Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 287, Issue 38, Pages 31905-31919Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.368712
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Funding
- Ministry of Education, Culture, Sports, Science, and Technology of Japan
- Japan Society for the Promotion of Science
- Kagawa University Faculty of Medicine Specially Promoted Research Fund
- Fund for Kagawa University Young Scientists
- Ichiro Kanehara Foundation
- Sumitomo Foundation
- Uehara Memorial Foundation
- Suzuken Memorial Foundation
- Grants-in-Aid for Scientific Research [24590355] Funding Source: KAKEN
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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 [C-14]NAPE and [C-14]NAE when cells were metabolically labeled with [C-14]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.
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