Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities

Genetic knockout of hormone-sensitive lipase in mice has implicated the presence of other intracellular triacylglycerol (TAG) lipases mediating TAG hydrolysis in adipocytes. Despite intense interest in these TAG lipases, their molecular identities thus far are largely unknown. Sequence data base searches for proteins containing calcium-independent phospholipase A(2) ( iPLA(2)) dual signature nucleotide ((G/A) XGXXG) and lipase (GXSXG) consensus sequence motifs identified a novel subfamily of three putative iPLA(2)/lipase family members designated iPLA(2)epsilon, iPLA(2)zeta, and iPLA(2)eta (previously named adiponutrin, TTS-2.2, and GS2, respectively) of previously unknown catalytic function. Herein we describe the cloning, heterologous expression, and affinity purification of the three human isoforms of this iPLA(2) subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA(2)epsilon, iPLA(2)zeta, and iPLA(2)eta also possess acylglycerol transacylase activity utilizing mono-olein as an acyl donor which, in the presence of mono-olein or diolein acceptors, results in the synthesis of diolein and triolein, respectively. (E)-6-(Bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2- one, a mechanism-based suicide substrate inhibitor of all known iPLA(2)s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC50 = 0.1 - 0.5 muM). Quantitative PCR revealed dramatically increased expression of iPLA(2)epsilon and iPLA(2)zeta transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA(2) isoforms in human SW872 liposarcoma cells. Collectively, these results identify three novel TAG lipases/acylglycerol transacylases that likely participate in TAG hydrolysis and the acyl-CoA independent transacylation of acylglycerols, thereby facilitating energy mobilization and storage in adipocytes.