Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.
The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.
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Division of Clinical Pharmacology, Vanderbilt University, Nashville, TN 37232.
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D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000097:blood / Q000235:genetics*; D001091:Arachidonate Lipoxygenases / Q000235:genetics*; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology*; D002460:Cell Line; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D001091:Arachidonate Lipoxygenases; D001092:Arachidonate 12-Lipoxygenase