Generation of N-acylphosphatidylethanolamine by members of the phospholipase A/acyltransferase (PLA/AT) family

J Biol Chem. 2012 Sep 14;287(38):31905-19. doi: 10.1074/jbc.M112.368712. Epub 2012 Jul 23.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / chemistry*
  • Animals
  • Base Sequence
  • COS Cells
  • Chlorocebus aethiops
  • Gene Expression Regulation*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Multigene Family
  • Peroxisomes / metabolism
  • Phosphatidylethanolamines / chemistry*
  • Phospholipase D / metabolism
  • Phospholipases A / chemistry*
  • Phospholipids / chemistry
  • RNA Interference
  • Tandem Mass Spectrometry / methods

Substances

  • Phosphatidylethanolamines
  • Phospholipids
  • Acyltransferases
  • Phospholipases A
  • Phospholipase D