Abstract
Recently, oxidized phospholipid species have emerged as important signaling lipids in activated immune cells and platelets. The canonical pathway for the synthesis of oxidized phospholipids is through the release of arachidonic acid by cytosolic phospholipase A2α (cPLA2α) followed by its enzymatic oxidation, activation of the carboxylate anion by acyl-CoA synthetase(s), and re-esterification to the sn-2 position by sn-2 acyltransferase activity (i.e. the Lands cycle). However, recent studies have demonstrated the unanticipated significance of sn-1 hydrolysis of arachidonoyl-containing choline and ethanolamine glycerophospholipids by other phospholipases to generate the corresponding 2-arachidonoyl-lysolipids. Herein, we identified a pathway for oxidized phospholipid synthesis comprising sequential sn-1 hydrolysis by a phospholipase A1 (e.g. by patatin-like phospholipase domain-containing 8 (PNPLA8)), direct enzymatic oxidation of the resultant 2-arachidonoyl-lysophospholipids, and the esterification of oxidized 2-arachidonoyl-lysophospholipids by acyl-CoA-dependent sn-1 acyltransferase(s). To circumvent ambiguities associated with acyl migration or hydrolysis, we developed a synthesis for optically active (d- and l-enantiomers) nonhydrolyzable analogs of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC). sn-1 acyltransferase activity in murine liver microsomes stereospecifically and preferentially utilized the naturally occurring l-enantiomer of the ether analog of lysophosphatidylcholine. Next, we demonstrated the high selectivity of the sn-1 acyltransferase activity for saturated acyl-CoA species. Importantly, we established that 2-15-hydroxyeicosatetraenoic acid (HETE) ether-LPC sn-1 esterification is markedly activated by thrombin treatment of murine platelets to generate oxidized PC. Collectively, these findings demonstrate the enantiomeric specificity and saturated acyl-CoA selectivity of microsomal sn-1 acyltransferase(s) and reveal its participation in a previously uncharacterized pathway for the synthesis of oxidized phospholipids with cell-signaling properties.
Highlights
Oxidized phospholipid species have emerged as important signaling lipids in activated immune cells and platelets
To circumvent ambiguities associated with acyl migration or hydrolysis, we developed a synthesis for optically active (D- and L-enantiomers) nonhydrolyzable analogs of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC). sn-1 acyltransferase activity in murine liver microsomes stereospecifically and preferentially utilized the naturally occurring L-enantiomer of the ether analog of lysophosphatidylcholine
The results showed that 1-stearoyl-2arachidonoyl-sn-PC-d9 (18:0/20:4-PC-d9) was synthesized from 2-AA-LPC-d9 by hepatic microsomal sn-1 acyltransferase activity, whereas 18:0/15-hydroxyeicosatetraenoic acid (HETE)-PC-d9 was synthesized from 2–15-HETE-LPC-d9 (Fig. 2A)
Summary
To determine the potential presence of this pathway in mammalian cells, we used a stable isotope labeling approach to determine the metabolic fate of 2-AA-LPC-d9 or 2–15-HETELPC-d9 incubated with murine hepatic microsomes in the presence of stearoyl-CoA. To determine whether the sn-1 acyltransferase activity was enantioselective, we compared the initial rates of acyl-CoA– dependent acylation of D- and L-2-AA-ether-LPCs. Incubation of 10 M L- or D-2-AA-ether-LPC with murine hepatic microsomes in the presence of 10 M stearoyl-CoA demonstrated that the formation of PC from L-2-AA-ether-LPC was approximately 3 times higher than that from D-2-AA-ether-LPC (Fig. 6). Incubation of 10 M L- or D-2-AA-ether-LPC with murine hepatic microsomes in the presence of 10 M stearoyl-CoA demonstrated that the formation of PC from L-2-AA-ether-LPC was approximately 3 times higher than that from D-2-AA-ether-LPC (Fig. 6) These results demonstrate that the acyl-CoA– dependent sn-1 acyltransferase reaction catalyzed by hepatic microsomal acyltransferase(s) is stereoselective for the naturally occurring form of lysophospholipids (i.e. L-lysophosphatidylcholine). Values are the average of four independent preparations ϮS.D
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