCTTCTCCTAC TATTTAATAGGGAAAGGAAGTITGGTGATGGAGGGGCAGAAACACCTGAACTCTAAGAAGAAAGGACTGAAA GCCTCCTTCTCTCTCTCACTGACCTTCACTTCGAGACTGGCCCCTGATCCTTCCCTGGTGATCTATGCCATT TTTCCCAGTGGAGGTGTTGTAGCTGACAAAATTCAGTTCTCAGTCGAGATGTGCTTTGACAATCAGCAGCTT CCAGGAGCAGAAGTGGAGCTGCAGCTGCAGGCAGCTCCCGGATCCCTGTGTGCGCTCCGGGCGGTGGATGAG AGTGTCTTACTGCTTAGGCCAGACAGAGAGCTGAGCAACCGCTCTGTCTATGGGATGTTTCCATTCTGGTAT GGTCACTACCCCTATCAAGTGGCTGAGTATGATCAGTGTCCAGTGTCTGGCCCATGGGACTTTCCTCAGCCC CTCATTGACCCAATGCCCCAAGGGCATTCGAGCCAGCGTTCCATTATCTGGAGGCCCTCGTTCTCTGAAGGC ACGGACCTTTTCAGCTTTTTCCGGGACGTGGGCCTGAAAATACTGTCCAATGCCAAAATCAAGAAGCCAGTA GATTGCAGTCACAGATCTCCAGAATACAGCACTGCTATGGGTGGCGGTGGTCATCCAGAGGCTTTTGAGTCA TCAACTCCTTTACATCAAGCAGAGGATTCTCAGGTCCGCCAGTACTTCCCAGAGACCTGGCTCTGGGATCTG TTTCCTATTGGTAACTCGGGGAAGGAGGCGGTCCACGTCACAGTTCCTGACGCCATCACCGAGTGGAAGGCG ATGAGTTTCTGCACTTCCCAGTCAAGAGGCTTCGGGCTTTCACCCACTGTTGGACTAACTGCTTTCAAGCCG TTCTTTGTTGACCTGACTCTCCCTTACTCAGTAGTCCGTGGGGAATCCTTTCGTCTTACTGCCACCATCTTC AATTACCTAAAGGATTGCATCAGGGTTCAGACTGACCTGGCTAAATCGCATGAGTACCAGCTAGAATCATGG GCAGATTCTCAGACCTCCAGTTGTCTCTGTGCTGATGACGCAAAAACCCACCACTGGAACATCACAGCTGTC AAATTGGGTCACATTAACTTTACTATTAGTACAAAGATTCTGGACAGCAATGAACCATGTGGGGGCCAGAAG GGGTTTGTTCCCCAAAAGGGCCGAAGTGACACGCTCATCAAGCCAGTTCTCGTCAAACCTGAGGGAGTCCTG GTGGAGAAGACACACAGCTCATTGCTGTGCCCAAAAGGAGGAAAGGTGGCATCTGAATCTGTCTCCCTGGAG CTCCCAGTGGACATTGTTCCTGACTCGACCAAGGCTTATGTTACGGTTCTGGGAGACATTATGGGCACAGCC CTGCAGAACCTGGATGGTCTGGTGCAGATGCCCAGTGGCTGTGGCGAGCAGAACATGGTCTTGTTTGCTCCC ATCATCTATGTCTTGCAGTACCTGGAGAAGGCAGGGCTGCTGACGGAGGAGATCAGGTCTCGGGCAGTGGGT TTCCTGGAAATAGGGTACCAGAAGGAGCTGATGTACAAACACAGCAATGGCTCATACAGTGCCTTTGGGGAG CGAGATGGAAATGGAAACACATGGCTGACAGCGTTTGTCACAAAATGCTTTGGCCAAGCTCAGAAATTCATC TTCATTGATCCCAAGAACATCCAGGATGCTCTCAAGTGGATGGCAGGAAACCAGCTCCCCAGTGGCTGCTAT GCCAACGTGGGAAATCTCCTTCACACAGCTATGAAGGGTGGTGTTGATGATGAGGTCTCCTTGACTGCGTAT GTCACAGCTGCATTGCTGGAGATGGGAAAGGATGTAGATGACCCAATGGTGAGTCAGGGTCTACGGTGTCTC AAGAATTCGGCCACCTCCACGACCAACCTCTACACACAGGCCCTGTTGGCTTACATTTTCTCCCTGGCTGGG GAAATGGACATCAGAAACATTCTCCTTAAACAGTTAGATCAACAGGCTATCATCTCAGGAGAATCCATTTAC TGGAGCCAGAAACCTACTCCATCATCGAACGCCAGCCCTTGGTCTGAGCCTGCGGCTGTAGATGTGGAACTC ACAGCATATGCATTGTTGGCCCAGCTTACCAAGCCCAGCCTGACTCAAAAGGAGATAGCGAAGGCCACTAGC ATAGTGGCTTGGTTGGCCAAGCAACACAATGCATATGGGGGCTTCTCTTCTACTCAGGATACTGTAGTTGCT CTCCAAGCTCTTGCCAAATATGCCACTACCGCCTACATGCCATCTGAGGAGATCAACCTGGTTGTAAAATCC ACTGAGAATTTCCAGCGCACATTCAACATACAGTCAGTTAACAGATTGGTATTTCAGCAGGATACCCTGCCC AATGTCCCTGGAATGTACACGTTGGAGGCCTCAGGCCAGGGCTGTGTCTATGTGCAGACGGTGTTGAGATAC AATATTCTCCCTCCCACAAATATGAAGACCTTTAGTCTTAGTGTGGAAATAGGAAAAGCTAGATGTGAGCAA CCGACTTCACCTCGATCCTTGACTCTCACTATTCACACCAGTTATGTGGGGAGCCGTAGCTCTTCCAATATG GCTATTGTGGAAGTGAAGATGCTATCTGGGTTCAGTCCCATGGAGGGCACCAATCAGTTACTTCTCCAGCAA CCCCTGGTGAAGAAGGTTGAATTTGGAACTGACACACTTAACATTTACTTGGATGAGCTCATTAAGAACACT CAGACTTACACCTTCACCATCAGCCAAAGTGTGCTGGTCACCAACTTGAAACCAGCAACCATCAAGGTCTAT GACTACTACCTACCAGGTTCTTTTAAATTATCTCAGTACACAATTGTGTGGTCCATGAACAATGACAGCATA GTGGACTCTGTGGCACGGCACCCAGAACCACCCCCTTTCAAGACAGAAGCATTTATTCCTTCACTTCCTGGG AGTGTTAACAACTGATAGCTACCA
The disclosed NOV2 nucleic acid sequence, localized to chromsome 12, has 840 of 1324 bases (63%) identical to a Rattus non'egicus Alpha-2-Macroglobulin Precursor mRNA (GENBANK-ID: RATA2M) (E = 1.3e'1 19).
A NOV2 polypeptide (SEQ ID NO: 10) encoded by SEQ ID NO:9 has 1492 amino acid residues and is presented using the one-letter code in Table 2B. Signal P, Psort and/or Hydropathy results predict that NOV2 does contain a signal peptide and is likely to be localized extraceliularly with a certainty of 0.3703. In other embodiments, NOV2 may also be localized to the lysosome (lumen) with a certainty of 0.1900, the microbody (peroxisome) with a certainty of 0.1585, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The most likely cleavage site for a NOV2 peptide is between amino acids 17 and 18, at: AlA- EE.
Table 2B. Encoded NOV2 protein sequence (SEQ ID NO:10). AQLbLGMLALSPAlAEE PNYLVTLPARl- NFPSVQKVC--jD]-jSPGYSDVKFTVTLETKDKTQKIιLEYSGLK KRHLHCISFLVPPPAGGTEEVATIRVSGVGNNISFEEKKKVLIQRQGNGTFVQTDKPLYTPGQQVYFRIVTM DSNFVPVNDKYSMVELQDPl.SNRIAQ --αEWPEQGrVDLSFQLAPEA LGTYTVAVAEGKTFGTFSVEEYVL S PFLLLLSSVbPKFKVEWEPKELSTVQESFLVKICCRYTYGKPMLGAVQVSVCQKANTYWYREVEREQLPD KCRNLSGQTDKTGCFSAPVDMATFDLIGYAYSHQINIVATWEEGTGVEANATQNIYI S PQMGSMTFEDTSN FYHPNFPFSGKM bKFPQGGV]-jPC I.H--4VFIιVIYGTNGTFN0TlιV-rDNNG APFTXjETSGWNGTDVS--jEGKF QMEDbVYMPEQVPRYYQNAYI HLRPFYSTTRS FLGIHRLNGPLKCGQPGEVLVDYYIDPADAS PDQEI SFSY YLIGKGS VMEGQKHLNSKKKGIjKASFS-ϋSLTFTSRLAPDPSLVIYAI FPSGGVVADKIQFSVEMCFDNQQl-j PGAEVELQ QAAPGSIiCA RAVDESVDLI/RPDRELSNRSVYGMFPFWYGHYPYOVAEYDQCPVSGP DFPQP IDPMPQGHSSQRSII RPSFSEGTDIjFSFFRDVGDKIIiSNAKIK PVDCSHRSPEYSTAMGGGGHPEAFES STPJ- HQAEDSQVRQYFPETW WDI-iFPIGNSGKEAVHVTVPDAITEWKAMSFCTSQSRGFGIjSPTVGLTAFKP FFVDLTDPYS RGEΞ FRLTATl FNYDKDCIRVQTDbAKSHEYQDESWADSQTSSCLCADDAKTHHWNITAV KLGHINFTISTKIbDSNEPCGGQKGFVPQKGRSDTDIKPVLVKPEGVLVEKTHSSbLCPKGGKVASESVSLE J-jPVDXVPDSTKAYVTVIiGDIMGTA QNLDG-jVQMPSGCGEQNMVLFAPTIYVLQYIiEKAGL TEEIRSRAVG FLiEIGYQKELMYKHSNGSYSAFGBRDGNGNT LTAFVTKCFGQAQKFIFIDPKNIQDA K MAGNQ PSGCY ANVGNLIJHTAMKGGVDDEVSLTAYVTAA LEMGKDVDDPMVSQG RCDKNSATSTTNLYTQAIIJ AYIFSIJAG EMDIRNILIjKQIiDQQAIISGESIY SQKPTPSΞNASP SEPAAVDVEIiTAYA ljAQl-jTKPSLTQKEIAKATS IVA AKQHNAYGGFSSTQDTλn/A QAbAKYATTAYMPSEEINLWKSTENFQRTFNIQSVNRLVFQQDTIiP VPGMYTLEASGQGCVYVQTV RYNII-iPPTNMKTFSl-jSVE-rGKARCEQPTSPRSIjTLTIHTSYVGSRSSSNM AIVEVK-MLSGFSPMEGTNQL LQQPLVKKVEFGTDTl NIYLDEl-jIK-NTQTYTFTrSQSVLVTNliKPATIK-VY DYYLPGSFKLSQYTIV SMMNDSIVDSVARHPEPPPFKTEAFIPS PGSVNN
The disclosed NOV2 amino acid sequence has 595 of 1450 amino acid residues (41%) identical to, and 873 of 1450 residues (60 %) positive with, the 1474 amino acid residue Alpha-2-Macroglobuhn Precursor protein from Homo sapiens (ptnπSPTREMBL- ACC:P01023) (E = 2.0e"279).
NOV2 is expressed in at least the following tissues: Hematopoietic tissues, blood plasma, fetal lung, and the coronary artery.
NOV2 also has homology to the ammo acid sequences shown in the BLASTP data listed in Table 2C.
Figure imgf000042_0001
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 2D.
Table 2D. ClustalW Analysis of NOV2
1 ) NOV2 t SEQ ID NO : 10 )
2) gi|l476571θ(xef [XP_00692Ξ. 1 alpha 2 macroglobulm precursor [Homo sapiens] (SEQ ID N0.35) 2) gi I 4557225 ||ref |NP_000005.11 alpha 2 macroglobulin precursor [Homo sapiens] (SEQ
ID NO: 36)
3) gi|224053 |pr j [1009174A macroglobulin alρha2 [Homo sapiens] (SEQ ID N0:37) 4i gi|6978425 ||ref |NP_036620.l| alpha-2-macroglobulrn [Rattus norvegicus1 ' S""E"Q" ID
NO: 3 ga.12144118 lpir| I JCΞ143 alpha-macroglobulin precursor - guinea pig (SEQ ID NO:39)
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
1450 1460 1470 1480
N0V2 ll QQP_ BK^
93- [14765710
Figure imgf000046_0001
1530 1540
ARHPEPPPFKTEAFIPSLPGSWN
Figure imgf000046_0002
Tables 2E-F list the domain description from DOMAIN analysis results against NOV2.
This indicates that the NOV2 sequence has properties similar to those of other proteins known to contain this domain.
Table 2E Domain Analysis of NOV2 gnl [ Pfam [pfam00207, A2M, Alpha-2-macroglobulin family. This family includes the C-termrnal region of the alpha-2 -macroglobulin mily. (SEQ ID NO:80)
CD-Length = 751 residues, 98.5% aligned Score = 563 bits (1451), Expect = 2e-161
Query: 728 EDSQVRQYFPETWLWD FPIGNSGKEAVHVTVPDAITEWKAMSFCTSQSRGFGI-SPTVG 787
+ | +| 1III + IH++ + I μ+1 + ιι + ιι μ ++ | ++| ++ | |
Sbjct: 4 DDITIRSYFPES JJWEVEEVDRSPVLTVNITIJPDSITT EILAVS SNTKGLCVADPVEL S3
Query: 788 TAFKPFFVDLTLPYSWRGESFRLTATIFNYL-KDCIRVQTDLAKSHEYQLESWADSQTS 84S
I 1+ II++I lltliitll i I ++MI ! + l Ni I
Sbjct- 64 TVFQDFFLELRiPySWRGEQVEIiRAVLYNYLPSQDIKV WQE-EVEPLCQAG 115
Query: 847 SCI1CADDAKTHHWNITAVKLGHINFTISTKII-JDSNEPCGGQKGFVPQKGRSDT--JIKPVLV 906
I I -+ [ ++ 1 + ( I, 1 μ ( ++( + (
Sbjct: 116 FCSLATQRTRSSQSVRPKSLSSVSFPWWPLASGLSLVEWASVPEFFVKDAWKILKV 175
Query: 907 KPEGVLVEKTHSSI-LCP KGGKVASESVSLELPVDΪVPD-STK-AYVTVI--GDIMGIALQ 962
+ 111 M llll I II + I I III +1 ++1 11 + ! + l,
Sbjct; 176 EPEGARKEETVSSL- r-PPEHI-GGGDEVSEVPAl-jKI-PDDVPDTEAEAVISVQGDPVAQAIQ 235
Query: 963 N LDGl-VQMPSGCGEQNMVl-FAPII YVbQYI-jEKAGI-I-'IE EIRSRAVGFLEIG 1013
! 1+ II +111 II++ + + + +μ + 1
St : 236 NTLSGEGIjNNLLRIjPSGCGEQNMIYMAPTVYVLHYLDETWQ E PGTKKKQKAIDI-jINKG 295
Query: 1014 YQKELMYKHSNGSYSAFGERDGNGNTWLTAFVtKCFGQAQ FIFIDPKNIQDAljKW-MAG 1072
II+-.I μ ++MI+ II i +1111 III I I 11+ ++II I +-+! (+ 11 -t-
Sbjct: 296 YQRQLNYRKADGSYAAFLHRA- -SSTWLTAFVLKVFSQARNYVFIDEEHICGAVKWLIIjN 353
Query 1073 NQLPSGCYANVGNLDHIAMKGGVDD EVSLTAYVTAALIjEMGKDVDDPMVSQG-jRCL 1128
! ++! Illll I, l + l I Sbjct: 354 QQKDDGVFRESGPVIHNEMKGGVGDDAEVEVTLTAFITIALLEAKIjVCISPWA ALSIL 413
Query: 1129 KNSATSTTN .LYTQALLAYIFSLAGEMDIRNILLKQLDQQAIISGESIYWS--QK 1180
I I I +1! II II! +11! + +11 I ++ + ÷ll II
Sbjct. 414 KASDYLLENYANGQRVYTLALTAYALALAGVLHKLKEILKSLKEELYKALVKGHWERPQK 473
Query: 1181 PTPSSNASPWSEPAAVDVELTAYALLAQLTKPSLTQKEIAKATSIVAWLAKQHNAYGGFS 1240
I + +1, I !l + l + liliill I! I ++ I +l II +! Ill
Sbjct. 474 PKDAPGHPYSPQPQAAAVE TSYALLA]jLT--IιLPFPKVEMAPKVVKWLTEQQYYGGGFG 531
Query: 1241 STQDTWALQALAKYATTAYMPSE-EINLWKSTEN-FQRTFNIQSVNRLVFQQDTLP-N 1297
IIMII + llMll + ll II +++ ++I !+ Il I + I + + 11 1
Sbjct. 532 STQDTVMALQALIS YGIATPTHKEKN SVTIQSPSGSFKSHFQILNNNAFLLRPVEIJPIJN 591
Query: 1298 VPGMYTLEASGQGCVYVQTVLRYNILPPTNMKTFSLSVEIGKARCEQPTSPR-SLTLTIH 1356
I + +1(1 + + I II +1 I I I +1 I +1 + I (+(
Sbjct: 592 EGFTVTAKVTGQGTLTLVTTYRYKVLDKKNTFCFDLKIETVPDTCVEPKGAKNSDYLSIC 651
Query: 1357 TSYVGSRSSSNMAIVEVKMLSGFSPMEGT~~NQ-DiLQQPLVKKVEFGTDTLNIYLDELIK 1414 i i mi i in ++ ii+ii ι++ i i i + + +m++
Sbjct. 652 TRYAGSRSDSGMAIADISMLTGFIPLKPDLKKLENGVDRYVSKYEIDGNHVLLYLDKVSH 711
Query. 1415 -N 1445
Figure imgf000047_0001
Sbjct: 712 SETECVGFKIHQDFEVGLLQPASVKVYDYYEP 743
Table 2F Domain Analysis of NOV2 gnl (p m(pfam01835 , A2M_N, Alpha-2-macroglobulin family N-terminal region. This family includes the N-terminal region of the alpha-2- macroglobulin family (SEQ ID NO: 81) CD-Length = 620 residues, 98.4% aligned Score = 236 bits (603) , Expect = 5e-63
Query: 5 LLLGMLALSPAIAEEL--PNYLVTLPARLNFPSVQKVCLDLSPGYSDVKFTVTLETKDKT 52
II +1 I1 I I I + l +!+ I + +111+ ! ! !l + ) +
Sbjct: 2 LLWLLLLLLLFFDSSLQKPRYMVIVPSILRTETPEKVCVQIJHDLNETVTVTVSLHSFPGK 61
Query 63 QKLLEYSGLK KRHLHCISFLVPPPA- - -GGTEEVATIRVSGVGNNISFEEKKKVLIQ 116
+ I + 1 II+II II I I + + I I +1+11 11+
Sbjct: 62 RNLSSLFTVLLSSKDLFHCVSFTVPQPGLFKΞSKGEESFVWQVKGPTHTFKEKVTVLVS 121
Query. 117 RQGNGTFVQTDKPLYTPGQQVYFRIVTMDSNFVPVNDKYSMVELQDPNΞNRIAQWLEWP 176 l + l II Ikl II II I +1+ ++I I 1 + 1+ +1 ++II 11+ II
Sbjct: 122 SRRGLVFIQTDKPIYTPGQTVRYRVFSVDENLRPLNELI-LVYIEDPEGNRVDQWEVNKL 180
Query: 177 EQGIVDLSFQLAPEAMLGTYTVAV---AEGKTFGT- -FSVEEY VbSPFIihhLSSVLPKFK 231
I II in i I + |μ + + ++ I I, l+l!
Sbjct: 181 EGGIFQtiSFPIPSEPIQGTWKIVARYESGPESNYTHYFEVKEY VLPSFEVS 231
Query 232 VEWEPKELSTVQESFLVKICCRYTYGKPMLGAVQVSVCQKANTYWYREVEREQLPDKCR 291
+ +1 + I I II IIIMII-. I II I I +++I
Sbjct: 232 ITPPKPFIYYDNFKEFEVTICARYTYGKPVPGVAYVRFGVK DEDGKKELLAGLE 285
Query: 292 NBSGQTDKTG--CFSAPVDMATFDLIGYAY-SHQINIVATWE-SGTGVBANA-TQNIYIS 347
+ I I M I + I + + l+! I I ! I
Sbjct. 286 ERAKLLDGNGEICLSQEVLLKELQLKNEDLEGKSLYVAVAVIESEGGDMEEAELGGIKIV 345
Query 348 PQMGSMTFEDTSNFYHPNFPFSGKMLLKFPQGGVLPCKNHLVFLVIYGTNGTFNQTLVTD 407
+ 1 I + + I II 1 + 1+ I I ( I, I + + ++ II
Sbjct: 346 RSPYKLKFVKTPSHFKPGIPFFLKVLWDPDGS--PAPNVPVK--VSAQDASYYSNGTTD 401
Query. 408 NNGLAPFTLETSGWNGTDVSLEGKFQMEDLVYNPEQVPRYYQNAYLHLRPFYSTTRSFLG 467
+ | 11 (++ ( i + +μ + ++ 1 + 11 + I + I
Sbjct. 402 EDGLAQFSINTS--GISSLSITVRTNHKELPEEVQAHAEAQATAYSTVSL--SKSYIHLS 457 Query . 468 IHRLNGPLKCGQPQEVLVDYYIDPADASPDQEISFSYYLIGKGSLVMEGQKHLNSKKKGL 527
I I I I I ++ + + + 1 II ++ 1 1 + 1 I ++ ++
Sbj ct : 458 IER TLPCGPGVGEQANFILRGKSLGELKILHFYYLIMSKGKIVKTGRE PREPG 510
Query : 528 KASFSLSIiTF-TSRLAPDPSLVIYAIFPSGGWADKIQFSVEMCFDN QQL 576
+ 1 1 1 1 + I I I I I I I I I M i l l + ( I I I + 1
Sbjct. 511 QGLFSLSIPVTPDLAPSFRLVAYYILPQGEWADSV IDVEDCCA KLDLSFSPSKDYRL 570 Query: 577 PGAEVELQLQAAPGSLCALRAVDESVLLLRPDRELSNRSVY 617
I +I+I+++I I II, ||||f(++| MM +11 II
Sbj ct ' 571 PAQQVKLRVEADPQSLVALRAVDQAVYLLKPKAKLSMSKVY 611
The proteinase-binding alpha-macroglobulins (A2M) [1] are large glycoproteins found in the plasma of vertebrates, in the hemolymph of some invertebrates and in reptilian and avian egg white. A2M-like proteins are able to inhibit all four classes of proteinases by a 'trapping' mechanism. They have a peptide stretch, called the 'bait region', which contains specific cleavage sites for different proteinases. When a proteinase cleaves the bait region, a conformational change is induced in the protein, thus trapping the proteinase. The entrapped enzyme remains active against low molecular weight substrates, whilst its activity toward larger substrates is greatly reduced, due to steric hindrance. Following cleavage in the bait region, a thiol ester bond, formed between the side chains of a cysteine and a glutamine, is cleaved and mediates the covalent binding ofthe A2M-like protein to the proteinase.
Alpha2 -Macroglobulin (A2M) is a proteinase inhibitor found in association with senile plaques (SP) in Alzheimer's disease (AD). A2M has been implicated biochemically in binding and degradation of the amyloid beta (Abeta) protein which accumulates in SP. We studied the relationship between Alzheimer's disease and a common A2M polymorphism, VallOOO (GTCyilelOOO (ATC), which occurs near the thiolester active site o the molecule. In an initial exploratory data set (90 controls and 171 Alzheimer's disease) we noted an increased frequency of the G/G genotype from 0.07 to 0.12. We therefore tested the hypothesis that the G/G genotype is over-represented in Alzheimer's disease in an additional independent data set: a group of 359 controls and 566 Alzheimer's disease patients. In the hypothesis testing cohort, the G/G genotype increased from 0.07 in controls to 0.12 in Alzheimer's disease (P < 0.05, Fisher's exact test). The odds ratio for Alzheimer's disease associated with the G/G genotype was 1.77 (1.16-2.70, P < 0.01) and in combination with APOE4 was 9.68 (95% CI 3.91 -24.0, P < 0 001). The presence of the G allele was associated with an increase in Abeta burden in a small series. The A2M receptor, A2M-r/L P, is a multifunctional receptor whose ligands include apolipoprotein E and the amyloid precursor protein. These four proteins have each been genetically linked to Alzheimer's disease, suggesting that they may participate in a common disease pathway. Six alpha 2-macroglobuliπ cDNA clones were isolated from two liver cDNA libraries produced from rats undergoing acute inflammation. The coding sequence for rat alpha 2- macroglobulin including its 27-residue signal peptide and the 3' - and part of he 5' nontranslated regions were detemiined. The mature protein consisting of 1445 amino acids is coded for by a 4790 +/- 40 nucleotide messenger RNA. It contains a typical internal thiol ester region and 25 cysteine residues which are conserved between rat and human alpha 2- macroglobulin. Although the amino acid sequences of rat and human alpha 2-macroglobuIin share 73% identity, two small divergent areas of 17 and 38 residues were found, corresponding to 29 and 11% identity, respectively. These areas are located in the bait region and, therefore, may confer specific proteinase recognition capabilities on rat alpha 2- macroglobulin. Following an inflammatory stimulation, rat alpha 2-macroglobulin mRNA levels increased 214-fold over control values and reached a maximum at 1 8 h. By 24 h the levels had decreased to less than 30% o the maximum value. Transcription rates from the alpha 2-macroglobulin gene as measured in nuclear run-on experiments showed a less than 3- fold increase in nuclei from acutely inflamed rats as compared to controls. These results suggest that the accummulation of alpha 2M mRNA is due to the combined effects of increased transcription rates and post-transGriptional processing.
The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) BMBO I. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain ofHeymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.
Alpha-2-macroglobulin is a serum pan-protease inhibitor. A possible correlation to this gene is the following reference on Rhinovirus infections causing exacerbations of eosinophilic airway disease. The acute effects of allergen-challenge on nasal interleukin-8 (IL- 8), eosinophil cationic protein (ECP), and alpha2-macroglobulin were examined in atopic subjects with common cold symptoms. Twenty-three patients with seasonal allergic rhinitis were inoculated with human rhinovirus 16 outside the pollen season. Diluent and allergen challenges, followed by nasal lavages, were carried out about 3 months before and 4 days after virus inoculation. Seventeen patients developed significant common cold symptoms with increased nasal lavage fluid levels of alpha2-macroglobulin, IL-8, and ECP at baseline (p<0.001 -0.05 versus before inoculation), and were further increased by allergen challenge (p< 0.001 -0.05); IL-8 and ECP levels were correlated. See Eur RespirJ \999 Jan;13(l):41-7. Allergen challenge-induced acute exudation of IL-8, ECP and alpha2-macroglobulin in human rhinovirus-induced common colds. Greiff L, Andersson M, Svensson C, Linden M, Myint S, Persson CG Dept of Otorhinolaryngology, Head and Neck Surgery, University Hospital, Lund, Sweden.
In the CNS areaAlpha-2-macroglobulin has been implicated in Alzheimer disease (AD) based on its ability to mediate the clearance and degradation of A-beta, the major component of amyloid beta deposits. Blacker et al. (1998) analyzed a deletion in the A2M gene at the 5-prime splice site of 'exon II' o the bait region (exon 18) and found that inheritance of the deletion, designated A2M-2, conferred increased risk for AD.
The disclosed NOV2 nucleic acid of the invention encoding a Alpha-2-macroglobulin precursor-like protein includes the nucleic acid whose sequence is provided in Table 2A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 2A while still encoding a protein that maintains its Alpha-2-macroglobulin precursor -like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability o the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 40% percent of the bases may be so σhanged. The disclosed NOV2 protein of the invention includes the Alpha-2-macroglobulin precursor -like protein whose sequence is provided in Table 2B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2B while still encoding a protein that maintains its Alpha-2- macroglobulin precursor -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 62% percent of the residues may be so changed.
The NOV2 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in asthma, allergy and psoriasis, Alzheimer disease, Emphysema, pulmonary disease, immune disorders and Cancer and/or other pathologies and disorders. The NOV2 nucleic acid encoding Alpha-2-macroglobulin precursor-like protein, and the Alpha-2-macroglobulin precursor-like protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
NOV2 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. The disclosed NOV2 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV2 epitope is from about amino acids 80 to 100. In another embodiment, a NOV2 epitope is from about amino acids 110 to 200. In additional embodiments, NOV2 epitopes are from about amino acids 290 to 340, from about 380 to 400, from about amino acids 410 to 580, from about amino acids 620 to 730, from about amino acids 810 to 900, from about amino acids 990 to 1 100, from about ammo acids 1 190 to 1210, from about amino acids 1240 to 1320, and from about amino acids 1400 to 1450. These novel proteins can be used in assay systems for functional analysis of various human disorders, which are useful in understanding of pathology of the disease and development of new drug targets for various disorders. NOV3
A disclosed NOV3 nucleic acid of 987 nucleotides (also referred to as GMAC079237_A) encoding a novel Heal Sodium/Bile Acid Cotransporter-hke protein is shown in Table 3 A. An open reading frame was identified beginning with a ATG initiation codon at nucleotides 1 -3 and ending with a TGA codon at nucleotides 979-981. The start and stop codons are in bold letters, and the 3' untranslated region is underlined.
Table 3 A. NOV3 Nucleotide Sequence (SEQ ID NO:ll)
ATGAGAGCCAATTGTTCCAGCAGCTCAGCCTGCCCTGCCAACAGTTCAGAGGAGGAGCTGCCAGTGGGACT GGAGGTGCATGGAAACCTGGAGCTCGTTTTCACAGTGGTGTCCACTATCATGATGGGGCTGCTCATGTTCT CTTTGGGATGTTCCGTGGAGATCCGGAAGCTGTGGTCGCACATCAGGAGACCCTGGGGCATTGCTGTGGGA CTGCTCTGCCAGTTTGGGCTCATGCCTTTTACAGCTTATCTCCTGGCCATTAGCTTTTCTCTGAAGCCAGT CCAAGCTATTGCTGTTCTCATCATGGGCTGCTGCCGGGGGGCACCATCTCTAACATTTTCACCTTCTGGGT TGATGGAGATATGGATCTCAGGTGCCCTGGGAATGATGCCACTCTGCATTTATCTCTACACCTGGTCCTGG AGTCTTCAGCAGAATCTCACCATTCCTTATCAGAACATAGGTCTGTCTTTAGGAATTACCCTTGTGTGCCT GACCATTCCTGTGGCCTTTGGTGTCTATGTGAATTACAGATGGCCAAAACAATCCAAAATCATTCTCAAGG CCGTTGTTGGTGGGGTCCTCCTTCTGGTGGTCGCAGTTGCTGGTGTGGTCCTGGCGAAAGGATCTTGGAAT TCAGACATCACCCTTCTGACCATCAGTTTCATCTTTCCTTTGATTGGCCATGTCACGGGTTTTCTGCTGGC ACTTTTTACCCACCAGTCTTGGCAAAGGACCTTGCCTATCTTTTTAGGTTTAGCITTCAAGACACCCTGΪG ATACCCTACTCGCAATGACTTCGTGTCCTGAATGTTCCAGGCTCATCTATGCCTTCATTCCTCTGCTATAT GGACTCTTCCAGCTGATAGATGGATTTCTTATTGTTGAAGAGAGAACAGAAGATACAGACTGCGATGGTTC ACCTTTACCTGAGTATTTTACTGAGGTAACAATAATACCTAAACAACCTAGGATATGACAGCTT
The disclosed NOV3 nucleic acid sequence maps to the q33 region of chromosome 13 and has 257 of 382 bases (67%) identical to a O cuniculus ileal sodium-dependent bile acid transporter mRNA from O.cimiculus (GENBANK-ID: Z54357) (E = 4.9e"34).
A disclosed NOV3 protein (SEQ ID NO: 12) encoded by SEQ ID NO: 1 1 has 326 amino acid residues, and is presented using the one-letter code in Table 3B. Signal P, Psort and/or Hydropathy results predict that NOV3 does not have a signal peptide, and is likely to be localized to the plasma membrane with a certainty of 0.6000. In other embodiments NOV3 is also likely to be localized to Golgi body with a certainty of 0.4000, to the mitochondrial inner membrane with a certainty of 0.3815, or to the endoplasmic reticulum (membrane) with a certainty of 0.3000.
Table 3B. Encoded NOV3 protein sequence (SEQ ID NO:12). RANCSSSSACPANSSEEELPVGLEVHGNLEDVFTWSTIMMGLLMFSLGCSVEIRKLWSHIRRPWGIAVG LLCQFGLMPFTAYLLAISFSLKPVQAIAVLIMGCCRGAPSLTFSPSGLMEI ISGALGMMPLCIYLYT S SLQQNLTIPYQNIGLSLGITLVCLTIPVAFGVYVNYR PKQSKIILKAWGGVLLLWAVAGWLAKGSWN SDITLLTISFIFPLIGHVTGFLLALFTHQS QRTLPIFLGLAFKTPCDTLLAMTSCPECSRLIYAFIPLLY GLFQLIDGFLIVEERTEDTDCDGSPLPEYFTEVTIIPKQPRI The disclosed NOV3 amino acid has 129 of 331 amino acid residues (38%) identical to, and 198 of 331 amino acid residues (59%) similar to, the 348 amino acid residue ileal sodium/bile acid cotransporter protein from Cricetulus griseus (Chinese hamster) (Q60414) (E= 1 6e"56).
The NOV3 sequence is predicted to be expressed in ileum because of the expression pattern of (GENBANK-ID: NTCI_HUMAN) a closely related ILEAL SODIUM/BILE ACID COTRANSPORTER homolog in species Homo sapien.
TaqMan data for NOV3 can be found below in Example 2. NOV3 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 3E.
Figure imgf000053_0001
gi 11 675553 0 I ref I NP_0 solute 34 ? 125/313 191/313 4 e- 52 35518 . l |l carrier ( 39% ) ( 60% ) family 10 , member 2 [Mus musculus] gi t 284263 l ! sp | Q6041 ILEAL 346 121/306 185/306 4e-52 4 ll NTCI CRIGR SODIUM/BILE (39%) (59%) ACID
COTRANSPORTE R (ILEAL
Figure imgf000054_0001
ACID
COTRANSPORTE R) (NA+ DEPENDENT ILEAL BILE ACID
TRANSPORTER) (ILEAL SODIUM- DEPENDENT BILE ACID TRANSPORTER) (ISBT)
(SODXUM/TAUR OCHOLATE COTRANSPORTI NG
POLYPEPTIDE, ILEAL)
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 3F
Table 3F. ClustalW Analysis of NOV3
1) NOV3 (SEQ ID NO: 12)
3) gill2858115|dbj ||BAB31203.1 | (AK018423) putative [Mus musculus] (SEQ ID NO.40)
4) gi I 3024224 |sp ||Q28727 |NTCI_RABIT ILEAL SODIUM/BILE ACID COTRANSPORTER (ILEAL NA(+)/BILE ACID COTRANSPORTER) (NA+ DEPENDENT ILEAL BILE ACID TRANSPORTER) (ILEAL SODIUM-DEPENDENT BILE ACID TRANSPORTER) (ISBT) (SODIUM/TAUROCHOLATE COTRANSPORTING POLYPEPTIDE, ILEAL) (SEQ ID NO;41)
5) gi|839428l|ref | NPJ058918.1 | solute carrier family 10, member 2 [Rattus norvegicus] (SEQ ID N0.42)
6) gι|6755530|ref |NP_035518.l| solute carrier family 10, member 2 [Mus musculus] (SEQ ID NQ.43)
7) g± I 67S5B 0 |ref |NP_035518.1 | solute carrier family 10, member 2 [Mus musculus] (SEQ ID NO -44)
8) gi|284263lIsp(Q60414 [NTCI_CRIGR ILEAL SODIUM/BILE ACID COTRANSPORTER (ILEAL NAi+./BILE ACID COTRANSPORTER) (NA+ DEPENDENT ILEAL BILE ACID TRANSPORTER) (ILEAL SODIUM-DEPENDENT BILE ACID TRANSPORTER) (ISBT) (SODIUM/TAUROCHOLATE COTRANSPORTING POLYPEPTIDE, ILEAL) (SEQ ID N0.45)
Figure imgf000054_0002
50 60 70 80 NOV3
Figure imgf000055_0002
Figure imgf000055_0001
Figure imgf000055_0003
Figure imgf000056_0001
Table 3G list the domain description from DOMAl-N analysis results against NOV3. This indicates that the NOV3 sequence has properties similar to those of other proteins known to contain this domain.
Table 3G Domain Analysis of NOV3 gnlJ Eam|p£am01758 , SBF, Sodium Bile acid symporter family. This family consists of Na+/bile acid co-transporters. These transmembrane proteins function in the liver in the uptake of bile acids from portal blood plasma a process mediated by the co-transport of Na+ . Also in the family is ARC3 from S. cerevisiae this is a putative transmembrane protein involved in resistance to arsenic compounds. (SEQ ID NO: 82) CD-Length = 186 residues, 80.1% aligned Score = 75.9 bits (185), Expect = 3e-l5
Query. 39 TΪMMGLLMFSLGCSVEIRKLWSHIRRPWGIAVGLLCQFGLMPFTAYLLAIS- FSLKPVQA 97
+ + M l l + li I ! + 1 1 1 + + 1 1 1 ! + + 1 1 ++ 1 1 i l l
Sbjct: 1 ALGLFLMMFSMGLKVRFEDLKEALRRPKALILGLLLQWIIMPLLMFILAWLLLRLPPELA 60 Query: 98 TAVLIMGCCRG APSLTFSPSGL EIWIS GALGMMPLCIYLYTWSWSLQQN 147
++++II I! + 1+ I +1+ + II + II +1
Sbjct: 61 TGLILVGCAPGGAMSNV TYLA GDVELSWMVALSTLLAPLVTPLLSFLLAGLL 115
Query: 148 LTIPYQNIGLSLGITLVCLTIPVAFGVYVNYRWP 181
+ + + + I M 1 1+ 1 + I I
Sbj ct : 116 VHVDAVSPWSLIKSVLVYVIIPLIAGMLTRYFLP 149
Bile acids are synthesized from cholesterol in the liver and secreted into the small intestine, where they facilitate absorption of fat-soluble vitamins and cholesterol. Wong et al. (1996) noted that, rather than being excreted, the majority of bile acids are reabsorbed from the intestine and returned to the liver via the portal circulation. In the liver, bile acids are quantitatively extracted and resecreted into bile, thereby eliminating the need for substantial de novo hepatic bile acid synthesis. The ileum is the major site of active uptake of bile acids from Ihe intestine, where the sodium-gradient-driven transporter has been identified in the ileal enterocyte. Mutations in the transporter or other ileal genes that participate in the transepithelial transport of bile acids are predicted to affect bile acid and cholesterol metabolism significantly. For example, Heubi et l. (1982) described an apparent familial defect in active ileal bile acid transport. Using homologous sequences from hamster and rat, Wong et al. (1995) cloned a cDNA encoding an ileal sodium/bile acid cotransporter gene (designated ISBT by them). They also isolated a genomic clone for human ISBT. The gene encodes a 348-amino acid polypeptide with 7 predicted transmembrane domains and a predicted molecular mass of 38 kD. The native human protein has a relative molecular mass of 40 kD on SDS gel electrophoresis due to N- linked glycosylation. Wong et al. (1 95) demonstrated a dysfunctional mutation (P290S) in the ileal sodium-bile acid cotransporter gene in the course of cloning the human cDNA.
Wong et al. (1996) mapped the SLC10A2 gene to chromosome 13 by study o a human/rodent cell hybrid mapping panel and refined the localization to 13q33 by fluorescence in situ hybridization. The ileal sodium-bile acid cotransporter gene is clearly distinct from the hepatic sodium-bile acid cotransporter gene (SLC10A1; 182396) which maps to chromosome 14. Lammert et al. (1998) mapped the Slcl0a2 gene to mouse chromosome 8 in a region homologous to chromosome 13q33.
Primary bile acid malabsorption (PBAM) is an idiopathic intestinal disorder associated with congenital diarrhea, steatorrhea, interruption o the enterohepatic circulation of bile acids, and reduced plasma cholesterol levels. Oelkers et al. (1997) screened the S C10A2 gene for PBAM-associated mutations using SSCP analysis. Four polymorphisms were identified and sequenced in a family with congenital PBAM. One allele encoded an A171S missense mutation and a mutated donor splice site for exon 3 (601295.0002). The other allele encoded 2 missense mutations at conserved amino acid positions, 243P and T262M (601295.0001). In transfected COS cells, the L243P, T262M, and double mutant (L243P/T262M) did not affect transporter protein expression or trafficking to the plasma membrane; however, transport of taurocholate and other bile acids was abolished. In contrast, the A171S mutation had no effect on taurocholate uptake. The dysfunctional mutations were not detected in 104 unaffected control subjects, whereas the A171S was present in 28% of that population. The findings of Oelkers et al. (1997) established that SLC10A2 mutations can cause PBAM and underscored the ileal Na(+)/bile acid cotransporter's role in intestinal reclamation of bile acids. Autosomal recessive inheritance had been suggested by earlier studies of PBAM patients and was supported by the findings that the proband's son, who inherited an SLC10A2 allele encoding the L243P and T262M missense mutations, was free of clinical symptoms. The authors stated this was the second reported defect of a Na(+)/solute cotransporter, the first having been SLC5A1 (182380), the cotransporter defective in glucose/galactose malabsorption.
