Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity

• Main Authors: Derek McMahon, Anna Dinh, Daniel Kurz, Dharika Shah, Gil-Soo Han, George M. Carman, Dawn L. Brasaemle
• Format: Article
• Language: English
• Published: Elsevier 2014-08-01
• Series: Journal of Lipid Research
• Subjects: phosphatidylinositol 3-phosphate; phosphatidylinositol 5-phosphate; adipose triglyceride lipase; Chanarin-Dorfman syndrome; neutral lipid storage disorder
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520353384

Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/β hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli. Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity.