Optimized expression and purification of adipose triglyceride lipase improved hydrolytic and transacylation activities in vitro

Abstract

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E . coli , the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro , the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

ATGL is a biosynthetic enzyme for fatty acid esters of hydroxy fatty acids

“…Full-length ATGL was expressed with an N-terminal 6xHis-tag in Expi293F cells (Thermo Fisher Scientific) and purified by affinity chromatography using an Akta pure chromatography system (Cytiva). Mouse ATGL-288 (amino acids 1–288) and ATGL-288(S47A) were immunopurified using the following method: mouse ATGL-288 and mouse ATGL-288(S47A) with an N-terminal 6×His-tag and a C-terminal Strep-tag II were expressed in Escherichia coli Arctic Express cells at 10 °C and purified by two-step affinity chromatography using an Äkta avant 25 chromatography system (Cytiva) 38 . We obtained highly purified WT ATGL (His–mAT288–Strep) and catalytically dead mutant (His–mAT288(S47A)–Strep) ATGL as assessed by Coomassie staining of SDS–polyacrylamide gel electrophoresis gels (Extended Data Fig.…”

“…4a, b ) and comparative gene identification-58 (CGI-58), an ATGL cofactor that increases its basal activity 37 , with 9-HSA and a triglyceride acyl donor, TG(18:1). ATGL-288 is a recently described form of ATGL that retains biological activity which is augmented by CGI-58 and inhibited by ATGLi 38 . We used ATGL-288 for all but one experiment because we can obtain large quantities of this owing to optimization of the purification and expression of this form of ATGL in bacteria 38 .…”

“…ATGL-288 is a recently described form of ATGL that retains biological activity which is augmented by CGI-58 and inhibited by ATGLi 38 . We used ATGL-288 for all but one experiment because we can obtain large quantities of this owing to optimization of the purification and expression of this form of ATGL in bacteria 38 . CGI-58 had no FAHFA biosynthetic activity by itself (Fig.…”

Microsomal triglyceride transfer protein regulates intracellular lipolysis in adipocytes independent of its lipid transfer activity

“…To further validate the interaction between MTP and ATGL, we coexpressed Xpress-tagged human ATGL (hATGL), mouse ATGL (mATGL), or a truncated mATGL variant lacking 198C-terminal amino acids (mATGL 288*) along with either Flag-tagged MTP, CGI58 or empty Flag (eFlag) in HEK-293 T cells. The mATGL 288* has been shown to retain TG hydrolase activity and it interacts with CGI-58 and G0S2 [46][47][48]. These proteins, respectively, enhance and reduce ATGL activity.…”

The role of lipotoxicity in cardiovascular disease

“…This is further confirmed by myocardial lipotoxicity in PPARα-CKO mice. [42] Knocking out downstream genes of PPARα, such as CPT-1, [43,44] ATGL [45][46][47] or DGAT, [48][49][50] also resulted in lipid-mediated cardiac dysfunction in mice. In addition, PPARγ-overexpressing mice had larger lipid droplet sizes in the myocardium.…”