HIGH SPECIFICITY OF HUMAN SECRETORY CLASS-II PHOSPHOLIPASE A(2) FOR PHOSPHATIDIC-ACID

Lysophosphatidic acid (LPA) is a potent lipid second messenger which s timulates platelet aggregation, cell proliferation and smooth-muscle c ontraction. The phospholipase A(2) (PLA(2))-catalysed hydrolysis of ph osphatidic acid (PA) is thought to be a primary synthetic route for LP A. Of the multiple forms of PLA, present in human tissues, human secre tory class-II PLA(2) (hs-PLA(2)) has been implicated in the production of LPA from platelets and whole blood cells challenged with inflammat ory stimuli. To explore further the possibility that hs-PLA(2) is invo lved 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 ov er other phospholipids found in the mammalian plasma membrane includin g phosphatidylserine (PS), phosphatidylcholine (PC) and pho phosphatid ylethanolamine (PE). The order of preference is PA much greater than P E 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 ph ospholipid head group in the X-ray-crystallographic structure of the h s-PLA(2)-transition-state-analogue complex. The K69Y mutant showed sel ective inactivation toward PA whereas the E56K mutant displayed a most pronounced inactivation to PE. Thus it appears that Lys-69 is at leas t 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 s tudy [Fourcade, Simon, Viode, Rugani, Leballe, Ragab, Fournie, Sarda a nd 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.