Elucidation of proinflammatory roles of human group V phospholipase A(2) by in vitro and in vivo structure-function analysis

Human group V phospholipase A₂ (hVPLA₂) is involved in eicosanoid formation in such inflammatory cells as macrophages and mast cells. In the first part of the work, we found that the steric and electrostatic properties in the catalytic site of hVPLA₂ allow for effective binding and hydrolysis of a bulky cationic choline head group of PC through rigorous mutagenesis. The high specificity of hVPLA₂ implies that hVPLA₂ can hydrolyze the mammalian cellular membranes leading to the production of fatty acids and lysophospholipid and release of LTB₄.; In the second part of work, we showed how hVPLA₂ interacts with cell membranes. hVPLA₂ binds heparin with high affinity and has a well defined heparin-binding site (HBS) that is spatially distinct from its interfacial binding surface (IBS). Our results show that hVPLA₂ acts directly on the outer plasma membranes of neutrophils to induce the cellular production of AA and LTB₄. Independently, the HSPG-mediated internalization and degradation of protein serves as a cell protection mechanism.; The last part of the work examined the signal transduction pathways induced by the addition of hVPLA₂. We found that the addition of hVPLA ₂ to human neutrophils caused a marked increase of cPLA₂ activity. To determine the signal transduction pathways leading to induce cellular responses, pharmacological and immunological analysis of signaling cascade were performed. These results suggest that hVPLA₂-induced neutrophil activation is mediated by a MAPK dependent pathway. To investigate the mechanism by which hVPLA₂ interacts with cell membrane to induce cellular responses, we used a series of well-defined mutant proteins. These studies show that the abilities of the exogenously-added mutant proteins to induce cellular responses correlated with their activities on PC membranes but not with their affinity for heparin or receptor. Taken together, these results indicate that the catalytic activity and the affinity to the PC membranes are major determining factors of hVPLA₂-induced cellular responses in human neutrophils.