Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca-dependent N-acyltransferase.

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-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A (cPLAε) was recently identified as a Ca-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLAε function as Ca-NAT. We next purified both mouse recombinant cPLAε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1 mM CaCl and lowered the EC value of Ca >8-fold. Using a PS probe, we showed that cPLAε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLAε with PS in living cells. Finally, we found that the Ca-ionophore ionomycin increased [C]NAPE levels >10-fold in [C]ethanolamine-labeled cPLAε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca-dependent activity and human cPLAε isoforms also functioned as Ca-NAT.