Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase

Abstract N -acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N -acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca 2+ -dependent or -independent N -acyltransferases. The e isoform of mouse cytosolic phospholipase A 2 (cPLA 2 e) was recently identified as a Ca 2+ -dependent N -acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA 2 e function as Ca-NAT. We next purified both mouse recombinant cPLA 2 e and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca 2+ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1 mM CaCl 2 and lowered the EC 50 value of Ca 2+ >8-fold. Using a PS probe, we showed that cPLA 2 e largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA 2 e with PS in living cells. Finally, we found that the Ca 2+ -ionophore ionomycin increased [ 14 C]NAPE levels >10-fold in [ 14 C]ethanolamine-labeled cPLA 2 e-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca 2+ -independent N -acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca 2+ -dependent activity and human cPLA 2 e isoforms also functioned as Ca-NAT.