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 A2 (iPLA2) dual signature nucleotide ((G/A)XGXXG) and lipase (GXSXG) consensus sequence motifs identified a novel subfamily of three putative iPLA2/lipase family members designated iPLA2epsilon, iPLA2zeta, and iPLA2eta (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 iPLA2 subfamily in Sf9 cells, and we demonstrate that each possesses abundant TAG lipase activity. Moreover, iPLA2epsilon, iPLA2zeta, and iPLA2eta 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 iPLA2s, inhibits the triglyceride lipase activity of each of the three isoforms similarly (IC50=0.1-0.5 microm). Quantitative PCR revealed dramatically increased expression of iPLA2epsilon and iPLA2zeta transcripts during the hormone-induced differentiation of 3T3-L1 cells into adipocytes and identified the presence of all three iPLA2 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.
Jenkins
Christopher M
CM
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mancuso
David J
DJ
Yan
Wei
W
Sims
Harold F
HF
Gibson
Beverly
B
Gross
Richard W
RW
D041721:3T3-L1 Cells; D000217:Acyltransferases / Q000378:metabolism*; D017667:Adipocytes / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002845:Chromatography; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D051048:Diacylglycerol O-Acyltransferase; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D054522:Group VI Phospholipases A2; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D008049:Lipase / Q000378:metabolism*; D050356:Lipid Metabolism; D008080:Liposarcoma / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics*; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011753:Pyrones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013997:Time Factors; D014304:Triolein / Q000494:pharmacology
D018076:DNA, Complementary; D004075:Diglycerides; D008565:Membrane Proteins; D009281:Naphthalenes; D010726:Phosphodiesterase Inhibitors; D020033:Protein Isoforms; D011506:Proteins; D011753:Pyrones; D011994:Recombinant Proteins; C436316:adiponutrin; D014304:Triolein; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D000217:Acyltransferases; C494882:Dgat1 protein, mouse; D051048:Diacylglycerol O-Acyltransferase; D008049:Lipase; C494794:PNPLA2 protein, human; C091246:PNPLA4 protein, human; D010741:Phospholipases A; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; D054467:Phospholipases A2; C516476:Pla2g6 protein, mouse; D002118:Calcium; C013965:diolein