Extended Data Fig. 5: sn2, sn2’ esterification position of S,S BMP confers resistance to PLA2G15-mediated hydrolysis in vitro.
From: PLA2G15 is a BMP hydrolase and its targeting ameliorates lysosomal disease
a, PLA2G15 efficiently deacylates BMP independent of stereoconfiguration. Hydrolysis of 1 µM BMP stereoisomers by 100 nM recombinant PLA2G15 for 30 s under acidic conditions (pH=5.0). LPG intermediate was measured with LC-MS. Data are mean ± s.d. of n = 3 independent replicates. b, A representative image of the extracted ion chromatogram of 2,2’ and 3,3’ positional BMP isomers used in this paper. Each compound has a minor amount of 2,3’ isomer. c, LPG monitoring shows minimal difference in the activity of PLA2G15 against positional BMP isomers. Same experiment as in (a) using 1 µM of either 3,3’ S,S BMP or 2,2’ S,S BMP as substrate incubated for 30 secs. Data are mean ± s.d. of n = 3 independent replicates. d,e, Time course of BMP hydrolysis shows 2,2’ BMP positional isomer confers resistance to PLA2G15 degradation. Hydrolysis of 20 µM 2,2’ S,S BMP or 3,3’ S,S BMP positional isomers as well as equimolar amount of both by 100 nM recombinant PLA2G15 over 10 h under acidic conditions (pH=5.0). Controls had similar reaction buffers except with no enzyme. BMP hydrolase activity was determined using LC-MS by measuring the abundance of LPG intermediate in (d) and GPG product in (e). Data are mean ± s.d. of n = 3 independent replicates. f, DiffDock of 3,3’ S,S BMP onto AlphaFold human PLA2G15 structure. Catalytic triad residues Ser198, Asp360 and His392 of the active site of PLA2G15 are annotated. g, BMP binds to PLA2G15 S198A at low micromolar concentration. Binding affinity was determined using microscale thermophoresis for 3,3’ S,S BMP. Data presented as mean ± s.e.m. from three measurements. h, Chemical structures of other BMP lipids used in this study. i, Time dependent hydrolysis of BMP stereoisomers confirms S,S stereochemistry of 2,2’ BMP contributes to resistance to PLA2G15-dpendent degradation. Assay as in (d) using indicated BMP isomers over 1 hour under acidic conditions (pH=5.0). Data are mean ± s.d. of n = 3 independent replicates.
