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. 2016 Sep;12(9):669-71.
doi: 10.1038/nchembio.2127. Epub 2016 Jul 11.

A Calcium-Dependent Acyltransferase That Produces N-acyl Phosphatidylethanolamines

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Free PMC article

A Calcium-Dependent Acyltransferase That Produces N-acyl Phosphatidylethanolamines

Yuji Ogura et al. Nat Chem Biol. .
Free PMC article

Abstract

More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells.

Figures

Figure 1
Figure 1
Characterization of calcium-dependent N-acyltransferase (Ca-NAT) activity in mouse brain. (a) Metabolic routes for N-acyl phosphatidylethanolamines (NAPEs) and N-acyl ethanolamines (NAEs). (b) Ca-NAT activity in mouse brain lysates (40 µg protein). Total homogenate (Total) and soluble (Sol.) and membrane (Mem.) brain lysates were incubated with sn-1, sn-2-dipalmitoyl-phosphatidylcholine (DPPC; 250 μM) and sn-1, sn-2-dioleoyl-phosphatidylethanolamine (DOPE, 250 µM) for 1 h at 37 °C with or without CaCl2 (3 mM) and N-C16:0 DOPE was measured. (c) Inhibition of Ca-NAT activity in mouse brain by EDTA and fluorophosphonate (FP) probes. Mouse brain membrane Ca-NAT activity was measured as in part b (+ 3 mM CaCl2) with or without the addition of EDTA (10 µM) or after pre-treatment with MAFP (3 µM), FP probes (1 µM each), or THL (3 µM) for 30 min at 25 °C. Data in parts b and c represent mean values ± s. d. for 3 biological replicates. (d) Correlation of spectral counts for PLA2G4E (determined for each fraction obtained by sucrose gradient (5-40%) centrifugation of detergent-solubilized mouse brain membrane lysates by labeling with FP-biotin (5 µM) followed by avidin enrichment and LC-MS analysis) with Ca-NAT activity of each fraction. A Pearson correlation coefficient of 0.99 was calculated for PLA2G4E. Data represent activity measurements from one experiment representative of two biological replicates.
Figure 2
Figure 2
Recombinant PLA2G4E exhibits Ca-NAT activity and generates NAPEs and NAEs in mammalian cells. (a) Gel-based ABPP analysis demonstrating calcium-enhanced FP-rhodamine labeling of recombinant mouse PLA2G4E in transfected, detergent-solubilized HEK293T cell lysates. See Supplementary Fig. 4 for quantification of FP-labeled PLA2G4E band intensities. (b) Ca-NAT activity of transfected HEK293T cell lysates (2 µg) as measured by N-C16:0 DOPE production in reactions with DPPC (40 μM) and DOPE (75 µM) for 30 min at 37 °C with or without CaCl2 (3 mM) added to the reaction mixture. (c) Comparison of phospholipase vs. NAT activity for recombinant PLA2G4E. PLA2G4E-transfected HEK293T lysates were incubated with the indicated combination of sn-1, sn-2-diheptadecanoyl-phosphatidyl choline (PC; 40 µM), DOPE (PE; 75 µM), and CaCl2 (3 mM) for 30 min at 37 °C. The production of C17:0 fatty acid and N-C17:0 DOPE was measured to determine the relative phospholipase and NAT activities, respectively. (d) Ca-NAT activity of WT- and S420A-PLA2G4E-transfected HEK293T cell lysates (10 µg) as measured in b. (e) Production of 13C16:0-containing NAPEs, GP-NAEs, and NAEs in mock, WT-PLA2G4E, and S420A-PLA2G4E-transfected HEK293T cells. Cells were simultaneously fed 250 μM 13C16-palmitic acid and incubated in the presence or absence of 2 μM ionomycin for 4 h. Lipids were then extracted and analyzed by LC-MS/MS. For a-e, data represent mean values ± s. d. for 3 biological replicates. For e, **, p < 0.01, *** p < 0.001 by two-sided Student’s t-test for ionomycin-treated vs control (DMSO)-treated WT-PLA2G4E-transfected cells.

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