High specificity of human secretory class II phospholipase A(2) for phosphatidic acid

Lysophosphatidic acid (LPA) is a potent lipid second messenger which stimulates platelet aggregation, cell proliferation and smooth-muscle contraction. The phospholipase A(2) (PLA(2))-catalysed hydrolysis of phosphatidic acid (PA) is thought to be a primary synthetic route for LPA. Of the multiple forms of PLA, present in human tissues, human secretory class-II PLA(2) (hs-PLA(2)) has been implicated in the production of LPA from platelets and whole blood cells challenged with inflammatory stimuli. To explore further the possibility that hs-PLA(2) is involved in the production of LPA, we rigorously measured the phospholipid head group specificity of hs-PLA(2) by a novel PLA(2) kinetic system using polymerized mixed liposomes. Kinetic analysis of recombinant hs-PLA(2) demonstrates that hs-PLA(2) strongly prefers PA as substrate over other phospholipids found in the mammalian plasma membrane including phosphatidylserine (PS), phosphatidylcholine (PC) and pho phosphatidylethanolamine (PE). The order of preference is PA much greater than PE approximate to PS > PC. To identify amino acid residues of hs-PLA(2) that are involved in its unique substrate specificity, we mutated two residues, Glu-56 and Lys-69, which were shown to interact with the phospholipid head group in the X-ray-crystallographic structure of the hs-PLA(2)-transition-state-analogue complex. The K69Y mutant showed selective inactivation toward PA whereas the E56K mutant displayed a most pronounced inactivation to PE. Thus it appears that Lys-69 is at least partially involved in the PA specificity of hs-PLA(2) and Glu-56 in the distinction between PE and PC. In conjunction with a recent cell study [Fourcade, Simon, Viode, Rugani, Leballe, Ragab, Fournie, Sarda and Chap (1995) Cell 80, 919-927], these studies suggest that hs-PLA(2) can rapidly hydrolyse PA molecules exposed to the outer layer of cell-derived microvesicles and thereby produce LPA.