Identification, Cloning, Expression, and Purification of Three Novel Human Calcium-independent Phospholipase A2 Family Members Possessing Triacylglycerol Lipase and Acylglycerol Transacylase Activities

Abstract

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 (GX-SXG) consensus sequence motifs identified a novel subfamily of three putative iPLA 2 /lipase family members designated iPLA 2 ⑀, iPLA 2 , and iPLA 2 (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 ⑀, iPLA 2 , and iPLA 2 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 (IC 50 ‫؍‬ 0.1-0.5 M). Quantitative PCR revealed dramatically increased expression of iPLA 2 ⑀ and iPLA 2 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.Obesity and its associated clinical sequelae (e.g. type 2 diabetes, atherosclerosis, and hypertension) represent the major and most rapidly expanding health epidemic in industrialized nations (1-4). Obesity results from an abnormal increase in white adipose tissue mass, primarily in the form of triglycerides, and in humans is thought to be caused by a complex array of genetic, environmental, and hormonal factors (1, 4). Under conditions of obesity, serum nonesterified fatty acids are elevated, contributing to the accumulation of triglycerides in nonadipose tissues (e.g. hepatic, myocardial, and pancreatic) and to the development of the type 2 metabolic syndrome (5). The combined effects of excess cellular triglycerides, fatty acylCoAs, and free fatty acids are believed to be primary mediators of the lipotoxic effects of obesity that include decreased insulin sensitivity, increased oxidative stress, reduced metabolic capacity, and increased rates of cellular apoptosis in multiple organ systems (5-8).Triacylglycerol/fatty acid recycling is an important mechanism by which adipocytes modulate fatty acyl flux in response to changing metab...

Variant in PNPLA3 is associated with alcoholic liver disease

“…The biochemical function of the PNPLA3 protein is unclear, though it appears to have lipogenic transacetylase activity 12 . The gene is highly expressed in adipocytes and liver 7 , and the protein may have a role in energy homeostasis 13 .…”

Human adipose triglyceride lipase (PNPLA2) is not regulated by obesity and exhibits low in vitro triglyceride hydrolase activity

“…HSL and ATGL glyceride hydrolase activities HSL and ATGL both possess triglyceride hydrolase activity [9,10,17]. We wished to determine the relative contribution of the two enzymes to the in vitro hydrolysis of triolein.…”

“…However, HSL knock-out mice have residual lipolytic activity [4,5], which may be attributed to an additional lipase with high affinity for triglycerides [6,7]. Accordingly, a novel adipocyte-specific lipase termed adipose triglyceride lipase (ATGL, now known as patatin-like phospholipase domain containing 2 [PNPLA2]), the product of Pnpla2, and alternatively designated iPLA 2 ζ or desnutrin, was recently identified using different strategies [8][9][10]. The murine ATGL-protein consists of 486 amino acids, whereas the corresponding human form is a 506-amino acid protein.…”

Obesity and polymorphisms in genes regulating human adipose tissue

“…94 Adiponutrin (ADPN) is a protein expressed at high levels in human adipose tissue and displaying triacylglycerol lipase activity. 95,96 Obese subjects have been reported to have either increased or unchanged levels of abdominal adipose tissue ADPN mRNA levels. 95,97 Two ADPN intronic polymorphisms are associated with obesity.…”