Fig. 3: Members of the pancreatic lipase family catalyze extracellular hemi-BMP and BMP synthesis. | Nature Communications

Fig. 3: Members of the pancreatic lipase family catalyze extracellular hemi-BMP and BMP synthesis.

From: Functionally overlapping intra- and extralysosomal pathways promote bis(monoacylglycero)phosphate synthesis in mammalian cells

Fig. 3

A Hemi-BMP formation in DMEM/10% FBS, DMEM/10% heat-inactivated FBS (hiFBS), and DMEM/10%FBS/40 µM Orlistat (n = 3 biological replicates). The reaction mix contained 50 µM di-oleoyl PG and samples were incubated for 4 h at 37 °C in a CO2 incubator. B Screening for hemi-BMP synthase activity using supernatants of Expi293F cells expressing mouse lipid hydrolases/transacylases (n = 1 biological replicate). The reaction mix (100 µl total) contained 20 µl conditioned supernatant, 0.32 mM di-oleoyl PG substrate, 1% FA-free BSA, and 33 mM bis-tris propane buffer (pH 7.4). The insert shows hemi-BMP synthase activity detected in supernatants of cells expressing wild-type LIPG and its catalytically inactive S169A variant (n = 3 biological replicates). C Representative TLC showing hemi-BMP synthase activity of PNLIP family members in the presence of PG. Note that BMP signals were barely visible. The reaction mix (80 µl total) contained 70 µl conditioned supernatant, 0.125 mM substrate, 1% FA-free BSA and 20 mM HEPES (pH 7.4). D Densitometric analysis of hemi-BMP formation shown in C (n = 3 biological replicates). E, F Hemi-BMP and BMP synthase activity of PNLIP family members shown in C was confirmed by LC-MS-based analysis of reaction products (n = 3 biological replicates). G Representative TLC showing hemi-BMP hydrolase activity of PNLIP family members. The reaction mix (40 µl total) contained 20 µl conditioned supernatant, 0.5 mM trioleoyl hemi-BMP substrate, 1% FA-free BSA, and 20 mM HEPES (pH 7.4). H Densitometric analysis of BMP and PG formation shown in G (n = 3 biological replicates). I, J Extracellular and K, L cell-associated hemi-BMP and BMP content of HEK293 cells overexpressing indicated members of the PNLIP family after 4 h of PG supplementation (50 µM). Experiments shown in I–L were performed in DMEM medium containing 10% heat-inactivated FBS (n = 3 biological replicates). Data are presented as mean ± SD. Statistically significant differences were determined by one-way ANOVA, followed by corrections for multiple comparisons versus control using Bonferroni post hoc test. Source data are provided as a Source Data file.

Back to article page