Bacterial expression and characterization of human secretory class V phospholipase A2

Mammalian secretory class V phospholipase A(2) (PLA(2)) is a newly discovered PLA(2) that is implicated in eicosanoid formation in inflammatory cells. As a first step towards understanding the structure, function and regulation of this PLA(2), we constructed a bacterial expression vector for human secretory class V PLA(2) (hV-PLA(2)), over-expressed and purified the protein, and determined its physical and kinetic properties. When compared with human class IIa enzyme (hIIa-PLA(2)), hV-PLA(2) has several distinct properties. First, hV-PLA(2) can catalyse the hydrolysis of phosphatidylcholine more effectively than hIIa-PLA(2) by two orders of magnitude. Secondly, hV-PLA(2) has much higher binding affinity and activity for compactly packed phosphatidylcholine bilayers than hIIa-PLA(2). Finally, hV-PLA(2) has much reduced thermal stability compared with hIIa-PLA(2). These data suggest that hV-PLA(2) is better suited than hIIa-PLA(2) for acting on the outer cellular membrane and liberating arachidonic acid from membrane phospholipids. Also, the unusually low thermal stability of hV-PLA(2) might contribute to tighter regulation of its activities in extracellular media.