Mechanism of human group V phospholipase A2 (PLA2)-induced leukotriene biosynthesis in human neutrophils. A potential role of heparan sulfate binding in PLA2 internalization and degradation.
Human group V phospholipase A(2) (hVPLA(2)) has been shown to have high activity to elicit leukotriene production in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism by which hVPLA(2) interacts with cell membranes to induce leukotriene formation, we mutated surface cationic residues and a catalytic residue of hVPLA(2) and measured the interactions of mutants with model membranes, immobilized heparin, and human neutrophils. These studies showed that cationic residues, Lys(7), Lys(11), and Arg(34), constitute a part of the interfacial binding surface of hVPLA(2), which accounts for its moderate 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 spatially distinct from its interfacial binding surface. Importantly, the activities of the mutants to hydrolyze cell membrane phospholipids and induce leukotriene biosynthesis, when enzymes were added exogenously to neutrophils, correlated with their activities on phosphatidylcholine membranes but not with their affinities for anionic membranes and heparin. These results indicate that hVPLA(2) acts directly on the outer plasma membranes of neutrophils to release fatty acids and lysophospholipids. Further studies suggest that products of hVPLA(2) hydrolysis trigger the cellular leukotriene production by activating cellular enzymes involved in leukotriene formation. Finally, the temporal and spatial resolution of exogenously added hVPLA(2) and mutants suggests that binding to cell surface heparan sulfate proteoglycans is important for the internalization and clearance of cell surface-bound hVPLA(2).
Kim
K P
KP
Department of Chemistry, University of Illinois, Chicago, Illinois 60607, USA.
Rafter
J D
JD
Bittova
L
L
Han
S K
SK
Snitko
Y
Y
Munoz
N M
NM
Leff
A R
AR
Cho
W
W
D001692:Biological Transport; D006493:Heparin; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D015289:Leukotrienes / Q000096:biosynthesis*; D009154:Mutation; D009504:Neutrophils / Q000378:metabolism*; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D015398:Signal Transduction
D006493:Heparin; D006497:Heparan Sulfate; D015289:Leukotrienes; D010741:Phospholipases A; D054467:Phospholipases A2