As reviewed by Small (1997), the enterohepatic circulation (EHC) is an in vivo ecologic system for the conservation of bile salts, allowing them to be used over and over for the absorption of fat. The EHC confines the bile salt pool to the liver, bile ducts, gallbladder, small intestine, and portal vein. There is a virtual absence of bile salts in the systemic circulation. A minimum of 6 (known or postulated) molecules (3 in the ileal mucosal cells and 3 analogous molecules in the hepatocytes) constitutes the active players in the EHC (Dawson and Oelkers, 1995). The polar ileal enterocytes and the hepatocytes each have 3 unique molecules: a receptor that binds bile salts on one surface and translocates them into the cell, a cellular bile salt binding protein that moves them across the cell, and an exit molecule that moves bile salts out of the other side ofthe cell. In the intestine, ileal sodium/bile salt transporter (ISBT) is present on the brush boarders of the ileum but not the jejunum. It binds bile salts in the gut lumen and transports them across the brush boarder membrane and hands them to the ileal lipid-binding protein (I BP; 600422) which binds bile acid in the cytoplasm of the cell. ILBP allows the bile salts to move through the cytoplasm to the basolateral membrane o the ileal intestinal epithelial cell, where a sodium-independent organic ion exchange system secretes bile salts into portal capillaries. Bile salts in portal capillaries bind to albumin and flow to the liver. There they are recognized by a transporter with high homology to ISBT, the sodium taurocholate cotransporting polypeptide (SLClOAl). Small et al. (1972) suggested that a genetic defed in the predicted bile acid receptor in the ileum would lead to diarrhea and/or steatorrhea and suggested that bile acid turnover and fecal bile acid excretion be studied in patients with unexplained diarrhea. Hess Thaysen and Pedersen (1976) described several patients who had diarrhea and excessive bile acid loss, without other ileal pathology. Heubi et al. (1979, 1982) reported a case study of a boy who presented 48 hours after birth with severe diarrhea, steatorrhea, and malabsorption. Intestinal absorption of bile acid was nearly absent and resulted in a small pool size, a low interluminal bile acid concentration, and severe malabsorption of water and fat. Ileal biopsies had no active bile acid transport. Parenteral nutrition was necessary to sustain the child. At the other extreme, a child with marked bile acid malabsorption but with almost normal development, nearly normal fat absorption, and a moderately well-maintained bile acid pool, was described by Jonas et al. ( 1986). This patient had a 15-fold increase in bile acid synthesis that was adequate to maintain pool size, interluminal bile adds, and fat absorption, Thus, the clinical phenotype apparently can vary from severe diarrhea, fat malabsorption- and malnutrition, to modest diarrhea without significant fat malabsorption. Small (1997) suggested that the bile acid malabsorption and the variable severity could represent mutations in any of the 3 main players in the ileal transport. The disclosed NOV3 nucleic acid of the invention encoding a Ileal Sodium/Bile Acid Cotransporter-like protein includes the nucleic acid whose sequence is provided in Table 3A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 3A while still encoding a protein that maintains its Ileal Sodium/Bile Acid Cotransporter-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleiG acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 33% percent ofthe bases may be so changed. The disclosed NOV3 protein ofthe invention includes the Ileal Sodium/Bile Acid
Cotransporter-like protein whose sequence is provided in Table 3B or 3D. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 3B or 3D while still encoding a protein that maintains its Ileal Sodium/Bile Acid Cotransporter-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 61% percent of the residues may be so changed.
The protein similarity information, expression pattern, and map location for the Heal Sodium/Bile Acid Cotransporter-like protein and nucleic acid (NOV3) disclosed herein suggest that NOV3 may have important structural and/or physiological functions characteristic of the citron kinase-like family. Therefore, the NOV3 nucleic acids and proteins o the invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount o the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
The NOV3 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from Primary bile acid malabsorption (PBAM, an idiopathic intestinal disorder), congenital diarrhea, steatorrhea, refractory infantile diarrhea, interruption of the enterohepatic circulation of bile acids, reduced plasma cholesterol levels, crohn's disease, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Cirrhosis, Transplantation,
Hypercalceimia, Ulcers, growth failure and/or other pathologies. The NOV3 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic cid or the protein are to be assessed.
NOV3 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example the disclosed NOV3 protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated NOV3 epitope is from about amino acids 5 to 30. In another embodiment, a NOV3 epitope is from about amino acids 55 to 60. In additional embodiments, NOV3 epitopes are from about amino acids 140 to 150, from about amino acids 180 to 190, and from about amino acids 280 to 330. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV4
A disclosed NOV4 nucleic acid of 850 nucleotides (designated CuraGen Ace. No. AL161453_A) encoding a novel Prohibitin -like protein is shown in Table 4A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 6-8 and ending with a TGA codon at nucleotides 822-824. A putative untranslated regions upstream of the initiation codon and downstream from the termination codon are underlined in Table 4A, and the start and stop codons are in bold letters. Table 4A. NOV4 Nucleotide Sequence (SEQ ID NO:13)
AAAACATGGCTGCCAAAATGTTTGAGTTCATCGGCAAGTTTGGCCTGGCCTTAGTTGTTGCAG GAGGCGTGGTGAACTCTGCCTTATATAGTGTGGATGCTGGGCACAGAGCTGTCGTCTTTGACC GATTCCGTGGAGTGCAGGACATTGTGGTAGGCAAAGGGACTCACTGTCTCATCCCATGGTTAC AGAAATCAATAATCTTTGACTGCCGTTCTCAGCCACGTAATGTGCCAGTCATCACCGGTAGCA AAGATTTACAGAATGTCAACCTCACACTGCGCATCATCTTCCGGCCCGTAGCTAGCCAGCTTC CTCACATCTTCACCAGCAGCGGAGAGGACCATGATGAGCGTGTGCCGCCGTCCATCACGAACAAGA TCCTCAAGTCAGTGGTGGCTCGCTTTGAAGCTGGAGAACTAATCACCCAGAGAGAGCAGATCTCCAG GCAGGTGAGCGATGACCTTACGGAGCCAGCAGCCACCTTTGGGCTCATTCTGGACGACGTGTC CTTGACATATCTGACCTTCGGGAAGGAGTTCATAGAAGCGGTGGAAGCCAAACAGATAGCTCA GCAGGAAGCAGAGAGGGCCAGATTTGTGGTGGAAAAGGCTGAGCAGCAGAAAAAGGCGGCCAT CATTTCTGCTGAGGGCGACTCCAAGGTGGCCGAGCTGATCACCAACTCACTGGCCACAGCAGG GGACGCCCTGATCGAGCTGCGCAAGCTGGAAGCTGTGGAGGACATCACCTACCAGCTTTTACG CTCTCGGAACATCACCTACCTGCGGGCAGGGCAGTCCATGCCCCTGCAGCTGCGCTGGTGAGG GCCCACCCTGCCTGCACCTCCGAG
The nucleic acid sequence of NOV4, localized on chromosome 9, has 782 of 849 bases (92%) identical to a Prohibitin mRNA from Homo sapiens (GENBANK-ID: S85655) (E = 7 2e"158).
A NOV4 polypeptide (SEQ ID NO: 14) encoded by SEQ ID NO: 13 is 272 amino acid residues and is presented using the one letter code in Table 4B. Signal P, Psort and/or Hydropathy results predict that NOV4 has a signal peptide and is likely to be localized at the endoplasmic reticulum (membrane) with a certainty of 0 5500. In other embodiments, NOV4 may also be localized to the lysosome (lumen) with a certainty of 0.2631, the endoplasmic reticulum (lumen) with a certainty of 0.1000, or extraceliularly with a certainty of 0.1000. The most likely cleavage site for a NOV4 peptide is between amino acids 25 and 26, at: VNS-AL.
Table 4B. NOV4 protein sequence (SEQ ID NO:14)
MAAK FEFIGKFGLALWAGGV SALYSVDAGHRAWFDRFRGVQDI GKGTHCLIPWDQKSIIF DCRSQPRNVPVITGSKDLQNVNLTLRIIFRPVASQIiPHIFTSSGEDH-DERVPPSITNKILKSWARF EAGEIJlTQREQtSRQVSDDLTEPAATFGLILDDVSLIYLTFGKEFIEAVEAKQIAQQEAERARFVVE KAEQQKKAAIISAEGDSKVAELITNSLAXAGDALIEIjRKLEAVEDITYQLLRSRNIXyLRAGQSMPL QLR
The full amino acid sequence of the protein of the invention was found to have 236 of 270 amino acid residues (87%) identical to, and 251 of 270 amino acid residues (92%) similar to, the 272 amino acid residue Prohibitin protein from Homo sapiens ACC: P35232) (E = 3.0e- ).
The disclosed NOV4 protein is widely expressed in a variety of tissues TaqMan data for NOV4 can be found below in Example 2. NOV4 also has homology to the ammo acid sequences shown in the BLASTP data listed in Table 4C.
Figure imgf000062_0002
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 4D.
Table 4D ClustalW Analysis of NOV4
1) N0V4 (SEQ ID NO: 14)
2) gi I 4505773 | re£ ||NP__002625 1 ]| prohibitin (Homo sapiens ( SEQ ID NO : 46 ) 3! gi|92643 lpir| |A39682 prohibitin - rat (SEQ ID N0.47'
4) gi|729854g)gb|AAF53765.ll AE003661 ) l (2 ) 37Cc gene product (Drosophila melanogaster] (SEQ ID N0.48)
5) gi[2055454|gb|,AAB53231.l| U97204 ) prohibi tin-like molecule TC-PRO-1 (Toxocara canis) (SEQ ID NO; 49)
6) gi[l3491275lgb|AAK27865.1 (AC087079 ) Hypothetical protein Y37E3 . 9 [Caenorhabditis elegans] (SEQ ID NO: 50
Figure imgf000062_0001
Figure imgf000063_0001
Tables 4E-F list the domain description from DOMAIN analysis results against NOV4. This indicates that the NOV4 sequence has properties similar to those of other proteins known to contain this domain.
Table 4E. Domain Analysis of NOV4 gnl ( Pfam 1 pf m01145 , Band_7, SPFH domain / Band 7 family. This family also includes proteins with high blast scores to known Band 7 protein:
HflC from E. coli HflK from E. coli, and Prohibitin family members
(SEQ ID NO: 83)
CD-Length = 191 residues, 91.6% aligned
Score = 157 bits (397), Expect = 7e-40
Query: 28 YSVDAGHRAWFDRFRGVQDIWGKGTHCLIPWEIQKSIIFDCRSQPRNVPVITGSKDLQN 87
I I I-*-! 1 1+ lit Ii 1 +1I++ ! 1+ !!
Sbjct: 17 KIVKEYERGVIFRLGRYVRQ-WGPGLHFIIPFIDTVKKVDIJRT YDVPSQEIITKDNV 75
Query. 88 VNLTLRIIFRPVASQLPHIFTSSGEDHDERVPPSITNKILKSWARFEAGELITQREQTS 147
I + +++ i i ++ 11 1 + μ+l+ +1 μ+μιμμ
Sbjct: 76 WIVDAWYYRWDPLKAVYEVED AERALPQLAQTTLRNVIGQFTLDEILTERERIN 132
Query: 148 RQVSDDLTEPAATFGLIIiuDVSLTYLTFGKEFIEAVEAKQIAQQEAERARFVVEKAEQQK 207
I + + I I + 1 + ++ i + + + 1 I + i + μ+ 11 I μ + + I μ
Sbjct: 133 SQLREΪLDEATDPWGIKVERVEIKDΪRLPEEVQRAMAAQMEAEREA-RAKILEAEGEQEA 191 Table 4F. Domain Analysis of NOV4 gnl I Smart | smart00244 , PHB, prohibitin homologues; prohibitin homologues (SEQ ID NO 84)
CD-Length = 160 residues, 98.8% aligned
Score = 97.4 bits (241), Expect = 9e-22
Query: 28 YSVDAGHRAWFDRFRGVQDIWGKGTHCLIPWLQKSIIFDCRSQPRNVPVITG-SKDLQ 86
+ i i + ι i i+ i I i + n++ i μ ιι + ι ι +m
Sbjct: 3 FYVIGEGERGWERLGRVLK-VLGPGLHFVIPFIDDVKRVDLRAQTDDVPPQEVITKDNV 61 Query: 87 NVNLTLRIIFRPVASQLPHIFTSSGEDHDERVPPSITNKILKSVVARFEAGELIT-QREQ 145 j ++ + + | i j ++ M i + μ j μ + | | + | + | μ
Sbj ct : 62 TVSVDAWYYR-VLDPLKAVYGV- -LDADYRALRQLAQTTLRSVIGKRTL.DELLTDEREK 118 Query : 146 ISRQVSDDLTEPAATFGLILDDVSLTYLTFGKEFIEAVEAKQ 187
I I + ++ lι I I + | + ++ | | + + + 1 I l + l ll + l
Sbj ct : 119 ISEN1REELNEAAEPWGIEVEDVEIKDIRLPEEIKEAMEAQQ 160
Genes that negatively regulate proliferation inside the cell are of considerable interest because of the implications in processes such as development and cancer. Prohibitin, a novel cytoplasmic anti- proliferative protein widely expressed in a variety of tissues, inhibits DNA synthesis. Studies have suggested that Prohibitin may be a suppressor gene and is associated with tumor development and/or progression of at least some breast cancers. Sequence comparisons suggest that the Prohibitin gene is an analogue of Cc, a Drosophila gene that is vital for normal development.
Prohibitin is a 30-kD intracellular, antiproliferative protein. White et al. (1991) mapped the gene to chromosome 17 by analysis of human-mouse somatic cell hybrid cell lines using a genomic fragment of human Prohibitin DNA isolated from a library using the rat Prohibitin cDNA clone. By a study of cell lines containing portions of human chromosome 17, they determined that the PHB gene was located in the 17qll.2-q23 region. By in situ hybridization, they localized the gene to 17q21. Sato et al. (1992) isolated the human homolog of the rat Prohibitin gene and mapped it to 17ql2-q21 by in situ hybridization. Sato et al. (1993) showed that the human Prohibitin gene family consists of 1 functional PHB gene on 7q21 and 4 processed pseudogenes, each on a different chromosome: PHBP1 on 6q25, PHBP2 on 1 l pl l.2, PHBP3 on l p3 .3, and PHBP4 on 2q21.
By DNA sequence analysis of 2 exons in the PHB gene in 23 sporadic breast cancers that showed loss of heterozygosity on 17q or developed in patients 35 years old or younger, they identified 4 cases of somatic mutation: 2 of these were missense mutations, 1 showed a 2- bp deletion resulting in truncation ofthe gene product due to frameshift, and the fourth had a C-to-T transition in an intron adjacent to an intron-exon boundary. Sato et al. (1993) found no mutations in the PHB gene in other forms of tumors, namely, those of ovary, liver, and lung. The disclosed NOV4 nucleic acid ofthe invention encoding a Prohibitin -like protein includes the nucleic acid whose sequence is provided in Table 4A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 4A while still encoding a protein that maintains its Prohibitin -like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications indude, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability o the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOV4 protein of the invention includes the Prohibitin -like protein whose sequence is provided in Table 4B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 4B while still encoding a protein that maintains its Prohibitin -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 43% percent of the residues may be so changed.
The protein similarity information, expression pattern, and map location for the Prohibitin-like protein and nucleic acid (NOV4) disclosed herein suggest that this NOV4 protein may have important structural and/or physiological functions characteristic ofthe Prohibitin family. Therefore, the NOV4 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount of the nucleic acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo. The NOV4 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from breast cancer (In a sporadic breast cancer, Sato et al. (1992) found a missense mutation from valine (GTC) to alanine (GCC) at codon 88 of the PHB gene), and/or other pathologies. The NOV4 nucleic acids, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV4 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example, the disclosed NOV4 protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOV4 epitope is from about amino acids 40 to 45. In another embodiment, a NOV4 epitope is from about amino acids 60 to 75. In additional embodiments, NON4 epitopes are from about amino acids 100 to 130, from about amino acids 140 to 160,, from about amino acids 180 to 220, and from about amino acids 240 to 260. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
ΝOV5
A disclosed NOV5 nudeic acid of 2011 nucleotides (also referred to as dj l 182al4__dal) encoding a novel Macrophage Stimulating Protein Precursor -like protein is shown in Table 5A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1-3 and ending with a TAG codon at nucleotides 1999-2001. A putative untranslated region downstream from the termination codon is underlined in Table 5A, and the start and stop codons are in bold letters.
Table 5A. NOV5 Nucleotide Sequence (SEQ ID NO:15) ATGGGGTGGCTCCCACTCCTGCTGCTTCTGACTCAATGCTTAGGGGTGCCTGGTCAGCGCTC GCCATTGAATGACTTCCAAGTGCTCCGGGGCACAGAGCTACAGCACCTGCTACATGCGGTGG TGCCCGGGCCTTGGCAGGAGGATGTGGCAGATGCTGAAGAGTGTGCTGGTCGCTGTGGGCCC TTAATGGACTGCCGGGCCTTCCACTACAACGTGAGCAGCCATGGTTGCCAACTGCTGCCATG GACTCAACACTCGCCCCACACGAGGCTGCGGCGTTCTGGGCGCTGTGACCTCTTCCAGAAGA AAGACTACGTACGGACCTGCATCATGAACAATGGGGTTGGGTACCGGGGCACCATGGGCACG ACCGTGGGTGGCCTGCCCTGCCAGGCTTGGAGCCACAAGTTCCCAAATGATCACAAGTACAC GCCCACTCTCCGGAATGGCCTGGAAGAGAACTTCTGCCGTAACCCTGATGGCGACCCCGGAG GTCCCTGGTGCTACACAACAGACCCTGCTGTGCGCTTCCAGAGCTGCGGCATCAAATCCTGC CGGGAGGCCGCGTGTGTCTGGTGCAATGGCGAGGAATACCGCGGCGCGGTAGACCGCACGGA GTCAGGGCGCGAGTGCCAGCGCTGGGATCTTCAGCACCCGCACCAGCACCCCTTCGAGCCGG GCAAGTTCCTCGACCAAGGTCTGGACGACAACTATTGCCGGAATCCTGACGGCTCCGAGCGG CCATGGTGCTACACTACGGATCCGCAGATCGAGCGAGAGTTCTGTGACCTCCCCCGCTGGGG TTCCGAGGCACAGCCCCGCCAAGAGGCCACAACTGTCAGCTGCTTCCGCGGGAAGGGTGAGG GCTACCGGGGCACAGCCAATACCACCACCGCGGGCGTACCTTGCCAGCGTTGGGACGCGCAA ATCCCGCATCAGCACCGATTTACGCCAGAAAAATACGCGTGCAAGGACCTTCGGGAGAACTT CTGCCGGAACCCCGACGGCTCAGAGGCGCCCTGGTGCTTCACACTGCGGCCCGGCATGCGCG CGGCCTTTTGCTACCAGATCCGGCGTTGTACAGACGACGTGCGGCCCCAGACTGCTACCACG GCGCAGGGGAGCAGTACCGCGGCACGGTCAGCAAGACCCGCAAGGGTGTCCAGTGCCAGCGC TGGTCCGCTGAGACGCCGCACAAGCCGCAGTTCACGTTTACCTCCGAACCGCATGCACAACT GGAGGAGAACTTCTGCCGGAACCCAGATGGGGATAGCCATGGGCCCTGGTGCTACACGATGG ACCCAAGGACCCCATTCGACTACTGTGCCCTGCGACGCTGCGCTGATGACCAGCCGCCATCA ATCCTGGACCCCCCAGACCAGGTGCAGTTTGAGAAGTGTGGCAAGAGGGTGGATCGGCTGGA TCAGCGGCGTTCCAAGCTGCGCGTGGTTGGGGGCCATCCGGGCAACTCACCCTGGACAGTCA GCTTGCGGAATCGGΪ TGCTGCCTCTCACGGGCTATGAGGTATGGTTGGGCACCCTGTTCCA GAACCCACAGCATGGAGAGCCAAGCCTACAGCGGGTCCCAGTAGCCAAGATGGTGTGTGGGC CCTCAGGCTCCCAGCTTGTCCTGCTCAAGCTGGAGAGATCTGTGACCCTGAACCAGCGTGTG GCCCTGATCTGCCTGCCCCCTGAATGGTATGTGGTGCCTCCAGGGACCAAGTGTGAGATTGC AGGCTGGGGTGAGACCAAAGGTACGGGTAATGACACAGTCCTAAATGTGGCCTTGCTGAATG TCATCTCCAACCAGGAGTGTAACATCAAGCACCGAGGACGTGGTGACTACGGGGGCCCACTT GCCTGCTTTACCCACAACTGCTGGGTCCTGGAAGGAATTATAATCCCCAACCGAGTATGCGC AAGGTCCTGCTGGCCAGCTGTCTTCACGCGTGTCTCTGTGTTTGTGGACTGGATTCACAAGG TCATGAGACTGGGTTAGGCCCAGCCTT
The NOV5 nucleic acid was identified on the q21 region of chromosome 3 and has 1508 of 1524 bases (98%) identical to a gb: GENBANK-ID :HUMMST1 A|acc:L11924 mRNA from Homo sapiens (Homo sapiens macrophage-stimulating protein (MSTl) mRNA, complete eds (E = 0.0).
A disclosed NOV5 polypeptide (SEQ ID NO: 16) encoded by SEQ ID NO: 15 is 666 amino acid residues and is presented using the one-letter code in Table 5B. Signal P, Psort and/or Hydropathy results predict that NOV5 has a signal peptide and is likely to be localized in the lysosome (lumen) with a certainty of 0.5493. In other embodiments, NOV5 may also be localized to extraceliularly with acertainty of 0.3700, the microbody (peroxisome) with a certainty of 0.1588, or the endoplasmic reticulum (membrane) with a certainty of 0.1000. The most likely cleavage site for a NOV5 peptide is between amino acids 18 and 19, at: VPG-QR.
Table 5B. Encoded NOV5 protein sequence (SEQ ID NO:16)
MGWLPLLLLLTQCLGVPGQRSPLNDFQVLRGTELQHLLHA PGPWQEDVADAEECAGRCGPLMDCRAFHYN VSSHGCQLLP TQHSPHTRLRRSGRCDLFQKKDYVRTCIMNNGVGYRGT ATTVGGLPCQAWSHKFPNDHKY TPTLRNGLEENFCRNPDGDPGGPWCYTTDPAVRFQSCGIKSCREAACVWCNGEEYRGAVDRTESGRECQRWD LQHPHQHPFEPGKFLDQGLDDNYCRNPDGSERP CYTTDPQIEREFCDLPRCGSEAQPRQEATTVSCPRGKG EGYRGTA TTTAGVPCQRWDAQIPHQHRFTPEKYACKDLRENFCRNPDGSEAPWCFTLRPGMRAAFCYQIRR CTDDVRPQTATTAQGSSTAARSARPARVSSASAGPLRRRTSRSSRLPPNRMHN RRTSAGTQMGIAMGPGAT _RWTQGPHSTTVPCDAALMTSRHQSWTPQTRCSLRSVARGWIGWI5GVFSCAWLGAIRATHPGQSACGIGMLg_ LTGYEVWLGTLFQNPQHGEPSLQRVPVAKMVCGPSGSQLVLLKLERSVTLNQRVALICLPPE YWPPGTKC EIAG GETKGTGNDTVLNVALLNVISNQECNIKHRGRGDYGGPLACFTHNCWVLEGIIIPNRVCARSC PAV FTRVSVFVD IHKVMRLG
The disclosed NOV5 amino acid sequence has 368 of 368 amino acid residues (100%) identical to, and 368 of 368 amino acid residues (100%) similar to, the 711 amino acid residue ptnr:SPTREMBL-ACC:Q14870 protein from Homo sapiens (Human) (Macrophage- Stimulating Protein Precursor (E = 9.9e" ).
NOV5 is expressed in at least HepG2 (liver) In addition, the sequence is predicted to be expressed in the following tissues because o the expression pattern of (GENBANK-ID: HUMMSTlA|acc:L11924) a closely related {Homo sapiens macrophage-stimulating protein (MSTl) mRNA, complete eds homolog in species Homo sapiens : HepG2, and liver.
TaqMan data for NOV5 can be found below in Example 2. NOV5 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 5C.
Figure imgf000068_0001
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 5D. Table 5D Clustal W Sequence Alignment
1 ) N0V5 (SEQ ID NO 16 )
2) gι| 15294659 I ref |XP_054070 l|| macrophage stimulating 1 (hepatocyte growth factorlike) [Homo sapiens] (SEQ ID No 51)
3) gij 10337615 |ref |NP_066278 l| macrophage stimulating 1 (hepatocyte growth factorlike) [Homo sapiens] (SEQ ID NO 52)
4) go. I 123114 | sp | P26927 | HGFL_HUMAN HEPATOCYTE GROWTH FACTOR-LIKE PROTEIN PRECURSOR (MACROPHAGE STIMULATORY PROTEIN) (MSP) (MACROPHAGE STIMULATING PROTEIN) (SEQ ID
NO 53)
5) gi || 15299258 I ref |XP_017811 2 || hypothetical protein XP_017811 [Homo sapiens] (SEQ ID NO 54)
6) gi I 90615 Ipir I |A40332 macrophage-stimulating protein 1 precursor - mouse (SEQ ID NO 55)
Figure imgf000069_0001
Figure imgf000070_0001
290 300 310 320
NOV5 gi|l5294659 | ©ERQE^TT'.WS;0--?RGKGEG^GTAlCT?ITffiGW-|CQRW-0AQI gi|l0337615| -.QE QEAΕ SC RGKGEGϊ-RσϊAOTTTA fGQR BAQI gi 1123114 I QPRQEATTVSGFRGKGEΘΪBGTANIΓTTAGVPGQR DAQI gi |15299258 \ OPRQEATILVSGFRGKGEGYRGTAMTTTAGVSCQRWDAQI gi ] 90615 |
Figure imgf000070_0002
530 540 550 560
PLTGYEVWLGT FQNPQHG
SLVKEQWILTARQCFSSCHMPLTGYEVWLGTLFQNPQHGΪ
Figure imgf000070_0003
Figure imgf000071_0001
610 620 630 640
ϊ? gΞ g M!£ ϊ uSMMmFMgg£
Figure imgf000071_0002
Figure imgf000071_0003
Figure imgf000071_0004
Tables 5E-N list the domain description from DOMAIN analysis results against NOV5. This indicates that the NOV5 sequence has properties similar to those of other proteins known to contain this domain.
Table 5E. Domain Analysis of NOV5 qnl )P--.am|p£am00051, kringle, Kringle domain. Kringle domains have been found in plas inogen, hepatocyte growth factors, prothrombin, and apolipoprotein A. Structure is disulfide-rich, nearly all-beta. (SEQ
ID NO i 85)
CD-Length = 79 residues, 100.0% aligned
Score = 117 bits (292) , Expect = 3e-27
Query: 191 CVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPF-EPGKFLDQGIiDDNYCRNPDGSERP 249
I III III 11(1 Mill I ll + l I -H- -.11 +IIIIIIII III Sbjct 1 CYHGNGENYRGTASTTESGAPCQRWDSQTPHRHSKYTPERYPAKGLGENYCRNPDGDERP 60 Query 250 268
Sbjc 61
Figure imgf000072_0001
79
Table 5F. Domain Analysis of NOV5 gnl 1 Pf m|pfamO0051) kringle, Kringle domain. Kringle domains have been found in plasminogen, hepatocyte growth factors, prothrombin, and apolipoprotein A. Structure is disulfide-rich, nearly all-beta. (SEQ
ID NO: 85)
CD-Length = 79 residues, 100.0% aligned
Score = 112 bits (279) , Expect = 9e-26
Query: 283 CFRGKGEGYRGTANTTTAGVPCQRWDAQIPHQHRF-TPEKYACKDLRENFCRNPDGSEAP 341
1+ I II lllll + ll +1 HI MM ll + l MM I II I M 111, ! I I !
Sbjct- 1 CYHGNGENYRGTASTTEΞGAPCQR DSQTPHRHSKYTPERYPAKGLGENYCRNPDGDERP 60
Query 342 CFTLRPGMRAAFCYQIRRC 361
IM I +1 +1 I II
Sbjct 61 CYTTDPRVR EYC-DIPRC 79
Table 5G. Domain Analysis of NOV5 gnl I Pfam 1 pfaπ.00051, kringle, Kringle domain. Kringle domains have been found in plasminogen, hepatocyte growth factors, prothrombin, and apolipoprotein A. Structure is disulfide-rich, nearly all-beta. (SEQ
ID NO:8Ξ)
CD-Length = 79 residues, 100.0% aligned
Score = 104 bits (259) , Expect = 2e-23
Query. 110 CIMNNGVGYRGT.V-ATTVGGLPCQAWSHKFPNDH-KYTPT--LRNGLEEI-JFCRNPDGDPGG 166
I II Mill +111 I III I + 1+ 1 1111 III, I Ml III II sbjct- i CYHGNGENYRGTASTTESGAPCQRWDSQTPHRHSKYTPERYPAKGLGENYCRNPDGDE-R 59
Query. 167 P CYTTDPAVRFQΞCGIKSC 186
III Mill IM I I !
Sbjct. 60 P CYTTDPRVR EYCDIPRC 79
Table 5H. Domain Analysis of NOV5 gnl (Smar [smart00130, KR, Kringle domain; Named after a Danish pastry. Found in several serine proteases and in ROR-Iike receptors. Can occur in up to 38 copies (in apolipoprotein (a) ) . Plasminogen-like kringles possess affinity for free lysine and lysine- containing peptides. (SEQ ID NO: 86)
CD-Length = 83 residues, 97.6% aligned Score = 111 bits (278) , Expect ---. le-25
Query 191 CVWCNGEEYRGAVDRTESGRECQRWDLQHPHQHPFEPGKFLDQGLDDNYCRNPDG-SERP 249
II III ill 1 + 11+ 1,1 II II I Ml II ! I +1, + 11+ mm II II I Sbjct: 3 CYAGNGESYRGTASTTKΞGKPCQR DSQTPHLHRFTPERFPELGLEHNYCRNPDGDSEGP 62 Query 250 WCYTTDPQIEREFCDLPRCGS 270 Mi l ! ! ! I + l + l l + l + l I
Sb}ct: 63 WCYTTDPNVRWEYCDIPQCES
Table 51. Domain Analysis of NOV5 gnl j Smart | smart00130 , KR, Kringle domain,- Named after a Danish pastry
Found m several serine proteases and in ROR-like receptors. Can occur in up to 38 copies (in apolipoprotei (a) ) . Plasmmogen-like kringles possess affinity for free lysine and lysine- containing peptides.
(SEQ ID NO: 86)
CD-Length = 83 residues, 97.6% aligned
Score = 106 bits (265) , Expect = 4e-24
Query: 108 RTCIMNNGVGYRGTMATTVGG PCQAWSHKPPNDHKYTPTLRN--GLEENFCR PDGDPG 165
I I II MM1 +11 il l + 1+ MM Ml 1 + 1 MM 11
Sbjct: 1 RDCYAGNGESYRGTASTTKSGKPCQRWDSQTPHLHRFTPERFPELGLEHNYCRNPDGDSE 60 Query 166 GPWCYTTDPAVRFQSCGIKSC 186
HUM III IM i i t
Sbjct: 61 GPWCYTTDPNVRWEYCDIPQC 81
Table 5J. Domain Analysis of NOV5 gnl (Smart [ smart00130, KR, Krmgle domain; Named after a Danish pastry. Found in several serine proteases and in ROR-like receptors. Can occur in up to 38 copies (in apolipoprotein (a) ) . Plasminogen-like kringles possess affinity for free lysine and lysine- containing peptides. (SEQ ID NO; 86)
CD-Length = 83 residues, 97.6% aligned Score = 104 bits (260! , Expect = le-23
Query: 283 CFRGKGEGYRGTANTTTAGVPCQRWDAQIPHQHRFTPEKYACKDLRENFCRNPDG-SEAP 341
!+ I II I II I II 1 + 11 +1 MIIM + ll II IMMI1++ I l + l II III II I
Sbjct: 3 CYAGNGESYRGTASTTKSGKPCQRWDSQTPHLHRFTPERFPELGLEHNYCRNPDGDSEGP 62 TD 363
Figure imgf000073_0001
ES 83
Table 5K. Domain Analysis of NOV5 gnl I Smar (smart00020 , Tryρ_SPc, Trypsm-like serine protease; Many of these are synthesised as inactive precursor zymogens that are cleaved during limited proteolysis to generate their active orms. A few, however, are active as single chain molecules, and others are inactive due to substitutions of the catalytic triad residues. (SEQ ID NO: 87) CD-Length = 230 residues, 79 ι% aligned Score = 110 bits (274), Expect = 3e-25
Query; 504 PLTGYEVWLGTLFQNPQHGEPSLQRVPVAKMVCGPSGSQ- -LVLLKLERSVTLNQR 557
+ i Ii + + Ml I + ! ++ I + + + 11,11 IM +
Sbjct: 49 APSS1RVRLGSHDLSSGEET---QTVKVSKVIVHPNYNPSTYDNDIALLKLSEPVTLSDT 105 Query: 558 VALICLPPEWYWPPGTKCEIAGWGETKGTG--NDTVLNVALLNVISNQECNIKHRGR-- 613
II Mill I II III I ++IIII I + II + ++11 I + I
Sbjct: 106 VRPICLPSSGYNVPAGTTCTVSGWGRTSESSGSLPDTLQEVNVPIVSNATCRRAYSGGPA 165 Query 614 GDYGGPLACFTHNC VLEGIIIPNRV-CARSC PAVFTRVΞ 653
II lllli II III 11+ III I ill
Sbjct: 166 ITDNMLCAGGLEGGKDACQGDSGGPLVC-NDPR VLVGIVSWGSYGCARPNKPGVYTRVΞ 224
Query. 654 VFVDWI 659 ++|||
Sbjct. 225 SYLDWI 230
Table 5L. Domain Analysis of NOV5 gnlj Pfam|ρfam00089, trypsin, Trypsin. Proteins recognized include all proteins m families SI, S2A, S2B, S2C, and S5 in the classi ication of peptidases Also included are proteins that are clearly members, but that lack peptidase activity, such as haptoglobin and protein Z (PRTZ*) . (SEQ ID NO: 88) CD-Length = 217 residues, 77.9% aligned Score = 92.0 bits (227), Expect = 9e-20
Query: 508 YEV LGTLFQNPQHGEPSLQRVPVAKMV CGPSGSQLVLLKLERSVTLNQRVALICL 563
Ml I l + l+ l lι++ I + + 1111+ HI I III
Sbjct. 49 VRWLGE--HNLGTTEGTEQKFDVKKIIVHPNYNPDTNDIALLKLKSPVTLGDTVRPICL 106
Query: 564 PPE YWPPGTKCEIAGWGETKGTGNDTVLNVALLNVISNQECNIKHRG 612 I +| MI ! +.+ IH Ml I I ++ ++M I + 1
Sbjct: 107 PSASSDLPVGTTCSVSGWGRTKNLGTΞDTLQEWVPIVSRETCRSAYGGTVTDTMICAGA 166
Query: 613 RGDYGGPLACFTHNC VLEGIIIPNRVCARSC PAVFTRVSVFVD I 659
+ MI MM II II 11+ III +1 l + IIM ++MI
Sbjct: 167 LGGKDACQGDSGGPLVCSDG---ELVGIVS GYGCAVGNYPGVYTRVSRYLDWI 217
Table 5M. Domain Analysis of NOV5 gnl ] Smart ] smart00473 , PAN_AP, divergent subfamily of APPLE domains;
Apple-like domains present m Plasminogen, C elegans hypothetical
ORFs and the extracellular portion of plant receptor-like protein kinases. Predicted to possess protein- and/or carbohydrate-binding functions. (SEQ ID NO: 89)
CD-Length = 79 residues, 94.9% aligned
Score = 52.0 bits (123), Expect = le-07
Query: 25 DFQVLRGTELQHLLHA PGP QEDVADAEECAGRC-GPLMDCRAFHYNVSSHGCQLLP 83 I I IM I I! Mil +| ll + l II + I! I
Sbjct: 5 CFVRLPNTKL PDFSPIVISVASLEECAQKCLNSNCSCRSFTYNNDTKGCLL SE S8
Query: 84 TQHSPHTRLRRSGRCDLFQKK 104
+ 1 III I, ++I
Sbjct: 59 SSLGDARQLLPSGGVDYYEKI 79 Table 5N. Domain Analysis of NOV5 gnl |ι Pfam| pfam00024 , PAN, PAN domain. The PAN domain contains a conserved core of three disulphide bridges. In some members of the family there is an additional fourth disulphide bridge the links the N and C termini of the domain. The domain is found in diverse proteins, in some they mediate protein-protein interactions, in others they mediate protein-carbohydrate interactions. (SEQ ID NO: 159) CD-Length = 78 residues, 76.9% aligned Score = 50.1 bits (118), Expect = 4e-07
Query 45 P QEDVADAEECAGRCGPLMD-CRAFHYNVSSHGCQLLP TQHSPHTRLRRSGRCDLFQK 103
+ 1 Mi l l I I M M i l l + f ( M M+l
Sbj ct 17 ISWNVPSLEECAALCLEEPRVCRSFTYNNKSKQCLLKSESSGSLPRLKRPSQKVDYYEK
MaGrophage-stimulating protein (MSP) is an 80- D serum protein with homology to hepatocyte growth factor (HGF)(Sakamoto 0, etal.; J Clin Invest 1997 Feb 15;99(4):701-9). Its receptor, RON tyrosine kinase, is a new member of he HGF receptor family. The MSP- RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of
RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium o the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP -RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.
Macrophage stimulating protein (MSP; 142408), also known as hepatocyte growth factor-like protein (HGFL), is structurally related to hepatocyte growth factor. Gaudino et al. (EMBO J. 13 : 3524-3532, 1994) showed that the RON gene is expressed at the cell surface of several epithelial cell types in addition to granulocytes and monocytes. The RON mRNA is translated into a glycosylated precursor that is cleaved into a 185-kD heterodimer of 35-kD (alpha) and 150-kD (beta) subunits joined by the predicted disulfide linkage. Gaudino et al. ( 1994) further demonstrated that the beta chain undergoes tyrosine phosphorylation upon stimulation by MSP. By isotopic in situ hybridization, Ronsin et al. (Oncogene 8: 1 195-1202, 1993) mapped the RON gene to 3p21 , with the most probable location being 3p21.3. The gene encoding MSP is also located on 3p21 , a region of frequent deletion or mutation in small cell lung and renal carcinoma.
The genes encoding hepatocyte growth factor (HGF; 142409) and its MET receptor are located on 7q. The location of ligand and receptor on the same chromosome, and the structural similarities between MSP and HGF suggested that the ligand for RON might be MSP. Wang et al. (Science 266: 117- 1 19, 1994) showed that this is indeed the case. Their experiments established that the RON gene product is a specific cell surface receptor for MSP. A human hepatoma (HepG2) cell line library was screened with an oligonucleotide probe for macrophage stimulating protein (MSP) to clone an MSP cDNA(Yoshimura T, et.al.; J Biol Chem 1993 Jul 25;268(21): 15461-8). Deduced sequences of isolated clones were compared with peptide fragment sequences of MSP. MSP9 cDNA encoded most of the known sequence of MSP except for a small segment of the 5' end o the open reading frame. Consequently, a hybrid 2300-base pair cDNA that encoded the complete MSP amino acid sequence was constructed from 2 clones. Culture fluid from COS-7 cells transfected with this full-length MSP cDNA had MSP biological activity, and the expressed MSP was detected by immunoprecipitation with antibody against native MSP. The deduced amino acid sequence of MSP includes 4 kringle domains, which have been found in hepatocyte growth factor and several proteins of the blood coagulation system. Among them, MSP has the highest sequence similarity to hepatocyte growth factor (45% identity). The MSP cDNA hybridized strongly to mR A from liver, and to a lesser extent to mRNA from kidney and pancreas, suggesting that a cell type in the liver is the source of MSP. Several cloned and sequenced MSP cDNAs had insertions or deletions, suggesting that alternatively spliced MSP mRNAs may occur. This was reflected in Northern blots probed with an MSP cDNA, which showed more than one mRNA species. Furthermore, although the gene coding for MSP is on chromosome 3, the sequence of one o the cDNAs was identical with a unique sequence in chromosome 1, indicating that there may be a family of MSP genes, located on chromosomes 3 and 1.
Although the hepatocyte growth factor-like protein (HLP) shares a 50% homology with the hepatocyte growth factor, the biological function of HLP has remained unknown (Shimamoto A, et.al.; FEBS Lett 1993 Oct 25;333(l-2):61 -6). Addition of conditioned medium of COS-7 cells transfected with the expression plasmid for HLP cDNA to cultures of resident peritoneal macrophages induced specific activation of macrophages, and the factor which stimulates macrophages was purified from the conditioned medium. The purified protein showed M(r) of 85 kDa on SDS-PAGE, and this M(r) is in agreement with that of macrophage-stimulating protein (MSP) previously purified from human serum, as well as with the predicted M(r) of HLP. Amino acid composition of the purified protein coincided with the compositions of human HLP and MSP. Together with the finding that the partial amino acid sequences of MSP are highly homologous to that of HLP, we conclude that the biological function of HLP is to activate macrophages and that HLP and MSP are identical molecules. The disclosed NOV5 nucleic acid of the invention encoding a Macrophage Stimulating
Protein Precursor -like protein includes the nucleic acid whose sequence is provided in Table 5A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 5A while still encoding a protein that maintains its Macrophage Stimulating Protein Precursor -like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any o the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability o the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOV5 protein of the invention includes the Macrophage Stimulating Protein Precursor -like protein whose sequence is provided in Table 5B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 5B while still encoding a protein that maintains its Macrophage Stimulating Protein Precursor -like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 39% percent o the residues may be so changed.
The NOV5 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in Aicardi-Goutieres syndrome 1 ; Brugada syndrome; Deafness, autosomal recessive 6; Heart block, nonprogressive; Heart block, progressive, 2;
Ichthyosi forme erythroderma, congenital, nonbullous; Long QT syndrome-3; Night blindness; congenital stationary; Pituitary ACTH-secreting adenoma; Small-cell cancer of lung; Ventricular fibrillation, idiopathic; entricular tachycardia, idiopathic; HIV infection, susceptibility/resistance to; Von Hippel-Lindau (VHL) syndrome; Cirrhosis; Transplantation as well as other diseases, disorders and conditions. The NOV5 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
NOV5 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example the disclosed NOV5 protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated NOV5 epitope is from about amino acids 20 to 80. In other embodiments, NOV5 epitope is from about amino acids 90 to 120, from about amino acids 140 to 180, from about amino acids 190 to 340, from about amino acids 350 to 460, from about amino acids 500 to 530, from about amino acids 570 to 590, and from about amino acids 600 to 620, or from about amino acids 420 to 460. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV6
A disclosed NOV6 nucleic acid of 634 nucleotides (also referred to as GM382a20_A) encoding a novel Fatty Acid-Binding Protein-like protein is shown in Table 6A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 58-60 and ending with a TAA codon at nucleotides 460-462. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 6A, and the start and stop codons are in bold letters.
