Expression, purification and characterization of a human serine-dependent phospholipase A2 with high specificity for oxidized phospholipids and platelet activating factor.
Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.
Rice
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Department of Biopharmaceutical Research U.K., SmithKline Beecham Pharmaceuticals, New Frontiers Science Park North, Harlow, Essex, CM19 5AW, U.K.
Southan
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Boyd
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Terrett
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JA
MacPhee
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Moores
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Gloger
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Tew
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D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D017173:Caenorhabditis elegans; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D016384:Consensus Sequence; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010084:Oxidation-Reduction; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection
D010743:Phospholipids; D010972:Platelet Activating Factor; D011994:Recombinant Proteins; D010741:Phospholipases A; D054467:Phospholipases A2