Mechanism of human group V phospholipase A(2) (PLA(2))-induced leukotrienebiosynthesis in human neutrophils - A potential role of heparan sulfate binding in PLA(2) internalization and degradation

Human group V phospholipase A(2) (hVPLA(2)) has been shown to have high act ivity to elicit leukotriene production in human neutrophils (Han, S. K., Ki m, K. P., Koduri, R., Bittdva, L., Munoz, N. M., Leff, A. R., Wilton, D. C. , Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To dete rmine the mechanism by which hVPLA(2) interacts with cell membranes to indu ce leukotriene formation, we mutated surface cationic residues and a cataly tic residue of hVPLA(2) and measured the interactions Of mutants with model membranes, immobilized heparin, and human neutrophils. These studies showe d that cationic residues, Lys(7), Lys(11), and Arg(34), constitute:a part o f the interfacial binding surface of hVPLA(2), which accounts for its moder ate preference for anionic membranes. Additionally, hVPLA(2) binds heparin with high affinity and has a well defined heparin-binding site. The site is composed of Arg(100), Lys(101), Lys(107), Arg(108), and Arg(111), and is s patially distinct from its interfacial binding surface. Importantly, the ac tivities of the mutants to hydrolyze cell membrane phospholipids and induce leukotriene biosynthesis, when enzymes were added exogenously to neutrophi ls, correlated with their activities on phosphatidylcholine membranes but n ot with their affinities for anionic membranes and heparin. These results i ndicate that hVPLA(2) acts directly on the outer plasma membranes of neutro phils to release fatty acids and lysophospholipids. Further studies suggest that products of hVPLA(2) hydrolysis trigger the cellular leukotriene prod uction by activating cellular enzymes involved in leukotriene formation. Fi nally, the temporal and spatial resolution of exogenously added hVPLA(2) an d mutants suggests that binding to cell surface heparan sulfate proteoglyca ns is important for the internalization and clearance of cell surface-bound hVPLA(2).