Table 6A. NOV6 Nucleotide Sequence (SEQ ID NO:17)
CAAGCTGCCCACGCCGACGGCAACCCTGCTCTGCATGCCCGCCCGCCCGTGCCCACCATGGCCACAGTTC AGCAGCTGGGAGGAAGATGGCGCCTGGTGGACAGCAAACGCTTTGATGAATACATGAAGGAGGGAGGAGT GGGAACTGCTITGCGAAAAATGGACGCAATGGCCAAGCCAGATTGTATCATCACTTGTGATGGCAAAAAC CTCACCATAAAAACCGAGAGCACTTTGAAAACACAGTTTTCTTGTACCCTGGGAGAGAAGTTTGAAGAAA CCACAGCTGATGGCAGAAAAACTCAGACTGTGTGCAGCTTTGCAGATGGTGCATTGGTTCAGCATCAGGA GTGGGATGGGAAGGAAAACACAATAACAAGAAAACTGAAAGATGGGAAATTAGTGGTGTACTGTGTCATG AACAATGTCGCCTGTACTCGGATCTATGAAAAAGTAGAATAAAAATTCCATCATCACTTTGGACAGGAGT TAACTAATAGAATGATCAAGCTCAGTTCAATGAGCAAATCTCCATAGTGTTTTTTTTCATTACTGTGTTC AATTATCTTTATCACAAACGTTTCACATGCAGCTATTTCAAAGTGTCTTGGATTAATTAGGATCATCCCT TTGG The disclosed NOV6 nucleic acid sequence , located on chromosome 15, has 537 of 589 bases (91 %) identical to a Fatty Acid-Binding Protein mRNA from Homo sapiens (GENBANK-ID: HUMFABPHA) (E = 3.7e"102).
A disclosed NOV6 polypeptide (SEQ ID NO: 18) encoded by SEQ ID NO: 17 is 134 amino acid residues and is presented using the one-letter amino acid code in Table 6B. Signal P, Psort and/or Hydropathy results predict that NOV6 contains no signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.4500. In other embodiments, NOV6 is also likely to be localized to the mitochondrial matrix space with a certainty of 0.3600, or to the lysosome (lumen) with a certainty of 0.1000.
Table 6B. Encoded NOV6 protein sequence (SEQ ID NO.18). ATVQQLGGRWRLVDSKRFDEYMKEGGVGTALRKMDAMAKPDCIITCDGKNLTIKTESTLKTQFSCTLGEKFEET TADGRKTQTVCSFADGALVQHQE DGKENTITRKLKDGKLWYCVMNNVACTRIYEKVE
The disclosed NOV6 amino acid sequence has 124 of 135 amino acid residues (91 %) identical to, and 126 of 135 amino acid residues (93 %) similar to, the 135 amino acid residue Fatty Acid-Binding protein from Homo sapiens (Q01469) (E = 2.1e"51). NOV6 is expressed in Sensory System. Skin; Nervous System.Brain; Male
Reproductive System.Testis; Respiratory System.Lung, Larynx; Female Reproductive System; .Placenta; Whole Organism; Cardiovascular System.Heart; Endocrine System.Parathyroid Gland; Hematopoietic and Lymphatic System, Hematopoietic Tissues, Liver, Tonsils; Gastrointestinal/Digestive System. Large Intestine, Colon, Stomach, Oesophagus; Urinary System.Kidney. In addition, the sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBANK-ID: ACC:Q05816) a closely related Fatty Acid-Binding Protein homolog in species Mus musculus} : Sensory System. Skin; Nervous System.Brain; Male Reproductive System.Testis; Respiratory System.Lung, Larynx; Female Reproductive System; .Placenta; Whole Organism; Cardiovascular System.Heart; Endocrine System.Parathyroid Gland; Hematopoietic and Lymphatic System, Hematopoietic Tissues, Liver, Tonsils; Gastro-intestinal/Digestive System. Large Intestine, Colon, Stomach, Oesophagus; Urinary System.Kidney.
TaqMan data for NOV6 can be found below in Example 2. NOV6 also has homology to the amino acid sequences shown in the BLASTP data listed in Table 6C.
Table 6C. BLAST results for NOV6
Gene Index/ protein/ Organism Length Identity Positives Expect Identifier (aa) (%! (%)
Figure imgf000080_0002
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 6D.
Table 6D Information for the ClustalW proteins 1) NOV6 (SEQ ID NO: 18)
2) gi 113651468 | re JXP_016351.1 ( similar to GASTRIN/CHOLECYSTOKININ TYPE B RECEPTOR (CCK-B RECEPTOR) (CCK-BR) (H. sapiens) [Homo sapiens] (SEQ ID NO: 56)
3) gi I 4557581 [ref |NP_001435.l| fatty acid binding protein 5 (psoriasis-associated); E-FABP [Homo sapiens] (SEQ ID NO:57) 4) gi| 13651882 |ref|xP_0H655.5 I fatty acid binding protein 5 (psoriasis-associated) [Homo sapiens] (SEQ ID NO: 58)
5) gi j 136515631 ef |XP_015760,1 I similar to GASTRIN/CHOLECYSTOKININ TYPE B RECEPTOR (CCK-B RECEPTOR) (CCK-BR) (H. sapiens) [Homo sapiens] (SEQ ID NO:59)
6) gi|6648071 I sp|p55052 ||FABE_BOVIN FATTY ACID-BINDING PROTEIN, EPIDERMAL (E-FABP) (SEQ ID NO: 60)
Figure imgf000080_0001
90 100 110 120
Figure imgf000081_0001
Table 6E lists the domain description from DOMAIN analysis results against NOV6. This indicates that the NOV6 sequence has properties similar to those of other proteins known to contain this domain.
Table 6E. Domain Analysis of NOV6 gnl [ Pfaπ[pfam00061, lipocalin, Lipocalin / cytosolic fatty-acid binding protein family. Lipocalins are transporters for small hydrophobic molecules, such as lipids, steroid hormones, bilins, and retinoids. Alignment subsumes both the lipocalin and fatty acid binding protein signatures from PROSITE. This is supported on structural and functional grounds. Structure is an eight-stranded beta barrel. (SEQ ID NO: 90)
CD-Length = 145 residues, 76.6% aligned Score = 53.1 bits (125), Expect = le-08
Query : 9 GRWRLVDSKRFDEYMK-EGGVGTALRKMDAMAK-PDCIITCDGKNLTIKTESTLKTQFSC 66
M i l l I II + 1 I I I I I I I + I I I I μ M + Sbjct. 4 GK YLVASANFDPELKEELGVLEATRKEITPLKEGNLEIVFDGDKNGICEETFGKLEKTK 63
Query: 67 TLGEKFEETTADGRKTQTVCSFADGALVQHQE DGKENTITRKLKDGKLW 117
I I + μ I I I + + I I μ i l I + ! +1 + Sbjct- 64 KLGVEFDYYTGDNRFWLDTDYDNYLLVCVQKGDGNETSRTAELYGRTPEL 114
Fatty acid metabolism in mammalian cells depends on a flux of fatty acids, between the plasma membrane and mitochondria or peroxisomes for beta-oxidation, and between other cellular organelles for lipid synthesis. The fatty acid-binding protein (FABP) family consists of small, cytosolic proteins believed to be involved in the uptake, transport, and solubilization of their hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structures. Fatty acid-binding proteins were first isolated in the intestine (FABP2;
OMIM- 134640) and later found in liver (FABP1 ; OMIM- 134650), striated muscle (FABP3;
OMIM- 134651 ), adipocytes (FABP4; OMIM- 600434) and epidermal tissues (E-FABP; GDB ID: 136450 ). Epidermal fatty acid binding protein (E-FABP) was cloned by as a novel keratinocyte protein by Madsen et al (1992, PMID. 1512466) from skin of psoriasis patients. Later using quantitative Western blot analysis, Kingma et al. (1998, PMID: 9521644) have shown that in addition to the skin, bovine E-FABP is expressed in retina, testis, and lens. Since E-FABP was originally identified from the skin of psoriasis patients, it is also known as psoriasis- associated fatty acid-binding protein (PA-FABP). PA-FABP is a cytoplasmic protein, and is expressed in keratinocytes. It is highly up-regulated in psoriatic skin. It shares similarity to other members o the fatty acid-binding proteins and belongs to the fabp/p2/crbp/crabp family of transporter. PA-FABP is believed to have a high specificity for fatty acids, with highest affinity for cl 8 chain length. Decreasing the chain length or introducing double bonds reduces the affinity. PA-FABP may be involved in keratinocyte differentiation.
Immunohistochemical localization of the expression of E-FABP in psoriasis, basal and squamous cell carcinomas has been carried out in order to obtain indirect information, at the cellular level, on the transport of the fatty acidss. (Masouye et al, 1996, PMID: 8726632). E- FABP was localized in the upper stratum spinosum and stratum granulosum in normal and non-lesional psoriatic skin, hi contrast, lesional psoriatiG epidermis strongly expressed E- FABP in all suprabasal layers, like nonkeratinized oral mucosa. The basal layer did not express E-FABP reactivity in any of these samples. Accordingly, basal cell carcinomas were E-FABP negative whereas only well-differentiated cells of squamous cell carcinomas expressed E-FABP. This suggests that E-FABP expression is related to the commitment of keratinocyte differentiation and that the putative role of E-FABP should not be restricted to the formation of the skin lipid barrier. Since the pattern of E-FABP expression mimics cellular FA transport, our results suggest that lesional psoriatic skin and oral mucosa have a higher metabolism/transport for FAs than normal and non-lesional psoriatic epidermis. The disclosed NOV6 nucleic acid of the invention encoding a Fatty acid binding protein-like protein includes the nucleic acid whose sequence is provided in Table 6A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 6A while still encoding a protein that maintains its Fatty acid binding protein-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nuGleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOV6 protein of the invention includes the Fatty acid binding proteinlike protein whose sequence is provided in Table 6B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 6B while still encoding a protein that maintains its Fatty acid binding protein-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 14% percent o the residues may be so changed.
The above defined information for this invention suggests that these Fatty acid binding protein-like proteins (NOV6) may function as a member of a "Fatty acid binding proteinfamily". Therefore, the NOV6 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/Gytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here. The nucleic acids and proteins of NOV6 are useful in Cardiomyopathy, AtherosclerosiSjHypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Transplantation, Endometriosis, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Crohn's Disease, Hemophilia, hypercoagulation, Idiopathic thrombocytopenic purpura, immunodeficiencies, Osteoporosis, Hypercalceimia, Arthritis, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Asthma, allergy, ARDS, Lesch-TMyhan syndrome, Multiple sclerosis, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection Fertility, psoriasis, cancer including but not limited to basal and squamous cell carcinomas, obesity, diabetis, and/or other pathologies and disorders involving fatty acid transport of skin, oral mucosa as well as other organs. The novel NOV6 nucleic acid encoding NOV6 protein,, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.
NOV6 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example the disclosed NOV6 protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated NOV6 epitope is from about amino acids 10 to 35. In other embodiments, NON6 epitope is from about amino acids 40 to 45, from about amino acids 50 to 85, or from about amino acids 90 to 1 10. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
ΝOV7
A disclosed NOV7 nucleic acid of 822 nucleotides (also referred to sggc_draft_dj895c5_20000819) encoding a novel Gap junction beta-5 protein-like protein is shown in Table 7 A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 1 -3 and ending with a TAA codon at nucleotides 800-802. In Table 7 A, the 5' and 31 untranslated regions are underlined and the start and stop codons are in bold letters.
Table 7A. NOV7 Nucleotide Sequence (SEQ ID NO:19)
CATGAACTGGGCATTICTGCAGGGCCTGCTGAGTGGCGTGAACAAGTACTCCACAGTGCTGAGCCGCATCT GGCTGTCTGTGGTGTTCATCTTTCGTGTGCTGGTGTACGTGGTGGCAGCGGAGGAGGTGTGGGACGATGAG CAGAAGGACTTTGTCTGCAACACCAAGCAGCCCGGCTGCCCCAACGTCTGCTATGACGAGTTCTTCCCCGT GTCCCACGTGCGCCTCTGGGCCCTACAGCTCATCCTGGTCACGTGCCCCTCACTGCTCGTGGTCATGCACG TGGCCTACCGCGAGGAACGCGAGCGCAAGCACCACCTGAAACACGGGCCCAATGCCCCGTCCCTGTACGAC AACCTGAGCAAGAAGCGGGGCGGACTGTGGTGGACGTACTTGCTGAGCCTCATCTTCAAGGCCGCCGTGGA TGCTGGCTTCCTCTATATCTTCCACCGCCTCTACAAGGATTATGACAxGCCCCGCGTGGTGGCCTGCTCCG TGGAGCCTTGCCCCCACACTGTGGACTGTTACATCTCCCGGCCCACGGAGAAGAAGGTCTTCACCTACTTC ATGGTGACCACAGCTGCCATCTGCATCCTGCTCAACCTCAGTGAAGTCTTCTACCTGGTGGGCAAGAGGTG CATGGAGATCTTCGGCCCCAGGCACCGGCGGCCTCGGTGCCGGGAATGCCTACCCGATACGTGCCCACCAT ATGTCCTCTCCCAGGGAGGGCACCCTGAGGATGGGAACTCTGTCCTAATGAAGGCTGGGTCGGCCCCAGTG GATGCAGGTGGGTATCCATAACCTGCGAGATCAGCΛGATAA
The disclosed NOV7 nucleic acid sequence, localized to the p34.3-36.1 1 region of chromosome 1 , has 682 of 807 bases (84%) identical to a gb:GENBAN -
S9 ID;RNCON303|acc:X76168 mRNA from Rattus norvegicus (R.norvegicus mRNA for connexin 30 3) (E = 3.7e"125).
A disclosed NOV7 polypeptide (SEQ ID NO:20) encoded by SEQ ID NO: 19 is 266 amino acid residues and is presented using the one-letter amino acid code in Table 7B. Signal P, Psort and/or Hydropathy results predict that NOV7 has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.6000. In other embodiments, NOV7 is also likely to be localized to the Golgi body with a certainty of 0.4000, to the endoplasmic reticulum (membrane) with a certainty of 0.3000, or the nucleus with a certainty of 0.2400. The most likely cleavage site for a NOV7 peptide is between amino acids 40 and 41, at: VAA- EE.
Table 7B. Encoded NOV7 protein sequence (SEQ ID NO:20).
MN AFLQGI -- SGVNKYSTVLSRIWI SVVFI FRVIιVYWAAEEVWDDEQKDFVCNT QPGCPNVCyDEFFPV SHVRIi A QLIliVTCPSL LW HVAYREERERKHH KHGPNAPΞIiyDNI SKKRGGl-j WTYL SI IFKAAVD AGFI1YI FHR--JYKDYDMPRWACSVEPCPHTVDCYISRPTE VFTYFMVTTAAICI--JI1NI1SEVFY VGKRC MEI FGPRHRRPRCRECLPDTCPPYVLSQGGHPEDGNSVIiMKAGSAPVDAGGYP
The disclosed NOV7 amino acid sequence has 230 of 266 amino acid residues (86%) identical to, and 244 of 266 amino acid residues (91%) similar to, the 266 amino acid residue ptnr:SWISSPROT-ACC:Q02738 protein from Mus musculus (Mouse) (Gap Junction Beta-5 Protein (Connexin 30.3) (CX30.3) (4.4e"ι z9).
NOV7 is expressed in at least kidney tissue. The sequence is predicted to be expressed in the following tissues because of the expression pattern of (GENBATNOK-.-ID: RNCON303|acc:X76168) a closely related {R.norvegicus mRNA for connexin 30.3 homolog in species Rattus norvegicus : kidney and thymus.
SNP data for NOV7 can be found below in Example 3. NOV7 also has homology to the amino acid sequence shown in the BLASTP data listed in Table 7C.
Figure imgf000085_0001
91.6680015 I ref ||NP_03 gap junction 266 230/266 244/266 e-13
2153.l| membrane channel (86%) (91%) protein beta ; connexin 30.3 [Mus musculus] gι| 10835079 |ref|NP_0 gap junction 273 153/226 177/226 3e-B8 05259. l|| protein, beta 5 (67%) (77%) (connexin 31.1) [Homo sapiens] gi| 4009522 |gb|AAC954 connexin 31.1 273 152/226 176/226 le-87 72. l| (AF099731) [Homo sapiens] (67%) (77%)
The homology of these sequences is shown graphically in the ClustalW analysis shown in Table 7D.
Table 7D. Information for the ClustalW proteins
1) NOV7 (SEQ ID NO: 20)
2) gi |l222976l||sp|Q9NTQ9|CXB4__HUMAN GAP JUNCTION BETA-4 PROTEIN (CONNEXIN 30.3) (CX30.3) (SEQ ID NO: 61)
3) gi || 5441X8 I sp|P3638θ|CXB5_RAT GAP JUNCTION BETA-5 PROTEIN (CONNEXIN 30.3) (CX30.3) (SEQ ID NO:62)
4) gi |6680O15 I ref JNP_032153.11 gap junction membrane channel protein beta 4; connexin 30.3 [Mus musculus] (SEQ ID NO:63)
5) gi 110835079) ref |, NP_005259.1 [ gap junction protein, beta 5 (connexin 31.1) [Homo sapiens] (SEQ ID NO: 64)
6) gi (4009522 ||gb | AAC95472.11 (AF099731) connexin 31.1 [Homo sapiens] (SEQ ID NO:65)
Figure imgf000086_0001
170 180 190 200
Figure imgf000087_0001
Table 7E-F lists the domain description from DOMAIN analysis results against NOV7. This indicates that the NOV7 sequence has properties similar to those of other proteins known to contain this domain.
Table 7E. Domain Analysis of NOV7 qni I Pfam |p£am00029 , connexin, Connexin. (SEQ ID NO: 91) CD-Length = 218 residues, 100.0% aligned Score = 318 bits (814), Expect = 3e-88
Query. 1 MNWAFLQGLI-SGVNKYSTVI-SRIWI-S WFIFR ViVΥWAAEEV DDEQKDFVCNTKQPGC 60 l+l+l I II llll+ ll + +IIIII +IIII+ II Mil II III II 1111+11 II
Sbjct: 1 MDWSFLGRLLEG KHSTAIGKIWLSVLFIFRILVI.GVAAESVWGDEQSDFVCNTQQPGC 60 Query: 61 PΪWCYDEFFPVSHVR--JWALQLII-VTCPSLLWMHVAYREERERKHHI-KHGPNAPSI-YDNL 120
Sb^ct: 61 EN iViCiYiDi+QFiFiPi+ISiHiViRiLnVL mQLIiFV ι+STP mSLIiiY +LGH iVnAYn IN RVRREEKIiREK 1EEEH ++SKGL nYSEE 120 Query: 121 SKK RGGLWWTY- LSωFKAAVDAGFLYIFHRIiYK-DYDMPR ACSVEPC 169
+ 1! 111111111+ I + III+ + till + 11 + ! +! 1! II
Sbjct: 121 AKKRCGSEDGKVRIRGGDWWTYVFSIIFKSIFEVGFLYGQYLL-Y-GFTMSPLVVCSRAPC 179 Query: 170 PHTVDCYISRPTEKKVFTYFMVTTAAICIL-I-NIjSEVFYLi 208
IIIII IIIII +ι ιι+ +111+1111+1+111
Sb]Ct: 180 PHTVDCFVSRPTEKTIFIVFMLWSAICLliLNLAEl-αFYL 218 Table 7F. Domain Analysis of NOV7 gnl I Smar | smart00037 , CNX, Connexin homologues; Connexin channels participate in the regulation of signaling between developing and differentiated cell types. (SEQ ID NO:160) CD-Length = 34 residues, 100.0% aligned Score = 79.0 bits (193), Expect = 3e-i6
Query: 42 EVWDDEQKDFVCNTKQPGCPNVCYDEFFPVSHVR 75 i i I I I I I i i i+ i i n i i i n+ i i i+ i n i
Sbj ct : 1 SVWGDEQSDFTCNTQQPGCENVCYDQFFPISHVR 34
Gap junctions are conduits that allow the direct cell-to-cell passage of small cytoplasmic molecules, including ions, metabolic intermediates, and second messengers, and thereby mediate intercellular metabolic and electrical communication. Gap junction channels consist of connexin protein subunits, which are encoded by a multigene family. Richard et al. (Nature Genet. 20: 366-369,1 98) identified 2 expressed sequence tags (ESTs) from the human EST database by their similarity to mouse Gjb3 (see 603324) and Gjb5. By radiation hybrid mapping, they placed them in proximity to a sequence tagged site (STS) that is linked to GJA4 (121012) at lp35.1. Richard et al. (1998) determined the cDNA sequences of the genes from which both ESTs were derived. Sequence similarity to rodent connexin genes established them as human homologs of Gjb3 and Gjb5, encoding Cx32 and Cx31.1, respectively. Richard et al. (1998) excluded GJB5 as a candidate gene for erythrokeratodermia variabilis (133200) by sequence analysis. Gap junctions are intercellular channels which connect adjacent cells and allow direct exchange of molecules of low molecular weight between them (Alves LA et.al; Braz J Med Biol Res 2000 Apr;33(4):457-65). Such a communication has been described as fundamental in many systems due to its importance in coordination, proliferation and differentiation. Recently, it has been shown that gap junctional intercellular communication (GJIC) can be modulated by several extracellular soluble factors such as classical hormones, neurotransmitters, interleukins, growth factors and some paracrine substances. Herein, we discuss some aspects o the general modulation of GJIC by extracellular messenger molecules and more particularly the regulation of such communication in the thymus gland. Additionally, we discuss recent data concerning the study of different neuropeptides and hormones in the modulation of GJIC in thymic epithelial cells. We also suggest that the thymus may be viewed as a model to study the modulation of gap junction communication by different extracellular messengers involved in non-classical circuits, since this organ is under bidirectional neuroimmunoendocrine control. The intercellular signaling system mediated by connexin channels is crucial for maintaining tissue homeostasis, growth control, development, and synchronized response of cells to stimuli (Richard G; Exp Dermatol 2000 Apr;9(2):77-96). This review summarizes the structure, assembly, and properties of the components of the complex and diverse connexin system, and their biological functions in skin. The importance of gap junctional intercellular communication for normal development and differentiation of human epidermis as well as the hearing function of the inner ear is illustrated by the examples of erythrokeratodermia variabilis and palmoplantar keratoderma associated with hearing loss. These autosomal dominant inherited disorders are caused by germline mutations in the connexin genes GJB3 (encoding connexin-31) and GJB2 (encoding connexin-26), respectively. Recent functional studies of individual connexin mutations suggest that they may exert a dominant inhibitory effect on normal connexin channel function and perturb gap junctional intercellular communication, resulting in phenotypic manifestation in patients with these disorders. Gap junction channels are sites of cytoplasmic communication between contacting cells. In vertebrates, they consist of protein subunits denoted connexins (Cxs) which are encoded by a gene family (Saez JC et.al; Braz J Med Biol Res 2000 Apr;33(4):447-55). According to their Cx composition, gap junction channels show different gating and permeability properties that define which ions and small molecules permeate them. Differences in Cx primary sequences suggest that channels composed of different Cxs are regulated differentially by intracellular pathways under specific physiological conditions. Functional roles of gap junction channels could be defined by the relative importance of permeant substances, resulting in coordination of electrical and/or metabolic cellular responses. Cells of the native and specific immune systems establish transient homo- and heterocellular contacts at various steps o the immune response. Morphological and functional studies reported during the last three decades have revealed that many intercellular contacts between cells in the immune response present gap junctions or "gap junction-like" structures. Partial characterization of the molecular composition of some of these plasma membrane structures and regulatory mechanisms that control them have been published recently. Studies designed to elucidate their physiological roles suggest that they might permit coordination of cellular events which favor the effective and timely response ofthe immune system. Antitumor suicide gene therapy is one of the emerging strategies against cancer (Mesnil et. al.; Cancer Res 2000 Aug l ;60(15):3989-99) . It consists of the introduction into cancer cells of a gene capable of converting a nontoxic prodrug into a cytotoxic drug. Because this therapeutic gene cannot be easily introduced into the whole cell population of a tumor, the successful eradication of tumors depends on a phenomenon called the "bystander effect," by which the introduced gene can affect even cells in which it is not itself present, From a therapeutic point of view, it may be crucial to enhance this phenomenon through various means to achieve tumor eradication. One such suicide gene, the thymidine kinase gene from the herpes simplex virus, in combination with the prodrug ganciclovir, has been extensively and successfully used in some animal models exhibiting a strong bystander effect. Among the mechanisms involved in this phenomenon, gap junctional intercellular communication (GJIC) is directly involved in the transfer of the toxic metabolites of ganciclovir, which pass directly from herpes simplex virus thymidine kinase-expressing cells to surrounding cells that do not express it. Because GJIC appears to be a mediator ofthe bystander effect both in vitro and in vivo, here we review possible molecular strategies for enhancing the extent of tumor cell death by increasing the intratumoral GJIC capacity. Synapses are classically defined as close connections between two nerve cells or between a neuronal cell and a muscle or gland cell across which a chemical signal (i.e., a neurotransmitter) and/or an electrical signal (i.e., current-carrying ions) can pass (Rozental R et.al; Brain Res Brain Res Rev 2000 Apr;32(l);l 1-5). The definition of synapse was developed by Charles Sherrington and by Ramon y Cajal at the beginning o this Gentury and refined by John Eccles and Bernard Katz 50 years later; in this collection of papers, the definition of synapses is discussed further in the chapter by Mike Bennett, who provided the first functional demonstration of electrical transmission via gap junction channels between vertebrate neurons. As is evidenced by the range of topics covered in this issue, research dealing with gap junctions in the nervous system has expanded enormously in the past decade, major findings being that specific cell types in the brain expresses specific types of connexins and that expression patterns coincide with tissue compartmentalization and function and that thesecompartments change during development. Connexins, the protein molecules forming gap junction channels, are reduced in number or redistributed from intercalated disks to lateral cell borders in a variety of cardiac diseases (Jongsma HJ et.al; Circ Res 2000 Jun 23;86( 12): 1 193-7). This "gap junction remodeling" is considered to be arrhythmogenic. Using a simple model of human ventricular myocardium, we found that quantitative remodeling data extracted from the literature gave rise to only small to moderate changes in conduction velocity and the anisotropy ratio. Especially for longitudinal conduction, cytoplasmic resistivity (and thus cellular geometry) is much more important than commonly realized. None of the remodeling data gave rise to slow conduction on the order of a few centimeters per second. Physical signals, in particular mechanical loading, are clearly important regulators of bone turnover (Donahue HJ; Bone 2000 May;26(5):417-22). Indeed, the structural success of the skeleton is due in large part to the bone's capacity to recognize some aspect of its functional environment as a stimulus for achievement and retention of a structurally adequate morphology. However, while the skeleton's ability to respond to its mechanical environment is widely accepted, identification of reasonable mechanism through which a mechanical "load" could be transformed to a signal relevant to the bone cell population has been elusive. In addition, the downstream response of bone cells to load-induced signals is unclear. In this work, we review evidence suggesting that gap junctional intercellular communication (GJIC) contributes to mechanotransduction in bone and, in so doing, contributes to the regulation of bone cell differentiation by biophysical signals. In this context, mechanotransduction is defined as transduction of a load-induced biophysical signal, such as fluid flow, substrate deformation, or electrokinetic effects, to a cell and ultimately throughout a cellular network. Thus, mechanotransduction would include interactions of extracellular signals with cellular membranes, generation of intracellular second messengers, and the propagation of these messengers, or signals they induce, through a cellular network. We propose that gap junctions contribute largely to the propagation of intracellular signals.
The disclosed NOV7 nucleic acid of the invention encoding a Gap Junction Beta-5 Protein-like protein includes the nucleic acid whose sequence is provided in Table 7A or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 7A while still encoding a protein that maintains its Gap Junction Beta-5 Protein-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 16% percent of the bases may be so changed. The disclosed NOV7 protein of the invention includes the Gap Junction Beta-5 Protein-like protein whose sequence is provided in Table 7B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 7B while still encoding a protein that maintains its Gap Junction Beta- 5 Protein-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 33% percent of the residues may be so changed.
The protein similarity information, expression pattern, and map location for the Gap Junction Beta-5 Protein-like protein and nucleic acid (NOV7) disclosed herein suggest that NOV7 may have important structural and/or physiological functions characteristic of the Gap Junction Beta-5 Protein-like family. Therefore, the NOV7 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications. These include serving as a specific or selective nucleic acid or protein diagnostic and/or prognostic marker, wherein the presence or amount ofthe nucleiϋ acid or the protein are to be assessed, as well as potential therapeutic applications such as the following: (i) a protein therapeutic, (ii) a small molecule drug target, (iii) an antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) a nucleic acid useful in gene therapy (gene delivery/gene ablation), and (v) a composition promoting tissue regeneration in vitro and in vivo.
The NOV7 nucleic acids and proteins of the invention are useful in potential diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies. For example, the compositions of the present invention will have efficacy for treatment of patients suffering from Deafness, autosomal dominant 2; Elliptocytosis-1 ; Fucosidosis; Hypophosphatasia (adult, childhood, infantile); Muscle-eye- brain disease; Neuropathy, paraneoplastiϋ sensory; Porphyria Gutanea tarda; Porphyria, hepatoerythropoietic; Schwartz-Jampel syndrome; Thrombocytopenia, congenital amegakaryocytic; Charcot-Marie-Tooth neuropathy-2A; GalaGtose epimerase deficiency; Glucose transport defect, blood-brain barrier; Kostmann neutropenia; Muscular dystrophy, congenital, with early spine rigidity; Myopathy due to succinate dehydrogenase deficiency; SCID due to LCK deficiency; Colorectal cancer, resistance to; Bartter syndrome, type 3; Breast cancer, ductal; Corneal dystrophy, crystalline, Schnyder; Hyperprolinemia, type II; Inflammatory bowel disease 7; Malignant melanoma, cutaneous; Neuroblastoma; Prostate cancer-brain cancer susceptibility; erythrokeratodermia variabihs; palmoplantar keratoderma; diseases and disorders involving intercellular metabolic and electrical communication; diseases and disorders involving coordination, proliferation and differentiation; diseases and disorders involving maintainance of tissue homeostasis, growth control, development, and synchronized response of cells to stimuli; diseases and disorders involving the the immune system; diseases and disorders involving regulation of bone cell differentiation, and/or other pathologies/disorders. The NOV7 nucleic acid, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount o the nucleic acid or the protein are to be assessed.
NOV7 nucleic acids and polypeptides are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti-NOVX Antibodies" section below. For example the disclosed NOV7 protein have multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, contemplated NOV7 epitope is from about amino acids 40 to 70. In other embodiments, NOV7 epitope is from about amino acids 90 to 140, from about amino acids 170 to 180, or from about amino acids 220 to 255. This novel protein also has value in development of powerful assay system for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders.
NOV8
A disclosed NOV8 nucleic acid of 546 nucleotides (also referred to as 56072181_dal) encoding a novel MT-like protein is shown in Table 8A. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 214-216 and ending with a TGA codon at nucleotides 397-399. A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 8A. The start and stop codons are in bold letters.
Table 8A. NOV8 nucleotide sequence (SEQ ID NO:21).
CAATCTGACATGTAACTGTAAAATCACTGGGCCCGAGGAGTGACTGTAATTAAGGGCTTACGTTGAAAATGT ATAAACAGAGCTTGATTCTTAAGTTTCGAAAATTCTTCACAGTGATGGTGTGAACTAAATCCACAGCCACAT AATAATCAAAACAGAAAAGCAAAAGAAAGCCACTTCAGGAAATACCACGGTCACACCTCAGTTGGCTTCATG CTACAGATTATAGAAAATATGTTGCTGCCCGGGCCACCAATCTGTTGGTTCACATTACTACGTGAGCAATGT AAGTGTTTGCAAGAAGCCATCCACTATCTAAATATCAGATATAGATGCTCCAAAGCAGCTACGTCAGTGATG AGAACAGAGAAAATACGTAGCAACATTTCATTAAGTTGAATTCTAATACTTAAAAGGCTCCTTTTAGTACTG ACATTCTGGATTTTAAAAGTTATGTTGACCGCATGTTCTCACTCACAAGTGGGAGTTGAACAATGAGAACAC ACGGACACGGGGAAGGGAACATCACACACCAGGGCCTGTCAG
The NOV8 nucleic acid sequence is located on the ql 3 region of chromsome 16. The disclosed NOV8 polypeptide (SEQ ID NO:22) encoded by SEQ ID NO:21 has 61 amino acid residues and is presented in Table SB using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOV8 has no signal peptide and is likely to be localized in the cytoplasm with a certainty of 0.6500. In other embodiments, NOV8 may also be localized to the mitochondrial matrix space with a certainty of 0.3100, or the lysosome (lumen) with a certainty of 0.1000.
Table 8B. Encoded NOV8 protein sequence (SEQ ID NO:22).
MLQIIENMLLPGPPIC FTL REQCKC QEΑIHY-DNIRYRCSKAATSVMRTEKIRSNISLS
A search of sequence databases reveals that the NOV8 amino acid sequence has 16 of
41 amino acid residues (39%) identical to, and 25 of 41 amino acid residues (60%) similar to, the 48 amino acid residue Metallothionein protein from Rhizomucor racemosus (Mucor cirάnelloides f, lusitanicus) (SPTREMBL-ACC: Q9Y762) (E = 0.049). SNP data for NOV8 can be found below in Example 3. Masters et al. (Proc. Nat. Acad. Sci. 91: 584-588, 1994) described metallothioneins
(MTs) as a family of low molecular weight, heavy metal-binding proteins characterized by a high cysteine content and lack of aromatic amino acids. MTs bind 7 to 12 heavy metal atoms per molecule of protein. They are ubiquitous in the animal and plant kingdoms and are found in prokaryotes. In mammals, the cysteine residues are absolutely conserved and serve to coordinate heavy metal atoms such as zinc, cadmium, and copper via mercaptide linkages. In human liver, MTs occur in 2 major forms, MT-I and MT-II (156360). In HeLa cells, MT synthesis is induced by either ionized zinc or ionized cadmium and by glucocorticoid hormones. In man, metallothioneins are encoded by at least 10 to 12 genes separated into 2 groups designated MT-I and MT-II. Masters et al. (1994) noted that, unlike MT-I and MT-II, which are expressed in most organs, MT-III (139255) expression appears to be restricted to the brain, and MT-IV is only expressed in certain stratified squamous epithelia.
MTs have been postulated to detoxify metals; to play a role in zinc and copper homeostasis during development; to regulate synthesis, assembly, or activity of zinc metalloproteins; and to protect against reactive oxygen species. MTs may also protect against copper toxicity in the Menkes disease (309400) and murine Mottled' phenotypes (X-linked diseases resulting in copper deficiency) as well as in Wilson disease (277900); see also the Animal Models section.
Karin and Richards (Nature 299: 797-802, 1982) described the molecular cloning and sequence analysis of human metallothionein transcripts. Karin et al. (1984) characterized DNA sequences that are involved in the induction of MT gene expression by cadmium and glucocorticoids. MAPPING
Karin et al. (Proc. Nat. Acad. Sci. 81 : 5494-5498, 1984) used several different hybridization probes derived from cloned and functional human MTl and MT2 genes to map the genes in somatic cell hybridization studies. They concluded that most of the human genes are clustered on chromosome 16. Analysis of RNA from somatic cell hybrids indicated that all hybrids that contain human chromosome 16 express both MTl and MT2 mRNA and that expression is regulated by both heavy metal ions and glucocorticoid hormones.
In the mouse, the metallothionein genes are on chromosome 8, which has other homology to human chromosome 16; by somatic cell hybridization, Cox and Palmiter (Hum. Genet. 64: 61-64, 1983) assigned the MT-1 structural gene to mouse chromosome 8, which also carries glutathione reductase in the mouse. (By chance the human 8 also carries glutathione reductase.)
Schmidt et al. (Science 224: 1 104-1106, 1984) concluded that MTl is located between PGP (172280) and DIA4 (125860) and is probably on the long arm 16cen-16q21 because APRT (102600), a 16q marker, and MTl are both on mouse chromosome 8, whereas HB alpha (141800), a 16p marker, is on mouse chromosome 11. They stated that analysis o the involvement of the MT genes in Wilson disease (277900) and in acrodermatitis enteropathica (201100) would be of great interest. By gel transfer hybridization analysis of the DNA from human-rodent cell hybrids,
Schmidt et al. (J. Biol. Chem. 260: 7731-7737, 1985) showed that chromosome 16 contains a cluster of metallothionein sequences, including 2 functional metallothionein I genes (156351 and 156352) and a functional metallothionein II gene. The remaining sequences, including a processed pseudogene, are dispersed to at least 4 other autosomes. The absence of metallothionein sequences from the X chromosome indicates that the Menkes disease mutation affects metallothionein expression by a 'trans-acting' mechanism. The processed pseudogene is on chromosome 4 and shows allelic variation (Karin and Richards, Nucleic Acids Res. 10: 3165-3173, 1982). Two MT genes are on chromosome 1 but not close together: one is on the distal two-thirds of the short arm and the second probably on the long arm. One metallothionein gene is on chromosome 20 and another is on chromosome 18.
By in situ hybridization, Le Beau et al. (Nature 313: 709-711 , 1985) assigned the metallothionein gene cluster to 16q22. This band is a breakpoint in 2 specific rearrangements, inv(16)(pl 3q22) and t(16; l 6)(pl 3;q22), found in a subgroup of patients with aGute myelomonocytic leukemia. Hybridization of an MT probe to malignant cells from patients with one or the other of these rearrangements showed that the breakpoint at 16q22 splits the MT gene cluster. The findings were interpreted as indicating that the MT genes or their regulatory regions may function as an 'activating' sequence for an as yet unidentified cellular gene located at 16p l 3. Band 16p22 carries 2 fragile sites: the rare FRA16B and the common FRA16C. Simmers et al. (Science 236: 92-94, 1987) showed that the specific leukemic break that is situated in the metallothionein gene cluster lies proximal to both fragile sites; therefore, neither of these fragile sites could have played a role in the breakage.
Using high-resolution in situ hybridization, Sutherland et al. (Cytogenet. Cell Genet. 51 : 1087 , 1989, Genomics 6; 144-148, 1990) corrected the mapping of the human metallothionein gene complex to 16ql3. They found, furthermore, that the complex is not disrupted by the rearrangement breakpoint on 16q in the patients with myelomonocytic leukemia with abnormal eosinophils, as had previously been reported. They showed that the order is cen— MT-FRA16B-D16S4-inversion breakpoint-HB-qter.
Foster et al. (J. Biol. Chem. 263: 11528-11535, 1988) indicated that 4 functional MTl genes had been identified and mapped to 16q: MTlA, MTIB (156349), MTIE (156351), and MT1F (156352). They also characterized a fifth MT gene, MT1G (156353). West et al. (Genomics 8: 513-518, 1990) mapped the cluster of MT genes in an 82.1-kb region of 16ql3. Of the 14 tightly linked genes, 6 had not previously been described. The mapped genes included the single MT2 gene, MT2A, and at least 2 pseudogenes, MT1C and MT1D. The genes were flanked by the single MT2A gene at one end and a gene labeled MT1X (156359) at the other. The order of genes, beginning at the MT2A end, was 1L--1E— 1K-1J— 1A— ID— I C— 1B--1F--1 G— 1H--1 I. This was also the 5-prime to 3-prime direction of transcπption for all the genes except MT1G, which had a tail-to-tail, head-to-head orientation to MT1F and MT1H, respectively.
ANTMAL MODEL
To test the proposed detoxification and homeostasis functions of mammalian MTs in vivo, Masters et al. (1994) inactivated both alleles of the Mtl and Mt2 genes in embryonic stem cells and generated mice homozygous for these mutant alleles. These mice were viable and reproduced normally when reared under normal laboratory conditions. They were, however, more susceptible to hepatic poisoning by cadmium. This suggested to Masters et al. ( 1 994) that these widely expressed MTs are not essential for development but do protect against cadmium toxicity, Human Menkes disease (309400) and the murine 'Mottled' phenotype are X-linked diseases that result from copper deficiency due to mutations in ATP7A, a copper-effluxing ATPase (30001 1 ). Male mice with the Mottled-Brindled allele accumulate copper in the intestine, fail to export copper to peripheral organs, and die a few weeks after birth. Much of the intestinal copper is bound by metallothionein. To determine the function of MT in the presence of Atp7a deficiency, Kelly and Palmiter (Nature Genet. 13: 219-222, 1996) crossed Mottled-Brindled females with males that bear a targeted disruption of the Mtl and Mt2 genes. On the metallothionein-deficient background most Mottled males as well as heterozygous Mottled females died before embryonic day 11. The authors explained the lethality in females by preferential inactivation of the paternal X chromosome in extra embryonic tissues and resultant copper toxicity in the absence of MT.
In support of this hypothesis, Kelly and Palmiter (1996) found that cell lines derived from metallothionein deficient, Mottled embryos were very sensitive to copper toxicity. They concluded that MT is essential to protect against copper toxicity in embryonic placenta, providing a second line of defense when copper effluxers are defective. They also stated that MT probably protects against hepatic copper toxicity in Wilson disease and the LEC rat model in which a similar copper effluxer, ATP7B (277900), is defective, because MT accumulates to high levels in the liver in those diseases.
