title: A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs.
abstract: We report the purification, molecular cloning, and expression of a novel cytosolic calcium-independent phospholipase A2 (iPLA2) from Chinese hamster ovary cells, which lacks extended homology to other phospholipases. iPLA2 is an 85-kDa protein that exists as a multimeric complex of 270-350 kDa with a specific activity of 1 micromol/min/mg. The full-length cDNA clone encodes a 752-amino acid cytoplasmic protein with one lipase motif (GXS465XG) and eight ankyrin repeats. Expression of the cDNA in mammalian cells generates an active 85-kDa protein. Mutagenesis studies show that Ser465 and the ankyrin repeats are required for activity. We demonstrate that iPLA2 selectively hydrolyzes the sn-2 over sn-1 fatty acid by 5-fold for 1,2-dipalmitoyl phosphatidylcholine in a mixed micelle. Moreover, we found the fatty acid preference at the sn-2 position to be highly dependent upon substrate presentation. However, iPLA2 does have a marked preference for 1,2-dipalmitoyl phosphatidic acid presented in a vesicle, generating the lipid second messenger lysophosphatidic acid. Finally the enzyme is able to hydrolyze the acetyl moiety at the sn-2 position of platelet-activating factor.
authors.affiliation: Genetics Institute, Cambridge, Massachusetts 02140, USA.
authors.forename: J
authors.initials: J
authors.lastname: Tang
authors.forename: R W
authors.initials: RW
authors.lastname: Kriz
authors.forename: N
authors.initials: N
authors.lastname: Wolfman
authors.forename: M
authors.initials: M
authors.lastname: Shaffer
authors.forename: J
authors.initials: J
authors.lastname: Seehra
authors.forename: S S
authors.initials: SS
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D016415:Sequence Alignment
chemical_list: D017487:Ankyrins; D007527:Isoenzymes; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium