Jenkins Christopher M, Mancuso David J, Yan Wei, Sims Harold F, Gibson Beverly, Gross Richard W
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2004 Nov 19;279(47):48968-75. doi: 10.1074/jbc.M407841200. Epub 2004 Sep 10.
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.
在小鼠中对激素敏感性脂肪酶进行基因敲除表明，脂肪细胞中存在其他介导三酰甘油（TAG）水解的细胞内三酰甘油脂肪酶。尽管人们对这些三酰甘油脂肪酶极为关注，但迄今为止它们的分子身份在很大程度上仍不为人所知。通过在序列数据库中搜索含有钙非依赖性磷脂酶A2（iPLA2）双重特征核苷酸（(G/A)XGXXG）和脂肪酶（GXSXG）共有序列基序的蛋白质，鉴定出一个由三个推定的iPLA2/脂肪酶家族成员组成的新亚家族，分别命名为iPLA2ε、iPLA2ζ和iPLA2η（之前分别称为脂肪营养蛋白、TTS-2.2和GS2），其催化功能此前未知。在此，我们描述了该iPLA2亚家族的三种人类同工型在Sf9细胞中的克隆、异源表达和亲和纯化，并证明每种同工型都具有丰富的三酰甘油脂肪酶活性。此外，iPLA2ε、iPLA2ζ和iPLA2η还具有以单油精作为酰基供体的酰基甘油转酰基酶活性，在存在单油精或二油精受体的情况下，分别导致二油精和三油精的合成。(E)-6-(溴亚甲基)-3-(1-萘基)-2H-四氢吡喃-2-酮是所有已知iPLA2的基于机制的自杀底物抑制剂，它对三种同工型的甘油三酯脂肪酶活性的抑制作用相似（IC50 = 0.1 - 0.5微摩尔）。定量PCR显示，在3T3-L1细胞激素诱导分化为脂肪细胞的过程中，iPLA2ε和iPLA2ζ转录本的表达显著增加，并确定在人SW872脂肪肉瘤细胞中存在所有三种iPLA2同工型。总体而言，这些结果鉴定出三种新的三酰甘油脂肪酶/酰基甘油转酰基酶，它们可能参与三酰甘油的水解以及酰基甘油的酰基辅酶A非依赖性转酰基作用，从而促进脂肪细胞中的能量动员和储存。