Beattie et al. (Proc. Nat. Acad. Sci. 95: 358-363, 1998) noted that mice with targeted disruption of the metallothionein-I and metallothionein-II genes were more sensitive to toxic metal and oxidative stress. In addition they were larger than most strains of mice, becoming significantly heavier at age 5 to 6 weeks. At age 14 weeks, the body weight and food intake of MT-null mice was 16 and 30% higher, respectively, compared with control mice. Most 22- to 39-week-old male MT-null mice were obese. Seven-week-old MT-null also had significantly higher levels of plasma leptin (601694) and elevated expression of OB (164160), lipoprotein lipase (238600), and CCAAT enhancer binding protein alpha (189965) genes as compared with age-matched control mice. Abnormal accretion of body fat and adipocyte maturation was initiated at 5 to 7 weeks of age, possibly coincident with sexual maturation. Beattie et al. ( 1998) concluded that a link between MT and the regulation of energy balance is implied by these observations. They noted the possibility that obesity and the associated biochemical changes in the MT-null mice may be caused by factors other than lack of MT. For example, disruption of MT genes by homologous recombination with DNA containing various modi fications may have affected other genes around this locus or may have had downstream effects on gene expression. The disclosed NOV8 nucleic acid of the invention encoding a MT-like protein includes the nucleic acid whose sequence is provided in Table 8A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 8A while still encoding a protein that maintains its MT-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic aσid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic add, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 10% percent of the bases may be so changed.
The disclosed NOV8 protein of the invention includes the MT-like protein whose sequence is provided in Table 8B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2 while still encoding a protein that maintains its MT-like activities and physiological functions, or a functional fragment thereof. In the mutant or variant protein, up to about 61% percent of the residues may be so changed.
The invention further encompasses antibodies and antibody fragments, such as Fab or (Fab)2, that bind immunospecifically to any of the proteins o the invention.
The above defined information for this invention suggests that this MT-like protein (NOV8) may function as a member of a "MT family". Therefore, the NOV8 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below. The potential therapeutic applications for this invention include, but are not limited to: protein therapeutic, small molecule drug targel, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vitro of all tissues and cell types composing (but not limited to) those defined here.
The NOV8 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to Inflamation, Autoimmune disorders, Aging and Cancer For example, a cDNA encoding the MT like protein (NOV8) may be useful in gene therapy, and the MT-like protein (NOV8) may be useful when admmisteied to a subject in need thereof By way of nonlimiting example, the compositions of the piesent invention will have efficacy for treatment of patients suffering fiom Gitelman syndiome, Menkes disease, Wilson's disease, aciodermatitis enteropathica, myelomonocytic leukemia, eosinophil disorders, hepatic disorders such as hepatic copper toxicity and other such conditions The NOV8 nucleic acid encoding MT-like protein, and the MT-hke protein of the invention, or fiagments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed
NOV8 nucleic acids and polypeptides are further useful in the generation of antibodies that bind lmmuno-specifically to the novel NOV8 substances for use in therapeutic or diagnostic methods These antibodies may be generated according to methods known in the art, using prediction from hydrophobidty charts, as described in the "Anti-NOVX Antibodies" section below The disclosed NOV8 protein has multiple hydrophilic regions, each of which can be used as an immunogen In one embodiment, a contemplated NOV8 epitope is from about amino acids 25 to 40 In another embodiment, a NOV8 epitope is from about ammo acids 45 to 55 These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders
NOV9
A disclosed NOV9 nucleic acid of 2309 nucleotides (also referred to as 2855519_0_19_dal) encoding a novel CfP4-hke protein is shown in Table 9A An open reading frame was identified beginning with an ATG initiation codon at nucleotides 78-80 and ending with a TGA codon at nucleotides 1719-1721 A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 9A The start and stop codons are in bold letters Table 9A. NOV9 nucleotide sequence (SEQ ID NO:23).
GAGTGAGAGGTCGGACAGACTGTGGAGCCGACAGACTGAAGGACAGCGGCACCGCCAGACGGCCAGAAAGTT CCGCCATGAGCTGGGGCACGGAGCTGTGGGATCAGTTCGACAGCTTAGACAAGCATACACAATGGGGAATTG ACTTCTTGGAAAGATATGCCAAATTTGTTAAAGAGAGGATAGAAATTGAACAGAACTATGCGAAACAATTGA GAAATCTGGTTAAGAAGTACTGCCCCAAACGTTCATCCAAAGATGAAGAGCCACGGTTTACCTCGTGTGTAG CCTTTTTTAATATCCTTAATGAGTTAAATGACTATGCAGGACAGCGAGAAGTTGTAGCAGAAGAAATGGCGC ACAGAGTGTATGGTGAATTAATGAGATATGCTCATGATCTGAAAACTGAAAGAAAAATGCATCTGCAAGAAG GACGAAAAGCTCAACAATATCTTGACATGTGCTGGAAACAGATGGGTAATAGTAAAAAGAAGTTTGAAAGAG AATGTAGAGAGGCAGAAAAGGCACAACAGAGTTATGAAAGATTGGATAATGATACTAATGCAACCAAGGCAG ATGTTGAAAATGCCAAACAGCAGTTGAATCTGCGTACGCATATGGCCGATGAAAATAAAAATGCATATGCTG CACAATTACAAAACTTTAATGGAGAACAACATAAACATTTTTATGTAGTGATTCCTCAGATTTACAAGCAAC TACAAGAAATGGACGAACGAAGGACTATTAAACTCAGTGAGTGTTACAGAGGATTTGCTGACTCAGAACGCA AAGTTATTCCCATCATTTCAAAATGTTTGGAAGGAATGATTCTTGCAGCAAAATGAGTTGATGAAAGAAGAG ACTCTCAAATGGTGGTAGACTCCTTCAAATCTGGTTTTGAACCTCCAGGAGACTTTCCATTTGAAGATTACA GTCAACATATATATAGAACCATTTCTGATGGGACTATCAGTGCATCCAAACAGGAGAGTGGGAAGATGGATG CCAAAACCCCAGTAGGAAAGGCCAAGGGCAAATTGTGGCTCTTTGGAAAGAAGCCAAAGGGCCCAGCACTAG AAGATTTCAGTCATCTGCCACCAGAACAGAGACGTAAAAAACTACAGCAGCGCATTGATGAACTTAACAGAG AACTACAGAAAGAATCAGACCAAAAAGATGCACTCAACAAAATGAAAGATGTATATGAGAAGGATCCACAAA TGGGGGATCCAGGGAGTTTGCAGCCTAAATTAGCAGAGACCATGAATAACATTGACCGCCTACGAATGGAAA TCCATAAGAATGAGGCTTGGCTCTCTGAAGTCGAAGGCAAAACAGGTGGGAGAGGAGACAGAAGACATAGCA GTGACATAAATCATCTTGTAACACAGGGACGAGAAAGTCCTGAGGGAAGTTACACTGATGATGCAAACCAGG AAGTCCGTGGGCCACCCCAGCAGCATGGTCACCACAATGAGTTTGATGATGAATTTGAGGATGATGATCCCT TGCCTGCTATTGGACACTGCAAAGCTATCTACCCTTTTGATGGACATAATGAAGGTACTCTAGCAATGAAAG AAGGTGAAGTTCTCTACATTATAGAGGAGGACAAAGGTGACGGATGGACAAGAGCTCGGAGACAGAACGGTG AAGAAGGCTACGTTCCCACGTCATACATAGATGTAACTCTAGAGAAAAACAGTAAAGGTTCCTGAAGAGGGT TTCTGAGGAAATGGGCAAGATGTTGAAGGAGGTTACATGCAGCTGCTTTTGGGGGAGGGTATTAGAGTTGTC AGGCTCAAAGAGAGTGAGAGAAGCAAGTTGCATGAGTGCATGCAGACATGATTTTTTTTTTACTAACTTCAT TAGCATTTCCATACATTGTTTTTAAAAATCATAATACCAACCCTTAAGTTCCTAGTTCACAGTTATTCCCAC AAAAGAAAAAGCCAACAATAGTGTACCATTTTTCTATTTTATTTTATTGCTGTCTAATCAATAAAGAATGCA GAGCTGTCAAAAAATGTGTCTTACATTTAGCTGTCCCAACAGGATTGTCTTCCCTCCCAGCTCTGGTTTTAA TTGGCTTTTAGACCCACTATCTGTCAGATCCTTGCCATCTGTCAGTGTCTGCCTGCGCCACCTCCGTGCTTG CCTAACATCCTGTTGCATGTCTAGCGTGATTGAGCNAGATTTTCAGGCATGTCTTTAGAATCCCCTGGTNCT GTCAAAGCCTGGTTTGGTTTACATTGGTNGTGCAATCNCTTTGTCAACATCTCCAGCACTATNGTTCCNTCT TAGGT
The disclosed NOV9 nucleic acid sequence, localized to the p21.2-22.2 region of cliromosome 1, has 916 of 1460 bases (62%.) identical to a 2001 bp cdc42-interactmg protein 4 (CfP4 mRNA from Homo sapiens (GENBANK-ID HSCrP4|acc:AJ000414) (E = 1.3e"97).
The disclosed NOV9 polypeptide (SEQ ID NO:24) encoded by SEQ ID NO:23 has 547 amino acid residues and is presented in Table 9B using the one-letter amino add code. Signal P, Psort and/or Hydropathy results predict that NOV9 has no signal peptide and is likely to be localized in the nucleus with a certainty of 0.7000 In other embodiments, NOV9 may also be localized to the microbosy (peroxisome) with a certainty of 0.3000, the mitochondrial matrix space with a certainty of 0.1000, or the lysosome (lumen) with a certainty of 0.1000.
Table 9B. Encoded NOV9 protein sequence (SEQ ID NO:24).
MSWGTE DQFDSLDKHTQ GIDFLERyAKFVKERIEIEQNXAKQLRNLVKKYCPKRSSKDEEP RFTSCVAFFNI NEIJNDYAGQREVVAEEMAHRVYGELMRYAHDIJKTERKMH QEGRKAQQYLDM C KQMGNS KKFBRECREAEKAQQSYERIiDNDTNATKADVENAKQQLNIiRTHMADENK AYAAQ LQNFNGEQHKHFYWIPQIYKQ QEMDERRTIKLSECYRGFADSERKVIPIISKCLEGMirjAAK SVDERRDSQMWDSFKSGFEPPGDFPFEDYSQHIYRTISDGTISASKQESGK DAKTPVGKAKG D LFGKKPKGPA EDFΞHLPPEQRRKKLQQRIDELNRELQKESDQKDAIiNKMKDVYEKDPQMG DPGSLQPKLAETMN IDRLRMEIHK-NEAWIjSEVEGKTGGRGDRRHSSDINHIjVXQGRESPEGSY TDDANQEVRGPPQQHGHHNEFDDEFEDDDP PAIGHCKAIYPFDGHNEGT AMKEGEV YIIEE DKGDGWTRARRQNGEEGYVPTSYIDVTLEKNSKGΞ
A search of sequence databases reveals that the NOV9 amino acid sequence has 303 of 544 amino acid residues (55%) identical to, and 403 of 544 amino acid residues (74%) similar to, the 545 amino acid residue CDC42-Interacting Protein 4 protein from Homo sapiens (SPTREMBL-ACC;015184) (E = 8.0e"l δ0).
TaqMan data for NOV9 can be found below in Example 2. The disclosed NOV9 polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table 9C.
Table 9C. BLAST results for NOV9
Gene Index/ Protein/ Organism Length Identity Positives Expect Identifier (aa) (%) (%) gi|l359153S|erab|CAC dJ1033H22.1 434 373/430 375/430 0.0 36351. ll (AL109613) (KIAA0554 (86%) (86%) protein) [Homo sapiens) gi 18923249 |ref |NP__O hypothetical 330 328/330 329/330 e-175
60207.1) protein FIιJ20275 (99%) ( 99%) |Homo sapiens] gi|l0435680|db | BAB unnamed protein 592 319/595 432/595 e-160 14538. ll (AK023681) product [Homo (53%) (71%) sapiens] gi|l3936547|gb|AAK4 formin-binding 679 307/624 422/624 e-14E 9824.1 |AF265550_1 protein 17 [Homo (49%) (67%) (AF265S50) sapiens] gi|3043632|dbj |BAA2 KIAA0554 protein 674 307/624 422/624 e-14!. 5480. l| (ABQ11126J [Homo sapiens] (49%) (67%)
The homology between these and other sequences is shown graphically in the ClustalW analysis shown in Table 9D. In the ClustalW alignment of the NOV9 protein, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function.
Table 9D. ClustalW Analysis of NOV9 1) Novel NOV9 (SEQ ID NO; 24)
3) gi|l3591Ξ36||emb||CAC363Ξl.l| (Al-109613. dσi033H22 , 1 (KIAA0554 protein) [Homo sapiens] (SEQ ID NO: 66)
4) gi|8923249|ref |NP_060207.l| hypothetical protein FIUJ20275 [Homo sapiens] (SEQ ID NO: 67) 5) gi|l0435680 |db |BAB14638.l| (AK023681) unnamed protein product [Homo sapiens] (SEQ ID NO: 68)
6) gi|l3936547|gb|AAK49824.llAF265550_l (AF265Ξ50) formin-b nding protein 17 [Homo sapiens] [SEQ ID NO:69)
7) gi| 3043632 ||dbj I BAA25480.l| (AB011126) IAA0554 protein [Homo sapiens] (SEQ ID NO: 70)
10 20 30 40
NOV9 gi|l3591536| gi j 8923249 || gi j 10435680 I gi|l3936547 | WNGGEEEPPRRPRARΞCEPEEAARTPGFPPSRGSRGAKG gi| 3043632 | EEEPPRRPRARSCEPEEAARTPGPPPSRGSRGA G
Figure imgf000102_0001
170 180 190 200
Figure imgf000102_0002
210 220 230 240
0
Figure imgf000102_0003
Figure imgf000103_0003
Figure imgf000103_0001
410 420 430 440
NOV9 gi|l3591536| --QSPPLTPTSLFTSSTPNGSQFITFSIjgVHYCM-glK2 gi|8923249 | gi |l0435680 | KSPKQQKgLSH FraFM-3 gi |13936547 j PHQPPPPPPASASPSAVPNGPQSPKQQKSI.SHRFSFM^ gi (3043632 | PHQPPPPPPASASPSAVPNGPQSPKQQKJSLSHRFMFMI
Figure imgf000103_0002
gi |3043632 I gGLYDSQ PPTVNNCAgDJ^^D^^EEQS^S EM
Figure imgf000104_0001
Table 9E-H lists the domain description from DOMAIN analysis results against NOV9. This indicates that the NOV9 sequence has properties similar to those of other proteins known to contain this domain.
Table 9E. Domain Analysis of NOV9 qnl I Smart | smart00326 , SH3 , Src homology 3 domains; Src homology 3
(SH3) domains bind to target proteins through sequences containing proline and hydrophobic amino acids. Pro-containing polypeptides may bind to SH3 domains in 2 different binding orientations. (SEQ ID
HO: 92)
CD-Length = 59 residues, 88.1% aligned
Score = 64.7 bits (156), Expect = le-11
Query : 484 HCKΛIYPFDGHNEGTLA.MKEGEVLYIIEEDKGDGWTRARRQNGEEGYVPTSYI 536
+ 1 + 1 + + l + l + l +++ ++ ( + 1 1 1 + I Ii + 1 1 I ++ 1 +
Sbj ct . 4 QVRALYDYTAQDPDELSFKKGDIITVLEKS -DDGWWKGRLGTGKEGLFPSNYV 55 Table 9F. Domain Analysis of NOV9 gnl |pfam|pfam00018 , SH3 , SH3 domain. SH3 (Src homology 3) domains are often indicative of a protein involved in signal transduction related to cytoskeletal organization First described in the Src cytoplasmic tyrosine kinase. The structure is a partly opened beta barrel. (SEQ
ID NO: 93)
CD-Length = 57 residues, 91.2% aligned
Score = 63.5 bits (153), Expect = 3e-ll
Query: 486 KAIYPFDGHNEGTLAMKEGEVLYIIEEDKGDGWTRARRQNGEEGYVPTSYID 537
A+Y + L+ K+G+++ ++E+ GW + R + +EG +P++Y++ SbjCt: 4 VALYDYQARESDELSFKKGDIIIVLEKSDDGGWWKGRI-KGTKEGLIPSNYVE 55
Table 9G. Domain Analysis of NOV9 gnl |ι Smart | smart00055, FCH, Fes/ciP4 homology domain; Alignment extended from original report. Highly alpha-helical. Also known as the RAEYB motif or the S. pombe Cdcl5 N-terminal domain. (SEQ ID NO: 94) CD-Length = 91 residues, 97.8% aligned Score = 58.2 bits (139), Expect = le-09
Query: 1 MSWGTELWDQFDSLDKHTQWGIDFLERYAKFVKERIEIEQNYAKQLRNLVKKYCPKRSSK 60
I + + 1 1 I I ++ I + 1+ I I I I ++ I I + 1 1 + 1 ( 1+ 1 + I I I +
Sbjct: 1 MGFWSELDDGFEALLSRLKNGLRLLEDLKKFMRERAKIEEEYAKKI1QKLSKK--LRAVRD 58 Query: 61 DEEPRFTSCVAFFNILNELNDYAGQREWAE 91
I + II + + 1 + 1 + I II ++ !
Sbjct. 59 TESELGSLRKAWEVLLSETDALAKQHIjQLΞE 89
Table 9H. Domain Analysis of NOV9 gnl |p am|pfam00611, FCH, Fes/CIP4 homology domain. Alignment extended from. Highly alpha-helical. (SEQ ID NO: 95) CD-Length = 94 residues, 97.9% aligned Score = 40.0 bits (92), Expect = 3e-04
Query 1 MSWGTELWDQ-FDSLDKΗTQWGIDFI-ERYAKFVKERIEIEQNYAKQLRNLVKKYCPKRSS 59
I + II + 11 + +1 + II I 11 + 11 + 11 + 11 + I ++ 1 + ++
Sb^ct. 1 MGFGSELCPEGH ALLSRQDNELRLbEEMKKFMAERAKIEKBYAGK-LQHLSAQVGKGPAT 60
Query: 60 KDEEPRFTSCVAFFNILNELNDYAGQREWAE 91
+ | + 1 ++ 11 + 1 + ++ 1
Sb ct: 61 AEGEDELΞSLKS AVILSETEQQΞKIHLQISE 92
The thyroid hormone receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes. They must specifically interact with a number of proteins as they progress from their initial translation and nuclear translocation to hεterodimerization with retinoid X receptors (RXRs), functional interactions with other transcription factors and the basic transcriptional apparatus, and eventually, degradation. To help elucidate the mechanisms that under lie the transcriptional effects and other potential functions of TRs, Lee et al. (Molec. Endocr. 9: 243-254, 1995) used the yeast interaction trap, a version of the yeast 2-hybrid system, to identify proteins that specifically interact with the ligand-binding domain of rat TR-beta (THRB; 190160). They isolated HeLa cell cDNAs encoding several different TR-interacting proteins (TRIPs), including TRIP 10. TRIP 10 interacted with rat Thrb only in the presence of thyroid hormone. It also showed a ligand- dependent interaction with RXR-alpha (RXRA; 180245), but did not interact with the glucocorticoid receptor (NR3C1 ; 138040) under any condition. TRIP10 contains a Src homology-3 (SH3) domain and shows sequence similarity to a Xenopus fyn homolog (see 137025) and chicken src (see 190090). Northern blot analysis detected a 2.6-kb TRIP 10 transcript in several tissues, with highest expression in skeletal muscle.
Using the yeast 2-hybrid system to identify proteins that bind to a constitutively active mutant of CDC42 (1 16952), Aspenstrom (1997) isolated a human B-cell cDNA encoding CDC42-interacting protein-4 (CTP4), which is identical to TRIP 10. The predicted 545-amino acid CIP4 protein contains an N-terminal domain that bears resemblance to the nonkinase domain of the FER (176942) and Fes/Fps family of tyrosine kinases, and a C-terminal SH3 domain. In addition, CIP4 shares sequence similarity with a number of proteins that have roles in regulating the actin cytoskeleton. Aspenstrom (Curr. Biol. 7: 479-487, 1997) demonstrated that CIP4 can bind to activated CDC42 in vitro and in vivo. Overexpression of CIP 4 in fibroblasts reduced the amount of stress fibers in these cells. Recombinant CfP4 protein accumulated at the cell periphery, particularly in areas that exhibited membrane ruffling. Coexpression of activated CDC42 and CIP4 led to clustering of CUM to a large number of foci at the dorsal side of the cells. Northern blot analysis showed a major 2.2-kb CIP4 transcript that was abundant in skeletal muscle, heart, and placenta, present at lower levels in pancreas, lung, liver, and kidney, and barely detectable in brain. Minor transcripts of 3.5 and 5 kb were also detected.
Aspenstrom (1997) concluded that CIP4 is a downstream target of activated GTP- bound CDC42, and is similar in sequence to proteins involved in signaling and cytoskeletal control. The author suggested that CIP4 may act as a link between CDC42 signaling and regulation of the actin cytoskeleton.
The Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency characterized by thrombocytopenia, eczema, and a tendency toward lymphoid malignancy (Tian L, et.al.; J Biol Chem 2000 Mar 17;275(11 ):7854-61). Lymphocytes from affected individuals have cytoskeletal abnormalities, and monocytes show impaired motility. The Wiskott-Aldrich syndrome protein (WASP) is a multi-domain protein involved in cytoskeletal organization. In a two-hybrid screen, we identified the protein Cdc42-interacting protein 4 (CIP4) as a WASP interactor. CIP4, like WASP, is a Cdc42 effector protein involved in cytoskeletal organization. We found that the WASP-CIP4 interaction is mediated by the binding ofthe Src homology 3 domain of CIP4 to the proline-rich segment of WASP. Cdc42 was not required for this interaction. Co-expression of CIP4 and green fluorescent protein- WASP in COS-7 cells led to the association of WASP with microtubules. In vitro experiments showed that CIP4 binds to microtubules via its NH(2) terminus. The region of CTP4 responsible for binding to active Cdc42 was localized to amino acids 383-417, and the mutation I398S abrogated binding. Deletion of the Cdc42-binding domain of CIP4 did not affect the colocalization of WASP with microtubules in vivo. We conclude that CIP4 can mediate the association of WASP with microtubules. This may facilitate transport of WASP to sites of substrate adhesion in hematopoietic cells. The disclosed NOV9 nucleic acid of the invention encoding a CfP4-like protein includes the nucleic acid whose sequence is provided in Table 9 A, or a fragment thereof. The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 9A while still encoding a protein that maintains its CIP4-like activities and physiological functions, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described. The invention additionally includes nucleiG acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic adds whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability o the modified nucleic acid, siiGh that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to about 38% percent o the bases may be so changed. The disclosed NOV9 protein of the invention includes the CIP4-like protein whose sequence is provided in Table 9B. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 2 while still encoding a protein that maintains its CIP4-like activities and physiological functions, or a functional fragment thereof In the mutant or variant protein, up to about 51% percent ofthe residues may be so changed
The invention further encompasses antibodies and antibody fragments, such as Fab or (FJ )2 that bind immunospecifically to any of the proteins of the invention The above defined information for this invention suggests that this CIP4-hke protein
(NOV9) may function as a member of a "MT family" Therefore, the NO V9 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below The potential therapeutic applications for this invention include, but are not limited to protein therapeutic, small molecule drug target, antibody target (therapeutiG, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in xitro of all tissues and cell types composing (but not limited to) those defined here
The NOV9 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to Inflamation,
Autoimmune disorders, Aging and Cancer For example, a oDNA encoding the CIP4-lιke protein (NOV9) may be useful in gene therapy, and the CIP 4-lιke protein (NOV9) may be useful when administered to a subject in need thereof By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from Wiskott-Aldπch syndrome, immunodeficiency, thrombocytopenia, eczema, lymphoid malignancy cytoskeletal abnormalities, impaired monocyte motihty, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis, Von Hippel-Lindau (VHL) syndrome, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy, Lesch-Nyhan syndrome, Ataxia-telangiectasia, Leukodystrophies, Behavioral disorders, Addiction, Anxiety, Pam, Neuroprotection, Fertility, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis , Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis, Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Tuberous sclerosis, Scleroderma, Obesity, Transplantation, Diabetes, Pancreatitis, Obesity, Systemic lupus erythematosus , Autoimmune disease, Asthma, Emphysema, Scleroderma, allergy, ARDS, Cirrhosis, Transplantation, Diabetes, Autoimmune disease, Renal artery stenosis, Inteistitial nephritis, Glomerulonephπtis, Polycystic kidney disease, Renal tubulai acidosis, IgA nephropathy, Hypercalceimia, or other pathologies or conditions The NOV9 nucleic acid encoding CIP4-hke piotein, and the CIP4-hke protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed
NOV9 nucleic acids and polypeptides are further useful in the generation of antibodies that bind lmmuno-specifically to the novel NOV9 substances for use in therapeutic or diagnostic methods These antibodies may be generated according to methods known in the art, using prediction from hydiophobicity charts, as described in the "Anti-NOVX Antibodies" section below The disclosed NOV9 piotein has multiple hydrophilic regions, each of which can be used as an immunogen In one embodiment, a contemplated NOV9 epitope is from about ammo acids 25 to 40 In another embodiment, a NOV9 epitope is from about amino acids 45 to 55 These novel proteins Gan be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology of the disease and development of new drug targets for various disorders
NOV10 NOV10 includes three novel hepsin/plasma transmembrane serine protease-like proteins disclosed below. The disclosed sequences have been named NOV 10a and NOV 10b NOVlOa
A disclosed NOVl 0a nucleic acid of 1787 nucleotides (also referred to as 129297354JEXT) encoding a novel hepsin/plasma transmembrane serine protease-hke protein is shown in Table 10A An open reading frame was identified beginning with an ATG initiation codon at nucleotides 54-56 and ending with a TAA codon at nucleotides 1470-1472 A putative untranslated region upstream from the initiation codon and downstream from the termination codon is underlined in Table 10A The start and stop codons are in bold letters
Table 10A. NOVlOa nucleotide sequence (SEQ ID NO:25).
GCGGAACATTGCCTAGTAGACCCTGAGGCTTTACAACAGTGCCACTGACCCCTATGAGCCTGATGCTGGATG ACCAACCCCCTATGGAGGCCCAGTATGCAGAGGAGGGCCCAGGACCTGGGATCTTCAGAGCAGAGCCTGGAG ACCAGCAGCATCCCATTTCTCAGGCGGTGTGCTGGCGTTCCATGCGACGTGGCTGTGCAGTGCTGGGAGCCC TGGGGCTGCTGGCCGGTGCAGGTGTTGGCTCATGGCTCCTAGTGCTGTATCTGTGTCCTGCTGCCTCTCAGC CCATTTCCGGGACCTTGCAGGATGAGGAGATAACTTTGAGCTGCTCAGAGGCCAGCGCTGAGGAAGCTCTGC TCCCTGCACTTCCCAAAACAGTATCTTTCAGAATAAACAGCGAAGACTTCTTGCTGGAAGCGCAAGTGAGGG ATCAGCCACGCTGGCTCCTGGTCTGCCATGAGGGCTGGAGCCCCGCCCTGGGGCTGCAGATCTGCTGGAGCC TTGGGCATCTCAGACTCACTCACCACAAGGGAGTAAACCTCACTGACATCAAACTCAACAGTTCCCAGGAGT TTGCTCAGCTCTCTCCTAGACTGGGAGGCTTCCTGGAGGAGGCGTGGCAGCCCAGTAGGACTACTGAGGCTG TTAGGAACAACTGCACTTCTGGTCAAGTTGTTTCCCTCAGATGCTCTGAGTGTGGAGCGAGGCCCCTGGCTT CCCGGATAGTTGGTGGGCAGTCTGTGGCTCCTGGGCGCTGGCCGTGGCAGGCCAGCGTGGCCCTGGGCTTCC GGCACACGTGTGGGGGCTCTGTGCTAGCGCCACGCTGGGTGGTGACTGCTGCACATTGTATGCACAGTTTCA GGCTGGCCCGCCTGTCCAGCTGGCGGGTTCATGCGGGGCTGGTCAGCCACAGTGCCGTCAGGCCCCACCAAG GGGCTCTGGTGGAGAGGATTATCCCACACCCCCTCTACAGTGCCCAGAATCATGACTACGACGTCGCCCTCC IGAGGCTCCAGACCGCTCTCAACTTCTCAGACACTGTGGGCGCTGTGTGCCTGCCGGCCAAGGAACAGCATT TTCCGAAGGGCTCGCGGTGCTGGGTGTCTGGCTGGGGCCACACCCACCCTAGCCATACTTACAGCTCGGATA TGCTCCAGGACACGGTGGTGCCCCTGCTCAGCACTCAGCTCTGCAACAGCTCTTGCGTGTACAGCGGAGCCC TCACCCCCCGCATGCTΪTGCGCΪGGCTACCTGGACGGAAGGGCTGATGCATGCCAGGGAGATAGCGGGGGCC CCCTAGTGTGCCCAGATGGGGACACATGGCGCCTAGTGGGGGTGGTCAGCTGGGGGCGTGGCTGCGCAGAGC CCAATCACCCAGGTGTCTACGCCAAGGTAGCTGAGTTTCTGGACTGGATCCATGACACTGCTCAGGTGAGTG TGGGGGCAGGAGTAGGGCAGGGAGATTTCTAAAGGACCTGCCCTCGAATGCAAGGAACCTTACCCCTTAGGC CCGGGCCCTGCTGGGGACTGGGGAGGGTGCTAGGACATATTCCCCAGAGTGAGTGGAGGAAGAAGTGAAGCT TAAACATGGAATCCATTGGATTTCTATCAGTTTAAGGATGAACTGGGTAAGAGTATGCCTGAGTTTGTATCC CAGATCTACCATTTCCTGTGTCGACCTTTGGCAAATTTCTAACTTTGTTAAACCTTAATTTCCTGATAATAA CCATGATGGCTACTTATATGCTATTGTTATATGCTATTAAATAAGACCCGTACAATGCC
The reverse complement of NOVl Oa is shown in Table 10B.
Table 10B. NOVlOa reverse complement sequence (SEQ ID NO:26).
GGCATTGTACGGGTCTTATTTAATAGCATATAACAATAGCATATAAGTAGCCATCATGGTTATTATCAGGAA ATTAAGGTTTAACAAAGTTAGAAATTTGCCAAAGGTCGACACAGGAAATGGTAGATCTGGGATACAAACTCA GGCATACTCTTACCCAGTTCAICCTTAAACTGATAGAAATCCAATGGATTCCAIGTTTAAGCTTCACTTCTT CCTCCACTCACTCTGGGGAATATGTCCTAGCACCCTCCCCAGTCCCCAGCAGGGCCCGGGCCTAAGGGGTAA GGTTCCTTGCATTCGAGGGCAGGTCCTTTAGAAATCTCCCTGCCCTACTCCTGCCCCCACACTCACCTGAGC AGTGTCATGGATCCAGTCCAGAAACTCAGCTACCTTGGCGTAGACACCTGGGTGATTGGGCTCTGCGCAGCC ACGCCCCCAGCTGACCACCCCCACTAGGCGCCATGTGTCCCCATCTGGGCACACTAGGGGGCCCCCGCTATC TCCCTGGCATGCATCAGCCCITCCGTCCAGGTAGCCAGCGCAAAGCATGCGGGGGGTGAGGGCTCCGCTGTA CACGCAAGAGCTGTTGCAGAGCTGAGTGCTGAGCAGGGGCACCACCGTGTCCTGGAGCAIATCCGAGCTGTA AGTATGGCTAGGGTGGGTGTGGCCCCAGCCAGACACCCAGCACCGCGAGCCCTTCGGAAAATGCTGTTCCTT GGCCGGCAGGCACACAGCGCCCACAGTGTCTGAGAAGTTGAGAGCGGTCTGGAGCCTCAGGAGGGCGACGTC GTAGTCATGATTCTGGGCACTGTAGAGGGGGTGTGGGATAATCCTCTCCACCAGAGCCCCTTGGTGGGGCCT GACGGCACTGTGGCTGACCAGCCCCGCATGAACCCGCCAGCTGGACAGGCGGGCCAGCCTGAAACTGTGCAT ACAATGTGCAGCAGTCACCACCCAGCGTGGCGCTAGCACAGAGCCCCCACACGTGTGCCGGAAGCCCAGGGC CACGCTGGCCTGCCACGGCCAGCGCCCAGGAGCCACAGACTGCCCACCAACTATCCGGGAAGCCAGGGGCCT CGCTCCACACTCAGAGCATCTGAGGGAAACAACTTGACCAGAAGTGCAGTTGTTCCTAACAGCCTCAGTAGT CCTACTGGGCTGCCACGCCTCCTCCAGGAAGCCTCCCAGTCTAGGAGAGAGCTGAGCAAACTCCTGGGAACT GTTGAGTTTGATGTCAGTGAGGTTTACTCCCTTGTGGTGAGTGAGTCTGAGATGCCCAAGGCTCCAGCAGAT CTGCAGCCCCAGGGCGGGGCTCCAGCCCTCATGGCAGACCAGGAGCCAGCGTGGCTGATCCCTCACTTGCGC TTCCAGCAAGAAGTCTTCGCIGTTTATTCTGAAAGATACTGTTTTGGGAAGTGCAGGGAGCAGAGCTICCTC AGCGCTGGCCTCTGAGCAGCTCAAAGTTATCTCCTCATCCTGCAAGGTCCCGGAAATGGGCTGAGAGGCAGC AGGACACAGATACAGCACTAGGAGCCATGAGCCAACACCTGCACCGGCCAGCAGCCCCAGGGCTCCCAGCAC TGCACAGCCACGTCGCATGGAACGCCAGCACACCGCCTGAGAAATGGGATGCTGCTGGTCTCCAGGCTCTGC TCTGAAGATCCCAGGTCCTGGGCCCTCCTCTGCATACTGGGCCTCCATAGGGGGTTGGTCATCCAGCATCAG GCTCATAGGGGTCAGTGGCACTGTTGTAAGCCTCAGGGTCTACTAGGCAATGTTCCGC
In a search of public sequence databases, the NOV l Oa nucleic acid sequence has 424 ases (60%) identical to a gb:GENBAN -ID:AF243500||acc:AF243500 transmembrane protein X mRNA, complete eds, 1735 bp. mRNA from mouse/ Mus musculus (E = 4.9e"19). Public nucleotide databases include all GenBank databases and the GeneSeq patent database. The disclosed NOV 10a polypeptide (SEQ ID NO:27) encoded by SEQ ID NO:25 has 472 amino acid residues and is presented in Table 10B using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOVlOa has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.7900. In other embodiments, NOVlOa may also be localized to the microbody (peroxisome) with acertainty of 0.5425, the Golgi body with a certainty of 0.3000, or in the endoplasmic reticulum (membrane) with a certainty of 0.2000. The most likely cleavage site for a NOVlOa peptide is between amino acids 13and 14, at: MEA-QY.
Table 10B. Encoded NOVlOa protein sequence (SEQ ID NO:27).
MS MLDDQPPMEAQYAEEGPGPGIF-RAEPGDQQHPISQAVCWRSMRRGCAVLGALG LAGAGVGSWLLVLYL CPAASQPISGTLQDEEITLSCSEASAEEALLPAIjPKTVSFRINSEDFLLEAQVRDQPR LLVCHEG SPALG LQICWSLGHLR THHKGVNLTDIK NSSQEFAQLSPRLGGFiEEA QPSRTTEAVRNNCTSGQ SLRCSEC GARPLASRIVGGQSVAPGRWPWQAΞVAbGFRHTCGGSVLAPRWWTAAHCMHSFRLAR SSWRVHAGLVSHS AVRPHQGALVERIIPHPLYSAQNHDYDVALLRBQTAIjNFSDTVGAVCItPAKEQHFPKGSRCWVSGWGHTHPS HTYSSDMLQDTWPLLSTQIjCNSSCVYSGABTPR LCAGYLDGRADACQGDSGGPLVCPDGDTWRLVGWS GRGCAEPNHPGVYAKVAEFIDWIHDTAQVSVGAGVGQGDF
A search of sequence databases reveals that the NOVlOa amino acid sequence has 130 of 346 amino acid residues (37%) identical to, and 190 of 346 amino acid residues (54%) similar to, the 417 amino acid residue Serine Protease Hepsin (EC 3.4.21.-) (Transmembrane Protease, Serine 1) protein from Homo sapiens (ptnr:SWISSPROT-ACC:P05981 (E = 3.5e"56). Public amino acid databases include the GenBank databases, SwissProt, PDB and PIR.
NOVlOa is expressed in at least the adrenal gland.
NOVlOb
A disclosed NOVlOb nucleic acid of 2148 nucleotides (also referred to as CGI 06783- 02) encoding a novel Spinesin-like protein is shown in Table IOC. An open reading frame was identified beginning with an ATG initiation codon at nucleotides 156-158 and ending with a TGA codon at nucleotides 1410-1412. A putative untranslated region upstream from the initiation codon is underlined in Table IOC. The start and stop codons are in bold letters. Table IOC. NOV] Ob nucleotide sequence (SEQ ID NO:28).
ACGCGGGATACAGGGAGGGGCCATGTGCGAACCAGGGAGACCTCATCTTCCAACCAAGCTTGCTGGGCTTGC ATTTAATCAATGCATGGCCAGAGAACAGGAGCGGAACATTGCCTAGTAGACCCTGAGGCTTTACAACAGTGC TACTGACCCCTATGAGCCTGATGCTGGATGACCAACCCCCTATGGAGGCCCAGTATGCAGAGGAGGGCCCAG GACCTGGGATCTTCAGAGCAGAGCCTGGAGACCAGCAGCATCCCATTTCTCAGGCGGTGTGCTGGCGTTCCA TGCGACGTGGCTGTGCAGTGCTGGGAGCCCTGGGGCTGCTGGCCGGTGCAGGTGTTGGCTCATGGCTCCTAG TGCTGTATCTGTGTCCTGCTGCCTCTCAGCCCATITCCGGGACCTTGCAGGATGAGGAGATAACTTTGAGCT GCTCAGAGGCCAGCGCTGAGGAAGCTCTGCTCCCTGCACTCCCCAAAACAGTATCTTTCAGAATAAACAGCG AAGACTTCTTGCTGGAAGCGCAAGTGAGGGATCAGCCACGCTGGCTCCTGGTCTGCCATGAGGGCTGGAGCC CCGCCCTGGGGCTGCAGATCTGCTGGAGCCTTGGGCATCTCAGACTCACTCACCACAAGGGAGTAAACCTCA CTGACATCAAACTCAACAGTTCCCAGGAGTTTGCTCAGCTCTCTCCTAGACTGGGAGGCTTCCTGGAGGAGG CGTGGCAGCCCAGGAACAACTGCACTTCTGGTCAAGTTGTTTCCCTCAGATGCTCTGAGTGTGGAGCGAGGC CCCTGGCTTCCCGGATAGTTGGTGGGCAGTCTGTGGCTCCTGGGCGCTGGCCGTGGCAGGCCAGCGTGGCCC TGGGCTTCCGGCACACGTGTGGGGGCTCTGTGCTAGCGCCACGCTGGGTGGTGACTGCTGCACATTGTATGC ACAGTGCCCAGAATCATGACTACGACGTCGCCCTCCTGAGGCTCCAGACCGCTCTCAACTTCTCAGACACTG TGGGCGCTGTGTGCCTGCCGGCCAAGGAACAGCATTTTCCGAAGGGCTCGCGGTGCTGGGTGTCCGGCTGGT GCCACACCCACCCTAGCCATACTTACAGCTCGGATATGCTCCAGGACACGGTGGTGCCCCTGCTCAGCACTC AGCTCTGCAACAGCTCTTGCGTGTACAGCGGAGCCCTCACCCCCCGCATGCTTTGCGCTGGCTACCTGGACG GAAGGGCTGATGCATGCCAGGGAGATAGCGGGGGCCCCCTAGTGTGCCCAGATGGGGACACATGGCGCCTAG TGGGGGTGGTCAGCTGGGGGCGTGGCTGCGCAGAGCCCAATCACCCAGGTGTCTACGCCAAGGTAGCTGAGT TTCTGGACTGGAτCCATGACACTGCTCAGGACτCCCTCCTCTGAGTCCTGCτGττTCCτCCAGTCTCACTGC ACACCACTGCCTCATGCTTCCTGGGGCCTCCAGCAGCTCCACTAATGGAGGAGAGGCAGTAGCCTCCGACAC AGAACGCATGGACCTCCTACTACTGTGTGTGAGGAACAGTCACTACCCACTGGCCAGCCACCCAGCCAACAG GTCTCTCCTCTTGGGCCCTGATTTCAGAGTCCTCTTTCTCACTAGAGACTCAATGACAGAAGAGAGGCTGGG ACTTGGTTGGGCATGCTGTGGTTGCTGAGGGATGAGGGGGAGGAGAGAGGTAGGAGCTGGAGATGAAGAGAC TGCTAGAAGCAGCAGGAAGCCTGCCCTTCTGCCCTCTCCCCTCCCTGCCCCTGTGTGAGTCTTTTAGGGAGG GTGACTGGGAGGTGCCCCCCGTCCCACCTTTTTCCTGTGCTCTAGGTGGGCTAAGTGCCTCCCTAGAGGACT CCATGGCTGAGAGGCTCCTGGGCAGATGGGGTCAAGGCTGGGCCAGTCCCAGATGAAGCCTATGGGAGTCAG GACCCTCTCCACTCTCCCTCTCCACTCCCCTTCCTGTTCTCACCTGGCTGTGGCTGGCCCTGTGTGGGGTGG GTACACTGGAAAACAAGAAGGτTGGAGTTGGTCTAGGACATTGGττTTAAATGACAGTTOTGTGAACτGGTC CAAGGAGGTTCTGTTATTAAAGTGATATATGGTCTTGAAAAAAAAAAAAAAAAAAAAAAA
In a search of public sequence databases, the NOVlOb nucleic acid sequence, located on chromosome 7 has 1343 of 1446 bases (92%) identical to a gb:GENBANK- ID:AB028140|acc:AB028140.1 mRNA from Homo sapiens (Homo sapiens mRNA for spinesin, complete eds) (E = 3.2e"268). Public nucleotide databases include all GenBank databases and the GeneSeq patent database.
The disclosed NOVlOb polypeptide (SEQ ID NO:29) encoded by SEQ ID NO:28 has 418 amino acid residues and is presented in Table 10D using the one-letter amino acid code. Signal P, Psort and/or Hydropathy results predict that NOV 10a has a signal peptide and is likely to be localized in the plasma membrane with a certainty of 0.7900. In other embodiments, NOVlOa may also be localized to the Golgi body with acertainty of 0.3000, the microbody (peroxisome) with a certainty of 0.2036 or in the endoplasmic reticulum (membrane) with a certainty of 0.2000. The most likely cleavage site for a NOVl Oa peptide is between amino acids 13 and 14, at: MEA-QY.
Table 10D. Encoded NOVlOa protein sequence (SEQ ID NO:29).
MSLMLDDQP MEAQ AEBG GPGiFR EPGDQQHPisQAvcwRSMRRGCAVLGALGi--L GAGVGS L ViιYiι
CPAASQPISGTIJQDEEITLSCSEASAEEAILPALPKTVSFRINSEDFDLEAQVRDQPRW LVCHEGWΞPAIJG ]JQIC SLGHL1R1)THH GVNI.TDIKDNSSQEFAQDSPRLGGFLEEAWQPRNNCTSGQWSLRCSECGARP1AS RIVGGQSVAPGRWPWQASVALGFRHTCGGSVLAPR VVTAAHCMHSAQNHDYDVALLRLQTAINFSDTVGAV CLEAKEQHFPKGSRCWVSG CHTHPSHTYSSDMLQDTVVPLLSTQLCNSSCVYSGALTPRMLCAGY DGRAD ACQGDSGGFLVCPDGDTWRLVGVVSWGRGCAEPNHPGVYAKVAEFIDWIHDIAQPSLI-J
A search of sequence databases reveals that the NOVlOb amino acid sequence has 262 of 262 amino acid residues (100%) identical to, and 262 of 262 amino acid residues (100%) similar lo, the 457 amino acid residue ptnr:SWlSSNEW-ACC:Q9H3S3 protein from Homo sapiens (Human) (Transmembrane protease, serine 5 (EC 3.4.21.-) (Spinesin) (E = 8.7e" 33). Public amino acid databases include the GenBank databases, SwissProt, PDB and P1R.
NOVlOb is expressed in at least the following tissues: Colon, Brain, Placenta, Testis, Adrenal Gland/Suprarenal gland, Retina. Expression information was derived from the tissue sources ofthe sequences that were included in the derivation ofthe sequence of NOVlOb. The sequence is predicted to be expressed in the following tissues because ofthe expression pattern of (GENBANK-ID: gb:GENBA-NK-ID:AB028140|acc:AB028140.1) a closely related Homo sapiens mRNA for spinesin, complete eds homolog in species Homo sapiens : brain.
TaqMan data for NOVlOa can be found below in Example 2. The proteins encoded by the NOVlOa and 10b nucleotides are very closely homologous as is shown in the alignment in Table 10E.
Table 10E Alignment of NOVlOa and 10b.
NOVlOa
NOVlOb
Figure imgf000113_0001
60 70 80 90 100
■ ■■■I.... I......... I.._..1^^ L ... I .... I .... I .... I
NOVlOa AGfiGVGSW EV YLCPAASQPISGTLQDEEITLSCSEASA
NOVlOb ^ GA; GL AGAGVGS LLVLY CPAASQPISGTEQDEEITLSCSEASA
110 120 130 140 150 I
NOVlOa LLPA PKTVSFRINSEDFLLEAQVRDQPR VCHEG SPA GLQICWε
NOVlOb -- )?ALPKTVS;PRINSED
NOVlOa NOVlOb
Figure imgf000113_0002
210 220 230 240 250
.... i .... ) .... I .... ι_.._.. i^ [....I...
NOVlOa NNC.TSGQVVSLRCSECGARP ASRIVGGQSVAPGR P QASVALGFRHTC
NOVlOb NNCTSGQVVSIR.CSECGARPLASRIVGGQSVAPGR EWQASVA GFRHTC
260 270 280 290 300
■ ... I .... I ■ .. II ■ ■ ■ ■ I .... I .... II .... I .... I .... i .... t
NOVlOa EgJ ljaaajjwmWM«^SFRLARLSSWRVHAGLVSHSAVREHQGAl,VER
310 320 330 340 350
■ ■ • • I ■ ■ ■ ■ II .... I .... I .... I .... I ■ ■ ■ ■ I .... I .... II .... II NQVlOa IIPHPLYg «.il.iiV.JiV.ffl.M;Mriiι>i^ NOVlOb SAQNHDYDVALLR QTALNFSDTVGAVCI-PAKEQH PKC
NOVlOa NOVlOb
Figure imgf000114_0001
410 420 430 440 450
NOVlOa LDGRADACQGDSGGP VCPDGDTWRLVGVVSWGRG.CAEPNHP.GVYAKVA NOVlOb D-GRADAGOGDSGGPLVCPDGDTWRLVGVVS GRGCAEPNHP-GVYAK -
460 470
. I.... I.... I..
NOVlOa |vgVGAGVGQGDF (SEQ ID NO: 27) NOVlOb D| L (SEQ ID NO: 29)
Homologies to any ofthe above NOVIO proteins will be shared by the other NOVIO protein insofar as they are homologous to each other as shown above. Any reference to
NOVIO is assumed to refer to both ofthe NOV10 proteins in general, unless otherwise noted.
The disclosed NOVlOa polypeptide has homology to the amino acid sequences shown in the BLASTP data listed in Table 10F.
Figure imgf000114_0002
The homology between these and other sequences is shown graphically in the ClustalW analysis shown in Table 10G. In the ClustalW alignment ofthe NOV10 protein, as well as all other ClustalW analyses herein, the black outlined amino acid residues indicate regions of conserved sequence (i.e., regions that may be required to preserve structural or functional properties), whereas non-highlighted amino acid residues are less conserved and can potentially be altered to a much broader extent without altering protein structure or function. O 02 3
Table 10G. ClustalW Analysis of NOVIO
1) Novel NOVlOa (SEQ ID NO:27)
2) Novel NOVlOb (SEQ ID NO:29)
4) gi|13540535 I ef lNP_J-.10397.-L I transmembrane protease, serine 5; spinesin [Homo sapiens] (SEQ ID N0:71)
5) gi 113878822 I sp|IQ9ER04 I TMS5_MOUSE TRANSMEMBRANE PROTEASE, SERINE 5 (SPINESIN) (SEQ ID NO: 72)
6) gi|1350765 I ef|NP_109634.It transmembrane protease, serine 5 (spinesin) [Mus musculus] (SEQ ID NO:73)
7) gi 114770563 I ref | XP_041427.11 transmembrane protease, serine 5 [Homo sapiens] (SEQ ID N0:74)
8) gi|12248777|db lBAB20276.il (AB016229) type 2 spinesin fMus musculus] (SEQ ID NO: 75)
Figure imgf000115_0001
210 230 ?40
Figure imgf000116_0001
290 300 310 320
Figure imgf000116_0002
330 340 350 360
Figure imgf000116_0003
450 460 470
NOVlOa /SVGAGVGQGDF
NOVlOb VYA VAEFLDWIH-D DS Ii gil 13540535 I 'SVYAKVAEF D IHD --S L gi|13878822| PGVYAKVAEFLDWIH gil 13507652 I ifevϊAK aE raffiHD gil 14770563 I P - gil 122487771 Rl iS SLPGRKG 5eιW Ά* J* »» jtø { ;}f i {»jV Tables 1H-1 I lists the domain description from DOMAIN analysis results against NOVIO. This indicates that the NOVIO sequence has properties similar to those of other proteins known to contain this domain.
Table 10H Domain Analysis of NOVIO gnl | Smar lsmart00020, Tryp_SPc, Trypsin-like serine protease; Many of these are synthesised as inactive precursor zymogens that are cleaved during limited proteolysis to generate their active forms. A few, however, are active as single chain molecules, and others are inactive due to substitutions of the catalytic triad residues. (SEQ ID NO: 96) CD-length = 230 residues, 100.0% aligned Score = 266 bits (681), Expect = 2e-72
Query: 224 RIVGGQSVAPGRWPWQASVAL-GFRHTCGGSVLAPRWVVTAAHCMHSFRLARLSSWRVHA 282
Mill I +1 M 1+ I I I I II I+++I I ll+l I I I I++ M M Sbjct: 1 RIVGGSEANIGSFPWQVSLQYRGGRHFCGGSLISPRWVLTAAHCVYGSAP SSIRVRL 57
Query: 283 G VSHSAVRPHQGALVERIIPHPLYSAQNHDYDVALLRLQTALNFSDTVGAVCLPAKEQH 342
I 1+ I ll ++I 11 1+ +| |-| II |4-| 4- I I I I 4-1 I 1+ + Sbjct: 58 GSHDLSSGEETQTVKVSKVIVHPNYNPSTYDNDIALLKLSEPVTLSDTVRPICLPSSGYN 117
Query: 343 FPKGSRCWVSGWGHTHPSHIYSSDMLQDTVVPLI-STQLCNSSCVYSGALTPRMLCAGYLD 402
I 1+ I I I I I I I I 1 11+ III++I I + l+ l I 11)1 ι +
Sbjct: 118 VPAGTTCTVSGWGRTSESSGSIPDTLQBVNVPIVSNATCRRAYSGGPAITDNMLCAGGLE 177 Query: 403 GRADACQGDSGGPLVCPDGDΪWRLVGVVSWGR-GCAEENHPGVYAKVAEFLDWl 455
I II I II I III II II + I I ll+l I I I I II I I I I I I +1+ +11 I!
Sbjct: 178 GGKDACQGDSGGPLVC-NDPP.WVLVGIVSWGSYGCARPNKPGVYTRVSSYLDWI 230
Table 101. Domain Analysis of NOV10 gnl I Pfamlpfam00089, trypsin, Trypsin. Proteins recognized include all proteins xn families SI, S2A, S2B, S2C, and S5 in the classification of peptidases. Also included are proteins that are clearly members, but that lack peptidase activity, such as haptoglobin and protein 3
(PRTZ4-) . (SEQ ID NO: 97)
CD-Length = 217 residues, 100.0% aligned core = 211 bits (538), Expect = 6e-56
Query: 225 IVGGQSVAPGRWPWQASVA1.GFRHTCGGSVLAPR VVIAAHCMHSFRLARLSSWRVHAGL 284 Ml I 1+ I +11 I II 1+ + I II I ll I+++ I l+l I ll 1+ I Ml II I Sbjct : 1 IVGGREAQAGSFPWQVSLQVSSGHFCGGSl-ISENWVI-TAAHCVSG ASSVRVVLGE 55 Query: 285 VSHSAVRPH~QGALVSRIIPHPLYSAQNHDYDVALLRIQTAI.NFSDTVGAVCIjPAK--.QHF 343 + I I++I 1 l ÷ l+M I + 1++ + III +111+ Sbjct: 56 HNLGTTEGTEQKFDVKKIIVHPNYNPDT—NDIALL LKSPVTLGDTVRPICLPSASSDL 113 Query: 344 PKGSRC VSGWGHTHPSHTYSSDMLQDTVVPLLSTQLCNSSCVYSGALTPRMLCAGYLDG 403
I 1+ I I II II I + +1 I I 1+ II II II++I + II )+ l l +l ll+l l I I Sbjct: 114 PVGTTCSVSS GRTK-—N GTSDTLQE VVPIVSRETCRSA—-YGGTVTDTMICAGALGG 169 Query: 404 RADACQGDSGGPiVCPDGDTWRLVG VSWGRGCAEPNHPGVYAKVAEFLDWI 455
+ I I ! Ml I I Ml I I I I I I I I l + l i I I I I I l + l II I I +1+ + 1 I I I Sbjct: 170 K-DACQGDSGGPLVCSDG— -ELVG1VS GYGCAVGNYPGVΪTRVSRYLDWI 217
15 Failure to thrive, nutritional edema, and hypoproteinemia with normal sweat electrolytes were features of 2 affected male infants reported by Townes (1965) and Townes et al. (1967). A protein hydrolysate diet was beneficial. A male sib of the first patient reported by Townes (1965) had died, apparently of the same condition. Morris and Fisher (1967) reported an affected female who also had imperforate anus. The clinical picture in enterokinase deficiency (226200) is closely similar; however, the defect is not in the synthesis of trypsinogen but in the synthesis of the enterokinase which activates proteolytic enzymes produced by the pancreas. Oral pancreatin represents a therapeutically successful form of enzyme replacement (Townes, 1972). Trypsin (EC 3.4.21.4 ), like elastase (130120), is a member of the pancreatic family of serine proteases. MacDonald et al. (1982) reported nucleotide sequences of cDNAs representing 2 pancreatic rat trypsinogens. Using a rat cDNA probe, Honey et al. (1984, 1984) found that a 3.8-kb DNA fragment containing human trypsin- 1 gene sequences cosegregated with chromosome 1 , and assigned the gene further to 7q22- 7qter by study of hybrids with a deletion of this segment. The trypsin gene is on mouse chromosome 6 (Honey et al., 1984). Carboxypeptidase A (114850) and trypsin are a syntenic pair conserved in mouse and man. Emi et al. (1986) isolated cDNA clones for 2 major human trypsinogen isozymes from a pancreatic cDNA library. The deduced amino acid sequences had 89% homology and the same number of amino acids (247), including a 15-amino acid signal peptide and an 8-amino acid activation peptide. Southern blot analysis of human genomic
DNA with the cloned cDNA as a probe showed that the human trypsinogen genes constitute a family of more than 10, some of which may be pseudogenes or may be expressed in other stages of development.
Rowen et al. (1996) found that there are 8 trypsinogen genes embedded in the beta T- cell receptor locus or cluster of genes (TCRB; 186930) mapping to 7q35. In the 685-kb DNA segment that they sequenced they found 5 tandemly arrayed 10-kb locus-specific repeats (homology units) at the 3-prime end ofthe locus. These repeats exhibited 90 to 91% overall nucleotide similarity, and embedded within each is a trypsinogen gene. Alignment of pancreatic trypsinogen cDNAs with the germline sequences showed that these trypsinogen genes contain 5 exons that span approximately 3.6 kb. Further analyses revealed 2 trypsinogen pseudogenes and 1 relic trypsinogen gene at the 5-prime end o the sequence, all in inverted transcriptional orientation. They denoted 8 trypsinogen genes TI through T8 from 5-prime to 3-prime.
Rowen et al. (1996) found that only 2 of 3 pancreatically expressed trypsinogen cDNAs correspond to trypsinogen genes in the TCRB locus; T4 was denoted trypsinogen 1 and T8 was denoted trypsinogen 2 (601564). The third pancreatic cDNA, identified independently as trypsinogen 3 (Tani et al., 1990) and 4 (Wiegand et al., 1993), is distinct from the third apparently functional trypsinogen gene (T6) in the TCRB locus but related to the other pancreatic trypsinogens. Rowen et al. (1996) stated that the T6 gene is deleted in a common insertion-deletion polymoφhism; if it is functional, its function is apparently not essential. Some of the trypsinogen genes are expressed in nonpancreatic tissues where their function is unknown. Rowen et al. (1996) noted that the intercalation ofthe trypsinogen genes in the TCRB locus is conserved in mouse and chicken, suggesting shared functional or regulatory constraints, as has been postulated for genes in the major histocompatibility complex (such as class I, II, and III genes) that share similar long-term organizational relationships.
Rowen et al. (1996) mapped the gene corresponding to the third pancreatic trypsinogen cDNA by fluorescence in situ hybridization. They used a cosmid clone containing 3 trypsinogen genes. Strong hybridization to chromosome 7 and weaker hybridization to chromosome 9 were observed. They isolated and partially sequenced 4 cosmid clones from the chromosome 9 region. They found that the region represents a duplication and translocation of a DNA segment from the 3-prime end of the TCRB locus that includes at least 7 V(beta) elements and a functional trypsinogen gene denoted T9. The assignment ofthe PRSS1 gene to 7q35 is established by the demonstration of its sequence within the sequence ofthe 'locus' for the T-cell receptor beta-chain (Rowen et al., 1996). Since hereditary pancreatitis (167800) has been mapped rather precisely to 7q35 and since a defect in the trypsinogen gene has been identified in hereditary pancreatitis, the assignment of the trypsinogen gene can be refined from 7q32-qter to 7q35.
Whitcomb et al. (1996) stated that the high degree of DNA sequence homology (more than 91%) present among this cluster of 5 trypsinogen genes identified by Rowen et al. (1996) demanded that highly specific sequence analysis strategies be developed for mutational screening in families with hereditary pancreatitis. This was necessary to ensure that each sequencing run contained only the 2 alleles corresponding to a single gene, thereby permitting detection of heterozygotes in this autosomal dominant disorder, and not a dozen or more alleles from multiple related trypsinogen-like genes, which would make detection of heterozygotes nearly impossible. In a family with hereditary pancreatitis, Whitcomb et al. (1996) found that affected individuals had a single G-to-A transition mutation in the third exon of cationic trypsinogen (276000.0001 ). This mutation was predicted to result in an argl05-to- his substitution in the trypsin gene (residue number 1 17 in the more common chymotrypsin number system). Subsequently, the same mutation was found in a total of 5 different hereditary pancreatitis kindreds (4 from the U.S. and 1 from Italy) containing a total of 20 affected individuals and 6 obligate carriers. The mutation was found in none ofthe obligate unaffected members (individuals who married into the family). Subsequent haplotyping revealed that all 4 of the American families displayed the same high risk haplotype over a 4- cM region encompassing 7 STR markers, confirming the likelihood that these kindreds shared a common ancestor, although no link could be found through 8 generations. A fifth family from Italy displayed a unique haplotype indicating that the same mutation had occurred on at least 2 occasions. The G-to-A mutation at codon 117 created a novel enzyme recognition site for Afllll which provided a facile means to screen for the mutation. As with the obligate unaffected members ofthe pancreatitis kindreds, none of 140 controls possessed the G-to-A mutation as assayed by the lack of Afllll digestion of the amplified exonic DNA.
Ferec et al. (1999) studied 14 families with hereditary pancreatitis and found mutations in the PRSS1 gene in 8 families. In 4 of these families, the mutation (RI17H; 276000.0001) had been described by Whitcomb et al. (1996). Three mutations were described in 4 other families (276000.0002, 276000.0003, 276000.0005).
Sahin-Toth et al. (1999) studied the roles of the 2 most frequent PRSS1 mutations in hereditary pancreatitis, Rl 17H and N211 (276000.0002). They stated that the Rl 17H mutation is believed to cause pancreatitis by eliminating an essential autolytic cleavage site in trypsin, thereby rendering the protease resistant to inactivation through autolysis. Sahin-Toth et al. (1999) demonstrated that the Rl 17H mutation also significantly inhibited autocatalytic trypsinogen breakdown under Ca(2+)-free conditions and stabilized the zymogen form of rat trypsin. Taken together with findings demonstrating that the N2 II mutation stabilized rat trypsinogen against autoactivation and consequent autocatalytic degradation, the observations suggested a unifying molecular pathomechanism for hereditary pancreatitis in which zymogen stabilization plays a central role.
Sahin-Toth and Toth (2000) demonstrated that the Rl 17H and N211 mutations significantly enhance autoactivation of human cationic trypsinogen in vitro, in a manner that correlates with the severity of clinical symptoms in hereditary pancreatitis. In addition, the Rl 17H mutation inhibited autocatalytic inactivation of trypsin, while the N211 mutation had no such effect. Thus, increased trypsinogen activation in the pancreas is presumably the common initiating step in both forms of hereditary pancreatitis, whereas trypsin stabilization may also contribute to hereditary pancreatitis associated with the Rl 17H mutation. The disclosed NOVIO nucleic acid o the invention encoding a spinesin like protein includes the nucleic acid whose sequence is provided in Table 10A and 10D or a fragment theieof The invention also includes a mutant or variant nucleic acid any of whose bases may be changed from the corresponding base shown in Table 10A and 10D while still encoding a protein that maintains its spinesm like activities and physiological functions, or a fragment of such a nudeic acid The invention further includes nucleiG acids whose sequences are complementary to those just described, including nucleic acid fragments that are complementary to any of the nucleic acids just described The invention additionally includes nucleiG adds or nucleic acid fragments, or complements thereto, whose structures include chemical modifications Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject In the mutant or variant nucleic acids, and their complements, up to about 40% percent ofthe bases may be so changed
The disclosed NOVIO protein of the invention includes the spinesm-hke protein whose sequence is provided in Table 10B or 10E The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residue shown in Table 10B or 10E while still encoding a protein that maintains its spinesin like activities and physiological functions, or a functional fragment thereof In the mutant or vanant protein, up to about 22% percent of the residues may be so changed
The invention further encompasses antibodies and antibody fragments, such as Fab or (Fab)2 that bind immunospecifically to any of the proteins ofthe invention
The above defined information for this invention suggests that this spmesin- ke protein (NOV10) may function as a member of a "spmesin family" Therefore, the NOV10 nucleic acids and proteins identified here may be useful in potential therapeutic applications implicated in (but not limited to) various pathologies and disorders as indicated below The potential therapeutic applications for this invention include, but are not limited to protein therapeutic, small molecule drug target, antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), diagnostic and/or prognostic marker, gene therapy (gene delivery/gene ablation), research tools, tissue regeneration in vivo and in vit/o of all tissues and cell types composing (but not limited to) those defined here
The NOV10 nucleic acids and proteins of the invention are useful in potential therapeutic applications implicated in cancer including but not limited to various pathologies and disorders as indicated below. For example, a cDNA encoding the spinesin-like protein (NOVIO) may be useful in gene therapy, and the spinesin -like protein (NOVIO) may be useful when administered to a subject in need thereof. By way of nonlimiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from cancer, trauma, tissue regeneration (in vitro and in vivo), viral/bacterial/parasitic infections, immunological disease, respiratory disease, gastro-intestinal diseases, reproductive health, neurological and neurodegenerative diseases, bone marrow transplantation, metabolic and endocrine diseases, allergy and inflammation, nephrological disorders, cardiovascular diseases, muscle, bone, joint and skeletal disorders, hematopoietic disorders, urinary system disorders, Tissue and organ transplantation, Cardiomyopathy, Atherosclerosis, Hypertension, Congenital heart defects, Aortic stenosis, Atrial septal defect (ASD), Atrioventricular (A-V) canal defect, Ductus arteriosus, Pulmonary stenosis , Subaortic stenosis, Ventricular septal defect (VSD), valve diseases, Scleroderma, Obesity, Hypertension, Fibromuscular dysplasia, Stroke, Aneurysm, Myocardial infarction, Embolism, Bypass surgery, Anemia , Bleeding disorders, Adrenoleukodystrophy, Congenital Adrenal Hyperplasia, Diabetes, Von Hippel- Lindau (VHL) syndrome, Pancreatitis, Hypeφarathyroidism, Hypoparathyroidism, Hyperthyroidism and Hypothyroidism, SIDS, Endometriosis, infertility, Xerostomia, Hypercalceimia, Ulcers, Cirrhosis, Inflammatory bowel disease, Diverticular disease, Hirschsprung's disease, Crohn's Disease, Appendicitis, Hemophilia, hypercoagulation, autoimmume disease,allergies, immunodeficiencies, transplantation, Graft vesus host disease (GVHD), Ataxia-telangiectasia, Autoimmume disease, Hemophilia, Hypercoagulation, Idiopathic thrombocytopenic puφura, Immunodeficiencies, Lymphedema, Allergies, Hemophilia, hypercoagulation, idiopathic thrombocytopenic puφura, Lymphaedema, Tonsilitis, Osteoporosis, Hypercalceimia, Arthritis, Ankylosing spondylitis, Scohosis, Tendinitis, Muscular dystrophy, Lesch-Nyhan syndrome, Myasthenia gravis, Dental disease and infection, Alzheimer's disease, Stroke, Tuberous sclerosis, hypercalceimia, Parkinson's disease, Huntington's disease, Cerebral palsy, Epilepsy,Lesch-Nyhan syndrome, Multiple sclerosis, Ataxia-telangiectasia, Behavioral disorders, Addiction, Anxiety, Pain, Neuroprotection, Endocrine dysfunctions, Growth and reproductive disorders, Myasthenia gravis, Leukodystrophies, Pain, Neuroprotection, Systemic lupus erythematosus, Autoimmune disease, Emphysema, Scleroderma, ARDS, Pharyngitis, Laryngitis, Asthma, Hearing loss, Tinnitus, Psoriasis, Actinic keratosis, Tuberous sclerosis, Acne, Hair growth, allopecia, pigmentation disorders, endocrine disorders, cystitis, incontinence, Autoimmune disease, Renal artery stenosis, Interstitial nephritis, Glomerulonephritis, Polycystic kidney disease, Systemic lupus erythematosus, Renal tubular acidosis, IgA nephropathy, Hypercalceimia, Lesch-Nyhan syndrome, Vesicoureteral reflux, and other pathologies and conditions.. The NOVIO nucleic acid encoding the spinesin-like protein ofthe invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount ofthe nucleic acid or the protein are to be assessed.
NOVIO nucleic acids and polypeptides are further useful in the generation of antibodies that bind immuno-specifically to the novel NOVIO substances for use in therapeutic or diagnostic methods. These antibodies may be generated according to methods known in the art, using prediction from hydrophobicity charts, as described in the "Anti- NOVX Antibodies" section below. The disclosed NOVIO protein has multiple hydrophilic regions, each of which can be used as an immunogen. In one embodiment, a contemplated NOVIO epitope is from about amino acids 5 to 50. In another embodiment, a NOVIO epitope is from about amino acids 60 to 70. In additional embodiments, NOV10 epitope is from about amino acids 100 to 130, from about amino acids 140 to 210, from about amino acid 270 to 320, from about amino acid 340 to 360, from about amino acid 390 to 410, and from about amino acids 430 to 450. These novel proteins can be used in assay systems for functional analysis of various human disorders, which will help in understanding of pathology ofthe disease and development of new drug targets for various disorders.
NOVX Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that encode NOVX polypeptides or biologically active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify NOVX-encoding nucleic acids (e.g., NOVX mRNAs) and fragments for use as PCR primers for the amplification and/or mutation of NOVX nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double- stranded DNA.
An NOVX nucleic acid can encode a mature NOVX polypeptide. As used herein, a "mature" form of a polypeptide or protein disclosed in the present invention is the product of naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an ORF described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an ORF, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine, would have residues 2 through N remaining after removal of the N-terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or as many as approximately, e.g., 6,000 nt, depending upon the specific use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are generally obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one, which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NOVX nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized. A nucleic acid molecule ofthe invention, e.g., a nucleic acid molecule having the nucleotide sequence SEQ ID NOS: 1 , 3, 5, 1, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, and 28, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28 as a hybridization probe, NOVX molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, et al., (eds.), MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, lohn Wiley & Sons, New York, NY, 1993.) A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NOVX nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment ofthe invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides SEQ ID NOS: 1, 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, and 28, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, and 28, or a portion ofthis nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an NOVX polypeptide). A nucleic acid molecule that is complementary to the nucleotide sequence shown SEQ ID NOS:l , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, or 28 is one that is sufficiently complementary to the nucleotide sequence shown SEQ ID NOS : 1 , 3, 5, 1, 9, 11, 13, 15, 17, 19, 21 , 23, 25, or 28 that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown SEQ ID NOS:l , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. -Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species. Derivatives and analogs may be full length or other than full length, ifthe derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below. A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of NOVX polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms can be encoded by different genes. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an NOVX polypeptide of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations ofthe nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human NOVX protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS.l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, and 28, as well as a polypeptide possessing NOVX biological activity. Various biological activities ofthe NOVX proteins are described below.
An NOVX polypeptide is encoded by the open reading frame ("ORF") of an NOVX nucleic acid. An ORF corresponds to a nucleotide sequence that could potentially be translated into a polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by a stop codon. An ORF that represents the coding sequence for a full protein begins with an ATG "start" codon and terminates with one o the three "stop" codons, namely, TAA, TAG, or TGA. For the puφoses of this invention, an ORF may be any part of a coding sequence, with or without a start codon, a stop codon, or both. For an ORF to be considered as a good candidate for coding for a bonafide cellular protein, a minimum size requirement is often set, e.g., a stretch of DNA that would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human NOVX genes allows for the generation of probes and primers designed for use in identifying and/or doning NOVX homologues in other cell types, e.g. from other tissues, as well as NOVX homologues from other vertebrates. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, or 28; or an anti-sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, or 28; or of a naturally occurring mutant of SEQ ID NOS: l , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, and 28.
Probes based on the human NOVX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis- express an NOVX protein, such as by measuring a level of an NOVX-encoding nucleic acid in a sample of cells from a subject e.g., detecting NOVX mRNA levels or determining whether a genomic NOVX gene has been mutated or deleted. "A polypeptide having a biologically-active portion of an NOVX polypeptide" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity ofa polypeptide of the invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically- active portion of NOVX" can be prepared by isolating a portion SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, or 28, that encodes a polypeptide having an NOVX biological activity (the biological activities of the NOVX proteins are described below), expressing the encoded portion of NOVX protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion of NOVX.
NOVX Nucleic Acid and Polypeptide Variants The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21, 23, 25, and 28 due to degeneracy of the genetic code and thus encode the same NOVX proteins as that encoded by the nucleotide sequences shown in SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21 , 23, 25, and 28. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29.
In addition to the human NOVX nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 1, 9, 1 1 , 13, 15, 17, 19, 21, 23, 25, and 28, it will be appreciated by those skilled in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequences of the NOVX polypeptides may exist within a population (e.g., the human population). Such genetic polymoφhism in the NOVX genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an NOVX protein, preferably a vertebrate NOVX protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the NOVX genes. Any and all such nucleotide variations and resulting amino acid polymoφhisms in the NOVX polypeptides, which are the result of natural allelic variation and that do not alter the functional activity ofthe NOVX polypeptides, are intended to be within the scope o the invention.
Moreover, nucleic acid molecules encoding NOVX proteins from other species, and thus that have a nucleotide sequence that differs from the human SEQ ID NOS.T, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28 are intended to be within the scope ofthe invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the NOVX cDNAs of the invention can be isolated based on their homology to the human NOVX nucleic adds disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or 2000 or more nucleotides in length. In yet another embodiment, an isolated nucleic acid molecule ofthe invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
Homologs (i.e., nucleic acids encoding NOVX proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 °C lower than the thenrial melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm,
50% ofthe probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in Ausubel, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 M Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequences SEQ ID NOS.T, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS : 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28, or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1 % SDS at 37°C. Other conditions of moderate stringency that may be used are well-known within the art. See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990; GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequences SEQ ID NOS: l , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, and 28, or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl Acad Sci USA 78: 6789-6792.
Conservative Mutations
In addition to naturally-occurring allelic variants of NOVX sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences SEQ ID NOS:l, 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, and 28, thereby leading to changes in the amino acid sequences of the encoded NOVX proteins, without altering the functional ability of said NOVX proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29. A "non-essential" amino add residue is a residue that can be altered from the wild-type sequences of the NOVX proteins without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the NOVX proteins ofthe invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known within the art. Another aspect of the invention pertains to nucleic acid molecules encoding NOVX proteins that contain changes in amino acid residues that are not essential for activity. Such NOVX proteins differ in amino acid sequence from SEQ ID NOS: 1, 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, and 28 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous to the amino acid sequences SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29. Preferably, the protein encoded by the nudeic acid molecule is at least about 60% homologous to SEQ ID NOS. -2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29; more preferably at least about 70% homologous SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 21, or 29; still more preferably at least about 80% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 27, or 29; even more preferably at least about 90% homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 21, or 29; and most preferably at least about 95% homologous to SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 21, or 29. An isolated nucleic acid molecule encoding an NOVX protein homologous to the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOS:l, 3, 5, 1, 9, 11 , 13, 15, 17, 19, 21, 23, 25, and 28, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NOS:!, 3, 5, 1, 9, 11, 13, 15, I E 19, 21, 23,
25, and 28 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted, non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side Ghain. Families of amino acid residues having similar side chains have been defined within the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in the NOVX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an NOVX coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for NOVX biological activity to identify mutants that retain activity. Following mutagenesis SΕQ ID NOS:l, 3, 5, 7, 9, 1 1 , 13, 15, IE 19, 21 ,
23, 25, and 28, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined. The relatedness of amino acid families may also be determined based on side chain interactions. Substituted amino acids may be fully conserved "strong" residues or fully conserved "weak" residues. The "strong" group of conserved amino acid residues may be any one ofthe following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the single letter amino acid codes are grouped by those amino acids that may be substituted for each other. Likewise, the "weak" group of conserved residues may be any one of the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY, wherein the letters within each group represent the single letter amino acid code.
In one embodiment, a mutant NOVX protein can be assayed for (/) the ability to form protein.-protein interactions with other NOVX proteins, other cell-surface proteins, or biologically-active portions thereof, (//') complex formation between a mutant NOVX protein and an NOVX ligand; or (iii) the ability o a mutant NOVX protein to bind to an intracellular target protein or biologically-active portion thereof; (e.g. avidin proteins).
In yet another embodiment, a mutant NOVX protein can be assayed for the ability to regulate a specific biological function (e.g., regulation of insulin release).
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOS.T, 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, and 28, or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein (e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NOVX coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of an NOVX protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29, or antisense nucleic acids complementary to an NOVX nucleic acid sequence of SEQ ID NOS.T, 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21, 23, 25, and 28, are additionally provided. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding an NOVX protein. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid O 02/33087
molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding the NOVX protein. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions). Given the coding strand sequences encoding the NOVX protein disclosed herein, antisense nucleic acids ofthe invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NOVX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of NOVX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NOVX mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability ofthe duplex formed between the antisense and sense nucleic acids (e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isoρentenyladenine, l-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an NOVX protein to thereby inhibit expression of the protein (e.g., by inhibiting transcription and/or translation). The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface (e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens). The antisense nucleic acid molecules can also be delivered to cells using the vedors described herein. To achieve sufficient nucleic acid molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control ofa strong pol π or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl. Acids Res. 15: 6625-6641. The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (See, e.g., Inoue, et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric R.NA-DNA analogue (See, e.g., Inoue, et al., 1987. FEBS Lett. 215: 327-330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability ofthe modified nucleic acid, such that they may be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach 1988. Nature 334: 585-591) can be used to catalytically cleave NOVX mRNA transcripts to thereby inhibit translation of NOVX mRNA. A ribozyme having specificity for an NOVX-encoding nucleic acid can be designed based upon the nucleotide sequence of an NOVX cDNA disclosed herein (i.e., SEQ ID NOS:l , 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21 , 23, 25, and 28). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an NOVX-encoding mRNA. See, e.g., U.S. Patent 4,987,071 to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. NOVX mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Barrel et al, (1993) Science 261 :1411-1418.
Alternatively, NOVX gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NOVX nucleic acid (e.g., the NOVX promoter and/or enhancers) to form triple helical structures that prevent transcription of the NOVX gene in target cells. See, e.g., Helene, 1991. Anticancer Drug Des. 6: 569-84; Helene, et al. 1992. Ann. N.Y. Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the NOVX nucleic acids can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nudeic acids. See, e.g., Hyrup, et al., 1996. Bioorg Med Chem 4: 5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nudeic acid mimics (e.g., DNA mimics) in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe, et al, 1996. Proc. Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of NOVX can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NOVX can also be used, for example, in the analysis of single base pair mutations in a gene (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S\ nucleases (See, Hyrup, el al., \996.supra); or as probes or primers for DNA sequence and hybridization (See, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al., 1996. supra). In another embodiment, PNAs of NOVX can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NOVX can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (see, Hyrup, et al., 1996. supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup, et al, 1996. supra and Finn, et al., 1996. Nucl Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA. See, e.g., Mag, et al, 1989. Nucl Acid Res 17: 5973-5988. PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment. See, e.g., Finn, et al., 1996. supra. Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g., Petersen, et al, 1975. Bioorg. Med. Chem. Lett. 5: 1119-1 1124. In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger, et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556; Lemaitre, et al., 1987. Proc. Natl Acad. Sci. 84: 648-652; PCT Publication No. WO88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol, et al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988. Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
NOVX Polypeptides
A polypeptide according to the invention includes a polypeptide including the amino acid sequence of NOVX polypeptides whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29 while still encoding a protein that maintains its NOVX activities and physiological functions, or a functional fragment thereof. In general, an NOVX variant that preserves NOVX-like function includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further include the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
One aspect of the invention pertains to isolated NOVX proteins, and biologically- active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NOVX antibodies. In one embodiment, native NOVX proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NOVX proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, an NOVX protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NOVX protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of NOVX proteins in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly-produced. In one embodiment, the language "substantially free of cellular material" includes preparations of NOVX proteins having less than about 30% (by dry weight) of non-NOVX proteins (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-NOVX proteins, still more preferably less than about 10% of non-NOVX proteins, and most preferably less than about 5% of non-NOVX proteins. When the NOVX protein or biologically-active portion thereof is recombinantly-produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the NOVX protein preparation. The language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis ofthe protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of NOVX proteins having less than about 30% (by dry weight) of chemical precursors or non-NOVX chemicals, more preferably less than about 20% chemical precursors or non-NOVX chemicals, still more preferably less than about 10% chemical precursors or non-NOVX chemicals, and most preferably less than about 5% chemical precursors or non-NOVX chemicals. Biologically-active portions of NOVX proteins include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the NOVX proteins (e.g., the amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29) that include fewer amino acids than the full-length NOVX proteins, and exhibit at least one activity of an NOVX protein. Typically, biologically-active portions comprise a domain or motif with at least one activity ofthe NOVX protein. A biologically-active portion of an NOVX protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native NOVX protein.
In an embodiment, the NOVX protein has an amino acid sequence shown SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29. In other embodiments, the NOVX protein is substantially homologous to SEQ ID NOS :2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29, and retains the functional activity o the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail, below. Accordingly, in another embodiment, the NOVX protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29, and retains the functional activity ofthe NOVX proteins of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 21, or 29.
Determining Homology Between Two or More Sequences
To determine the percent homology of two amino add sequences or of two nucleic acids, the sequences are aligned for optimal comparison puφoses (e g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970. J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the Goding region ofthe analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part ofthe DNA sequence shown in SEQ ID NOS:l , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
Chimeric and Fusion Proteins
The invention also provides NOVX chimeric or fusion proteins. As used herein, an NOVX "chimeric protein" or "fusion protein" comprises an NOVX polypeptide operatively- linked to a non-NOVX polypeptide. An "NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to an NOVX protein SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, or 29, whereas a "non-NOVX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the NOVX protein, e.g., a protein that is different from the NOVX protein and that is derived from the same or a different organism. Within an NOVX fusion protein the NOVX polypeptide can correspond to all or a portion of an NOVX protein. In one embodiment, an NOVX fusion protein comprises at least one biologically-active portion of an NOVX protein. In another embodiment, an NOVX fusion protein comprises at least two biologically-active portions of an NOVX protein. In yet another embodiment, an NOVX fusion protein comprises at least three biologically-active portions of an NOVX protein. Within the fusion protein, the teπn "operatively-linked" is intended to indicate that the NOVX polypeptide and the non-NOVX polypeptide are fused in-frame with one another. The non-NOVX polypeptide can be fused to the N-terminus or C-terminus ofthe NOVX polypeptide.
In one embodiment, the fusion protein is a GST-NOVX fusion protein in which the NOVX sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant NOVX polypeptides.
In another embodiment, the fusion protein is an NOVX protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of NOVX can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is an NOVX-immunoglobulin fusion protein in which the NOVX sequences are fused to sequences derived from a member ofthe immunoglobulin protein family. The NOVX-immunoglobulin fusion proteins ofthe invention can be incoφorated into pharmaceutical compositions and administered to a subject to inhibit an interaction between an NOVX ligand and an NOVX protein on the surface ofa cell, to thereby suppress NOVX-mediated signal transduction in vivo. The NOVX-immunoglobulin fusion proteins can be used to affect the bioavailability of an NOVX cognate ligand. Inhibition of the NOVX ligand/NOVX interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the NOVX-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NOVX antibodies in a subject, to purify NOVX ligands, and in screening assays to identify molecules that inhibit the interaction of NOVX with an NOVX ligand. An NOVX chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, e.g., Ausubel, et al (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An NOVX-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NOVX protein.
NOVX Agonists and Antagonists
The invention also pertains to variants ofthe NOVX proteins that function as either NOVX agonists (le., mimetics) or as NOVX antagonists. Variants o the NOVX protein can be generated by mutagenesis (e.g., discrete point mutation or truncation of the NOVX protein). An agonist of the NOVX protein can retain substantially the same, or a subset of, the biological activities ofthe naturally occurring form of the NOVX protein. An antagonist of the NOVX protein can inhibit one or more ofthe activities of the naturally occurring form of the NOVX protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the NOVX protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form o the NOVX proteins. Variants of the NOVX proteins that function as either NOVX agonists (i.e., mimetics) or as NOVX antagonists can be identified by screening combinatorial libraries of mutants (e.g., truncation mutants) ofthe NOVX proteins for NOVX protein agonist or antagonist activity. In one embodiment, a variegated library of NOVX variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of NOVX variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential NOVX sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of NOVX sequences therein. There are a variety of methods which can be used to produce libraries of potential NOVX variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all ofthe sequences encoding the desired set of potential NOVX sequences. Methods for synthesizing degenerate oligonucleotides are well-known within the art. See, e.g., Narang, 1983. Tetrahedron 39: 3; Itakura, et al., 1984. Annu. Rev. Biochem. 53: 323; Itakura, et al, 1984. Science 198: 1056; Ike, et al., 1983. Nucl. Acids Res. 11 : 477.
Polypeptide Libraries
In addition, libraries of fragments ofthe NOVX protein coding sequences can be used to generate a variegated population of NOVX fragments for screening and subsequent selection of variants of an NOVX protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an NOVX coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double-stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Sj nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression libraries can be derived which encodes N-terminal and internal fragments of various sizes of the NOVX proteins.
Various techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of NOVX proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation ofthe vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify NOVX variants. See, e.g., Arkin and Yourvan, 1992. Proc. Natl. Acad. Sci. USA 89: 781 1 -7815; Delgrave, et al., 1993. Protein Engineering 6:327-331.
