Fig. 1: Accumulation of hemi-BMP and LPG precedes BMP synthesis.
A Di-oleoyl PG supplementation induces BMP formation in the indicated cell lines (n = 3 biological replicates). Cells were incubated in DMEM containing 10% FBS in the presence and absence of PG (50 µM) for 18 h. B Incorporation of head-group labeled di-oleoyl 13C-PG (50 µM) and UL-13C-glucose (4.5 g/l) into BMP stores of COS-7 cells. M/z = 773 and m/z = 776 correspond to unlabeled and labeled di-oleoyl BMP, respectively (n = 3 biological replicates). Cells were incubated for 18 h in DMEM containing 10% FBS under the indicated conditions. C Schematic depiction of BMP synthesis starting from its precursor PG. Acyl group positions and the stereoconfiguration of metabolites are described in the discussion section. D–G Time course of cellular BMP, hemi-BMP, LPG, and PG concentrations in HEK293 cells upon 13C-PG supplementation (50 µM, n = 4 biological replicates). Total (labeled plus unlabeled) as well as 13C-labeled metabolites were determined by LC-MS. Unlabeled hemi-BMP was not detectable under the applied conditions (E). H, I pH-dependent formation of BMP and hemi-BMP using Expi293F cell lysates as a source of enzymatic activity and sn-1-oleoyl-LPG or di-oleoyl-PG as substrate (n = 3 biological replicates). J, K Hemi-BMP and BMP formation by Expi293F lysates using the indicated substrates (n = 3 biological replicates). All phospholipids were esterified with oleic acid. PG-induced formation of hemi-BMP/BMP was detected in the absence and presence of oleoyl-CoA (100 µM). Transacylation reactions depicted in H–K were conducted for a period of 1 h at a temperature of 37 °C. The reaction mix (100 µl total) contained 40 µg cell protein, 0.32 mM phospholipid substrate, 1% FA-free BSA, and 33 mM reaction buffer (dependent on the pH). Data are presented as mean ± SD. Statistically significant differences were determined by two-tailed unpaired multiple t-tests for A without corrections and one-way ANOVA for J and K, corrected for multiple comparisons against control by Bonferroni post hoc test. Source data are provided as a Source Data file.
