Human cyclooxygenase-3 enzyme and uses thereof

Abstract

The present invention provides nucleic acid and polypeptide sequences describing a novel isozyme of the cyclooxygenase enzyme, herein named as human cyclooxygenase-3. The isolated nucleic acid or polypeptide molecule of the invention can be used in detection assays, gene therapy, and screening assays.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule comprising a nucleotide sequence having at least an 80% identity to SEQ ID NO:3 or SEQ ID NO:5, or the complementary sequence thereof.  
     
     
         2 . An isolated nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having at least a 70% identity to SEQ ID NO:4 or SEQ ID NO:6, or the complementary sequence thereof.  
     
     
         3 . An isolated nucleic acid molecule comprising at least 12 sequential bases of SEQ ID NO:3 or SEQ ID NO:5, or the complementary sequence thereof.  
     
     
         4 . The isolated nucleic acid molecule of  claim 2 , comprising a nucleotide sequence encoding a cyclooxygenase-3 enzyme, wherein the enzyme comprises an amino acid sequence having at least about 70% amino acid sequence identity to SEQ ID NO:4 or SEQ ID NO:6, or the complementary sequence thereof.  
     
     
         5 . The isolated nucleic acid molecule of  claim 2 , comprising a nucleotide sequence encoding a cyclooxygenase-3 enzyme wherein the enzyme comprises an amino acid sequence having at least about 80% amino acid sequence identity to SEQ ID NO: 4 or SEQ ID NO:6, or the complementary sequence thereof.  
     
     
         6 . The isolated nucleic acid molecule of  claim 2 , comprising a nucleotide sequence encoding a cyclooxygenase-3 enzyme, wherein the enzyme comprises an amino acid sequence having at least about 90% amino acid sequence identity to SEQ ID NO: 4 or SEQ ID NO:6, or the complementary sequence thereof.  
     
     
         7 . The isolated nucleic acid molecule of  claim 2 , comprising a nucleotide sequence encoding a cyclooxygenase-3 enzyme wherein the enzyme comprises an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6, or the complementary sequence thereof.  
     
     
         8 . The isolated nucleic acid molecule of  claim 2 , comprising a nucleotide sequence encoding a cyclooxygenase-3 enzyme wherein the enzyme comprises an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:11, or the complementary sequence thereof.  
     
     
         9 . The isolated nucleic acid molecule of  claim 2 , comprising a nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5, or the complementary sequence thereof.  
     
     
         10 . The isolated and purified nucleic acid molecule of  claim 2 , comprising a nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:10, or the complementary sequence thereof.  
     
     
         11 . An expression vector comprising the nucleic acid molecule of  claim 1 .  
     
     
         12 . An expression vector comprising the nucleic acid molecule of  claim 2 .  
     
     
         13 . An expression vector comprising the nucleic acid molecule of  claim 3 .  
     
     
         14 . A recombinant host cell comprising the expression vector of  claim 11 .  
     
     
         15 . A recombinant host cell comprising the expression vector of  claim 12 .  
     
     
         16 . A recombinant host cell comprising the expression vector of  claim 13 .  
     
     
         17 . A substantially purified polypeptide comprising an amino acid sequence having at least about 70% identity to SEQ ID NO:4 or SEQ ID NO:6, wherein said polypeptide is capable of converting arachidonic acid to prostaglandin H 2 .  
     
     
         18 . The substantially purified polypeptide of  claim 17  comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:4 or SEQ ID NO:6.  
     
     
         19 . The substantially purified polypeptide of  claim 17  comprising an amino acid sequence having at least about 90% identity to SEQ ID NO:4 or SEQ ID NO:6.  
     
     
         20 . The substantially purified polypeptide of  claim 17  comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.  
     
     
         21 . The substantially purified polypeptide of  claim 17  comprising an amino acid sequence of SEQ ID NO:9 or SEQ ID NO:11.  
     
     
         22 . A method for expressing a human cyclooxygenase-3 protein in a recombinant host cell, comprising the steps of: 
 (a) introducing an expression vector capable of encoding a human cyclooxygenase-3 protein into a cell; and    (b) culturing the cells under conditions that allow expression of the human cyclooxygenase-3 protein from the expression vector.    
     
     
         23 . An antibody that selectively binds to a polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO:4 or SEQ ID NO:6.  
     
     
         24 . A kit comprising a nucleic acid probe that selectively hybridizes to a nucleic acid molecule encoding a polypeptide having at least a 80% sequence identity to SEQ ID NO:4 or SEQ ID NO:6.  
     
     
         25 . A kit comprising an antibody that selectively binds to a polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO:4 or SEQ ID NO:6.  
     
