Extended Data Fig. 7: Esterification position in BMP confers resistance to hydrolysis in cells.
From: PLA2G15 is a BMP hydrolase and its targeting ameliorates lysosomal disease
a, Generation and sequence validation of PLA2G15 KO on a background of CLN5 KO HEK293T cells (DKO). b, BMP signal is diminished in CLN5 PLA2G15 double KO HEK293T cells compared to wildtype. Fold changes of normalized BMP 18:1/18:1 abundance. Data are mean ± s.d. Statistical analysis was performed using two-tailed unpaired t-tests. ****p = 1.72×10−8. c, Supplementation of synthesized positional isomers to HEK293T cells demonstrates 2,2’ BMP as the major BMP form in cells. Representative smoothened graphs of the extracted ion chromatograms following addition of 2,2’ and 3,3’ BMP standards to CLN5 PLA2G15 double KO HEK293T cells. The retention times are shown in the list mode (left) while the alignment of standards in overlaid mode (right) indicate 2,2’ BMP as the predominant endogenous form. d, Representative images of labeled PLA2G15 wildtype and mutant proteins (Alexa488) and lysosomes (Lysotracker) show successful uptake and trafficking in CLN5 PLA2G15 KO HEK293T cells. In the merged images, green and magenta represents the protein and Lysotracker channel respectively and white represents the colocalized spots. Scale bar = 5 µm. Each experiment was repeated three times. e, Curve represents intensity showing that labeled proteins (green) colocalizes with Lysotracker (red) as marker for the lysosome. f, Exogenous BMP feeding to cells was optimized for pulse-chase experiments. Normalized BMP abundance was measured following addition of 3,3’ BMP at indicated time points. Data are mean ± s.d. of n = 3 independent replicates. g, PLA2G15 WT hydrolyzes BMP in cells while cells supplemented with inactive PLA2G15 S198A exhibit reduced BMP hydrolysis capacity. 100 nM PLA2G15 proteins were supplemented in the chase period after delivery of 10 µM 2,2’ S,S BMP for two hours. h-j, Turnover of 3,3’ S,S BMP is rapid while 2,2’ BMP may require positional isomerization for efficient degradation in cells. h, The intensity of each BMP peak (2,2’ BMP peak; left, 2,3’ BMP peak; middle, 3,3’ BMP peak; right) was quantified following a 4-hour chase by PLA2G15 proteins or no enzyme control following addition of either 10 µM of 2,2’ S,S BMP (top) or 3,3’ S,S BMP (bottom). PLA2G15 quickly hydrolyzes 3,3’ and 2,3’ BMP after 3,3’ BMP supplementation while 2,2’ BMP is converted to 2,3’ and 3,3’ BMP. Data are mean ± s.d. of n = 3 independent replicates. i, Quantitation of endogenous BMP peaks during pulse-chase experiment in cells that were not supplemented with BMP in (h). These levels are minimal compared to cells supplemented with the lipids. Data are mean ± s.d. of n = 3 independent replicates. Statistical analysis was performed using two-tailed unpaired t-tests. ***p = 0.0003 and ****p < 0.0001. j, Quantitation of endogenous BMP peaks in cells that were not supplemented with BMP during pulse-chase experiment (Fig. 4d–f) using equimolar mixture of 2,2’ BMP and 3,3’ BMP. Data are mean ± s.d. of n = 3 independent replicates. Statistical analysis was performed using two-tailed unpaired t-tests. **p = 0.0011 and ***p < 0.001. k, BMP stereoisomers are degraded similarly in cells. Same experiment as in (h) using S,S BMP (left) and S,R BMP (right) to measure total BMP levels during chase. Data are mean ± s.d. of n = 3 independent replicates.