Anti-NOVX Antibodies
Also included in the invention are antibodies to NOVX proteins, or fragments of NOVX proteins. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fa , Fab- and F(ab')2 fragments, and an Fab expression library. In general, an antibody molecule obtained from humans relates to any ofthe classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subdasses and types of human antibody species.
An isolated NOVX-related protein of the invention may be intended to serve as an antigen, or a portion or fragment thereof, and additionally can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation. The full-length protein can be used or, alternatively, the invention provides antigenic peptide fragments of the antigen for use as immunogens. An antigenic peptide fragment comprises at least 6 amino acid residues of the amino acid sequence of the full length protein and encompasses an epitope thereof such that an antibody raised against the peptide forms a specific immune complex with the full length protein or with any fragment that contains the epitope. Preferably, the antigenic peptide comprises at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions ofthe protein that are located on its surface; commonly these are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by the antigenic peptide is a region of NOVX-related protein that is located on the surface of the protein, e.g., a hydrophilic region. A hydrophobicity analysis ofthe human NOVX-related protein sequence will indicate which regions ofa NOVX-related protein are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production. As a means for targeting antibody production, hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Erσc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle 1982, J. Mol Biol. 157: 105-142, each of which is incoφorated herein by reference in its entirety. Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein ofthe invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow and Lane, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incoφorated herein by reference). Some of these antibodies are discussed below.
Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the native protein, a synthetic variant thereof, or a derivative ofthe foregoing. An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein. Furthermore, the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target ofthe immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs thus contain an antigen binding site capable of immunoreacting with a particular epitope ofthe antigen characterized by a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell
(Goding, MONOCLONAL ANTIBODIES : PRINCIPLES AND PRACTICE, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., MONOCLONAL ANTIBODY PRODUCTION TECHNIQUES AND APPLICATIONS, Marcel Dekker, Inc., New York, (1987) pp. 51-63). The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Preferably, antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this puφose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,81 ,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
Humanized Antibodies
The antibodies directed against the protein antigens ofthe invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen- binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No. 5,225,539.) In some instances, Fv framework residues ofthe human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all ofthe CDR regions co espond to those of non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)).
Human Antibodies Fully human antibodies relate to antibody molecules in which essentially the entire sequences of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
In addition, human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in Marks et al. (Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild et al,( Nature Biotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication
WO94/02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incoφorated, for example, using yeast artificial chromosomes containing the requisite human DNA segments An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications The preferred embodiment of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096 This animal produces B cells which secrete fully human immunoglobulins The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells deπved from the animal, such as hybridomas producing monoclonal antibodies Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules
An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U S Patent No 5,939,598 It can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcπpt of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker, and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker
A method for producing an antibody of interest, such as a human antibody, is disclosed in U S Patent No 5,916,771 It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell The hybrid cell expresses an antibody containing the heavy chain and the light chain
In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen, and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication WO 99/53049
Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e g , U S Patent O 02/33087
No. 4,946,778). In addition, methods can be adapted for the construction of Fa expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(a ')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F Sb')2 fragment; (iii) an Fa fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for an antigenic protein of the invention. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al, 1991 EMBOJ., 10:3655-3659. Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding present in at least one ofthe fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Eniymology, 121 :210 (1986). According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') fragments. These fragments are reduced in the presence of the dithiol comProhibiting agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One ofthe Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production o a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al, J. Immunol. 148(5): 1547- 1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary V and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen ofthe invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this puφose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
Effector Function Engineering It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness o the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989).
Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAP II, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuchdes are available for the production of radioconjugated antibodies. Examples include 2,2Bi, ,3 ,I, '3 lIn, 90Y, and l 86Re.
Conjugates ofthe antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazomumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration o a "ligand" (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of an NOVX protein is facilitated by generation of hybridomas that bind to the fragment of an NOVX protein possessing such a domain. Thus, antibodies that are specific for a desired domain within an NOVX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-NOVX antibodies may be used in methods known within the art relating to the localization and/or quantitation of an NOVX protein (e.g., for use in measuring levels of the NOVX protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for NOVX proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds (hereinafter "Therapeutics").
An anti-NOVX antibody (e.g , monoclonal antibody) can be used to isolate an NOVX polypeptide by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-NOVX antibody can facilitate the purification of natural NOVX polypeptide from cells and of recombinantly-produced NOVX polypeptide expressed in host cells. Moreover, an anti-NOVX antibody can be used to detect NOVX protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NOVX protein. Anti-NOVX antibodies can be used diagnostically to monitor protein levels in tissue O 02/33087
as part of a clinical testing procedure, e.g., to, for example, determine the efficacy o a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include 11~ luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, 131I, 35S or 3H.
NOVX Recombinant Expression Vectors and Host Cells Another aspect ofthe invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an NOVX protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably-Iinked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NOVX proteins, mutant forms of NOVX proteins, fusion proteins, etc.). The recombinant expression vectors ofthe invention can be designed for expression of
NOVX proteins in prokaryotic or eukaryotic cells. For example, NOVX proteins can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vedors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three puφoses: (/) to increase expression of recombinant protein; (ii) to increase the solubility of the reGombinant protein; and (Hi) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 61: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET lid (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence ofthe nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (see, e.g., Wada, et al, 1992. Nucl Acids Res. 20: 2111-21 18). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques. In another embodiment, the NOVX expression vector is a yeast expression vector.
Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSecl (Baldari, et al, 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al, 1987. Gene 54: 113-123), pYES2 (Invitrogen Coφoration, San Diego, Calif), and picZ (InVitrogen Coφ, San Diego, Calif). Alternatively, NOVX can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al, 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al, 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, 1989. In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pmkert, et al, 1987. Genes Dev. 1 : 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al, 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741 -748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al, 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively-linked to a regulatory sequence in a manner that allows for expression (by transcription ofthe DNA molecule) of an RNA molecule that is antisense to NOVX mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form ofa recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see, e.g., Weintraub, et al, "Antisense RNA as a molecular tool for genetic analysis," Reviews-Trends in Genetics, Vol. 1(1) 1986.
Another aspect o the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, m fact, be identical to the parent cell, but are still included within the scope ofthe term as used herein
A host cell can be any prokaryotic or eukaryotic cell For example, NOVX protein can be expressed in bacterial cells such as E coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells) Other suitable host cells are known to those skilled m the art Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e g , DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al (MOLECULAR CLONING A LABORATORY MANUAL 2nd ed , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y , 1989), and other laboratory manuals
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome In order to identify and select these integrants, a gene that encodes a selectable marker (e g , resistance to antibiotics) is generally introduced into the host cells along with the gene of interest Various selectable maikers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding
NOVX or can be introduced on a separate vector Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e g , cells that have incoφorated the selectable marker gene will survive, while the other cells die)
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (; e , express) NOVX protein Accordingly, the invention further provides methods for producing NOVX protein using the host cells of the invention In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NOVX protein has been introduced) in a suitable medium such that NOVX protein is produced. In another embodiment, the method further comprises isolating NOVX protein from the medium or the host cell.
Transgenic NOVX Animals
The host cells o the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which NOVX protein-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NOVX sequences have been introduced into their genome or homologous recombinant animals in which endogenous NOVX sequences have been altered. Such animals are useful for studying the function and/or activity of NOVX protein and for identifying and/or evaluating modulators of NOVX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NOVX gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell ofthe animal, prior to development of the animal.
A transgenic animal ofthe invention can be created by introducing NOVX-encoding nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NOVX cDNA sequences SEQ ID NOS:!, 3, 5, 1, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28 can be introduced as a transgene into the genome ofa non-human animal. Alternatively, a non-human homologue o the human NOVX gene, such as a mouse NOVX gene, can be isolated based on hybridization to the human NOVX cDNA (described further supra) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably-linked to the NOVX transgene to direct expression of NOVX protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191 ; and Hogan, 1986. In: MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NT. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NOVX transgene in its genome and/or expression of NOVX mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene- encoding NOVX protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an NOVX gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NOVX gene. The NOVX gene can be a human gene (e.g., the cDNA of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, and 28), but more preferably, is a non-human homologue of a human NOVX gene. For example, a mouse homologue of human NOVX gene of SEQ ID NOS.T , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28 can be used to construct a homologous recombination vector suitable for altering an endogenous NOVX gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NOVX gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NOVX gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NOVX protein). In the homologous recombination vector, the altered portion ofthe NOVX gene is flanked at its 5 - and 3'-termini by additional nucleic acid of the NOVX gene to allow for homologous recombination to occur between the exogenous NOVX gene carried by the vector and an endogenous NOVX gene in an embryonic stem cell. The additional flanking NOVX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini) are included in the vector. See, e.g., Thomas, et al, 1987. Cell 51 : 503 for a description of homologous recombination vectors. The vector is ten introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NOVX gene has homologously-recombined with the endogenous NOVX gene are selected. See, e.g., Li, et al, 1992. Cell 69: 915.
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See, e.g., Bradley, 1987. In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 1 13-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells ofthe animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, 1991. Curr. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI. For a description ofthe cre/loxP recombinase system, See, e.g., Lakso, et al, 1992. Proc. Natl Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae. See, O'Gorman, et al, 1991. Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression ofthe transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wil ut, et al, 1997. Nature 385: 810-813. In brief, a cell (e.g., a somatic cell) from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell (e.g., the somatic cell) is isolated. Pharmaceutical Compositions
The NOVX nucleic acid molecules, NOVX proteins, and anti-NOVX antibodies (also referred to herein as "active compounds") of the invention, and derivatives, fragments, analogs and homologs thereof, can be incoφorated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and anti ungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incoφorated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incoφorated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be endosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL'U (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absoφtion of the injectable compositions can be brought about by including in the composition an agent which delays absoφtion, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incoφorating the active compound (e.g., an NOVX protein or anti-NOVX antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the puφose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Coφoration and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
The nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al, 1994. Proc. Natl Acad. Sci. USA 91 : 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Screening and Detection Methods The isolated nucleic acid molecules ofthe invention can be used to express NOVX protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect NOVX mRNA (e.g., in a biological sample) or a genetic lesion in an NOVX gene, and to modulate NOVX activity, as described further, below. In addition, the NOVX proteins can be used to screen drugs or compounds that modulate the NOVX protein activity or expression as well as to treat disorders characterized by insufficient or excessive production of NOVX protein or production of NOVX protein forms that have decreased or aberrant activity compared to NOVX wild-type protein (e.g.; diabetes (regulates insulin release); obesity (binds and transport lipids); metabolic disturbances associated with obesity, the metabolic syndrome X as well as anorexia and wasting disorders associated with chronic diseases and various cancers, and infectious disease(possesses anti-microbial activity) and the various dyslipidemias. In addition, the anti-NOVX antibodies ofthe invention can be used to detect and isolate NOVX proteins and modulate NOVX activity. In yet a further aspect, the invention can be used in methods to influence appetite, absoφtion of nutrients and the disposition of metabolic substrates in both a positive and negative fashion. The invention further pertains to novel agents identified by the screening assays described herein and uses thereof for treatments as described, supra. Screening Assays
The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that bind to NOVX proteins or have a stimulatory or inhibitory effect on, e.g., NOVX protein expression or NOVX protein activity. The invention also includes compounds identified in the screening assays described herein.
In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity ofthe membrane-bound form of an NOVX protein or polypeptide or biologically-active portion thereof. The test compounds ofthe invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. See, e.g., Lam, 1997 '. Anticancer Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt, et al, 1993. Proc. Natl Acad. Sci. U.S.A. 90: 6909; Erb, et al, 1994. Proc. Natl. Acad. Sci. U.S.A. 91 : 1 1422; Zuckermann, et al, 1994. J. Med. C em. 37: 2678; Cho, et al, 1993. Science 261 : 1303; Carrell, et al, 1994. Angew. Chem. Int. Ed. Engl. 33: 2059; Carell, et al, 1994. Angew. Chem. Int. Ed. Engl 33: 2061; and Gallop, et al, 1994. J. Med. Chem. 37: 1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992. Biotechniques 13: 412-421 ), or on beads (Lam, 1991. Nature 354: 82-84), on chips (Fodor, 1993. Nature 364: 555-556), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner, U.S. Patent 5,233,409), plasmids (Cull, et al, 1992. Proc. Natl. Acad. Sci. USA 89: 1865- 1869) or on phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249: 404-406; Cwirla, et al, 1990. Proc. Natl Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol 222: 301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to an NOVX protein determined. The cell, for example, can of mammalian origin or a yeast cell. Determining the ability o the test compound to bind to the NOVX protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding ofthe test compound to the NOVX protein or biologically-active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 1251, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability of the test compound to interact with an NOVX protein comprises determining the ability of the test compound to preferentially bind to NOVX protein or a biologically-active portion thereof as compared to the known compound.
In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NOVX protein, or a biologically-active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NOVX protein or biologically-active portion thereof. Determining the ability of the test compound to modulate the activity of NOVX or a biologically- active portion thereof can be accomplished, for example, by determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule. As used herein, a "target molecule" is a molecule with which an NOVX protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses an NOVX interacting protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. An NOVX target molecule can be a non-NOVX molecule or an NOVX protein or polypeptide of the invention. In one embodiment, an NOVX target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g. a signal generated by binding ofa compound to a membrane-bound NOVX molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NOVX.
Determining the ability of the NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by one ofthe methods described above for determining direct binding. In one embodiment, determining the ability ofthe NOVX protein to bind to or interact with an NOVX target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger ofthe target (i.e. intracellular Ca , diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of a reporter gene (comprising an NOVX-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
In yet another embodiment, an assay ofthe invention is a cell-free assay comprising contacting an NOVX protein or biologically-active portion thereof with a test compound and detennining the ability of the test compound to bind to the NOVX protein or biologically- active portion thereof. Binding ofthe test compound to the NOVX protein can be determined either directly or indirectly as described above. In one such embodiment, the assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein detennining the ability of the test compound to interact with an NOVX protein comprises determining the ability ofthe test compound to preferentially bind to NOVX or biologically-active portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising contacting NOVX protein or biologically-active portion thereof with a test compound and detennining the ability ofthe test compound to modulate (e.g-. stimulate or inhibit) the activity ofthe NOVX protein or biologically-active portion thereof. Determining the ability ofthe test compound to modulate the activity of NOVX can be accomplished, for example, by detennining the ability ofthe NOVX protein to bind to an NOVX target molecule by one of the methods described above for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of NOVX protein can be accomplished by determining the ability ofthe NOVX protein further modulate an NOVX target molecule. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the NOVX protein or biologically-active portion thereof with a known compound which binds NOVX protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with an NOVX protein, wherein determining the ability ofthe test compound to interact with an NOVX protein comprises determining the ability o the NOVX protein to preferentially bind to or modulate the activity of an NOVX target molecule.
The cell-free assays of the invention are amenable to use of both the soluble form or the membrane-bound form of NOVX protein. In the case of cell-free assays comprising the membrane-bound form of NOVX protein, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NOVX protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-l 14, Thesit®, Isotridecypoly(ethylene glycol ether)n, N-dodecyl~N,N-dimethyl-3-ammonio-l -propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-l-propane sulfonate (CHAPSO).
In more than one embodiment of the above assay methods ofthe invention, it may be desirable to immobilize either NOVX protein or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation ofthe assay. Binding of a test compound to NOVX protein, or interaction of NOVX protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST-NOVX fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NOVX protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described, supra. Alternatively, the complexes can be dissociated from the matrix, and the level of NOVX protein binding or activity detemiined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the NOVX protein or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NOVX protein or target molecules can be prepared from biotin-NHS
(N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NOVX protein or target molecules, but which do not interfere with binding ofthe NOVX protein to its target molecule, can be derivatized to the wells ofthe plate, and unbound target or NOVX protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NOVX protein or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NOVX protein or target molecule.
In another embodiment, modulators of NOVX protein expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of NOVX mRNA or protein in the cell is determined. The level of expression of NOVX mRNA or protein in the presence of the candidate compound is compared to the level of expression of NOVX mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NOVX mRNA or protein expression based upon this comparison. For example, when expression of NOVX mRNA or protein is greater (i.e., statistically significantly greater) in the presence o the candidate compound than in its absence, the candidate compound is identified as a stimulator of NOVX mRNA or protein expression. Alternatively, when expression of NOVX mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of NOVX mRNA or protein expression. The level of NOVX mRNA or protein expression in the cells can be determined by methods described herein for detecting NOVX mRNA or protein. In yet another aspect of the invention, the NOVX proteins can be used as "bait proteins" in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos, et al, 1993. Ce// 72: 223-232; Madura, et al, 1993. J. Biol. Chem. 268: 12046-12054; Barrel, et al, 1993. Biotechniques 14: 920-924; Iwabuchi, et al, 1993. Oncogene 8: 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NOVX ("NOVX-binding proteins" or "NOVX-bp") and modulate NOVX activity. Such NOVX-binding proteins are also likely to be involved in the propagation of signals by the NOVX proteins as, for example, upstream or downstream elements ofthe NOVX pathway. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one constmct, the gene that codes for NOVX is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an NOVX-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription ofa reporter gene (e.g., LacZ) that is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the protein which interacts with NOVX.
The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
Detection Assays Portions or fragments ofthe cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. By way of example, and not of limitation, these sequences can be used to: (/) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (///) aid in forensic identification of a biological sample. Some of these applications are described in the subsections, below. Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe NOVX sequences, SEQ ID NOS.T , 3, 5, 1, 9, 11 , 13, 15, 17, 19, 21, 23, 25, and 28, or fragments or derivatives thereof, can be used to map the location of the NOVX genes, respectively, on a chromosome. The mapping ofthe NOVX sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease. Briefly, NOVX genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp in length) from the NOVX sequences. Computer analysis of the NOVX, sequences can be used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the NOVX sequences will yield an amplified fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but in which human cells can, the one human chromosome that contains the gene encoding the needed enzyme will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. See, e.g., D'Eustachio, et al, 1983. Science 220: 919-924. Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the NOVX sequences to design oligonucleotide primers, sub- localization can be achieved with panels of fragments from specific chromosomes.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical like colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
However, clones larger than 1 ,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1 ,000 bases, and more preferably 2,000 bases, will suffice to get good results at a reasonable amount of time. For a review of this technique, see, Verma, et al, HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping puφoses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position ofthe sequence on the chromosome can be correlated with genetic map data. Such data are found, e.g., in McKusick, MENDELIAN INHERITANCE IN MAN, available on-line through Johns Hopkins University Welch Medical Library). The relationship between genes and disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, e.g., Egeland, et al, 1987. Nature, 325: 783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the NOVX gene, can be determined. If a mutation is observed in some or all ofthe affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymoφhisms. Tissue Typing
The NOVX sequences of the invention can also be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. The sequences ofthe invention are useful as additional DNA markers for RFLP ("restriction fragment length polymoφhisms," described in U.S. Patent No. 5,272,057).
Furthermore, the sequences of the invention can be used to provide an alternative technique that determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the NOVX sequences described herein can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
Panels of conesponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences o the invention can be used to obtain such identification sequences from individuals and from tissue. The NOVX sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Much of the allelic variation is due to single nucleotide polymoφhisms (SNPs), which include restriction fragment length polymoφhisms (RFLPs).
Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences can comfortably provide positive individual identification with a panel of perhaps 10 to 1 ,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS. l , 3, 5, 1, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) puφoses to thereby treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining NOVX protein and/or nucleic acid expression as well as NOVX activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant NOVX expression or activity. The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. For example, mutations in an NOVX gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive puφose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with NOVX protein, nucleic acid expression, or biological activity.
Another aspect of the invention provides methods for determining NOVX protein, nucleic acid expression or activity in an individual to thereby select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype ofthe individual examined to determine the ability ofthe individual to respond to a particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NOVX in clinical trials.
These and other agents are described in further detail in the following sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of NOVX in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes NOVX protein such that the presence of NOVX is detected in the biological sample. An agent for detecting NOVX mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to NOVX mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length NOVX nucleic acid, such as the nucleic acid ofSEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NOVX mRNA or genomic DNA Other suitable probes for use in the diagnostic assays of the invention are described herein
Ai agent for detecting NOVX protein is an antibody capable of binding to NOVX protein, preferably an antibody with a detectable label Antibodies can be polyclonal, or more preferably, monoclonal An intact antibody, or a fragment thereof (e g , Fab or F(ab')2) can be used The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i e , physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject That is, the detection method ofthe invention can be used to detect NOVX mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of NOVX mRNA include Northern hybndizahons and in situ hybridizations In vitro techniques for detection of NOVX protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence In vitro techniques for detection of NOVX genomic DNA include Southern hybridizations Furthermore, in vivo techniques for detection of NOVX protein include introducing into a subject a labeled anti-NOVX antibody For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques
In one embodiment, the biological sample contains protein molecules from the test subject Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting NOVX protein, mRNA, or genomic DNA, such that the presence of NOVX protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of NOVX protein, mRNA or genomic DNA in the control sample with the presence of NOVX protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of NOVX in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting NOVX protein or mRNA in a biological sample; means for determining the amount of NOVX in the sample; and means for comparing the amount of NOVX in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect NOVX protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with NOVX protein, nucleic acid expression or activity. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disease or disorder. Thus, the invention provides a method for identifying a disease or disorder associated with aberrant NOVX expression or activity in which a test sample is obtained from a subject and NOVX protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant NOVX expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g. , serum), cell sample, or tissue.
Furthenriore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abenant NOVX expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Thus, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abenant NOVX expression or activity in which a test sample is obtained and NOVX protein or nucleic acid is detected (e.g., wherein the presence of NOVX protein or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with abenant NOVX expression or activity).
The methods of the invention can also be used to detect genetic lesions in an NOVX gene, thereby determining if a subject with the lesioned gene is at risk for a disorder characterized by abenant cell proliferation and/or differentiation. In various embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of an alteration affecting the integrity of a gene encoding an NOVX-protein, or the misexpression ofthe NOVX gene. For example, such genetic lesions can be detected by ascertaining the existence of at least one of: (/) a deletion of one or more nucleotides from an NOVX gene; (ii) an addition of one or more nucleotides to an NOVX gene; (iii) a substitution of one or more nucleotides of an NOVX gene, (iv) a chromosomal rearrangement of an NOVX gene; (v) an alteration in the level of a messenger RNA transcript of an NOVX gene, (vi) abenant modification of an NOVX gene, such as of the methylation pattern o the genomic DNA, (vii) the presence of a non-wild-type splicing pattern of a messenger RNA transcript of an NOVX gene, (viii) a non-wild-type level of an NOVX protein, (ix) allelic loss of an NOVX gene, and (x) inappropriate post-translational modification of an NOVX protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting lesions in an NOVX gene. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
In certain embodiments, detection of the lesion involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran, et αl, 1988. Science 241 : 1077-1080; and akazawa, et αl., 1994. Proc. Nαtl.
Acαd. Sci. USA 91: 360-364), the latter of which can be particularly useful for detecting point mutations in the NOVX-gene (see, Abravaya, et αl., 1995. Nucl. Acids Res. 23: 675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers that specifically hybridize to an NOVX gene under conditions such that hybridization and amplification of the NOVX gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step m conjunction with any ofthe techniques used for detecting mutations described herein
Alternative amplification methods include self sustained sequence replication (see, Guatelh, et al , 1990 Pioc Natl Acad Set USA 81 1874-1878), transcriptional amplification system (see Kwoh, et al , 1989 Pioc Nail Acad Sci USA 86 1173-1177), Qβ Replicase (see Lizardi, et al, 1988 BioTechnology 6 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art These detection schemes are especially useful for the detection of nudeic acid molecules if such molecules are present in very low numbers In an alternative embodiment, mutations in an NOVX gene from a sample cell can be identified by alterations in restnction enzyme cleavage patterns For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restnction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA Moreover, the use of sequence specific ribozymes (see, e g , U S Patent No 5,493,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site
In other embodiments, genetic mutations in NOVX can be identified by hybndizing a sample and control nucleic acids, e g , DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes See e g , Cronin, et al , 1996 Human Mutation 1 244-255, Kozal, et al , 1996 Nat Med 2 753-759 For example, genetic mutations in NOVX can be identified in two dimensional anays containing light-generated DNA probes as described in Cronin, et al , supra Briefly, a first hybridization anay of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear anays of sequential overlapping probes This step allows the identification of point mutations This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected Each mutation anay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene
In yet anothei embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the NOVX gene and detect mutations by comparing the sequence of the sample NOVX with the conesponding wild-type (control) sequence Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert, 1977 Proc Natl Acad Sci USA 74 560 or Sanger, 1977 Proc Natl Acad Sci USA 74 5463 It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (see, e g , Naeve, et al , 1995 Biotechniques 19 448), including sequencing by mass spectrometry (see, e g , PCT International Publication No WO 94/16101 , Cohen, et al , 1996 Adv Chromatogi aphy 36 127-162, and Griffin, et al , 1993 Appl Biochem Bwtechnol 38 147-159)
Other methods for detecting mutations in the NOVX gene include methods in which protection from cleavage agents is used to detect mismatched bases in R-NA/RNA or RNA DNA heteroduplexes See e g , Myers, et al , 1985 Science 230 1242 In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type NOVX sequence with potentially mutant RNA or DNA obtained from a tissue sample The double-stranded duplexes are treated with an agent that cleaves single stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Si nuclease to enzymatically digesting the mismatched regions In other embodiments, either DNA DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with pipendine in order to digest mismatched regions After digestion ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation See, e g Cotton, et al , 1988 P oc Natl Acad Sci USA 85 4397, Saleeba, et al , 1992 Methods Enzymol 217 286-295 In an embodiment, the control DNA or RNA can be labeled for detection
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in NOVX cDNAs obtained from samples of cells For example, the mufY enzyme of E coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches See, e g , Hsu, et al , 1994 Ca cinogenesis 15 1657-1662 According to an exemplary embodiment, a probe based on an NOVX sequence, e g , a wild-type NOVX sequence, is hybridized to a cDNA or other DNA product from a test cell(s) The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like See e j U S Patent No 5,459,039
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in NOVX genes For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids. See, e.g., Orita, et al, 1989. Proc. Natl Acad. Sci. USA: 86: 2766; Cotton, 1993. Mutat. Res. 285: 125-144; Hayashi, 1992. Genet. Anal. Tech. Appl. 9: 73-79. Single-stranded DNA fragments of sample and control NOVX nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In one embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility. See, e.g., Keen, et al, 1991. Trends Genet. 1: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE). See, e.g., Myers, et al, 1985. Nature 313: 495. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC damp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA. See, e.g., Rosenbaum and Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found. See, e.g., Saiki, et al, 1986. Nature 324: 163; Saiki, et al, 1989. Proc. Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA. Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, e.g., Gibbs, et al, 1 89. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, e.g., Prossner, 1993. Tibtech. 11 : 238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection. See, e.g., Gasparini, et al, 1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification. See, e.g., Barany, 1991. Proc. Natl. Acad. Sci. USA 88: 189- In such cases, ligation will occur only if there is a perfect match at the 3'-terminus of the 5' sequence, making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an NOVX gene.
Furthermore, any cell type or tissue, preferably peripheral blood leukocytes, in which NOVX is expressed may be utilized in the prognostic assays described herein. However, any biological sample containing nucleated cells may be used, including, for example, buccal mucosal cells.
Pharmacogenomics Agents, or modulators that have a stimulatory or inhibitory effect on NOVX activity
(e.g., NOVX gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (The disorders include metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, and hematopoietic disorders, and the various dyslipidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers.) In conjunction with such treatment, the phamiacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) ofthe individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration o the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NOVX protein, expression of NOVX nucleic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol, 23: 983-985; Linder, 1997. Clin. Chem., 43: 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can ocGur either as rare defects or as polymoφhisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymoφhisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymoφhisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymoφhic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite moφhine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of NOVX protein, expression of NOVX nudeic acid, or mutation content of NOVX genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment ofthe individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drug metabolizing enzymes to the identification of an individual's drug responsiveness phenotype This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an NOVX modulator, such as a modulator identified by one of the exemplary screening assays described herein
Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e g , drugs, compounds) on the expression or activity of NOVX (e g , the ability to modulate abenant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials For example, the effectiveness of an agent determined by a screening assay as described herein to increase NOVX gene expression, protein levels, or upregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting decreased NOVX gene expression, protein levels, or downregulated NOVX activity Alternatively, the effectiveness of an agent determined by a screening assay to decrease NOVX gene expression, protein levels, or downregulate NOVX activity, can be monitored in clinical trails of subjects exhibiting increased NOVX gene expression, protein levels, or upregulated NOVX activity In such clinical trials, the expression or activity of NOVX and, preferably, other genes that have been implicated in, for example, a cellular proliferation or immune disorder can be used as a "read out" or markers of the immune responsiveness of a particular cell
By way of example, and not of limitation, genes, including NOVX, that are modulated in cells by treatment with an agent (e g , compound, drug or small molecule) that modulates NOVX activity (e g , identified in a screening assay as described herein) can be identified Thus, to study the effect of agents on cellular proliferation disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NOVX and othei genes implicated in the disorder The levels of gene expression (i e , a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of NOVX or other genes In this manner, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of ( ) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an NOVX protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity ofthe NOVX protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NOVX protein, mRNA, or genomic DNA in the pre-administration sample with the NOVX protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NOVX to higher levels than detected, i.e., to increase the effectiveness ofthe agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NOVX to lower levels than detected, i.e., to decrease the effectiveness ofthe agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NOVX expression or activity. The disorders include cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, tuberous sclerosis, scleroderma, obesity, transplantation, adrenoleukodystrophy, congenital adrenal hypeφlasia, prostate cancer, neoplasm; adenocarcinoma, lymphoma, uterus cancer, fertility, hemophilia, hypercoagulation, idiopathic thrombocytopenic puφura, immunodeficiencies, graft versus host disease, AIDS, bronchial asthma, Crohn's disease; multiple sderosis, treatment of Albright Hereditary Ostoeodystrophy, and other diseases, disorders and conditions of the like. These methods of treatment will be discussed more fully, below.
Disease and Disorders
Diseases and disorders that arc characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to: (i) an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; (//') antibodies to an aforementioned peptide; (iii) nucleic acids encoding an aforementioned peptide; (iv) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences of coding sequences to an aforementioned peptide) that are utilized to "knockout" endogenous function of an aforementioned peptide by homologous recombination (see, e.g., Capecchi, 1989. Science 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide ofthe invention) that alter the interaction between an aforementioned peptide and its binding partner.
Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, an aforementioned peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of an aforementioned peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an abenant NOVX expression or activity, by administering to the subject an agent that modulates NOVX expression or at least one NOVX activity. Subjects at risk for a disease that is caused or contributed to by abenant NOVX expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration ofa prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe NOVX abenancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending upon the type of NOVX abenancy, for example, an NOVX agonist or NOVX antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods o the invention are further discussed in the following subsections.
Therapeutic Methods
Another aspect of the invention pertains to methods of modulating NOVX expression or activity for therapeutic puφoses. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more ofthe activities of NOVX protein activity associated with the cell. An agent that modulates NOVX protein activity can be an agent as described herein, such as a nudeic acid or a protein, a naturally-occurring cognate ligand of an NOVX protein, a peptide, an NOVX peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NOVX protein activity. Examples of such stimulatory agents include active NOVX protein and a nucleic acid molecule encoding NOVX that has been introduced into the cell. In another embodiment, the agent inhibits one or more NOVX protein activity. Examples of such inhibitory agents include antisense NOVX nucleic acid molecules and anti-NOVX antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an NOVX protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., up-regulates or down-regulates) NOVX expression or activity. In another embodiment, the method involves administering an NOVX protein or nucleic acid molecule as therapy to compensate for reduced or abenant NOVX expression or activity.
Stimulation of NOVX activity is desirable in situations in which NOVX is abnormally downregulated and/or in which increased NOVX activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by abenant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders). Another example of such a situation is where the subject has a gestational disease (e.g., preclampsia). Determination of the Biological Effect of he Therapeutic
In various embodiments of the invention, suitable in vύto or in vivo assays are performed to detenmne the effect of a specific Theiapeutic and whether its administration is indicated for treatment of the affected tissue In various specific embodiments, in v to assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given Therapeutic exerts the desired effect upon the cell type(s) Compounds for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, pnor to testing in human subjects Similarly, for in vivo testing, any of the animal model system known in the art may be used pnor to administration to human subjects
Prophylactic and Therapeutic Uses o he Compositions o the Invention
The NOVX nucleic acids and proteins of the invention are useful in potential prophylactic and therapeutic applications implicated m a vanety of disorders including, but not limited to metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer- associated cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyshpidemias, metabolic disturbances associated with obesity, the metabolic syndrome X and wasting disorders associated with chronic diseases and various cancers As an example, a cDNA encoding the NOVX protein ofthe invention may be useful in gene therapy, and the protein may be useful when administered to a subject m need thereof By way of non-limiting example, the compositions ofthe invention will have efficacy for treatment of patients suffering from metabolic disorders, diabetes, obesity, infectious disease, anorexia, cancer-associated cachexia, cancer, neurodegenerative disorders, Alzheimer's Disease, Parkinson's Disorder, immune disorders, hematopoietic disorders, and the various dyshpidemias
Both the novel nucleic acid encoding the NOVX protein, and the NOVX protein of the invention, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed A further use could be as an anti-bacterial molecule (/ e , some peptides have been found to possess anti-bacterial properties) These materials are further useful in the generation of antibodies, which immunospecifically-bmd to the novel substances of the invention for use in therapeutic or diagnostic methods The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Examples
Example 1. Identification of NOVX clones
The novel NOVX target sequences identified in the present invention were subjected to the exon linking process to confirm the sequence. PCR primers were designed by starting at the most upstream sequence available, for the forward primer, and at the most downstream sequence available for the reverse primer. Table 11A shows the sequences of the PCR primers used for obtaining different clones. In each case, the sequence was examined, walking inward from the respective termini toward the coding sequence, until a suitable sequence that is either unique or highly selective was encountered, or, in the case ofthe reverse primer, until the stop codon was reached. Such primers were designed based on in silico predictions for the full length cDNA, part (one or more exons) ofthe DNA or protein sequence of the target sequence, or by translated homology of the predicted exons to closely related human sequences from other species. These primers were then employed in PCR amplification based on the following pool of human cDNAs: adrenal gland, bone manow, brain - amygdala, brain - cerebellum, brain - hippocampus, brain - substantia nigra, brain - thalamus, brain -whole, fetal brain, fetal kidney, fetal liver, fetal lung, heart, kidney, lymphoma - Raji, mammary gland, pancreas, pituitary gland, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thyroid, trachea, uterus. Usually the resulting amplicons were gel purified, cloned and sequenced to high redundancy. The PCR product derived from exon linking was cloned into the pCR2.1 vector from Invitrogen. The resulting bacterial clone has an insert covering the entire open reading frame cloned into the pCR2.1 vector. Table 7B shows a list of these bacterial clones. The resulting sequences from all clones were assembled with themselves, with other fragments in CuraGen Coφoration's database and with public ESTs. Fragments and ESTs were included as components for an assembly when the extent of their identity with another component of the assembly was at least 95% over 50 bp. In addition, sequence traces were evaluated manually and edited for conections if appropriate. These procedures provide the sequence reported herein.
Table 1 1 A. PCR Primers for Exon Linking
Figure imgf000191_0001
[ 89
Figure imgf000192_0001
Physical clone: Exons were predicted by homology and the intron/exon boundaries were detemiined using standard genetic rules. Exons were further selected and refined by means of similarity detennination using multiple BLAST (for example, tBlastN, BlastX, and BlastN) searches, and, in some instances, GeneScan and Grail. Expressed sequences from both public and proprietary databases were also added when available to further define and complete the gene sequence. The DNA sequence was then manually corrected for apparent inconsistencies thereby obtaining the sequences encoding the full-length protein.
Table 1 IB. Physical Clones for PCR products
Figure imgf000192_0002
Example 2. Quantitative expression analysis of clones in various cells and tissues
The quantitative expression of various clones was assessed using microtiter plates containing RNA samples from a variety of normal and pathology-derived cells, cell lines and tissues using real time quantitative PCR (RTQ PCR). RTQ PCR was performed on a Perkin- Elmer Biosystems ABI PRISM® 7700 Sequence Detection System. Various collections of samples are assembled on the plates, and referred to as Panel 1 (containing normal tissues and cancer cell lines), Panel 2 (containing samples derived from tissues from normal and cancer sources), Panel 3 (containing cancer cell lines), Panel 4 (containing cells and cell lines from normal tissues and cells related to inflammatory conditions), Panel 5D/5I (containing human tissues and cell lines with an emphasis on metabolic diseases), Panel CNSD.01 (containing samples from normal and diseased brains) and CNS_neurodegeneration_panel (containing samples from normal and diseased brains).
RNA integrity from all samples is controlled for quality by visual assessment of agarose gel electropherograms using 28S and 18S ribosomal RNA staining intensity ratio as a guide (2:1 to 2.5: 1 28s:18s) and the absence of low molecular weight RNAs that would be indicative of degradation products. Samples are controlled against genomic DNA contamination by RTQ PCR reactions run in the absence of reverse transcriptase using probe and primer sets designed to amplify across the span of a single exon.
First, the RNA samples were normalized to reference nucleic acids such as conεtitutively expressed genes (for example, β-actin and GAPDH). Normalized RNA (5 ul) was converted to cDNA and analyzed by RTQ-PCR using One Step RT-PCR Master Mix Reagents (PE Biosystems; Catalog No. 4309169) and gene-specific primers according to the manufacturer's instructions. Probes and primers were designed for each assay according to Perkin Elmer Biosystem's Primer Express Software package (version I for Apple Computer's Macintosh Power PC) or a similar algorithm using the target sequence as input. Default settings were used for reaction conditions and the following parameters were set before selecting primers: primer concentration = 250 nM, primer melting temperature (Tm) range = 58°-60° C, primer optimal Tm = 59° C, maximum primer difference = 2° C, probe does not have 5' G, probe Tm must be 10° C greater than primer Tm, amplicon size 75 bp to 100 bp. The probes and primers selected (see below) were synthesized by Synthegen (Houston, TX, USA). Probes were double purified by HPLC to remove uncoupled dye and evaluated by mass spectroscopy to verify coupling of reporter and quencher dyes to the 5' and 3' ends of the probe, respectively. Their final concentrations were: forward and reverse primers, 900 nM each, and probe, 200nM.
PCR conditions: Normalized RNA from each tissue and each cell line was spotted in each well ofa 96 well PCR plate (Perkin Elmer Biosystems). PCR cocktails including two probes (a probe specific for the target clone and another gene-specific probe multiplexed with the target probe) were set up using 1 X TaqMan™ PCR Master Mix for the PE Biosystems 7700, with 5 mM MgC12, dNTPs (dA, G, C, U at 1 : 1: 1 :2 ratios), 0.25 U/ml AmpliTaq Gold™ (PE Biosystems), and 0.4 U/μl RNase inhibitor, and 0.25 U/μl reverse transcriptase. Reverse transcription was performed at 48° C for 30 minutes followed by amplification/PCR cydes as follows: 95° C 10 min, then 40 cycles of 95° C for 15 seconds, 60° C for 1 minute. Results were recorded as CT values (cycle at which a given sample crosses a threshold level of fluorescence) using a log scale, with the difference in RNA concentration between a given sample and the sample with the lowest CT value being represented as 2 to the power of delta CT. The percent relative expression is then obtained by taking the reciprocal of this RNA difference and multiplying by 100.
Panels 1, 1.1, 1.2, and 1.3D The plates for Panels 1 , 1.1, 1.2 and 1.3D include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in these panels are broken into 2 dasses: samples derived from cultured Gell lines and samples derived from primary normal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in these panels are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on these panels are comprised of samples derived from all major organ systems from single adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone marrow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.
In the results for Panels 1, 1.1, 1.2 and 1.3D, the following abbreviations are used: ca. = carcinoma,
* = established from metastasis, met = metastasis, s cell var = small cell variant, non-s = non-sm = non-small, squam = squamous, pi. eff = pi effusion = pleural effusion, glio = glioma, astro = astrocytoma, and neuro = neuroblastoma.
GENERAL SCREENING PANEL VI .4
The plates for Panel 1.4 include 2 control wells (genomic DNA control and chemistry control) and 94 wells containing cDNA from various samples. The samples in Panel 1.4 are broken into 2 classes: samples derived from cultured cell lines and samples derived from primary nonnal tissues. The cell lines are derived from cancers of the following types: lung cancer, breast cancer, melanoma, colon cancer, prostate cancer, CNS cancer, squamous cell carcinoma, ovarian cancer, liver cancer, renal cancer, gastric cancer and pancreatic cancer. Cell lines used in Panel 1.4 are widely available through the American Type Culture Collection (ATCC), a repository for cultured cell lines, and were cultured using the conditions recommended by the ATCC. The normal tissues found on Panel 1.4 are comprised of pools of samples derived from all major organ systems from 2 to 5 different adult individuals or fetuses. These samples are derived from the following organs: adult skeletal muscle, fetal skeletal muscle, adult heart, fetal heart, adult kidney, fetal kidney, adult liver, fetal liver, adult lung, fetal lung, various regions of the brain, the spleen, bone manow, lymph node, pancreas, salivary gland, pituitary gland, adrenal gland, spinal cord, thymus, stomach, small intestine, colon, bladder, trachea, breast, ovary, uterus, placenta, prostate, testis and adipose.
