Biosynthesis of N-acylethanolamine and lysophosphatidic acid by glycerophosphodiesterase GDE4 and GDE7

[Speaker] Kazuhito Tsuboi:1,2
[Co-author] Iffat As Rahman:2, Zahir Hussain:2, Ryouhei Yamashita:3, Toru Uyama:2, Yasuhiro Takenouchi:1, Tamotsu Tanaka:3, Yasuo Okamoto:1, Akira Tokumura:4, Natsuo Ueda:2
1:Department of Pharmacology, Kawasaki Medical School, Japan, 2:Department of Biochemistry, Kagawa University School of Medicine, Japan, 3:Institute of Biomedical Sciences, Tokushima University Graduate School, Japan, 4:Faculty of Pharmacy, Yasuda Women's University, Japan

[Background] N-Acylethanolamines are ethanolamides of long-chain fatty acids and include analgesic and anti-inflammatory palmitoylethanolamide, appetite-suppressing oleoylethanolamide, and endocannabinoid anandamide (arachidonoylethanolamide). Lysophosphatidic acids (LPAs) are lysophospholipids exerting physiological actions such as cell proliferation, cancer infiltration, platelet aggregation, and neurite retraction. These lipid mediators are biosynthesized on demand and released to the target cells. Thus, the enzymes responsible for their metabolism may be targets of novel therapeutic drugs. Here, we report two novel lysophospholipase D (lysoPLD)-type enzymes simultaneously generating N-acylethanolamines and LPAs from N-acyl-lysophosphatidylethanolamine (lysoNAPE), which is a lysophospholipid known as an intermediate metabolite in the biosynthetic pathways of N-acylethanolamines. We examined whether two members of the glycerophosphodiesterase (GDE) family have this lysoPLD activity.
[Methods] We overexpressed recombinant GDE4 and GDE7 from human and mouse origins in HEK293 cells. The membrane fractions were allowed to react with various [14C]lysophospholipids, and the products were analyzed by using thin-layer chromatography. The cells were also subjected to lipid extraction, followed by liquid chromatography-tandem mass spectrometry for analyzing N-acylethanolamine and LPA levels. RT-PCR was used to examine the tissue distributions of GDE4 and GDE7 mRNAs in humans and mice.
[Results] GDE4 and GDE7 of both human and mouse origins exhibited the lysoPLD activity towards [14C]lysoNAPE to form N-acylethanolamine and LPA. Both enzymes also hydrolyzed 1-alkenyl analog of lysoNAPE, N-acyl-lysoplasmalogen. In addition, they were active with lysophosphatidylcholine to form LPA. GDE4 activity was increased by 2 mM Mg2+, but not by 1-10 mM Ca2+. In contrast, GDE7 was stimulated by micromolar concentrations of Ca2+, but not by millimolar concentrations of Mg2+. Furthermore, intracellular levels of LPA were increased by GDE4 overexpression, and N-acylethanolamines and LPAs were elevated by GDE7 overexpression, suggesting that the two enzymes function as lysoPLDs in cells. GDE4 and GDE7 mRNAs were expressed in various tissues of humans and mice with higher expressions in brain and testis (GDE4) or in kidney (GDE7).
[Conclusions] We found GDE4 and GDE7 as novel lysoPLDs, which may be involved in the generation of N-acylethanolamines and LPAs.

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