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

Abstract

Bis(monoacylglycero)phosphate (BMP) is a major phospholipid constituent of intralumenal membranes in late endosomes/lysosomes, where it regulates the degradation and sorting of lipid cargo. Recent observations suggest that the Batten disease-associated protein CLN5 functions as lysosomal BMP synthase. Here, we show that transacylation reactions catalyzed by cytosolic and secreted enzymes enhance BMP synthesis independently of CLN5. The transacylases identified in this study are capable of acylating the precursor lipid phosphatidylglycerol (PG), generating acyl-PG, which is subsequently hydrolyzed to BMP. Extracellularly, acyl-PG and BMP are generated by endothelial lipase in cooperation with other serum enzymes of the pancreatic lipase family. The intracellular acylation of PG is catalyzed by several members of the cytosolic phospholipase A2 group IV (PLA2G4) family. Overexpression of secreted or cytosolic transacylases was sufficient to correct BMP deficiency in HEK293 cells lacking CLN5. Collectively, our observations suggest that functionally overlapping pathways promote BMP synthesis in mammalian cells.

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.

Fig. 2. Members of the cytosolic phospholipase A2 group IV enzyme family catalyze hemi-BMP and BMP synthesis.

A Screening for hemi-BMP synthase activity using a lipid-hydrolase/transacylase enzyme library (n = 1). The reaction mix (100 µl total) contained 40 µg cell protein, 0.32 mM di-oleoyl PG substrate, 1% FA-free BSA, and 33 mM M bis-tris propane buffer (pH 7.0). B TLC analysis of the reaction products of semi-purified PLA2G4D and PLA2G4E (1 µg/reaction) incubated with PG (1 mM). Reactions were conducted under the conditions described in A. C Densitometric quantitation of B (n = 3 biological replicates). D Hemi-BMP synthase activity of wild-type (WT) and mutant enzymes. The active serine of PLA2G4D and PLA2G4E was replaced with an alanine (S370A and S420A, respectively). Lysates of Expi293F cells expressing WT and mutant variants were used as source of enzymatic activity (n = 3 biological replicates). Reactions were conducted under the conditions described in A. E, F Hemi-BMP synthase activity of mouse and human PLA2G4 (hPLA2G4A-F) orthologues. G, H BMP synthase activity of mouse and human PLA2G4 orthologues. In E–H, lysates of Expi293F cells expressing recombinant enzymes were used as source of enzymatic activity. Assays were conducted as described in A in the absence and presence of 2 mM CaCl₂ (n = 3, representative of three independent experiments). I Gene expression analysis of PLA2G4 family members in HEK293 cells (n = 3 biological replicates). J Basal BMP content of control, PLA2G4B-KO, and PLA2G4C-KO cells (n = 6 biological replicates, representative of three independent experiments). K, L BMP and hemi-BMP content of control, PLA2G4B-KO, and PLA2G4C-KO cells after incubation with PG (50 µM) for 12 h (n = 6 biological replicates, representative of three independent experiments). Data are shown as mean ± SD. Statistically significant differences were determined by two-tailed unpaired multiple t-tests for D without corrections, two-way ANOVA for E–H, and one-way ANOVA for J–L, followed by corrections for multiple comparisons (* = compared to the respective control, ^# = comparison between groups) using Bonferroni post hoc test (levels of statistically significant differences are: *^,#p < 0.05, **^,##p < 0.01^, and ***^,###p < 0.001). Source data are provided as a Source Data file.

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

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.

Fig. 4. Members of the PLA2G4 and PNLIP family promote BMP synthesis independently of CLN5.

