title: Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity.
abstract: 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.
authors.affiliation: Rutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food Science.
authors.forename: Derek
authors.initials: D
authors.lastname: McMahon
authors.affiliation: Rutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food Science.
authors.forename: Anna
authors.initials: A
authors.lastname: Dinh
authors.affiliation: Rutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food Science.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kurz
authors.affiliation: Rutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food Science.
authors.forename: Dharika
authors.initials: D
authors.lastname: Shah
authors.affiliation: Rutgers, The State University of New Jersey, New Brunswick, NJ 08901.
authors.forename: Gil-Soo
authors.initials: GS
authors.lastname: Han
authors.affiliation: Rutgers, The State University of New Jersey, New Brunswick, NJ 08901.
authors.forename: George M
authors.initials: GM
authors.lastname: Carman
authors.affiliation: Rutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food Science.
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Brasaemle
mesh_terms: D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D006801:Humans; D008246:Lysophospholipids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D008246:Lysophospholipids; D011994:Recombinant Proteins; D000217:Acyltransferases; D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; C439972:ABHD5 protein, human; C492986:Abhd5 protein, mouse; C018764:2-acylglycerophosphate acyltransferase; C032881:lysophosphatidic acid
keywords: Chanarin-Dorfman syndrome; adipose triglyceride lipase; neutral lipid storage disorder; phosphatidylinositol 3-phosphate; phosphatidylinositol 5-phosphate