Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity
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 lysophosph...
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doaj-eece68f1eaa14a68b99994364081dc1a2021-04-28T05:59:56ZengElsevierJournal of Lipid Research0022-22752014-08-0155817501761Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activityDerek McMahon0Anna Dinh1Daniel Kurz2Dharika Shah3Gil-Soo Han4George M. Carman5Dawn L. Brasaemle6Rutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food ScienceRutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food ScienceRutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food ScienceRutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food ScienceRutgers, The State University of New Jersey, New Brunswick, NJ 08901Rutgers, The State University of New Jersey, New Brunswick, NJ 08901To whom correspondence should be addressed; Rutgers Center for Lipid Research and Department of Nutritional Sciences and Rutgers Center for Lipid Research and Department of Food Science; To whom correspondence should be addressedMutations 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.http://www.sciencedirect.com/science/article/pii/S0022227520353384phosphatidylinositol 3-phosphatephosphatidylinositol 5-phosphateadipose triglyceride lipaseChanarin-Dorfman syndromeneutral lipid storage disorder |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Derek McMahon Anna Dinh Daniel Kurz Dharika Shah Gil-Soo Han George M. Carman Dawn L. Brasaemle |
spellingShingle |
Derek McMahon Anna Dinh Daniel Kurz Dharika Shah Gil-Soo Han George M. Carman Dawn L. Brasaemle Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity Journal of Lipid Research phosphatidylinositol 3-phosphate phosphatidylinositol 5-phosphate adipose triglyceride lipase Chanarin-Dorfman syndrome neutral lipid storage disorder |
author_facet |
Derek McMahon Anna Dinh Daniel Kurz Dharika Shah Gil-Soo Han George M. Carman Dawn L. Brasaemle |
author_sort |
Derek McMahon |
title |
Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity |
title_short |
Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity |
title_full |
Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity |
title_fullStr |
Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity |
title_full_unstemmed |
Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity |
title_sort |
comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity |
publisher |
Elsevier |
series |
Journal of Lipid Research |
issn |
0022-2275 |
publishDate |
2014-08-01 |
description |
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. |
topic |
phosphatidylinositol 3-phosphate phosphatidylinositol 5-phosphate adipose triglyceride lipase Chanarin-Dorfman syndrome neutral lipid storage disorder |
url |
http://www.sciencedirect.com/science/article/pii/S0022227520353384 |
work_keys_str_mv |
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