Panels 2D and 2.2
The plates for Panels 2D and 2.2 generally include 2 control wells and 94 test samples composed of RNA or cDNA isolated from human tissue procured by surgeons working in close cooperation with the National Cancer Institute's Cooperative Human Tissue Network (CHTN) or the National Disease Research Initiative (NDRI). The tissues are derived from human malignancies and in cases where indicated many malignant tissues have "matched margins" obtained from noncancerous tissue just adjacent to the tumor. These are termed normal adjacent tissues and are denoted "NAT" in the results below. The tumor tissue and the "matched margins" are evaluated by two independent pathologists (the surgical pathologists and again by a pathologists at NDRI or CHTN). This analysis provides a gross histopathological assessment of tumor differentiation grade. Moreover, most samples include the original surgical pathology report that provides information regarding the clinical stage of the patient. These matched margins are taken from the tissue sunounding (i.e. immediately proximal) to the zone of surgery (designated "NAT", for normal adjacent tissue, in Table RR). In addition, RNA and cDNA samples were obtained from various human tissues derived from autopsies perfom ed on elderly people or sudden death victims (accidents, etc.). These tissues were ascertained to be free of disease and were purchased from various commercial sources such as Clontech (Palo Alto, CA), Research Genetics, and Invitrogen.
PANEL 3D
The plates of Panel 3D are comprised of 94 cDNA samples and two control samples. Specifically, 92 of these samples are derived from cultured human cancer cell lines, 2 samples of human primary cerebellar tissue and 2 controls. The human cell lines are generally obtained from ATCC (American Type Culture Collection), NCI or the German tumor cell bank and fall into the following tissue groups: Squamous cell carcinoma ofthe tongue, breast cancer, prostate cancer, melanoma, epidennoid carcinoma, sarcomas, bladder carcinomas, pancreatic cancers, kidney cancers, leukemias/lymphomas, ovarian/uterine/cervical, gastric, colon, lung and CNS cancer cell lines. In addition, there are two independent samples of cerebellum. These cells are all cultured under standard recommended conditions and RNA extracted using the standard procedures. The cell lines in panel 3D and 1.3D are ofthe most common cell lines used in the scientific literature.
Panels 4D, 4R, and 4.1D
Panel 4 includes samples on a 96 well plate (2 control wells, 94 test samples) composed of RNA (Panel 4R) or cDNA (Panels 4D/4.1D) isolated from various human cell lines or tissues related to inflammatory conditions. Total RNA from control normal tissues such as colon and lung (Stratagene, La Jolla, CA) and thymus and kidney (Clontech) were employed. Total RNA from liver tissue from cinhosis patients and kidney from lupus patients was obtained from BioChain (Biochain Institute, Inc., Hayward, CA). Intestinal tissue for RNA preparation from patients diagnosed as having Crohn's disease and ulcerative colitis was obtained from the National Disease Research Interchange (NDRI) (Philadelphia, PA).
Astrocytes, lung fibroblasts, dermal fibroblasts, coronary artery smooth muscle cells, small airway epithelium, bronchial epithelium, microvascular dermal endothelial cells, microvascular lung endothelial cells, human pulmonary aortic endothelial cells, human umbilical vein endothelial cells were all purchased from Clonetics (Walkersville, MD) and grown in the media supplied for these cell types by Clonetics. These primary cell types were activated with various cytokines or combinations of cytokines for 6 and/or 12-14 hours, as indicated. The following cytokines were used; IL-1 beta at approximately 1-5 ng/ml, TNF alpha at approximately 5-10 ng/ml, IFN gamma at approximately 20-50 ng/ml, IL-4 at approximately 5-10 ng/ml, IL-9 at approximately 5-10 ng/ml, IL-13 at approximately 5-10 ng/ml. Endothelial cells were sometimes starved for various times by culture in the basal media from Clonetics with 0.1 % serum.
Mononuclear cells were prepared from blood of employees at CuraGen Corporation, using Ficoll. LAK, cells were prepared from these cells by culture in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco/Life Technologies, Rockville, MD), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco) and Interleukin 2 for 4-6 days. Cells were then either activated with 10-20 ng/ml PMA and 1 -2 μg/ml ionomycin, IL- 12 at 5-10 ng/ml, IFN gamma at 20-50 ng/ml and IL-18 at 5-10 ng/ml for 6 hours. In some cases, mononuclear cells were cultured for 4-5 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco) with PHA (phytohemagglutinin) or PWM (pokeweed mitogen) at approximately 5 μg/ml. Samples were taken at 24, 48 and 72 hours for RNA preparation. MLR (mixed lymphocyte reaction) samples were obtained by taking blood from two donors, isolating the mononuclear cells using Ficoll and mixing the isolated mononuclear cells 1 : 1 at a final concentration of approximately 2x106 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol (5.5 x 10"5 M) (Gibco), and 10 mM Hepes (Gibco). The MLR was cultured and samples taken at various time points ranging from 1- 7 days for RNA preparation. Monocytes were isolated from mononuclear cells using CD14 Miltenyi Beads, +ve VS selection columns and a Vario Magnet according to the manufacturer's instructions. Monocytes were differentiated into dendritic cells by culture in DMEM 5% fetal calf serum (FCS) (Hyclone, Logan, UT), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco), 50 ng/ml GMCSF and 5 ng/ml IL-4 for 5-7 days. Macrophages were prepared by culture of monocytes for 5-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), 10 mM Hepes (Gibco) and 10% AB Human Serum or MCSF at approximately 50 ng/ml. Monocytes, macrophages and dendritic cells were stimulated for 6 and 12-14 hours with lipopolysaccharide (LPS) at 100 ng/ml. Dendritic cells were also stimulated with anti-CD40 monoclonal antibody (Pharmingen) at 10 μg/ml for 6 and 12-14 hours.
CD4 lymphocytes, CD8 lymphocytes and NK. cells were also isolated from mononuclear cells using CD4, CD8 and CD56 Miltenyi beads, positive VS selection columns and a Vario Magnet according to the manufacturer's instructions. CD45RA and CD45RO CD4 lymphocytes were isolated by depleting mononuclear cells of CD8, CD56, CD 14 and CD 19 cells using CD8, CD56, CD 14 and CD 19 Miltenyi beads and positive selection. Then CD45RO beads were used to isolate the CD45RO CD4 lymphocytes with the remaining cells being CD45RA CD4 lymphocytes. CD45RA CD4, CD45RO CD4 and CD8 lymphocytes were placed in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco) and plated at 10δ cells/ml onto Falcon 6 well tissue culture plates that had been coated overnight with 0.5 μg/ml anti-CD28 (Pharmingen) and 3 ug/ml anti-CD3 (OKT3, ATCC) in PBS. After 6 and 24 hours, the cells were harvested for RNA preparation. To prepare chronically activated CD8 lymphocytes, we activated the isolated CD8 lymphocytes for 4 days on anti-CD28 and anti-CD3 coated plates and then harvested the cells and expanded them in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2. The expanded CD8 cells were then activated again with plate bound anti-CD3 and anti-CD28 for 4 days and expanded as before. RNA was isolated 6 and 24 hours after the second activation and after 4 days ofthe second expansion culture. The isolated NK cells were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco) and IL-2 for 4-6 days before RNA was prepared. To obtain B cells, tonsils were procured from NDRI. The tonsil was cut up with sterile dissecting scissors and then passed through a sieve. Tonsil cells were then spun down and resupended at 10° cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco). To activate the cells, we used PWM at 5 μg/ml or anti-CD40 (Pharmingen) at approximately 10 μg/ml and IL-4 at 5-10 ng/ml. Cells were harvested for RNA preparation at 24,48 and 72 hours.
To prepare the primary and secondary Thl/Th2 and Trl cells, six-well Falcon plates were coated overnight with 10 μg/ml anti-CD28 (Pharmingen) and 2 μg/ml OKT3 (ATCC), and then washed twice with PBS. Umbilical cord blood CD4 lymphocytes (Poietic Systems,
5 6 Gennan Town, MD) were cultured at 10 - 10 cells/ml in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10" 5 M (Gibco), 10 mM Hepes (Gibco) and IL-2 (4 ng/ml). IL-12 (5 ng/ml) and anti-IL4 (1 Dg/ml) were used to direct to Thl , while IL-4 (5 ng/ml) and anti-IFN gamma (1 Dg/ml) were used to direct to Th2 and IL-10 at 5 ng/ml was used to direct to Trl . After 4-5 days, the activated Thl , Th2 and Trl lymphocytes were washed once in DMEM and expanded for 4-7 days in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 (Gibco), 10 mM Hepes (Gibco) and IL-2 (1 ng/ml). Following this, the activated Thl , Th2 and Trl lymphocytes were re-stimulated for 5 days with anti-CD28/OKT3 and cytokines as described above, but with the addition of anti- CD95L (1 Dg/ml) to prevent apoptosis. After 4-5 days, the Thl , Th2 and Trl lymphocytes were washed and then expanded again with IL-2 for 4-7 days. Activated Thl and Th2 lymphocytes were maintained in this way for a maximum of three cycles. RNA was prepared from primary and secondary Thl , Th2 and Trl after 6 and 24 hours following the second and third activations with plate bound anti-CD3 and anti-CD28 mAbs and 4 days into the second and third expansion cultures in Interleukin 2.
The following leukocyte cells lines were obtained from the ATCC: Ramos, EOL-1, KU-812. EOL cells were further differentiated by culture in 0.1 mM dbcAMP at 5 xlO5 cells/ml for 8 days, changing the media every 3 days and adjusting the cell concentration to 5 xlO5 cells/ml. For the culture of these cells, we used DMEM or RPMI (as recommended by the ATCC), with the addition of 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), 10 mM Hepes (Gibco). RNA was either prepared from resting cells or cells activated with PMA at 10 ng/ml and ionomycin at 1 μg/ml for 6 and 14 hours. Keratinocyte line CCD106 and an airway epithelial tumor line NCI-H292 were also obtained from the ATCC. Both were cultured in DMEM 5% FCS (Hyclone), 100 μM non essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), mercaptoethanol 5.5 x 10"5 M (Gibco), and 10 mM Hepes (Gibco). CCD1106 cells were adivated for 6 and 14 hours with approximately 5 ng/ml TNF alpha and 1 ng/ml IL-1 beta, while NCI-H292 cells were activated for 6 and 14 hours with the following cytokines: 5 ng/ml IL-4, 5 ng/ml IL-9, 5 ng/ml IL- 13 and 25 ng/ml IFN gamma.
For these cell lines and blood cells, RNA was prepared by lysing approximately 107 cells/ml using Trizol (Gibco BRL). Briefly, 1/10 volume of bromochloropropane (Molecular Research Corporation) was added to the RNA sample, vortexed and after 10 minutes at room temperature, the tubes were spun at 14,000 rpm in a Sorvall SS34 rotor. The aqueous phase was removed and placed in a 15 ml Falcon Tube. An equal volume of isopropanol was added and left at -20 degrees C overnight. The precipitated RNA was spun down at 9,000 φm for 15 min in a Sorvall SS34 rotor and washed in 70% ethanol. The pellet was redissolved in 300 μl of RNAse-free water and 35 μl buffer (Promega) 5 μl DTT, 7 μl RNAsin and 8 μl DNAse were added. The tube was incubated at 37 degrees C for 30 minutes to remove contaminating genomic DNA, extracted once with phenol chloroform and re-precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of 100% ethanol. The RNA was spun down and placed in RNAse free water. RNA was stored at -80 degrees C Panel CNSD.01
The plates for Panel CNSD.01 include two control wells and 94 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center. Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology.
Disease diagnoses are taken from patient records. The panel contains two brains from each of the following diagnoses: Alzheimer's disease, Parkinson's disease, Huntington's disease, Progressive Supemuclear Palsy, Depression, and "Normal controls". Within each of these brains, the following regions are represented: cingulate gyrus, temporal pole, globus palladus, substantia nigra, Brodman Area 4 (primary motor strip), Brodman Area 7 (parietal cortex), Brodman Area 9 (prefrontal cortex), and Brodman area 17 (occipital cortex). Not all brain regions are represented in all cases; e.g., Huntington's disease is characterized in part by neurodegeneration in the globus palladus, thus this region is impossible to obtain from confirmed Huntington's cases. Likewise Parkinson's disease is characterized by degeneration ofthe substantia nigra making this region more difficult to obtain. Normal control brains were examined for neuropathology and found to be free of any pathology consistent with neurodegeneration.
In the labels employed to identify tissues in the CNS panel, the following abbreviations are used:
PSP = Progressive supranuclear palsy Sub Nigra = Substantia nigra
Glob Palladus= Globus palladus Temp Pole = Temporal pole Cing Gyr = Cingulate gyrus BA 4 = Brodman Area 4
Panel CNS eurodegeneration V1.0
The plates for Panel CNSjNeurodegeneration VT .O include two control wells and 47 test samples comprised of cDNA isolated from postmortem human brain tissue obtained from the Harvard Brain Tissue Resource Center (McLean Hospital) and the Human Brain and Spinal Fluid Resource Center (VA Greater Los Angeles Healthcare System). Brains are removed from calvaria of donors between 4 and 24 hours after death, sectioned by neuroanatomists, and frozen at -80°C in liquid nitrogen vapor. All brains are sectioned and examined by neuropathologists to confirm diagnoses with clear associated neuropathology. Disease diagnoses are taken from patient records. The panel contains six brains from
Alzheimer's disease (AD) pateins, and eight brains from "Normal controls" who showed no evidence of dementia prior to death. The eight normal control brains are divided into two categories: Controls with no dementia and no Alzheimer's like pathology (Controls) and controls with no dementia but evidence of severe Alzheimer's like pathology, (specifically senile plaque load rated as level 3 on a scale of 0-3; 0 = no evidence of plaques, 3 = severe AD senile plaque load). Within each of these brains, the following regions are represented: hippocampus, temporal cortex (Broddmann Area 21), parietal cortex (Broddmann area 7), and occipital cortex (Brodmann area 17). These regions were chosen to encompass all levels of neurodegeneration in AD. The hippocampus is a region of early and severe neuronal loss in AD; the temporal cortex is known to show neurodegeneration in AD after the hippocampus; the parietal cortex shows moderate neuronal death in the late stages of the disease; the occipital cortex is spared in A and therefore acts as a "control" region within AD patients. Not all brain regions are represented in all cases.
In the labels employed to identify tissues in the CNS_Neurodegeneration_V1.0 panel, the following abbreviations are used:
AD = Alzheimer's disease brain; patient was demented and showed AD-like pathology upon autopsy
Control = Control brains; patient not demented, showing no neuropathology Control (Path) = Control brains; pateint not demented but showing sever AD-like pathology
SupTemporal Ctx = Superior Temporal Cortex
Inf Temporal Ctx = Inferior Temporal Cortex
NOVl a and NOVld
Expression of gene NOVl a and variant NOVl a was assessed using the primer-probe sets Ag4164, Agl 313b, Ag2197, and Ag708 described in Tables 12, 13, 14, and 15. Please note that Ag4164 contains a single mismatch in the probe relative to the NOVla and NOVld sequences. This mismatch is not predicted to alter the RTQ-PCR results. Results from RTQ- PCR runs are shown in Tables 16, 17, 18, and 19.
Table 12. Probe Name Ag4164
Figure imgf000202_0001
Table 13. Probe Name Agl313b
Figure imgf000202_0002
Table 14. Probe Name Ag2197
Figure imgf000202_0003
Table 15. Probe Name Ag708
Figure imgf000202_0004
Table 16. Panel 1.2
Figure imgf000202_0005
Figure imgf000203_0001
Figure imgf000204_0001
Table 17. Panel 1.3D
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Table 18. Panel 2D
Figure imgf000207_0002
Figure imgf000208_0001
Figure imgf000209_0001
Table 19. Panel 4D
Figure imgf000209_0002
Figure imgf000210_0001
Figure imgf000211_0001
Panel 1.2 Summary Ag708 Expression ofthe NOVla gene was assessed in two independent experiments using the same probe/primer set. There appears to be poor concordance between runs for some tissues but there is good concordance for others; only those results that are in agreement will be discussed here. In both experiments, highest expression of the NOVla gene in a sample derived from a glioblastoma cell line (CTs = 24- 26). Among normal tissues derived from the central nervous system, the NOVla gene is also expressed at moderate levels in the cerebral cortex, cerebellum and hippocampus.
Among tissues with metabolic function, the NOVla gene is expressed in the pancreas. Thus, this gene may be involved in the pathogenesis and/or treatment of diseases involving the pancreas, such as pancreatitis and diabetes. In addition, NOVl gene expression is decreased in a pancreatic cancer cell line. The NOVl a gene appears to be overexpressed in fetal kidney when compared to the adult kidney. This result suggests that the NOVla gene could be used to distinguish between adult and fetal kidney tissue and that this gene may play an important role in kidney development, growth and survival. Furthermore, NOVla gene expression is higher in normal ovary, mammary gland and lung when compared to the cancer cell lines obtained from these tissues suggesting that this can be used as a marker to differentiate malignant and normal tissue.
Panel 1.3D Summary Ag708/Agl313b/Ag2197 Three experiments with three different probe and primer sets produced results that were in very good agreement. One run, designated 1.3dx4tm5365t, appears to have lower absolute expression, but produces the same expression profile as the other two experiments. Highest expression of the NOVl A gene in all three runs is seen in a sample derived from a CNS cancer cell line (CTs=27). Certain glioblastoma and astrocytoma cell lines also express this gene as well so it may have a role in different types of brain cancer. Among normal tissues derived from the central nervous system, the NOVla gene is expressed in the amygdala, cerebellum, hippocampus and cerebral cortex.
Among tissues with metabolic function, the NOVla gene is expressed in the pancreas and in adipose. Interestingly, this gene is also expressed at higher levels in fetal heart and skeletal tissue when compared to the adult tissues.
The NOVla gene appears to be expressed at high levels in a sample from a renal cancer cell line and from a breast cancer cell line. In addition, the NOVla gene is expressed in ovarian tissue, but not significantly in cell lines derived from ovarian cancer.
Panel 2D Summary Ag708/Agl313b In two runs using two different probe and primer sets, highest expression of the NOVl A gene is seen in the normal ovary, colon and kidney. Furthermore, in all nine matched kidney pairs and in the matched tissue pair derived from the ovary, the NOVl A gene is expressed more highly in normal tissue than in the adjacent cancer samples. This result suggests that expression of the NOV1A gene could be used as a diagnostic marker for the presence of kidney and ovarian cancer. In addition, therapeutic upregulation of the gene activity of NOVl A could be effective in the treatment of kidney and ovarian cancer. The NOV l A gene is also expressed at higher levels in lung cancer samples, when compared to normal adjacent tissue in six out of seven matched tissue pairs.
Thus, therapeutic inhibition o the NOV l a gene, through the application of antibodies or small molecule drugs, could be effective in the treatment of lung cancer. Panel 4D Summary Ag2197 The NOVl a gene is expressed at a high level in astrocytes (CT 27.8) and its expression is down regulated upon treatment with TNF-a and IL¬ lb, suggesting that modulation ofthis protein could be beneficial in the treatment of CNS diseases-associated inflammation or neurodegeneration. The NOVl a gene is also expressed highly in the thymus (CT 28.4). More moderate expression ofthis gene is observed in the lung and in a muco-epidermoid cell line (H292). Thus, the protein encoded by the NOVl a gene could play an important role in the normal homeostasis of these tissues. Therapeutics designed with this protein could be important for maintaining or restoring normal function to these organs during inflammation.
Panel 4.1D Summary Ag4164 Expression of this gene is low/undetectable (CT values >35) among the samples on this panel (data not shown).
Panel CNS neurodegenerarion vl.O Summary Ag4164 Expression ofthis gene is low/undetectable (CT values >35) among the samples on this panel (data not shown).
NOVlb
The NOVlb sequence is a variant of gene NOVla annotated above. However, the NOVlb sequence only matches a subset of the probe and primer sets discussed above and is discussed independently below in section B. Expression of gene NOVlb was assessed using the primer-probe sets Ag4164 and Ag2197 described in Tables 20 and 21. Please note that Ag4164 contains a single mismatch in the probe relative to the NOVlb sequence. This mismatch is not predicted to alter the RTQ-PCR results. Results from RTQ-PCR runs are shown in Tables 22, 23, and 24.
Table 20. Probe Name Ag4164
Figure imgf000213_0001
21 Table 21. Probe Name Ag2197
Figure imgf000214_0001
Table 22. Panel 1.3D
Figure imgf000214_0002
Figure imgf000215_0001
Table 23. Panel 2D
Figure imgf000215_0002
Figure imgf000216_0001
Figure imgf000217_0001
Table 24. Panel 4D
Figure imgf000217_0002
Figure imgf000218_0001
Figure imgf000219_0001
Panel 1.3D Summary Ag21_97 Highest expression of the NOVla gene is seen in a sample derived from a CNS cancer cell line (CT=27). Certain glioblastoma and astrocytoma cell lines also express this gene as well so it may have a role in different types of brain cancer. Among normal tissues derived from the central nervous system, the NOVla gene is expressed in the amygdala, cerebellum, hippocampus and cerebral cortex.
Among tissues with metabolic function, the NOVla gene is expressed in the pancreas and in adipose. Interestingly, this gene is also expressed at higher levels in fetal heart and skeletal tissue when compared to the adult tissues.
The NOVl a gene appears to be expressed at high levels in a sample from a renal cancer cell line and from a breast cancer cell line. In addition, the NOVla gene is expressed in ovarian tissue, but not significantly in cell lines derived from ovarian cancer.
Panel 2D Summary Ag2197 Expression of the NOVlb gene is highest in normal colon tissue (CT=25). The gene is also expressed at high levels in the uterus, but not in uterine cancer. Thus, expression of NOVlb could be used as a diagnostic marker for the presence of uterine cancer. Furthermore, therapeutic upregulation of the activity of the protein product could potentially be useful in the treatment of uterine cancer. The NOVlb gene also appears to be expressed at higher levels in liver cancers (two out of two matched tissue pairs) and kidney cancers (seven out nine matched tissue pairs) when compared to normal adjacent tissue. Thus, expression of the NOVlb gene could be used to diffentiate between kidney and liver cancers and other cancers or normal tissue. Furthermore, thereapeutic inhibition ofthe activity ofthe protein encoded by the NOVlb gene, through the use of antibodies or small molecule drugs, could be effective in treating kidney and liver cancers.
Panel 4D Summary Ag2197 The NOVla gene is expressed at a high level in astrocytes (CT 27.8) and its expression is down regulated upon treatment with TNP-a and ILlb, suggesting that modulation o this protein could be beneficial in the treatment of CNS diseases-associated inflammation or neurodegeneration. The NOVl a gene is also expressed highly in the thymus (CT 28.4). More moderate expression ofthis gene is observed in the lung and in a muco-epidermoid cell line (H292). Thus, the protein encoded by the NOVla gene could play an important role in the normal homeostasis of these tissues. Therapeutics designed with this protein could be important for maintaining or restoring normal function to these organs during inflammation.
Panel 4.1D Summary Ag4164 Expression of this gene is low/undetectable (CT values >35) among the samples on this panel (data not shown).
Panel CNS_neurodegeneration_vl.O Summary Ag4164 Expression of this gene is low/undetectable (CT values >35) among the samples on this panel (data not shown).
NOVlc
Please note that the NOVlc sequence is a variant of gene NOVla annotated in section A. However, the NOVlc sequence only matches a subset of the probe and primer sets discussed above and is discussed independently below in section C. Expression of gene NOVlc was assessed using the primer-probe sets Ag2197, Ag708 and Agl313b described in Tables 25, 26, and 27. Results from RTQ-PCR runs are shown in Tables 28, 29, 30, and 31.
Table 25. Probe Name Ag2197
Figure imgf000220_0001
Reverse 5 ' -TTCATTTCTATGGGACCTCAGA-3 58.7 22 1086 128
Table 26. Probe Name Ag708
Figure imgf000221_0001
Table 27. Probe Name Agl313b
Figure imgf000221_0002
Table 28. Panel 1.2
Figure imgf000221_0003
Figure imgf000222_0001
Figure imgf000223_0001
Table 29. Panel 1.3D
Figure imgf000223_0002
Figure imgf000224_0001
2?7
Figure imgf000225_0001
Table 30. Panel 2D
Figure imgf000225_0002
Figure imgf000226_0001
Figure imgf000227_0001
NAT Stomach Clontech 9060394 0.3 1.8 1.1
Gastric Cancer Clontech 9060397 0.1 9.3 5.7
INAT Stomach Clontech 9060396 0.0 0.2 0.8
Gastric Cancer GENPAK 064005 0.4 0.4 1.5
Table 31. Panel 4D
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Panel 1.2 Summary Ag708 Expression ofthe NOVl a gene was assessed in two independent experiments using the same probe/primer set. There appears to be poor concordance between runs for some tissues but there is good concordance for others; only those results that are in agreement will be discussed here. In both experiments, highest expression of the NOVl gene in a sample derived from a glioblastoma cell line (CTs = 24- 26). Among normal tissues derived from the central nervous system, the NOVla gene is also expressed at moderate levels in the cerebral cortex, cerebellum and hippocampus. Among tissues with metabolic function, the NOVla gene is expressed in the pancreas.
Thus, this gene may be involved in the pathogenesis and/or treatment of diseases involving the pancreas, such as pancreatitis and diabetes. In addition, NOVla gene expression is decreased in a pancreatic cancer cell line.
The NOVla gene appears to be overexpressed in fetal kidney when compared to the adult kidney. This result suggests that the NOVla gene could be used to distinguish between adult and fetal kidney tissue and that this gene may play an important role in kidney development, growth and survival. Furthermore, NOVla gene expression is higher in normal ovary, mammary gland and lung when compared to the cancer cell lines obtained from these tissues suggesting that this can be used as a marker to differentiate malignant and normal tissue.
Panel 1.3D Summary Ag708/ Agl 313b/Ag2197 Three experiments with three different probe and primer sets produced results that were in very good agreement. One run, designated 1 .3dx4tm5365t, appears to have lower absolute expression, but produces the same expression profile as the other two experiments. Highest expression ofthe NOVla gene in all three runs is seen in a sample derived from a CNS cancer cell line (CTs=27). Certain glioblastoma and astrocytoma cell lines also express this gene as well so it may have a role in different types of brain cancer. Among normal tissues derived from the central nervous system, the NOVla gene is expressed in the amygdala, cerebellum, hippocampus and cerebral cortex.
Among tissues with metabolic function, the NOV l a gene is expressed in the pancreas and in adipose. Interestingly, this gene is also expressed at higher levels in fetal heart and skeletal tissue when compared to the adult tissues.
The NOVla gene appears to be expressed at high levels in a sample from a renal cancer cell line and from a breast cancer cell line. In addition, the NOVla gene is expressed in ovarian tissue, but not significantly in cell lines derived from ovarian cancer.
Panel 2D Summary Ag708/Agl313b/Ag2197 In two runs using two different probe and primer sets, highest expression of the NOVla gene is seen in the normal ovary, colon and kidney. Furthermore, in all nine matched kidney pairs and in the matched tissue pair derived from the ovary, the NOVla gene is expressed more highly in normal tissue than in the adjacent cancer samples. This result suggests that expression ofthe NOVla gene could be used as a diagnostic marker for the presence of kidney and ovarian cancer. In addition, therapeutic upregulation ofthe gene activity of NOVla could be effective in the treatment of kidney and ovarian cancer. The NOV la gene is also expressed at higher levels in lung cancer samples, when compared to normal adjacent tissue in six out of seven matched tissue pairs. Thus, therapeutic inhibition of the NOVla gene, through the application of antibodies or small molecule drugs, could be effective in the treatment of lung cancer.
Panel 4D Summary Ag2197 The NOVla gene is expressed at a high level in astrocytes (CT 27.8) and its expression is down regulated upon treatment with TNT-a and ILlb, suggesting that modulation of this protein could be beneficial in the treatment of CNS diseases-associated inflammation or neurodegeneration. The NOVl a gene is also expressed highly in the thymus (CT 28.4). More moderate expression ofthis gene is observed in the lung and in a muco-epidermoid cell line (H292). Thus, the protein encoded by the NOVla gene could play an important role in the normal homeostasis of these tissues. Therapeutics designed with this protein could be important for maintaining or restoring normal function to these organs during inflammation.
NOV3 Expression of gene NOV3 was assessed using the primer-probe set Agl534 described in Table 32. Results from RTQ-PCR runs are shown in Tables 33, 34, 35, and 36.
Table 32. Probe Name Agl534
Figure imgf000232_0001
Table 33. Panel 1.2
Figure imgf000232_0002
Figure imgf000233_0001
Table 34. Panel 1.3D
Figure imgf000233_0002
Figure imgf000234_0001
Table 35. Panel 2D
Figure imgf000234_0002
Figure imgf000235_0001
Figure imgf000236_0001
Table 36. Panel 3D
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Panel 1.2 Summary Agl 534 The NOV3 gene encodes a protein with homology to the ileal sodium/bile cotransporter. Highest expression of this gene is detected in the kidney (CT=28.1). The NOV3 gene appears to be expressed in clusters of cell lines derived from breast cancer, ovarian cancer, lung cancer, renal cancer and melanoma. Thus expression ofthis gene could be used to detect the presence of any of these cancers. Furthermore, therapeutic modulation o the expression of the NOV3 gene or the activity of its protein product may be beneficial in the treatment of breast cancer, ovarian cancer, lung cancer, renal cancer and melanoma.
Among tissues involved in metabolic function, the NOV3 gene is expressed in the thyroid, adrenal gland, heart, liver, and skeletal muscle. Thus, the protein encoded by the NOV3 gene could be involved in the pathogenesis and/or treatment of diseases that involve any of these tissues. Furthermore, the NOV3 gene is expressed at higher levels in adult heart tissue (CT=29.7) than in fetal heart tissue (CT=33.6). Therefore, expression o the NOV3 gene could also be used to differentiate between adult and fetal heart tissue.
The NOV3 gene is also widely expressed in tissues originating in the central nervous system. These tissues include the fetal brain, amygdala, hippocampus, fhalamus, cerebral cortex and spinal cord. This transporter gene most likely plays a role in the uptake of nutrients. Blockade o this transporter may decrease the loss of neurons due to excitotoxicity during ischemic stroke.
Panel 1.3D Summary Agl534 Highest expression of the NOV3 gene in Panel 1.3D is detected in the hippocampus (CT=31.2). This gene is also expressed in the amygdala and cerebral cortex. Please see Panel 1.2 summary for discussion of potential utility of this gene with respect to CNS function.
Among tissues with metabolic function, the NOV3 gene is expressed in the thyroid, pituitary gland, adipose, and fetal skeletal muscle. Interestingly, this gene is muGh more highly expressed in fetal skeletal muscle (CT=33.4) than in adult skeletal muscle (CT=40), suggesting that this gene could be used to distinguish the two. In addition, the increased NOV3 gene expression in fetal skeletal muscle when compared to adult suggests that the protein product may enhance muscular growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation of the NOV3 gene could be useful in treatment of muscular related disease. More specifically, treatment of weak or dystrophic muscle with the protein encoded by this gene could restore muscle mass or function. This gene is also more highly expressed in fetal kidney, lung and brain when compared to the corresponding adult tissues.
Panel 2D Summary Agl 534 The NOV3 gene is most highly expressed in a kidney cancer (CT=31.1). In general, however, this gene is more commonly expressed at higher levels in normal tissues than the adjacent tumor tissues. Specifically, this gene is expressed at higher levels in normal adjacent tissues next to thyroid cancer as well as adjacent to some kidney and lung cancers. These data indicate that NOV3 gene expression might be used to distinguish normal tissue from malignant tissue and also that therapeutic modulation of this gene product might be of use in the treatment of these types of cancer.
Panel 3D Summary Agl534 Highest expression of the NOV3 gene is detected in a cell line derived from a medulloblastoma (CT=30.1). Additionally there is expression in a chronic myelogenous leukemia (megokaryoblast) cell line, a gastric adenocarcinoma cell line, a clear cell renal carcinoma cell line, a pancreatic ductal adenocarcinoma cell line and a small cell lung cancer cell line. Thus, the expression of this gene could be used to distinguish some cancer cell lines from others. In addition, these data indicate that the expression of the NOV3 gene might be associated with these forms of cancer and thus, therapeutic modulation of this gene might be of use in the treatment of cancer.
References:
1. Oelkers P, Kirby LC, Heubi -IE, Dawson PA. Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2). J Clin Invest 1997 Apr 15;99(8): 1880-7.
Primary bile acid malabsorption (PBAM) is an idiopathic intestinal disorder associated with congenital diarrhea, steatorrhea, interruption o the enterohepatic circulation of bile acids, and reduced plasma cholesterol levels. The molecular basis of PBAM is unknown, and several conflicting mechanisms have been postulated. In this study, we cloned the human ileal Na+/bile acid cotransporter gene (SLC 10A2) and employed single-stranded conformation polymoφhism analysis to screen for PBAM-associated mutations. Four polymoφhisms were identified and sequenced in a family with congenital PBAM. One allele encoded an A171 S missense mutation and a mutated donor splice site for exon 3. The other allele encoded two missense mutations at conserved amino acid positions, L243P and T262M. In transfected COS cells, the L243P, T262M, and double mutant (L243P/T262M) did not affect transporter protein expression or trafficking to the plasma membrane; however, transport of taurocholate and other bile acids was abolished. In contrast, the Al 71 S mutation had no effect on taurocholate uptake. The dysfunctional mutations were not detected in 104 unaffected control subjects, whereas the A171S was present in 28% of that population. These findings establish that SLC10A2 mutations can cause PBAM and underscore the ileal Na+/bile acid cotransporter's role in intestinal reclamation of bile acids.
NOV4 Expression of gene NOV4 was assessed using the primer-probe sets Ag2432 and
Agl 250 described in Tables 37 and 38. Results from RTQ-PCR runs are shown in Tables 39, 40, 41, and 42.
Table 37. Probe Name Ag2432
Figure imgf000241_0001
Table 38. Probe Name Agl250
Figure imgf000241_0002
Table 39. Panel 1.2
Figure imgf000241_0003
Figure imgf000242_0001
Figure imgf000243_0001
Table 40. Panel 1.3D
Figure imgf000243_0002
Figure imgf000244_0001
Table 41. Panel 2D
Figure imgf000244_0002
Figure imgf000245_0001
Figure imgf000246_0001
Table 42. Panel 4D
Figure imgf000246_0002
Figure imgf000247_0001
Figure imgf000248_0001
Panel 1.2 Summary Agl 250 The NOV4 gene is expressed at high levels in lung cancer cell lines. Overall, there is a predominant expression pattern that shows higher expression of this gene in cancer cell lines when compared to normal tissues. Specifically, the NOV4 gene is expressed at higher levels in samples derived from colon cancer, ovarian cancer, breast cancer, prostate cancer and melanoma cell lines. Thus, expression ofthe NOV4 gene could be used to distinguish cultured cell lines from normal tissues. In addition, these data indicate that the expression ofthis gene might be associated with these forms of cancer and thus, therapeutic modulation of the NOV4 gene product might be of use in the treatment of these cancers.
Panel 1.3D Summary Ag2432 Expression of the NOV4 gene is limited to the hippocampus (CT=27.6), where it is expressed at high levels. Therefore, expression ofthis gene could be used to distinguish hippocampus from other tissues.
Panel 2D Summary Ag2432 Significant but low expression of the NOV4 gene is detected in normal colon and bladder tissues. Therefore, expression of this gene could be used to distinguish colon and bladder from other tissues.
Panel 4D Summary Agl 250 The NOV4 gene encodes a protein with homology to prohibitins, which are proteins that have been shown to be involved various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging (ref. 1). The NOV4 gene is expressed at moderate levels throughout the samples on this panel (CTs=30.8-33.6). Interestingly, however, this gene is expressed at highest levels in the thymus as well as in activated Thl and Th2 T cells. Given this expression pattern, the NOV4 gene product may play an important role in the normal homeostasis of the thymus and might be associated with the activation process of T cells. Therefore, modulation of this protein by small molecule drugs might be important for controlling T cell activation and could have some benefit for treatment of diseases associated with hyperactive T cells, such as autoimmune disease, delayed type hypersentivity, and other T cell mediated diseases (such as asthma and psoriasis). In addition, the NOV4 gene is also expressed in activated B cells and in a Ramos B cell line. It has been suggested that increased prohibitin expression is associated with and may facilitate B-cell maturation (ref. 2). Thus, modulation ofthis protein by small molecule drugs might be important for controlling B differentiation and the generation of immunoglobulins by B cells and could therefore have some therapeutic benefit in the treatment of hypoglobulinemia.
Panel CNS_neurodegeneration_vl.O Summary Ag2432 Expression of this gene is low/undetectable (CT values >35) among the samples on this panel (data not shown).
References:
1. Coates PJ, Nenutil R, McGregor A, Picksley SM, Crouch DH, Hall PA, Wright EG. Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence. Exp Cell Res 2001 May l;265(2):262-73
The two prohibitin proteins, Phbl p and Phb2p(BAP37), have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses The gene promoter sequences contain binding sites for the Myc oncoprotem and overexpression of Myc induces expression of the prohibitins The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging
PMID 11302691
2 Woodlock TJ, Bethlendy G, Segel GB Prohibitin expression is increased in phorbol ester-treated chronic leukemic B-lymphocytes Blood Cells Mol Dis 2001 Jan-Feb,27(l) 27-34
Chronic lymphocytic leukemia (CLL) is characterized by the gradual accumulation of immature B-lymphocytes CLL B-lymphocytes mature to a plasmacytoid phenotype when treated in vitro with phorbol esters CLL B-cell apparent maturation is associated with altered expression of specific plasma membrane and mitochondrial proteins including heightened expression of a 30-kDa heat shock protein 60 (hspόO) analog During our efforts to further characterize this hsp60 analog by mass spectrometry, we detected the mitochondπal protein prohibitin in phorbol-ester-matured CLL B-lymphocytes Prohibitin modulates cell proliferation and inhibits cell cycle traverse m several systems, although few data are available for lymphocytes A twofold increase in prohibitin concentration wasobserved in phorbol-ester- matured compared to resting CLL B-cells as determined by quantitative Western immunoblot analysis A similar increase in prohibitin was observed in phorbol-ester- treated normal human B-lymphocyte populations An antisense oligonucleotide complementary to the 5' coding region of the prohibitin gene blunted the increase in prohibitin protein in phorbol-ester-treated CLL B-cells by 42% These data suggest that increased prohibitin expression is associated with and may facilitate B-cell maturation
PMID 11 162143
NOV5
Expression of gene NOV5 was assessed using the pπmer-probe sets Ag3086 and Ag3797 described in Tables 43 and 44 Results from RTQ-PCR runs are shown in Tables 45, 46 47 48 49, and 50 Table 45. Probe Name Ag3086
Figure imgf000251_0001
Table 41. Panel 1.3D
Figure imgf000251_0002
Figure imgf000252_0001
Table 48. General Screening Panel _vl.4
Figure imgf000252_0002
Figure imgf000253_0001
Figure imgf000254_0001
Table 49. Panel 2.2
Figure imgf000254_0002
Figure imgf000255_0001
Table 50. Panel 4D
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0001
Table 51. Panel 4.1D
Figure imgf000258_0002
Figure imgf000259_0001
Figure imgf000260_0001
Table 52. Panel CNS_Neurodegeneration vl.O
Figure imgf000260_0002
Figure imgf000261_0001
Panel 1.3D Summary Ag3086 The NOV5gene is highly expressed in both fetal and adult liver tissue (CTs = 26) and liver cancer cell lines (CT = 27) The gene is also expressed at moderate to low levels in most of the other tissues in the panel Thus, since the NOV5gene appears to be highly expressed m liver tissue, it could therefore be used to distinguish liver derived tissue from other tissues The NOV5gene product may also be a potential therapeutic treatment of liver disease
Among tissues involved in the central nervous system, the NOV5gene is moderately expressed in the fetal and adult brain, including the adult thalamus, substantia nigra, hippocampus, amygdala and is also expressed at low but significant levels in the cerebellum and cerebral cortex This expression profile suggests that the NOV5gene has functional significance in the CNS The close homologue to the NOV5 gene product, hepatocyte growth factor, has numerous therapeutic applications in the CNS, including prevention of neuronal death in animal models of stroke and ischemia Hepatocyte growth factor has mitogenic activity, crossing the blood bram barrier when disrupted, and thus has potential application as a protein therapeutic to treat brain pathologies when administered directly to the cortico spmal fluid or systemically when the blood brain barriei is disrupted Hepatocyte growth factor like protein is a neurotrophic factor useful in the prevention of motoneuron atrophy upon axotomy Therefore the protein encoded by the NOV5gene may be useful as a therapeutic agent in treating stroke and neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and Huntington's disease The potential role of the NOV5gene or its protein product in bram plasticity and regeneration affords utility in treating brain damage and aging related disorders, such as memory impairment that has hippocampal dysfunction as its primary focus
General_Screening_Panel_l .4 Ag3797 The expression of the NOV5gene in panel 1 4 appears to be highest in a sample derived from a liver cancer cell line (HepG2) (CT = 25 3) In addition there is substantial expression of this gene associated with other liver derived material (adult liver CT=27 2, fetal liver CT=26 5) Thus, the expression of the NOVSgene could be used to distinguish liver derived specimens from other samples. In addition, therapeutic modulation of this gene might be of benefit in the treatment of liver related disorders, such as cirrhosis.
Panel 2.2 Summary Ag3086 The expression of the NOV5gene appears to be highest in a sample derived from a liver cancer specimen (CT=26) and is also significant in a number of samples derived from liver tissue. This result is consistent with what is seen in Panels 1.4 and 2D. In addition there appears to be substantial expression of this gene associated with normal kidney tissue (CT=27.2) when compared to adjacent kidney cancer specimens. Thus, this gene could be used to distinguish liver tissue from non-liver tissue as well as distinguish normal kidney tissue when compared to adjacent kidney cancer. Moreover, therapeutic modulation of the expression of the NOV5gene or function of its product might be of benefit in the treatment of kidney cancer.
Panel 4D Summary Ag3086 The NOV5gene is highly expressed in the thymus (CT =
24), colon (CT = 28.4), and IBD Colitis 2 (CT = 27.2) and is expressed at lower levels in mature T cells. The NOV5gene encodes a putative hepatocyte like growth factor homologue. There are reports that hepatocyte growth factor (HGF) is expressed in the thymus and colon. In the thymus, HGF may promote T cell production and in the colon, overexpression of HGF has been shown to leads to IBD like disease in mice. Therapies designed with the protein encoded for by the NOV5gene could be important in the regulation of T cell development and immune function and be useful in organ transplantation. In addition, blocking the function of the NOV5 gene product could help in the treatment of IBD colitis.
