Cytosolic phospholipase A2 epsilon
N-acyltransferase activity
Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca<sup>2+</sup>-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<sup>2+</sup>-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A<sub>2</sub> (cPLA<sub>2</sub>ε) was recently identified as a Ca<sup>2+</sup>-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA<sub>2</sub>ε function as Ca-NAT. We next purified both mouse recombinant cPLA<sub>2</sub>ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca<sup>2+</sup> for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1 mM CaCl<sub>2</sub> and lowered the EC<sub>50</sub> value of Ca<sup>2+</sup> >8-fold. Using a PS probe, we showed that cPLA<sub>2</sub>ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA<sub>2</sub>ε with PS in living cells. Finally, we found that the Ca<sup>2+</sup>-ionophore ionomycin increased [<sup>14</sup>C]NAPE levels >10-fold in [<sup>14</sup>C]ethanolamine-labeled cPLA<sub>2</sub>ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca<sup>2+</sup>-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca<sup>2+</sup>-dependent activity and human cPLA<sub>2</sub>ε isoforms also functioned as Ca-NAT.