Fig. 3.

PLA2G4D acylates mono- and diacylglycerol. (A) TLC analysis of m- and hPLA2G4D-derived reaction products incubated with rac-18:1 MAG (1 mM) in the absence and presence of 1 mM CaCl₂. The mS370A mutant was used as negative control. (B, C) Densitometric quantification of m- and hPLA2G4D-derived reaction products shown in (A). Data is presented as the relative ratio of products. (D) Schematic representation of MAG hydrolysis and transacylation reactions. (E) Time-dependent formation of glycerol and FFAs in the presence of mPLA2G4D using rac-18:1 MAG (1 mM) as substrate. The release of glycerol and FFAs was quantified using commercial kits. Linear regression was used to calculate the specific MMAT activity of the enzyme, based on the difference between glycerol and FFA release (insert). The dotted line indicates the 95% confidence interval (CI), and R² shows the coefficient of determination. (F) mPLA2G4D-derived reaction products using an equimolar substrate mixture of 2–18:1 MAG and 2-20:4 MAG (0.5 mM each). Assay was performed in the absence of 1 mM CaCl₂, and lipids were analyzed by HPLC-MS. DAG subspecies are labeled according to their acyl-chain composition. (G) Relative abundance of 18:1 and 20:4 acyl chains in total DAG, calculated from data shown in (F). (H) TLC analysis of mPLA2G4D-derived reaction products incubated with rac-18:1 MAG or 2-18:1 MAG (0.5 mM each) in the absence of CaCl₂. (I) Total DAG levels calculated as sum of sn-1,2 and sn-1,3-DAG band intensities shown in (H). (J) Abundance of sn-1,2 and sn-1,3-DAG isomers, based on band intensities shown in (H). (K–P) PLA2G4D-derived reaction products using an equimolar substrate mixture (0.5 mM each) of rac-18:1 MAG and sn-1-16:0-sn-2-20:4 PE, or rac-18:1 DAG and sn-1-16:0-sn-2-20:4 PE, respectively. Assays were performed in the absence of CaCl₂ and lipids were analyzed by HPLC-MS. All enzyme activity assays were carried out under conditions described in Figure 1 (n = 3). Data are presented as mean ± SD. Statistical comparisons in (F, K, L, N, O) were performed with one-way ANOVA followed by Bonferroni posthoc analysis and in (G, I, J) with unpaired two-tailed Student’s t test. Statistically significant differences are shown as: ∗P < 0.05; ∗∗P < 0.01; and ∗∗∗P < 0.001. DAG, diacylglycerol; MAG, monoacylglycerol; PE, phosphatidylethanolamine; PLA2G4, phospholipase A2 group IV; TAG, triacylglycerol.