Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase

Proc Natl Acad Sci U S A. 1990 Aug;87(15):5638-42. doi: 10.1073/pnas.87.15.5638.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arachidonate 12-Lipoxygenase / blood
  • Arachidonate 12-Lipoxygenase / genetics*
  • Arachidonate Lipoxygenases / genetics*
  • Base Sequence
  • Blood Platelets / enzymology*
  • Cell Line
  • Cloning, Molecular*
  • Gene Library
  • Humans
  • Leukemia, Erythroblastic, Acute / enzymology*
  • Leukemia, Erythroblastic, Acute / genetics
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Restriction Mapping
  • Sequence Homology, Nucleic Acid

Substances

  • Arachidonate Lipoxygenases
  • Arachidonate 12-Lipoxygenase

Associated data

  • GENBANK/M35418