| Phospholipases A2 (iPLA2) are a group of enzymes that serve very important roles in cellular signal transduction and lipid metabolism by catalyzing the hydrolysis of acyl ester bonds of membrane phospholipids releasing the lipid second messengers, lysophospholipids and free fatty acid. Previously, a novel human calcium-independent phospholipase A2, termed iPLA2gamma, was expressed in Sf9 cells in crude form. In this work, an N-terminal His-tagged iPLA2gamma was synthesized and purified by cobalt affinity chromatography. Remarkably, analyses of its catalytic regioselective properties using electrospray ionization mass spectrometry (ESI/MS) and tandem mass spectrometry (ESI/MS/MS) uncovered its unusual substrate selectivity that results in the highly selective generation of 2-arachidonoyl lysophosphatidylcholine. The physiologic relevance of this finding was substantiated by the selective accumulation of 2-arachidonoyl LPC during iPLA2gamma-mediated hydrolysis of naturally occurring peroxisomal membranes, a native subcellular compartment where iPLA2gamma is located. Since 2-arachidonyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, 2-archidonoyl glycerol and its COX-2 oxidized derivatives), these results identified a novel eicosanoid selective pathway that amplifies and diversifies the repertoire of biologic responses to cellular stimulation through 1PLA2gamma mediated 2-arachidonoyl LPC production. Another novel putative human iPLA2 member (previously known as GS2 protein) containing dual signature lipase and nucleotide binding motifs was recently identified by sequence database searching on the X chromosome. In this work, the cloning, heterologous expression, and affinity purification of this protein, now termed iPLA2eta, were accomplished, demonstrating that it possesses abundant TAG lipase and acylglycerol transacylase activities, in addition to its intrinsic PLA2 activity. These findings suggest that iPLA2eta may catalyze both TAG hydrolysis (lipase) and TAG synthesis (acyl-CoA independent transacylase) in adipocytes to facilitating energy mobilization and storage through TAG cycling. |
