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.