Panel 4. ID Summary Ag3797 Results from two experiments using the same probe and primer set are in very good agreement. In both experiments, highest expression of the NOV5gene is detected in kidney (CT=29, 27.4). Moderate expression is also detected in liver cirrhosis (CT=29.4, 30.7). Moderate to low expression of the gene is detected in many of the tissues in this panel. Thus, expression of the NOV5gene could be used to distinguish those tissues from other tissues.
Panel CNS_Neurodegeneration_vl.O Summary Ag3797 Highest expression of the
NOV5 gene is detected in the occipital cortex of a control patient (CT=31 .3). Moderate to low expression is detected throughought the tissue samples in this panel. Please see panel 1.3 for a discussion of potential utility of this gene with regards to the CNS. References:
1. Korhonen L, Sjoholm TJ, Takei N, Kern MA, Schirmacher P, Castren E, Lindholm D. (2000) Eur J Neurosci. 12:3453-61.
Hepatocyte growth factor-scatter factor (HGF) is expressed in different parts of the nervous system, and has been shown to exhibit neurotrophic activity. Here we show that c- Met, the receptor for HGF, is expressed in developing rat hippocampus, with the highest levels during the first postnatal weeks. To study the function of HGF, hippocampal neurons were prepared from embryonic rats and treated with different HGF concentrations. In these cultures, HGF increased the number of neurons expressing the 28-kDa calcium-binding protein (calbindin D) in a dose-dependent manner. The effect of HGF was larger than that observed with either brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), and cotreatment of the cultures with HGF and the neurotrophins was additive with respect to calbindin D neurons. Besides affecting the number of neurons, HGF significantly increased the degree of sprouting of calbindin D-positive neurons, suggesting an influence on neuronal maturation. BDNF and NT-3 stimulated neurite outgrowth of calbindin D neurons to a much smaller degree. In contrast to calbindin D neurons, HGF did not significantly increase the number of neurons immunoreactive with the neurotransmitter gamma-aminobutyric acid (GABA) in the hippocampal cultures. Immunohistochemical studies showed that c-Met-, calbindin D- and HGF-immunoreactive cells are all present in the dentate gyrus and partly colocalize within neurons. These results show that HGF acts on calbindin D-containing hippocampal neurons and increases their neurite outgrowth, suggesting that HGF plays an important role for the maturation and function of these neurons in the hippocampus.
PMID: 1 1029614
2, Powell EM, Mars WM, Levitt P. (2001) Neuron 30:79-89.
Cortical intemeurons arise from the proliferative zone of the ventral telencephalon, the ganglionic eminence, and migrate into the developing neocortex. The spatial patterns of migratory intemeurons reflect the complementary expression of hepatocyte growth factor/sGatter factor (HGF/SF) and its receptor, MET, in the forebrain. Scatter assays on forebrain explants demonstrate regionally specific motogenic activity due to HGF/SF. In addition, exogenous ligand disrupts normal cell migration. Mice lacking the urokinase-type plasminogen activator receptor (u-PAR), a key component of HGF/SF activation, exhibit deficient scatter activity in the forebrain, abnormal interneuron migration from the ganglionic eminence, and reduced intemeurons in the frontal and parietal cortex. The data suggest that HGF/SF motogenic activity, which is essential for normal development of other organ systems, is a conserved mechanism that regulates trans-telencephalic migration of intemeurons.
PMID: 1 1343646
3. Stella MC, Vercelli A, Repici M, Follenzi A, Comoglio PM. (2001) Mol Biol Cell 12:1341- 52.
Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family ofthe plasminogen-related growth factors (PRGFs). PRGFs are alpha/beta heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target ofthe hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration.
PMID: 11359926
4. Kern MA, Bamborschke S, Nekic M, Schubert D, Rydin C, Lindholm D, Schirmacher P. (2001) Cytokine 14: 170-6.
Hepatocyte growth factor (HGF) and its specific receptor, MET, are expressed in the developing and adult mammalian brain. Recent studies have shown a neurotrophic activity of
HGF in the nervous system. The present study focused on HGF concentrations in the cerebrospinal fluid (CSF) and serum in normal persons and in different central nervous system (CNS) diseases considering blood-CSF barrier (BCB) function. Concentrations of HGF were analyzed using an enzyme-linked immunosorbent assay (ELISA). HGF was present in normal human CSF (346+/-126 pg/ml) representing approximately half of the HGF serum concentrations. The CSF HGF levels were not significantly changed in chronic CNS disease and in aseptic meningitis (41947-71 pg/ml), but significantly increased in patients with bacterial meningitis (6101+/- 5200 pg/ml). The HGF levels in CSF were not influenced by increased serum concentrations in patients with normal or mildly affected BCB function. The results show that HGF is present in normal CSF and does not appear to cross the CSF barrier significantly unless it is severely disrupted. So far, strong increases of HGF concentration in CSF are only present in acute bacterial meningitis. Copyright 2001 Academic Press.
PMID: 1 1396995
5. Hayashi K, Morishita R, Nakagami H, Yoshimura S, Hara A, Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Sakai N. (2001) Gene Ther 8:1167-73.
To develop a novel strategy to prevent delayed neuronal death (DND) following transient occlusion of arteries, the gene of hepatocyte growth factor (HGF), a novel neurotrophic factor, was transfected into the subarachnoid space of gerbils after transient forebrain ischemia. Importantly, transfection of HGF gene into the subarachnoid space prevented DND, accompanied by a significant increase in HGF in the cerebrospinal fluid. Prevention of DND by HGF is due to the inhibition of apoptosis through the blockade of bax translocation from the cytoplasm to the nucleus. HGF gene transfer into the subarachnoid space may provide a new therapeutic strategy for cerebrovascular disease.
PMID: 11509947
6. Tamura S, Sugawara T, Tokoro Y, Taniguchi H, Fukao K, Nakauchi H, Takahama Y. (1998) Scand J Immunol. 47:296-301.
The c-Met oncoprotein is a cell-surface receptor for hepatocyte growth factor (HGF).
Signals through HGF and c-Met have been appreciated for their crucial roles in the development of many cell types, including liver cells. The present study examined whether c-
Met is expressed in the thymus and whether c-Met/HGF signals can regulate T-cell development in the thymus. We have found that mRNA transcripts encoding c-Met are expressed in mouse thymus. The c-Met transcripts were expressed at higher levels in fetal and neonatal thymus than in adult thymus, and were mostly expressed by lymphoid cells rather than by stromal cells. Interestingly, the addition of HGF to fetal thymus organ cultures increased the generation of mature T cells expressing high levels of T-cell antigen receptors. These results indicate that c-Met is expressed in the thymus during early ontogeny, and that c- Met/HGF signals can promote T-cell development.
PMID: 9600310
7. Takayama H, Takagi H, Larochelle WJ, Kapur RP, Merlino G. (2001) Lab Invest. 81 :297-305.
Hepatocyte growth factor/scatter factor (HGF/SF) can stimulate growth of gastrointestinal epithelial cells in vitro; however, the physiological role of HGF/SF in the digestive tract is poorly understood. To elucidate this in vivo function, mice were analyzed in which an HGF/SF transgene was overexpressed throughout the digestive tract. Nearly a third of all HGF/SF transgenic mice in this study (28 of 87) died by 6 months of age as a result of sporadic intestinal obstruction of unknown etiology. Enteric ganglia were not overtly affected, indicating that the pathogenesis of this intestinal lesion was different from that operating in Hirschsprung's disease. Transgenic mice also exhibited a rectal inflammatory bowel disease (D3D) with a high incidence of anoreotal prolapse. Expression of interleukin-2 was decreased in the transgenic colon, indicating that HGF/SF may influence regulation of the local intestinal immune system within the colon. These results suggest that HGF/SF plays an important role in the development of gastrointestinal paresis and chronic intestinal inflammation. HGF/SF transgenic mice may represent a useful model for the study of molecular mechanisms associated with a subset of IBD and intestinal pseudo-obstruction. Moreover, our data identify previously unappreciated side effects that may be encountered when using HGF/SF as a therapeutic agent.
PMID: 11310823
NOV6
Expression of gene NOV6 was assessed using the primer-probe set Ag2439 described in Table 53, Results from RTQ-PCR runs are shown in Tables 54, 55, 56, and 57. Table 53. Probe Name Ag2439
Figure imgf000267_0001
Table 54. Panel 1.3D
Figure imgf000267_0002
Figure imgf000268_0001
Table 55. Panel 2D
Figure imgf000268_0002
Figure imgf000269_0001
Figure imgf000270_0001
Table 56. Panel 4D
Figure imgf000270_0002
Figure imgf000271_0001
Figure imgf000272_0001
Table 57.Panel CNS neurodegeneration vl.O
Figure imgf000272_0002
Figure imgf000273_0001
Panel 1.3D Summary Ag2439 The NOV6 gene is widely expressed across the samples in this panel, with highest expression detected in the hippocampus (CT=28.1). This gene is also expressed at lower levels in the other tissues originating from the central nervous system, including the amygdala, cerebellum, cerebral cortex, substantia nigra, thalamus. and spinal cord. Thus, NOV6 gene expression may be used to distinguish hippocampus from other tissues. Please see CNS_neurodegeneration_panel_vl .0 summary for a discussion of the potential utility of this gene in CNS disorders. Among tissues involved in metabolic function, the NOV6 gene is also expressed in thyroid, adrenal gland, pituitary gland, pancreas, heart (adult and fetal), liver (adult and fetal), and adipose. Interestingly, this gene is more highly expressed in fetal skeletal muscle (CT = 29.4) than in adult skeletal muscle (CT = 33.4). This observation suggests that the NOV6 protein product may enhance muscular growth or development in the fetus and thus may also act in a regenerative capacity in the adult. Therefore, therapeutic modulation ofthe NOV6 gene could be useful in treatment of muscular related disease. More specifically, treatment of weak or dystrophic muscle with the protein encoded by this gene could restore muscle mass or function.
The NOV6 gene is also expressed at higher levels in gastric, colon, melanoma, lung, and colon cancer cell lines than in normal tissues. Therefore, this gene may be used as a marker for gastric cancer, colon cancer, melanoma, lung, and colon cancer cell lines. In addition, therapeutic modulation of the NOV6 gene product might be of use in the treatment of these cancers.
Panel 2D Summary Ag2439 The NOV6 gene is most highly expressed in a sample derived from normal colon (CT=26). However, in general this gene appears to be more highly expressed in cancers than in normal tissues. Specifically, NOV6 gene expression is slightly higher in lung cancer (squamous cell type), gastric cancer, ovarian cancer, a kidney cancer sample and a sample of breast cancer relative to the normal controls. Thus, the expression of this gene could be used to distinguish malignant colon, lung, stomach, ovary and some breast and kidney tissue from normal tissue from these organs. In addition, therapeutic modulation of the NOV6 gene product might be of use in the treatment of these cancers. Panel 4D Summary Ag2439 The N0V6 gene is most highly expressed in ionomycin- treated Ramos B cells (CT = 26). This gene is also expressed at moderate levels in T cells, monocytes, dendritic cells, endothelial cells, smooth muscle cells, and airway epithelial cells both under resting and cytokine-stimulated conditions. Therefore, this gene may be useful as a marker for these resting and activated cells.
Panel CNS_neurodegeneration_vl.0 Summary Ag2439 The NOV6 gene encodes a protein with homology to fatty acid binding protein and is expressed across the brain, although expression appears to be the highest in the hippocampus (Panel 1.3D). This gene does not appear to be differentially expressed in Alzheimer's disease based on the results from panel
CNS_Neurodegeneration_Vl .0, although the transcript is detected at low levels in many ofthe brain samples. Fatty acid binding protein expression is increased in development during axon growth and during the response to injury, probably for the transport of fatty acids for use as membrane components (ref. 1). Therefore, upregulation of the NOV6 gene or its protein product may be beneficial during neurite outgrowth and synaptogenesis in response to neuronal death or injury (Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, stroke, and head/spinal cord trauma).
References:
1 . Liu Y, Molina CA, elcher AA, Longo LD, De Leon M. Expression of DAI 1, a neuronal- injury-induced fatty acid binding protein, coincides with axon growth and neuronal differentiation during central nervous system development. J Neurosci Res 1997 Jun 1 5;48(6);551 -62.
DAI 1 is the first fatty acid binding protein (FABP) for which gene expression has been shown to be upregulated following neuronal injury in the adult peripheral nervous system. To understand better the potential regulatory role(s) of this unique FABP in axonal growth and neuronal differentiation, we undertook a temporal and spatial study of DAI 1 gene expression in the developing rat central nervous system (CNS). Transient upregulation of DAI 1 mRNA and protein levels in CNS tissues were quantified by Northern blot hybridization and Western immunoblot analyses at different developmental ages. Homogenates of embryonic and neonatal cerebral cortex, cerebellum, brain stem, and hippocampal tissues contained 100-fold more DAI 1 mRNA and protein than corresponding adult tissues. Significant increase in DAI 1 mRNA was observed as early as embryonic day (E) 14 in cerebral cortex and cerebellum and El 9 in brain stem and hippocampus. Postnatal levels of DAI 1 remained elevated through postnatal day (P) 10 in cerebral cortex, P14 in brain stem and hippocampus, and P20 in cerebellum. Localization of DAl l-like immunoreactivity to specific CNS tissues, cell types, and intracellular compartments at P9 revealed a spatial pattern of neuronal expression different than that reported for other FABPs. DAI 1 protein was detected in the nucleus, cytoplasm, axons, and dendrites of differentiating neurons in cerebral cortex, hippocampus, Gerebellum, brain stem, spinal cord, and olfactory bulb. The strong association of DAl 1 gene expression with development throughout the CNS suggests that this unique FABP plays an important role in axonal growth and neuronal differentiation in many different neuronal populations.
PMID: 9210525
NOV9
Expression of gene NOV9 was assessed using the primer-probe set Ag2771 described in Table 58. Results from RTQ-PCR runs are shown in Tables 59, 60, 61, and 62.
Table 58. Probe Name Ag2771
Figure imgf000275_0001
Table 59. Panel 1.3D
Figure imgf000275_0002
Figure imgf000276_0001
Table 60. Panel 2D
Figure imgf000277_0001
Figure imgf000278_0001
Table 61. Panel 4D
Figure imgf000278_0002
Figure imgf000279_0001
Figure imgf000280_0001
Table 62. Panel CNS neurodegeneration vl.O
Figure imgf000281_0001
Panel 1.3D Summary A 2771 Expression of the NOV9 gene is highest in normal bladder (CT=27.2). This gene is more highly expressed in colon cancer cell lines relative to normal colon as well as in some lung cancer cell lines relative to normal lung. Thus, expression of this gene could be used to distinguish between colon or lung cancer cell lines and other cell lines. Furthermore, therapeutic inhibition of the NOV9 gene or its protein product, throught the use of antibodies, small molecule or protein drugs, may be effective in the treatment of colon and lung cancers.
The NOV9 gene is expressed at moderate levels throughout CNS, with expression detected in fetal brain, amygdala, cerebellum, hippocampus, substantia nigra, thalamus, cerebral cortex and spinal cord (CTs=29-33). See CNS_neurodegeneration_panel_vl .O summary for potential utility of this gene in CNS disorders.
Among tissues with metabolic function, this gene shows low expression in pancreas, adrenal gland, pituitary gland, and liver (adult and fetal) with higher expression in thyroid, heart (adult and fetal), skeletal muscle (adult and fetal), and adipose. Therefore, the NOV9 gene product may play a role in the pathogenesis and/or treatment of metabolic diseases in any or all of these tissues, including obesity and diabetes.
Panel 2D Summary Ag2771 Highest expression of the NOV9 gene is found in a kidney cancer sample (CT=23). However, this gene is rather ubiquitously expressed at moderate levels in all the tissue samples on this panel. Interestingly, the level of NOV9 gene expression appears to be lower in liver and lung tissues when compared to other organs. In addition, this gene appears to be overexpressed in ovarian Gancers as well as in several colon cancers relative to the normal controls. The expression patterns suggest that this gene is required for the survival and proliferation ofthe majority of cell types.
Panel 4D Summary Ag2771 The NOV9 gene encodes protein with homology to CDC-42-interacting protein 4 and is highly expressed in resting HUVEC endothelial cells (CT=24-25), lung microvascular endothelium, bronchial epithelium, small airway epithelium, coronary artery smooth muscle cells, as well as in mucoepidermoid cells (NCI-H292). Basal expression of this gene in these cells appears to be decreased by various treatments with proinflammatory cytokines, such as IL-1 beta, IL-4, IL-1 , IFN-gamma, and TNF-alpha. CDC-42-interacting protein 4 is a Cdc42 effector protein involved in cytoskeletal organization (ref. 1 -2). Since cytokine-activated cells express lower levels ofthe NOV9 gene, increasing the activity of this gene product may reduce the pro-inflammatory effects of these cytokines. Therefore, the NOV9 gene product may be a useful target for agonistic small molecule therapeutics that increase activity of the protein, and such small molecule drugs may reduce the severity of symptoms of asthma and inflammatory bowel disease.
Panel CNS neurodegeneration vl.O Summary Ag2771 CDC42 has been implicated as a neuronal death effector in Alzheimer's disease and as playing an essential role in cerebellar granule neuron survival (refs. 4-6). Cdc42-interacting protein 4, which is homologous to the NOVO gene, has been identified as a substrate of CDC42 (ref. 2).
Therefore, drugs that inhibit NOV9 gene product activity may be effective in blocking processes downstream of CDC42, such as neuronal death in Alzheimer's disease. Since CDC42 can mediate both desirable (cerebellar neuronal survival) and undesirable (Alzheimer's disease) processes, it is likely that specifically targeting distinct downstream substrates may enable the effective targeting of distinct processes without affeGting other CDC42-mediated processes. The NOV9 gene is expressed in the human brain in all regions examined, including the cerebral cortex, hippocampus, amygdala, cerebellum, substantia nigra, spinal cord and thalamus (see Panel 1.3D). Additionally, this gene is expressed more highly in the hippocampus of some patients with Alzheimer's disease than in normal control brains, indicating a possible pathological role in neurodegenerative brain disease. Therefore, targeting the NOV9 gene product may have utility in selective targeting of undesirable CDC42-mediated processes, such as Alzheimer's disease.
References:
1. Linder S, Hufner K, Wintergerst U, Aepfelbacher M. Microtubule-dependent formation of podosomal adhesion structures in primary human macrophages. J Cell Sci 2000 Dec;l 13 Pt 23:4165-76.
Podosomes are unique actin-rich adhesion structures of monocyte-derived cells such as macrophages and osteoclasts. They clearly differ from other substratum-contacting organelles like focal adhesions in moφhological and functional regards. Formation of podosomes has been shown to be dependent on the small GTPase CDC42Hs and its effector Wiskott-Aldrich syndrome protein (WASp). In this study, we investigated the functional relation between podosomes and the microtubule system in primary human macrophages. We demonstrate that, in contrast to focal adhesions, assembly of podosomes in macrophages and their monocytic precursors is dependent on an intact microtubule system. In contrast, experiments using
Wiskott-Aldrich syndrome (WAS) macrophages indicate that the microtubule system is not reciprocally dependent on podosomes. A potential linker between podosomes and microtubules may be WASp itself, considering that microinjection of the WASp polyproline domain prevents podosome reassembly. This polyproline domain is thought to link WASp to microtubules via CDC42 interacting protein 4 (CIP4). Consistently, macrophages microinjected with CEP4 constructs deficient in either the microtubule- or the WASp-binding domain also fail to reassemble podosomes. In sum, our findings show that microtubules are essential for podosome formation in primary human macrophages and that WASp and CIP4 may be involved in this phenomenon. PMID 11069762
2 Tian L, Nelson DL, Stewart DM Cdc42-ιnteractιng protein 4 mediates binding ofthe Wiskott-Aldrich syndrome protein to microtubules J Biol Chem 2000 Mar 17,275(11) 7854 61
The Wiskott-Aldrich syndrome is an inherited X-linked immunodeficiency characterized by thrombocytopenia, eczema, and a tendency toward lymphoid malignancy Lymphocytes from affected individuals have cytoskeletal abnormalities, and monocytes show impaired motihty The Wiskott-Aldrich syndrome protein (WASP) is a multi domain protein involved in cytoskeletal organization In a two-hybrid scieen, we identified the protein Cdc42-mteractιng protein 4 (CIP 4) as a WASP mteractor CIP4, like WASP, is a Cdc42 effector protein involved in cytoskeletal organization We found that the WASP-CIP4 interaction is mediated by the binding of the Src homology 3 domain of CIP4 to the prolme-πch segment of WASP Cdc42 was not required for this interaction Co-expression of CfP4 and green fluorescent protein-
WASP m COS-7 cells led to the association of WASP with microtubules In vitro experiments showed that CIP4 binds to microtubules via its NH(2) terminus The region of CIP4 responsible for binding to active Cdc42 was localized to ammo acids 383-417, and the mutation I398S abrogated binding Deletion of the Cdc42-bιndmg domain of CfP4 did not affect the colocahzation of WASP with microtubules in vivo We conclude that CIP4 can mediate the association of WASP with microtubules This may facilitate transport of WASP to sites of substrate adhesion in hematopoietic cells
PMID 10713100
3 Aspenstrom P A Cdc42 target protein with homology to the non-kinase domain of FER has a potential role in regulating the actin cytoskeleton Curr Biol 1997 Jul 1 ,7(7) 479-87
BACKGROUND Members of the Rho family of small GTPases have been shown to have a diverse role in cell signalling events They were originally identified as proteins that, by regulating the assembly of the actin cytoskeleton, are important determinants of cell morphology, and have recently been shown to be involved in transcriptional activation by the JNK/SAPK signalling pathway In order to understand the mechanisms underlying the effects of Rho GTPases on these processes, the yeast two-hybrid system has been used to identify proteins that bind to an activated mutant of Cdc42, a Rho-family member. RESULTS: A cDNA encoding a previously unidentified Cdc42 target protein, CIP4, which is 545 amino- acids long and contains an SH3 domain at its Garboxyl terminus, was cloned from a human B- cell library. The amino terminus of CIP4 bears resemblance to the non-kinase domain of the FER and Fes/Fps family of tyrosine kinases. In addition, similarities to a number of proteins with roles in regulating the actin cytoskeleton were noticed. CIP4 binds to activated Cdc42 in vitro and in vivo and overexpression of CIP4 in Swiss 3T3 fibroblasts reduces the amount of stress fibres in these cells. Moreover, coexpression of activated Cdc42 and CIP4 leads to clustering of CIP4 to a large number of foci at the dorsal side of the cells. CONCLUSIONS: CIP4 is a downstream target of activated GTP-bound Cdc42, and is similar in sequence to proteins involved in signalling and cytoskeletal control. Together, these findings suggest that CfP4 may act as a link between Cdc42 signalling and regulation of the actin cytoskeleton.
PMID: 9210375
4. Mota M, Reeder M, Chernoff J, Bazenet CE. Evidence for a role of mixed lineage kinases in neuronal apoptosis. J Neurosci 2001 lul 15;21(14):4949-57
Superior cervical ganglion (SCG) sympathetic neurons die by apoptosis when deprived of nerve growth factor (NGF). It has been shown previously that the induction of apoptosis in these neurons at NGF withdrawal requires both the activity of the small GTP -binding protein Cdc42 and the activation of the c-Iun N-terminal kinase (INK) pathway. The mixed lineage kinase 3 (MLK3) belongs to a family of mitogen-activated protein (MAP) kinase kinase kinases. MLK3 contains a Cdc42/Rac interactive-binding (CRIB) domain and activates both the JNK and the p38 MAP kinase pathways. In this study the role of MLK3 in the induction of apoptosis in sympathetic neurons has been investigated. Overexpression of an active MLK3 induces activation of the JNK pathway and apoptosis in SCG neurons. In addition, overexpression of kinase dead mutants of MLK3 blocks apoptosis as well as c-Jun phosphorylation induced by NGF deprivation. More importantly, MLK3 activity seems to increase by 5 hr after NGF withdrawal in both differentiated PCI 2 cells and SCG neurons. We also show that MLK3 lies downstream of Cdc42 in the neuronal death pathway. Regulation of M LK3 in neurons seems to be dependent on MLK3 activity and possibly on an additional cellular component, but not on its binding to Cdc42. These results suggest that MLK3, or a closely related kinase, is a physiological element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal death. MLK3 therefore could be an interesting therapeutic target in a number of neurodegenerative diseases involving neuronal apoptosis.
PMID: 1 1438570
5. Linseman DA, Laessig T, Meintzer MK, McCIure M, Barth H, Aktories K, Heidenreich KA. An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival. J Biol Chem 2001 Aug 16; [epub ahead of print]
Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. C. difficile toxin B, a specific inhibitor of Rho, Rac and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the anti-apoptotic kinase Akt or its target glycogen synthase kιnase-3beta. The pro-apoptotic effects of toxin B were mimicked by C. sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with C. botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal INK and low p38 MAPK activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JTSIK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.
PMID: 1 1509562
6. Zhu X, Raina AK, Boux H, Simmons ZL, Takeda A, Smith MA.
Activation of oncogenic pathways in degenerating neurons in Alzheimer disease. Int J Dev Neurosci 2000 Jul-Aug; 18(4-5):433-7
A number of recent findings have highlighted the similarities between neurogenesis during development and neurodegeneration during Alzheimer disease. In fact, neuronal populations that are known to degenerate in Alzheimer disease exhibit phenotypic changes characteristic of cells re-entering the cell division cycle. In this study, we extended these findings by investigating components of the cell cycle, known to trigger progression through G I through activation of signal transduction cascades. Specifically, we found that proteins implicated in GI transition, namely Cdc42/Rac, are upregulated in select neuronal populations in cases of Alzheimer disease in comparison to age-matched controls. Importantly, Cdc42/Rac shows considerable overlap with early cytoskeletal abnormalities suggesting that these changes are an extremely proximal event in the pathogenesis ofthe disease. Given the functional role of Cdc42/Rac in various cellular processes known to be perturbed in Alzheimer disease, namely cytoskeletal organization, oxidative balance, and oncogenic signaling, it is likely that increased neuronal Cdc42/Rac is highly significant in relation to the pathogenic process and contributes to neuronal degeneration. In fact, these findings suggest that Alzheimer disease is an oncogenic process.
PMID: 10817927
NOVlOa
Expression of gene NOVlOa was assessed using the primer-probe set Agl674 described in Table 63. Results from RTQ-PCR runs are shown in Tables 64, 65, and 66.
Table 63. Probe Name Agl674
Figure imgf000287_0001
Table 64. Panel CNS 1
Figure imgf000287_0002
Figure imgf000288_0001
Figure imgf000289_0001
Figure imgf000290_0001
Figure imgf000291_0001
Table 66. Panel CNS neurodegeneration vl.O
Figure imgf000291_0002
Figure imgf000292_0001
Panel CNS l/CNS l.l/Panel CNS neurodegeneration vl.O Summary Agl674 The NOVlOa gene encodes a protein with homology to hepsin/plasma transmembrane serine proteases. This gene is more highly expressed in the substantia nigra, globus palladus, BAl 7, BA4, BA9 and cingulate gyrus region of a Parkinson's disease brain than in the control brains. In addition, expression of this gene is 5-fold higher in the substantia nigra and cingulate gyrus o a Parkinson's disease brain than in similar regions from the brains of patients with other neurodegenerative diseases, such as Alzheimer's or Huntington's disease; this observation suggests that overexpression of the NOVl Oa gene in these regions is particular to Parkinson's disease. The substantia nigra is particularly vulnerable in Parkinson's disease, indicating that the NOVl Oa gene product may be a component of the biological dysregulation that contributes to Parkinson's disease pathogenesis. In particular, the NOVlOa gene may be involved in the neuronal death that occurs in the substantia nigra in Parkinson's disease. The NOVlOa gene is also more highly expressed in the superior/inferior temporal cortex, globus palladus, and hippocampus of an Alzheimer's brain when compared to the control brains. This observation suggests that this gene may play a role the neurodegeneration of other diseases in addition to Parkinson's disease, such as Alzheimer's disease. Based on the proposed role of transmembrane proteases such as beta secretase in neurodegenerative disorders, agents that influence the activity of the NOVl Oa gene product may be useful in treating these disorders, especially Parkinson's disease in which this gene appears to be dramatically upregulated.
Example 3. SNP analysis of NOVX clones
SeqCallingTM Technology: cDNA was derived from various human samples representing multiple tissue types, normal and diseased states, physiological states, and developmental states from different donors. Samples were obtained as whole tissue, cell lines, primary cells or tissue cultured primary cells and cell lines. Cells and cell lines may have been treated with biological or chemical agents that regulate gene expression for example, growth factors, chemokines, steroids. The cDNA thus derived was then sequenced using CuraGen's proprietary SeqCalling technology. Sequence traces were evaluated manually and edited for corrections if appropriate. cDNA sequences from all samples were assembled with themselves and with public ESTs using bioinformatics programs to generate CuraGen's human SeqCalling database of SeqCalling assemblies. Each assembly contains one or more overlapping cDNA sequences derived from one or more human samples. Fragments and ESTs were included as components for an assembly when the extent of identity with another component of the assembly was at least 95% over 50 bp. Each assembly can represent a gene and/or its variants such as splice forms and/or single nucleotide polymorphisms (SNPs) and their combinations. Variant sequences are included in this application. A variant sequence can include a single nucleotide polymorphism (SNP). A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP originates as a cDNA. A SNP can arise in several ways. For example, a SNP may be due to a substitution of one nucleotide for another at the polymoφhic site. Such a substitution can be either a transition or a transversion. A SNP can also arise from a deletion of a nucleotide or an insertion of a nucleotide, relative to a reference allele. In this case, the polymoφhic site is a site at which one allele bears a gap with respect to a particular nucleotide in another allele. SNPs occurring within genes may result in an alteration of the amino acid encoded by the gene at the position of the SNP. Intragenic SNPs may also be silent, however, in the case that a codon including a SNP encodes the same amino acid as a result of the redundancy of the genetic code. SNPs occurring outside the region of a gene, or in an intron within a gene, do not result in changes in any amino acid sequence of a protein but may result in altered regulation of the expression pattern for example, alteration in temporal expression, physiological response regulation, cell type expression regulation, intensity of expression, stability of transcribed message
Method of novel SNP Identification: SNPs are identified by analyzing sequence assemblies using CuraGen's proprietary SNPTool algorithm SNPTool identifies variation in assemblies with the following criteria SNPs are not analyzed within 10 base pairs on both ends of an alignment, Window size (number of bases in a view) is 10, The allowed number of mismatches in a window is 2, Minimum SNP base quality (PHRED score) is 23, Minimum numbei of changes to score an SNP is 2/assembly position SNPTool analyzes the assembly and displays SNP positions, associated individual variant sequences in the assembly, the depth of the assembly at that given position, the putative assembly allele frequency, and the SNP sequence variation Sequence traces are then selected and brought into view for manual validation The consensus assembly sequence is imported into CuraTools along with vanant sequence Ghanges to identify potential ammo acid changes resulting from the SNP sequence variation Comprehensive SNP data analysis is then exported into the SNPCallmg database Method of novel SNP Confirmation: SNPs are confirmed employing a validated method know as Pyrosequencing (Pyrosequencing, Westborough, MA) Detailed protocols for Pyrosequencing can be found in Alderborn et al Determination of Single Nucleotide Polymoφhisms by Real-time Pyrophosphate DNA Sequencing (2000) Genome Reseat ch 10, Issue 8, August 1249 1265 In brief, Pyrosequencing is a real time primer extension process of genotyping This protocol takes double-stranded, biotinylated PCR products from genomic DNA samples and binds them to streptavidm beads These beads are then denatured producing single stranded bound DNA SNPs are characterized utilizing a technique based on an indirect bioluminometπc assay of pyrophosphate (PPi) that is released from each dNTP upon DNA chain elongation Following Klenow polymerase-mediated base mcoφoration, PPi is released and used as a substrate, together with adenosme 5'-phosphosulfate (APS), for ATP sulfurylase, which results in the formation of ATP Subsequently, the ATP accomplishes the conversion of luciferin to its oxi-deπvative by the action of luciferase The ensuing light output becomes proportional to the number of added bases, up to about four bases To allow processivity of the method dNTP excess is degraded by apyrase, which is also present in the starting reaction mixture, so that only dNTPs are added to the template during the sequencing The process has been fully automated and adapted to a 96-well format, which allows rapid screening of large SNP panels The DNA and protein sequences for the novel single nucleotide polymoφhic variants are reported Variants are reported individually but any combination of all or a select subset of variants are also included. In addition, the positions of the variant bases and the variant amino acid residues are underlined.
Results Variants are reported individually but any combination of all or a select subset of variants are also included as contemplated NOVX embodiments of the invention.
NOVl a SNP data:
NOVla has two SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOs:l and 2, respectively. The nucleotide sequence of the NOVla variant differs as shown in Table 67.
Figure imgf000295_0001
NOV4 SNP data: NOV4 has four SNP variants, whose variant positions for its nucleotide and amino acid sequences is numbered according to SEQ ID NOs: 13 and 14, respectively. The nucleotide sequence ofthe NOV4 variant differs as shown in Table 68.
Figure imgf000295_0002
NOV7 SNP data:
NOV7 has one SNP variant, whose variant position for its nucleotide and amino acid sequences is numbered according to SEQ ID NOs: 19 and 20, respectively. The nucleotide sequence of the NOV7 variant differs as shown in Table 69.
Figure imgf000296_0001
The SNP at nucleotide 746 has a putative allele frequency of 0.250.
?94 OTHER EMBODIMENTS
Although particular embodiments have been disclosed herein in detail, this has been done by way of example for puφoses of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, which follow In particular, it is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope o the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered to be within the scope of the following claims

Claims

WHAT IS CLAIMED IS:
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) a mature form of an amino acid sequence selected from the group consisting of SEQ 1D N0S:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 21, and 29;
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of the amino acid residues from the amino acid sequence of said mature form;
(c) an amino acid sequence selected from the group consisting SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29; and
(d) a variant of an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of amino acid residues from said amino acid sequence.
2 The polypeptide of claim 1, wherein said polypeptide comprises the amino acid sequence of a naturally-occurring allelic variant of an amino acid sequence selected from the group consisting SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29.
3. The polypeptide of claim 2, wherein said allelic variant comprises an amino acid sequence that is the translation of a nudeic acid sequence differing by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS: l , 3, 5, 1, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, and 28.
4. The polypeptide of claim 1 , wherein the amino acid sequence of said variant comprises a conservative amino acid substitution. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an ammo acid sequence selected from the group consisting of
(a) a mature form of an ammo acid sequence selected from the group consisting of SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29,
(b) a variant of a mature form of an amino acid sequence selected from the group consisting of SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 21, and 29, wherein one or more amino acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15%o of the amino add residues from the ammo acid sequence of said mature form,
(c) an amino acid sequence selected from the group consisting of SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29,
(d) a variant of an ammo acid sequence selected from the group consisting SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29, wherein one or more ammo acid residues in said variant differs from the amino acid sequence of said mature form, provided that said variant differs in no more than 15% of ammo acid residues from said ammo acid sequence,
(e) a nudeic acid fragment encoding at least a portion o a polypeptide comprising an amino acid sequence chosen from the group consisting of SEQ D NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29, or a variant of said polypeptide, wherein one or more ammo acid residues in said variant differs from the ammo acid sequence of said mature form, provided that said vanant differs in no more than 15%) of amino acid residues from said ammo acid sequence, and
(f) a nucleic acid molecule comprising the complement of (a), (b), (c), (d) or (e)
The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally-occurring allelic nucleic acid variant
The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide comprising the amino acid sequence of a naturally-occurring polypeptide variant The nucleic acid molecule of claim 5, wherein the nucleic acid molecule differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of SEQ ID NOS 1 , 3, 5, 7, 9, 1 1 , 13, 15, 11, 19, 21 , 23, 25, and 28
The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of
(a) a nucleotide sequence selected from the group consisting of SEQ ID NOS 1 , 3, 5, 1, 9, 11 , 13, 15, 17, 19, 21, 23, 25, and 28,
(b) a nucleotide sequence differing by one or more nucleotides from a nucleotide sequence selected from the group consisting of SEQ ID NOS 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 28, provided that no more than 20% of the nucleotides differ from said nucleotide sequence,
(c) a nucleic acid fragment of (a), and
(d) a nucleic acid fragment of (b)
The nucleic acid molecule of claim 5, wherein said nudeic acid molecule hybridizes under stringent conditions to a nucleotide sequence chosen from the group consisting SEQ ID NOS 1, 3, 5, 7, 9, 1 1 , 13, 15, 11, 19, 21 , 23, 25, and 28, or a complement of said nucleotide sequence
The nucleic acid molecule of claim 5, wherein the nucleic acid molecule compnses a nucleotide sequence selected from the group consisting of
(a) a first nucleotide sequence comprising a coding sequence differing by one or more nucleotide sequences from a coding sequence encoding said ammo acid sequence, provided that no more than 20%> of the nucleotides in the coding sequence in said first nucleotide sequence differ from said coding sequence,
(b) an isolated second polynucleotide that is a complement of the first polynucleotide, and
(c) a nucleic acid fiagment of (a) or (b)
A vector comprising the nucleic acid molecule of claim 1 1
The vector of claim 12, further comprising a promoter operably-hnked to said nucleic acid molecule
14. A cell comprising the vector of claim 12.
15. An antibody that binds immunospecifically to the polypeptide of claim 1
16. The antibody of claim 15, wheiein said antibody is a monoclonal antibody.
17. The antibody of claim 15, wherein the antibody is a humanized antibody.
18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with an antibody that binds immunospecifically to the polypeptide; and
(c) determining the presence or amount of antibody bound to said polypeptide, thereby determining the presence or amount of polypeptide in said sample.
19. A method for determining the presence or amount of the nucleic acid molecule of claim 5 in a sample, the method comprising:
(a) providing the sample;
(b) contacting the sample with a probe that binds to said nucleic acid molecule; and
(c) determining the presence or amount of the probe bound to said nucleic acid molecule, thereby determining the presence or amount o the nucleic acid molecule in said sample.
20 The method of claim 19 wherein presence or amount ofthe nucleic acid molecule is used as a marker for cell or tissue type.
21. The method of claim 20 wherein the cell or tissue type is cancerous.
22. A method of identifying an agent that binds to a polypeptide of claim 1, the method comprising:
(a) contacting said polypeptide with said agent; and b) determining whether said agent binds to said polypeptide. 23 The method of claim 22 wherein the agent is a cellular receptor or a downstream effector
24 A method for identifying an agent that modulates the expression or activity of the polypeptide of claim 1 , the method compns g
(a) providing a cell expressing said polypeptide,
(b) contacting the cell with said agent, and
(G) determining whether the agent modulates expression or activity of said polypeptide, whereby an alteration in expression or activity of said peptide indicates said agent modulates expression or activity of said polypeptide
25 A method for modulating the activity ofthe polypeptide of claim 1, the method comprising contacting a cell sample expressing the polypeptide of said claim with a compound that binds to said polypeptide in an amount sufficient to modulate the activity ofthe polypeptide
26 A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the polypeptide of claim 1 in an amount sufficient to treat or prevent said NOVX- associated disorder in said subject
27 The method of claim 26 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis
28 The method of claim 26 wherein the disorder is related to cell signal processing and metabolic pathway modulation
29 The method of claim 26, wherein said subject is a human
30 A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the nucleic acid of claim 5 in an amount sufficient to treat or prevent said NOVX- associated disorder in said subject.
31. The method of claim 30 wherein the disorder is selected from the group consisting of cardiomyopathy and atherosclerosis.
32. The method of claim 30 wherein the disorder is related to cell signal processing and metabolic pathway modulation.
33. The method of claim 30, wherein said subject is a human.
34. A method of treating or preventing a NOVX-associated disorder, said method comprising administering to a subject in which such treatment or prevention is desired the antibody of claim 15 in an amount sufficient to treat or prevent said NOVX- associated disorder in said subject.
35. The method of daim 34 wherein the disorder is diabetes.
36. The method of claim 34 wherein the disorder is related to cell signal processing and metabolic pathway modulation,
37. The method of claim 34, wherein the subject is a human.
38. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically-acceptable carrier.
39. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically-acceptable carrier.
40. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically-acceptable carrier.
41. A kit comprising in one or more containers, the pharmaceutical composition of claim
38.
42. A kit comprising in one or more containers, the pharmaceutical composition of claim 39.
43. A kit comprising in one or more containers, the pharmaceutical composition of claim 40.
44. A method for determining the presence of or predisposition to a disease associated with altered levels o the polypeptide of claim 1 in a first mammalian subject, the method comprising:
(a) measuring the level of expression of the polypeptide in a sample from the first mammalian subject; and
(b) comparing the amount of said polypeptide in the sample of step (a) to the amount of the polypeptide present in a control sample from a second mammalian subject known not to have, or not to be predisposed to, said disease; wherein an alteration in the expression level o the polypeptide in the first subject as compared to the control sample indicates the presence of or predisposition to said disease.
45. The method of claim 44 wherein the predisposition is to a cancer.
46. A method for detennining the presence of or predisposition to a disease associated with altered levels of the nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising:
(a) measuring the amount of the nucleic acid in a sample from the first mammalian subject; and
(b) comparing the amount of said nucleic acid in the sample of step (a) to the amount of the nucleic acid present in a control sample from a second mammalian subject known not to have or not be predisposed to, the disease; wherein an alteration in the level of the nucleic acid in the first subject as compared to the control sample indicates the presence of or predisposition to the disease.
47. The method of claim 46 wherein the predisposition is to a cancer.
48. A method of treating a pathological state in a mammal, the method comprising administering to the mammal a polypeptide in an amount that is sufficient to alleviate the pathological state, wherein the polypeptide is a polypeptide having an amino acid sequence at least 95% identical to a polypeptide comprising an amino acid sequence of at least one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, and 29, or a biologically active fragment thereof.
49. A method of treating a pathological state in a mammal, the method comprising administering to the mammal the antibody of claim 15 in an amount sufficient to alleviate the pathological state.
PCT/US2001/032496 2000-10-17 2001-10-17 Proteins and nucleic acids encoding same WO2002033087A2 (en)

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