     
         26 . A kit comprising a polypeptide that has an amino acid sequence having at least 70% identity to SEQ ID NO:4 or SEQ ID NO:6, an agent that is more potent at inhibiting human cyclooxygenase-3 activity than either cyclooxygenase-1 or -2, a substrate for cyclooxygenases, and a means to detect the cyclooxygenase activity.  
     
     
         27 . The kit of  claim 26 , wherein the agent that is more potent at inhibiting human cyclooxygenase-3 activity than either cyclooxygenase-1 or -2, is selected from acetaminophen, phenacetin, dipyrone, Aspirin, diclofenac, or ibuprofen.  
     
     
         28 . The kit of  claim 26 , wherein the substrate for cyclooxygenases is arachidonic acid.  
     
     
         29 . The kit of  claim 26 , wherein the substrate for cyclooxygenases comprises arachidonic acid coupled with a reducing agent cosubatrate that is chromagenic, fluorogenic, or capable of generating luminescent when catalyzed by the peroxidase activity of cyclooxygenases.  
     
     
         30 . A method of detecting a nucleic acid molecule of a human cyclooxygenase-3 gene, comprising the steps of: 
 (a) contacting a test sample with a nucleic acid probe that hybridizes under stringent conditions to the nucleic acid molecule encoding a polypeptide comprising an amino acid sequence having at least a 70% identity to SEQ ID NO:4 or SEQ ID NO:6, or the complementary sequence thereof; and    (b) detecting the probe-nucleic acid molecule complex.    
     
     
         31 . A method of detecting a human cyclooxygenase-3 protein, comprising the steps of: 
 (a) contacting a test sample with an antibody that selectively binds to a polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO:4 or SEQ ID NO:6; and    (b) detecting the protein-antibody complex.    
     
     
         32 . A method of determining human cyclooxygenase-3 activity, comprising the steps of: 
 a) incubating a test sample with an agent that is more potent at inhibiting cyclooxygenase-3 than either cyclooxygenase-1 or -2;    b) exposing a substrate for cyclooxygenase to the test sample;    c) determining the cyclooxygenase activity of the test sample and comparing it with that of a control wherein the test sample is only exposed to the substrate for cyclooxygenase but not to the agent that is more potent at inhibiting cyclooxygenase-3 than either cyclooxygenase-1 or -2.    
     
     
         33 . The method of  claim 32 , wherein the agent that is more potent at inhibiting cyclooxygenase-3 than either cyclooxygenase-1 or cyclooxygenase-2 is selected from acetaminophen, phenacetin, dipyrone, Aspirin, diclofenac, or ibuprofen.  
     
     
         34 . The method of  claim 32 , wherein the cyclooxygenase activity of the test sample is determined by the amount of prostanoids produced from the substrate arachidonic acid.  
     
     
         35 . The method of  claim 32 , wherein the cyclooxygenase activity of the test sample is determined by the amount of prostanoids selected from prostaglandin D 2 , prostaglandin E 2 , prostaglandin F 2α , prostaglandin I 2 , or thromboxane A 2 , and the prostanoids are produced from the substrate arachidonic acid.  
     
     
         36 . The method of  claim 32 , wherein the cyclooxygenase activity of the test sample is determined by the amount of oxygen consumption in the presence of the substrate.  
     
     
         37 . The method of  claim 32 , wherein the cyclooxygenase activity of the biological sample is determined by the peroxidase activity associated with the cyclooxygenase.  
     
     
         38 . The method of  claim 37 , wherein the peroxidase activity associated with the cyclooxygenase is determined using a reducing agent cosubstrate that is chromagenic, or fluorogenic.  
     
     
         39 . The method of  claim 38 , wherein the reducing agent cosubatrate is homovanillic acid or N,N,N′,N′-tetramethylphenylenediamine (TMPD).  
     
     
         40 . The method of  claim 37 , wherein the peroxidase activity associated with the cyclooxygenase is determined by a luminescence assay.  
     
     
         41 . The method of  claim 40 , wherein the luminescence assay is a real-time luminescence assay involving luminol reduction.  
     
     
         42 . A method of identifying variations in human cyclooxygenase-3 gene sequences in a sample, comprising the steps of: 
 (a) isolating a human cyclooxygenase-3 polynucleotide from the sample; and    (b) sequencing the human cyclooxygenase-3 polynucleotide to detect alterations in the gene sequence.    
     
     
         43 . A method for reducing human cyclooxygenase-3 levels in a cell, comprising contacting the cell with a therapeutically effective amount of a composition that decreases the expression of a cyclooxygenase-3 in the cell.  
     
     
         44 . The method of  claim 43  wherein the composition comprises an antisense nucleic acid or siRNA molecule specific for a human cyclooxygenase-3 gene and wherein the antisense nucleic acid or siRNA molecule specifically suppresses human cyclooxygenase-3 gene expression.  
     
     
         45 . A method of evaluating the mechanism of action of an analgesic/antipyretic drug in a cell, comprising the steps of: 
 (a) administering to the cell an effective amount of a composition that increases or decreases the expression or activity of a human cyclooxygenase-3 in the cell;    (b) administering to the cell a therapeutically effective amount of the analgesic/antipyretic drug;    (c) measuring a therapeutic effect of the analgesic/antipyretic drug on the cell, and    (d) comparing the therapeutic effect with that of a control.    
     
     
         46 . A method of identifying a compound that alters prostanoid synthesis catalyzed by a human cyclooxygenase-3, comprising the steps of: 
 a) contacting a human cyclooxygenase-3 protein, or a polypeptide comprising an active fragment of the human cyclooxygenase-3 protein, with a test compound and with a substrate for cyclooxygenase;    b) determining the cyclooxygenase activity in step(a), and comparing it with that of a control wherein the human cyclooxygenase-3 protein, or the polypeptide is exposed to the substrate for cyclooxygenase and is not exposed to the test compound.    
     
     
         47 . The method of  claim 46 , wherein the human cyclooxygenase-3 protein or polypeptide is isolated or substantially purified.  
     
     
         48 . The method of  claim 46 , wherein the human cyclooxygenase-3 protein or polypeptide is part of an isolated membrane preparation.  
     
     
         49 . The method of  claim 46 , wherein the human cyclooxygenase-3 protein or polypeptide is expressed from a recombinant host cell.  
     
     
         50 . The method of  claim 46 , wherein the cyclooxygenase activity is determined by the amount of prostanoids produced from the substrate arachidonic acid.  
     
     
         51 . The method of  claim 50 , wherein the cyclooxygenase activity is determined by the amount of prostanoids selected from prostaglandin D 2 , prostaglandin E 2 , prostaglandin F 2α , prostaglandin I 2 , or thromboxane A 2 , and the prostanoids are produced from the substrate arachidonic acid.  
     
     
         52 . The method of  claim 46 , wherein the cyclooxygenase activity of the biological sample is determined by the amount of oxygen consumption in the presence of the substrate for cyclooxygenase.  
     
     
         53 . The method of  claim 46 , wherein the cyclooxygenase activity is determined by the peroxidase activity associated with the cyclooxygenase.  
     
     
         54 . The method of  claim 53 , wherein the peroxidase activity associated with the cyclooxygenase is determined using a reducing agent cosubatrate that is chromagenic, or fluorogenic.  
     
     
         55 . The method of  claim 54 , wherein the reducing agent cosubatrate is homovanillic acid or N,N,N′,N′-tetramethylphenylenediamine.  
     
     
         56 . The method of  claim 53 , wherein the peroxidase activity associated with the cyclooxygenase is determined by a luminescence assay.  
     
     
         57 . The method of  claim 56 , wherein the luminescence assay is a real-time luminescence assay involving luminol reduction.  
     
     
         58 . The method of  claim 46  further comprising the step of testing whether the test compound changes the cyclooxygenase activity of another cyclooxygenase enzyme.  
     
     
         59 . The method of  claim 58 , wherein the other cyclooxygenase is selected from a human cyclooxygenase-1 or a human cyclooxygenase-2.  
     
     
         60 . A method of identifying a compound that binds to a human cyclooxygenase-3 protein, comprising the steps of: 
 (a) incubating a test compound with the human cyclooxygenase-3 protein or an active fragment thereof, and a labeled ligand for the human cyclooxygenase-3 protein;    (b) separating the human cyclooxygenase-3 protein or an active fragment thereof from unbound labeled ligand; and    (c) identifying a compound that inhibits ligand binding to the human cyclooxygenase-3 by a reduction in the amount of labeled ligand binding to the human cyclooxygenase-3 or an active fragment thereof.    
     
     
         61 . The method of  claim 60 , wherein said human cyclooxygenase-3 protein or an active fragment thereof is isolated or substantially purified.  
     
     
         62 . The method of  claim 60 , wherein said human cyclooxygenase-3 protein or an active fragment thereof is part of an isolated membrane preparation.  
     
     
         63 . The method of  claim 60 , wherein said human cyclooxygenase-3 protein or an active fragment thereof is expressed from a recombinant host cell.  
     
     
         64 . The method of  claim 60 , wherein said labeled ligand for the human cyclooxygenase-3 protein is selected from acetaminophen, phenacetin, dipyrone, Aspirin, diclofenac, or ibuprofen.