Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase Molecular and cell biology of lipids

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 Ca²⁺-dependent or -independent N-acyltransferases. The e isoform of mouse cytosolic phospholipase A2 (cPLA2e) was recently identified as a Ca²⁺-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA2e function as Ca-NAT. We next purified both mouse recombinant cPLA2e and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca²⁺ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1 mM CaCl2 and lowered the EC50 value of Ca²⁺ >8-fold. Using a PS probe, we showed that cPLA2e largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA2e with PS in living cells. Finally, we found that the Ca²⁺-ionophore ionomycin increased [¹⁴C]NAPE levels >10-fold in [¹⁴C]ethanolamine-labeled cPLA2e-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca²⁺-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca²⁺-dependent activity and human cPLA2e isoforms also functioned as Ca-NAT.