Abstract
Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1-5 as phospholipase A/acyltransferase (PLA/AT)-1-5. However, it was poorly understood whether these proteins were involved in the formation of NAPE in living cells. In the present studies, we first show that COS-7 cells transiently expressing recombinant PLA/AT-1, -2, -4, or -5, and HEK293 cells stably expressing PLA/AT-2 generated significant amounts of [(14)C]NAPE and [(14)C]NAE when cells were metabolically labeled with [(14)C]ethanolamine. Second, as analyzed by liquid chromatography-tandem mass spectrometry, the stable expression of PLA/AT-2 in cells remarkably increased endogenous levels of NAPEs and NAEs with various N-acyl species. Third, when NAPE-hydrolyzing phospholipase D was additionally expressed in PLA/AT-2-expressing cells, accumulating NAPE was efficiently converted to NAE. We also found that PLA/AT-2 was partly responsible for NAPE formation in HeLa cells that endogenously express PLA/AT-2. These results suggest that PLA/AT family proteins may produce NAPEs serving as precursors of bioactive NAEs in vivo.
Highlights
The mammalian enzymes that form N-acylphosphatidylethanolamines (NAPEs), precursors of bioactive N-acylethanolamines, are poorly understood
The ratio of N-acyltransferase activity to phospholipase A1/2 (PLA1/2) activity was largely different among phospholipase A/acyltransferase (PLA/AT)-1–5 (Fig. 2B)
These results indicated that NAPE generated by PLA/AT-2 can be metabolized by NAPE-PLD and that the resultant NAE is degraded by fatty acid amide hydrolase (FAAH) and NAE-hydrolyzing acid amidase (NAAA)
Summary
The mammalian enzymes that form N-acylphosphatidylethanolamines (NAPEs), precursors of bioactive N-acylethanolamines, are poorly understood. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1–5 as phospholipase A/acyltransferase (PLA/AT)-1–5. It was poorly understood whether these proteins were involved in the formation of NAPE in living cells. We show by metabolic labeling with [14C]ethanolamine and liquid chromatography-tandem mass spectrometry (LC-MS/MS) that the expression of PLA/AT family proteins in animal cells significantly increases intracellular levels of NAPE and NAE. These results suggest that PLA/AT proteins may function as NAPE-forming N-acyltransferases in living cells
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