Isolated and purified 12R-lipoxygenase polypeptide and polynucleic acid encoding the same

Abstract

Isolated and purified lipoxygenase proteins and nucleic acids are described. Particularly, a novel human 12R-lipoxygenase (12R-LO) protein and cDNA are described. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also described, along with methods of producing each. Isolated and purified antibodies to 12R-LO, and methods of producing the same, are also described.

What is claimed is:  
     
         1 . An isolated and purified biologically active vertebrate lipoxygenase polypeptide capable of converting arachidonic acid to 12R-hydroxyeicosatetraenoic acid.  
     
     
         2 . The polypeptide of  claim 1 , wherein the polypeptide comprises a mammalian 12R-LO.  
     
     
         3 . The polypeptide of  claim 2 , wherein the polypeptide comprises a human 12R-LO.  
     
     
         4 . The polypeptide of  claim 3 , wherein the 12R-LO comprises the amino acid sequence of SEQ ID NO:2.  
     
     
         5 . The polypeptide of  claim 1 , modified to be in detectably labeled form.  
     
     
         6 . An isolated and purified antibody capable of specifically binding to the polypeptide of  claim 1 .  
     
     
         7 . The antibody of  claim 6  which is a monoclonal antibody.  
     
     
         8 . The antibody of  claim 6  which is a polyclonal antibody.  
     
     
         9 . A hybridoma cell line which produces the monoclonal antibody of  claim 8 .  
     
     
         10 . An isolated and purified antibody capable of neutralizing the biological activity of the polypeptide of  claim 1 .  
     
     
         11 . The antibody of  claim 10  which is a monoclonal antibody.  
     
     
         12 . The antibody of  claim 10  which is a polyclonal antibody.  
     
     
         13 . A hybridoma cell line which produces the monoclonal antibody of  claim 11 .  
     
     
         14 . An isolated and purified nucleic acid segment comprising an isolated gene encoding a biologically active vertebrate lipoxygenase polypeptide capable of converting arachidonic acid to 12R-hydroxyeicosatetraenoic acid.  
     
     
         15 . The nucleic acid segment of  claim 14 , wherein the isolated gene encodes a mammalian 12R-LO.  
     
     
         16 . The nucleic acid segment of  claim 15 , wherein the isolated gene encodes a human 12R-LO.  
     
     
         17 . The nucleic acid segment of  claim 14 , wherein the isolated gene encodes a 12R-LO comprising the amino acid sequence of SEQ ID NO:2.  
     
     
         18 . The nucleic acid segment of  claim 17 , further defined as comprising the 12R-LO-encoding nucleic acid sequence of SEQ ID NO:1.  
     
     
         19 . The nucleic acid segment of  claim 14 , further defined as a DNA segment.  
     
     
         20 . The nucleic acid segment of  claim 14 , wherein the isolated gene is positioned under the control of a promoter.  
     
     
         21 . The nucleic acid segment of  claim 14 , further defined as a recombinant vector which comprises the isolated gene.  
     
     
         22 . The nucleic acid segment of  claim 21 , wherein the vector is a recombinant expression vector.  
     
     
         23 . The nucleic acid segment of  claim 21 , further defined as a nucleic acid fragment of up to 10,000 basepairs in length.  
     
     
         24 . The nucleic acid segment of  claim 23 , further defined as comprising at least a 1000 nucleotide long contiguous stretch of the nucleic acid sequence of SEQ ID NO:1.  
     
     
         25 . A recombinant host cell comprising the nucleic acid segment of  claim 14 .  
     
     
         26 . The recombinant host cell of  claim 25 , wherein the host cell is a procaryotic cell.  
     
     
         27 . The recombinant host cell of  claim 25 , wherein the host cell is a eukaryotic cell.  
     
     
         28 . A method of preparing a lipoxygenase polypeptide, comprising: transforming a cell with the nucleic acid segment of  claim 14  to produce a lipoxygenase under conditions suitable for the expression of said polypeptide.  
     
     
         29 . A process of detecting in a sample an RNA that encodes the lipoxygenase polypeptide encoded by the nucleic acid of  claim 14 , said process comprising the steps of: 
 (a) contacting said sample under hybridizing conditions with the nucleic acid segment of  claim 14  to form a duplex; and    (b) detecting the presence of said duplex.    
     
     
         30 . A process of producing an antibody immunoreactive with a lipoxygenase polypeptide, the process comprising steps of: 
 (a) transfecting a recombinant host cell with the a nucleic acid segment of  claim 14 , which encodes a lipoxygenase polypeptide;    (b) culturing the host cell under conditions sufficient for expression of the polypeptide;    (c) recovering the polypeptide; and    (d) preparing the antibody to the polypeptide.    
     
     
         31 . The process of  claim 30 , wherein the polypeptide comprises SEQ ID NO:2.  
     
     
         32 . The process of  claim 30 , wherein the nucleic acid segment comprises SEQ ID NO:1.  
     
     
         33 . An antibody produced by the process of  claim 30 .  
     
     
         34 . A process of detecting a lipoxygenase polypeptide, the process comprising immunoreacting the polypeptide with an antibody prepared according the process of  claim 30  to form an antibody-polypeptide conjugate; and detecting the conjugate.  
     
     
         35 . A process of detecting a messenger RNA transcript that encodes a lipoxygenase polypeptide, the process comprising the steps of hybridizing the messenger RNA transcript with the nucleic acid segment of  claim 14  to form a duplex; and detecting the duplex.  
     
