Glycerol-3-phosphate acyltransferase 2 preferentially esterifies arachidonyl-CoA to both glycerol-3-phosphate and 1-oleoylglycerol-3-phosphate

CATTÁNEO ER; PELLON-MAISON M; COLEMAN RA; GONZALEZ-BARO MR

Colorado, EEUU

Congreso; 2010 FASEB Summer Research Conferences; 2010

FASEB

Glycerol-3-phosphate acyltransferase 2 preferentially esterifies arachidonyl-CoA to both glycerol-3-phosphate and 1-oleoylglycerol-3-phosphate. Magalí Pellon-Maison1, Elizabeth R. Cattaneo1, Rosalind A. Coleman2 and María R. Gonzalez-Baró1. 1Instituto de Investigaciones Bioquímicas de La Plata (CONICET-UNLP), Argentina, 2University of North Carolina, Chapel Hill, NC 27599   Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first and committed step in de novo glycerolipid synthesis.  In mammals at least four isoforms have been described.  Each isoform is the product of a different gene, and differs in cellular location and kinetic properties.  GPAT1 prefers saturated acyl-CoAs and is resistant to inactivation by N-ethylmaleimide (NEM), whereas GPAT2 is sensitive to NEM.  The mitochondrial isoform GPAT2 is highly expressed in rat testis and its preference for acyl-CoA substrates had not been determined.  We hypothesized that GPAT2 would be involved in the synthesis of triacylglycerols rich in the unsaturated fatty acids that are required for the proper testicular function. GPAT2 cDNA was transiently overexpressed in CHOK1 cells, and GPAT activity was assayed with palmitoyl-CoA, oleoyl-CoA, linoleoyl-CoA and arachidonyl-CoA substrates and compared with activity from cells transfected with GPAT1 or an empty vector control.  Whereas GPAT1 overexpression increased NEM-resistant GPAT activity, especially with palmitoyl-CoA, overexpression of GPAT2 increased NEM-sensitive GPAT activity only when arachidonyl-CoA was used (~3-fold higher than GPAT1-overexpressing and control cells).  When acyl-glycerol-3-phosphate acyltransferase (AGPAT) activity was assayed using 1-oleoyl-lysophosphatidic acid as acyl-group acceptor, AGPAT activity did not increase in cells overexpressing GPAT1, but cells overexpressing GPAT2 increased AGPAT activity ~50% with oleoyl-CoA and 2-fold with arachidonyl-CoA. To determine the function of GPAT2 in incorporating exogenous fatty acid into glycerolipids, CHOK1 cells that overexpressed GPAT2 were incubated with 50 µM oleate, linoleate or arachidonate for 24 h.  GPAT2 enhanced the incorporation of these three fatty acids into triacylglycerol, but not into phospholipid.  Consistently, both testicular GPAT2 expression and arachidonic acid in testicular triacylglycerol increased in 30-day old rats. We conclude that GPAT2 is involved in synthesizing triacylglycerol that contains arachidonic acid, and that GPAT2 expresses both GPAT and AGPAT activities.  Supported by CONICET PIP0527 and R01-DK56598.