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.
Highlights
Mutations in the gene encoding comparative gene identification 58 (CGI-58)/␣/ hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues
The mouse CGI-58 cDNA sequence includes this motif as HYVYAD with a histidine residue in position 329 (H329) and aspartate in position 334 (D334); we and others have hypothesized that these amino acids comprise active site residues [10, 11]
Several new lines of evidence provide support for the conclusion that CGI-58 is not an lysophosphatidic acid acyltransferase (LPAAT): 1) in vitro LPAAT assays of cell extracts from SM2-1 (DE3) E. coli expressing recombinant CGI-58, but lacking endogenous plsC, show absence of LPAAT activity; 2) bacterial cell extracts with recombinant GST-CGI-58 lack LPAAT activity when membranes with endogenous plsC are removed by centrifugation; 3) purification of recombinant 12-His-CGI-58 over metal affinity resins yields CGI-58 free of LPAAT activity when E. coli membranes are removed by centrifugation and stringent wash conditions are used prior to elution of protein; and 4) cell-free extracts from yeast fail to show increased LPAAT activity when human CGI-58 (hCGI-58) is expressed
Summary
Mutations in the gene encoding comparative gene identification 58 (CGI-58)/␣/ hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. Mutations in the gene encoding comparative gene identification-58 [CGI-58; ␣/ hydrolase domain 5 (ABHD5)] cause Chanarin-Dorfman syndrome (CDS) [1], a neutral lipid storage disorder characterized by excessive accumulation of triacylglycerols (TAGs) in cells and tissues, including liver, skeletal muscle, intestinal epithelia, leukocytes, keratinocytes, and skin fibroblasts, leading to hepatomegaly, ichthyosis, and mild muscle weakness [2,3,4,5,6,7]. These early observations suggested that CGI-58 plays an important role in TAG homeostasis in multiple tissues.
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