     
         36 . A process of detecting a DNA molecule that encodes a lipoxygenase polypeptide, the process comprising the steps of hybridizing DNA molecules with the nucleic acid segment of  claim 14  to form a duplex; and detecting the duplex.  
     
     
         37 . A diagnostic assay kit for detecting the presence of a lipoxygenase polypeptide in a biological sample, the kit comprising a first container containing a first antibody capable of immunoreacting with a lipoxygenase polypeptide of  claim 1 , wherein the first antibody is present in an amount sufficient to perform at least one assay.  
     
     
         38 . The assay kit of  claim 37 , further comprising a second container containing a second antibody that immunoreacts with the first antibody.  
     
     
         39 . The assay kit of  claim 38 , wherein the first antibody and the second antibody comprise monoclonal antibodies.  
     
     
         40 . The assay kit of  claim 38 , wherein the first antibody is affixed to a solid support.  
     
     
         41 . The assay kit of  claim 38 , wherein the first and second antibodies each comprise an indicator.  
     
     
         42 . The assay kit of  claim 41 , wherein the indicator is a radioactive label or an enzyme.  
     
     
         43 . A diagnostic assay kit for detecting the presence, in biological samples, of a lipoxygenase polypeptide, the kit comprising a first container that contains a nucleic acid segment identical or complimentary to a segment of at least ten contiguous nucleotide bases of the nucleic acid segment of  claim 14 .  
     
     
         44 . A diagnostic assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a lipoxygenase polypeptide, the kit comprising a first container containing a lipoxygenase polypeptide of claim  1  that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.  
     
     
         45 . A method of screening candidate substances for their ability to modulate arachidonic acid metabolism, the method comprising the steps of: 
 (a) establishing replicate test and control samples that comprise arachidonic acid and a vertebrate lipoxygenase polypeptide capable of converting arachidonic acid to 12R-hydroxyeicosatetraenoic acid;    (b) administering a candidate substance to test sample but not the control sample;    (c) measuring 12R-hydroxyeicosatetraenoic acid levels in the test and the control samples; and    (d) determining that the candidate substance modulates arachidonic acid metabolism if the 12R-hydroxyeicosatetraenoic acid level measured for the test sample is greater or less than the 12R-hydroxyeicosatetraenoic acid level measured for the control sample.    
     
     
         46 . The method of  claim 45 , wherein the replicate test and control samples further comprise a cell that expresses a vertebrate lipoxygenase polypeptide capable of converting arachidonic acid to 12R-hydroxyeicosatetraenoic acid.  
     
     
         47 . A recombinant cell line suitable for use in the method of  claim 46 .  
     
     
         48 . A method of modulating vertebrate 12R-lipoxygenase polypeptide activity in a vertebrate, the method comprising the step of administering to the vertebrate an effective amount of a substance capable of modulating the 12R-lipoxygenase polypeptide activity in the vertebrate, whereby modulation of the 12R-lipoxygenase polypeptide activity is accomplished.  
     
     
         49 . The method of  claim 48 , wherein the step of administering further comprises administering an effective amount of a substance that modulates expression of a 12R-LO-encoding nucleic acid segment in the vertebrate.  
     
     
         50 . The method of  claim 49 , wherein the substance that modulates expression of a 12R-LO-encoding nucleic acid segment comprises an antisense oligonulceotide.  
     
     
         51 . The method of  claim 48 , wherein the 12R-lipoxygenase polypeptide activity comprises converting arachidonic acid to 12R-hydroxyeicosatetraenoic acid, and wherein the step of administering comprises administering to the vertebrate an effective 12R-LO-modulating amount of a substance capable of modulating 12R-LO metabolism of arachidonic acid to 12R-hydroxyeicosatetraenoic acid.  
     
     
         52 . The method of  claim 48 , wherein the vertebrate is a mammal.  
     
     
         53 . The method of  claim 52 , wherein the mammal is a human.  
     
     
         54 . A method of treating a patient suffering from a disorder associated with 12R-LO activity in the patient, the method comprising the step of administering to the patient an effective amount of a substance capable of modulating the 12R-LO activity in the patient, whereby treatment of the disorder is accomplished.  
     
     
         55 . The method of  claim 54 , wherein the step of administering further comprises administering an effective amount of a substance that modulates expression of a 12R-LO-encoding nucleic acid segment in the patient.  
     
     
         56 . The method of  claim 55 , wherein the substance that modulates expression of a 12R-LO-encoding nucleic acid segment comprises an antisense oligonulceotide.  
     
     
         57 . The method of  claim 54 , wherein the 12R-lipoxygenase polypeptide activity comprises converting arachidonic acid to 12R-hydroxyeicosatetraenoic acid, and wherein the step of administering comprises administering to the vertebrate an effective 12R-LO-modulating amount of a substance capable of modulating 12R-LO metabolism of arachidonic acid to 12R-hydroxyeicosatetraenoic acid.  
     
     
         58 . The method of  claim 54 , wherein the disorder comprises psoriasis.  
     
     
         59 . A transgenic non-human animal having incorporated into its genome a nucleic acid segment comprising an isolated gene encoding a biologically active vertebrate lipoxygenase polypeptide capable of converting arachidonic acid to 12R-hydroxyeicosatetraenoic acid, the nucleic acid segment being present in said genome in a copy number effective to confer expression in the animal of the lipoxygenase polypeptide.  
     
     
         60 . The trangenic non-human animal of  claim 59 , wherein said nucleic acid segment is further defined as a human 12R-LO encoding segment.  
     
     
         61 . The transgenic non-human animal of  claim 60 , wherein the expression of the lipoxygenase polypeptide is conferred in skin tissue of the animal.