A, B Basal LPG and BMP content in WT and CLN5-KO cells (n = 3 biological replicates). C, D LPG and BMP content of WT and CLN5-KO cells after supplementation with di-oleoyl PG (50 µM) for 6 h (n = 3 biological replicates). E PG-induced increase in BMP levels in CLN5-KO and WT cells (n = 3). F, G BMP content of CLN5-KO cells upon overexpression of indicated PLA2G4 and PNLIP family members under basal conditions and after 8 h PG supplementation (50 µM, n = 3 biological replicates). The WT control was transfected with empty vector. Note that cells used for experiments A–G were pre-cultivated in DMEM containing 10% FBS. For experiments (6-8 h), FBS was replaced by hiFBS to avoid serum-mediated acylation reactions. H, I LPG and BMP content of WT and CLN5-KO cells cultivated for two weeks in DMEM medium containing either 10% FBS or hiFBS (n = 3 biological replicates). J, K LPG and BMP content of WT and CLN5-KO cells cultured in DMEM with 10% hiFBS for one week and subsequently in DMEM with 10% FBS for another week (n = 3 biological replicates). L Secretion of LPG by WT and CLN5-KO HEK293 cells (n = 3 biological replicates). Cells were incubated in DMEM containing 2% BSA for 24 h and lipids were extracted from lyophilized conditioned media. Data are presented as mean ± SD and representative of at least two independent experiments. Statistically significant differences were determined by two-tailed unpaired t-test (B-E, L) without corrections for multiple comparisons, and one-way ANOVA (F, G) followed by corrections for multiple comparisons versus control using Bonferroni post hoc test. Source data are provided as a Source Data file.

Fig. 5. Serum transacylases produce hemi-BMP and BMP in vitro and in vivo.

A–C BMP, PG, and hemi-BMP synthesis by heparin-plasma of wild-type and Lipg-ko mice using sn-1-oleoyl LPG or di-oleoyl PG as substrate (n = 4). The reaction mix (50 µl total) contained 30 µl plasma, 0.5 mM substrate prepared in PBS (pH 7.4), and 1% FA-free BSA. Samples were incubated for 1 h at 37 °C. D–F Plasma PG, BMP, and hemi-BMP content of mice after intraperitoneal injection of di-oleoyl PG (wild-type n = 3; Lipg-ko n = 4) (5 mg/mouse). Blood samples of wild-type and Lipg-ko mice were collected before injection and at the indicated time points after injection (n = 3). G Total tissue BMP content of wild-type (n = 4) and Lipg-ko mice (n = 5). Data are presented as mean ± SD. Statistically significant differences were determined by unpaired two-tailed Student’s t-test, followed by corrections for multiple comparisons using the Holm-Sidak post hoc test. Source data are provided as a Source Data file.

Fig. 6. Circulating PG is incorporated into tissue BMP stores.

A Incorporation of ¹³C-labeled di-oleoyl PG into BMP stores of the indicated tissues of wild-type mice (n = 4 mice). B FA remodeling of ¹³C-labeled BMP. In A and B, tissues were collected 4 h after intraperitoneal injection of 0.2 mg ¹³C-PG/mouse (n = 4 wild-type mice). Data are presented as mean ± SD. Source data are provided as a Source Data file.

Fig. 7. BMP synthesis is promoted by functionally overlapping pathways.

A The acylation of the head group of the precursor lipids PG or LPG is a necessary step in BMP synthesis. In principle, this reaction can be catalyzed by secreted (PNLIP family), cytosolic (PLA2G4 family), and lysosomal (CLN5) enzymes. CLN5 is the major lysosomal BMP synthase in HEK293 cells and uses LPG as both donor and acceptor lipid. Most transacylases of the PNLIP and PLA2G4 family preferentially acylate PG leading to the formation of hemi-BMP. Some of these enzymes also show hemi-BMP and/or BMP hydrolase activity resulting in the formation or degradation of BMP.Gain-of function experiments indicate that PLA2G4D, LIPC, LIPG, LIPH, and PLA1A can enhance BMP synthesis independently of CLN5, suggesting that these enzymes generate BMP precursors that are transferred to LE/lysosomes. Precursor formation may occur extracellularly, followed by endocytosis, or at intracellular membranes accessible for PLA2G4D. The acylation of natural R,S - PG/LPG generates BMP precursors with R,S - configuration, which are then subjected to stereoconversion and FA remodeling. These reactions deliver mature S,S - BMP, which is primarily acylated in sn-2 positions. The mechanism and site of stereoconversion remain unknown. One possible mechanism is the formation of a cyclic intermediate followed by its stereospecific hydrolysis, as depicted in B. Figure A was created in BioRender. Bulfon, D. (2023) BioRender.com/a22i133.