The Malate Aspartate Shuttle is important for de novo serine biosynthesis. Broeks et al.
We hypothesized that all MAS defects lead to a secondary serine biosynthesis defect. To study whether MAS defects lead to diminished serine and glycine biosynthesis, we performed isotopic tracing experiments in 33 HEK293 cell lines with a genetic disruption of the MAS enzymes and transporters (GOT1, MDH1, SLC25A11, MDH2, GOT2, SLC25A12/13) and corresponding WT lines, including a double knockout of the AGC transporter. Cells were incubated with [U-13C]-glucose, as serine and glycine are synthesized de novo from the glycolytic intermediate 3-phosphoglycerate (3-PG). After 8 hours of incubation with [U-13C]-glucose, 13C3-serine and 13C2-glycine concentrations were most strongly decreased in MDH1 KO cells, followed by AGC, GOT1, GOT2, OGC and MDH2 KO cells when compared to control cells. The fraction of 13C3-serine and 13C2-glycine over time confirmed diminished de novo serine and glycine biosynthesis in all MAS KO cells, serine and glycine biosynthesis being only partially hampered in OGC and MDH2 KO cells. Overall, these findings demonstrate that all MAS defects lead to decreased de novo serine and glycine biosynthesis on a cellular level. [Figures 1 & S2-3] To further investigate the underlying causes of diminished de novo serine biosynthesis in MAS KO cells, we analyzed the intracellular metabolomes of the cells incubated with [U-13C]-glucose for 8 hours using direct infusion high resolution mass spectrometry (DI-HRMS). Our data demonstrates that the abundant availability of intermediates in the first steps of glycolysis leads to an increased flux to glycerol biosynthesis in MAS KO cells, while flux from glucose to pyruvate was decreased in all KO cells. The lactate (m+3)/pyruvate (m+3) ratio was increased in all KO cells and strongly correlated with serine and glycerol 3-P synthesis from glucose. Analysis of isotope enrichment in the TCA cycle and MAS demonstrated impaired flux through TCA cycle in most MAS defects. In addition, our findings indicated that a defect in the MAS cycle from GOT2 (-AGC-GOT1-) to MDH1 results in diminished metabolite flux through the NAD+ regenerating enzyme MDH1. [Figure 2, 3, S4 & S5]
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Figures 1 & S2-3: The datafile contains concentration values for serine, glycine, 13C3-serine and 13C2-glycine that were quantified by LC-MS/MS in methanol fractions from isotope tracing experiments. WT and KO HEK293 cells were cultured with [U-13C]-glucose containing medium for 2,4 and 8 hours. Following incubation, cells were washed with 2 ml cold PBS (4 ºC). Cells were collected by scraping twice in 0.25 ml pre-chilled 100% methanol (-80 ºC). The total 0.5 ml cellysate/methanol mixture was centrifuged (16,200 g, 10 min, 4 ºC), after which the supernatant was transferred to a new 1.5 ml Eppendorf tube. Peak integration and data analysis were performed using Waters MassLynx v4.2 software. The concentration for each analyte was calculated by linear regression analysis of the peak area/IS area using the calibration curve. Technical triplicates of cell lysates were measured and concentrations were normalized to total protein concentrations, obtained by the Pierce BCA protein assay. The datafile contains data corrected for total protein concentrations, that were obtained in parallel to the isotope tracing experiment. The manuscript presents the data as fractions and relative values to experimental controls, which are also reported in the datafile. The data expressed as relative to experimental controls are measured within the same run to control for interrun and experimental day variation. Figures 2,3,S4&S5: The datafile contains intensity data from DI-HRMS. Methanol samples collected after 8 hours incubation with [U-13C]-glucose were analyzed by DI-HRMS. A peak calling pipeline, developed in R programming language, annotated the raw mass spectrometry data according to the Human Metabolome DataBase (HMDB, version 3.6) with a range of 2 ppm (https://github.com/UMCUGenetics/DIMS). ). Only metabolites that were endogenously relevant (according to HMDB) and with the following adduct ions were selected: [M+H]+, [M+Na]+, [M+K]+ (positive mode), [M-H]- and [M+Cl]- (negative mode). As DI-HRMS is unable to separate isomers, the resulting mass peak intensities consist of summed intensities of these isomers. In order to extract isotopologues of metabolites from the resulting data, the theoretical m/z value for the annotated metabolite in the negative or ionization mode was used to determine the expected m/z of the 13C labelled metabolite. Potential isobaric compounds are reported in supplemental table 2 (Table S2). All samples were measured in a single run. The fractional enrichment of each isotopologue is calculated as a percentage of the sum of all possible isotopologues. Isotopologues range from M+0 to M+n (1-6), in which M+0 reflects the unlabeled 12C carbons and M+n the labelled 13C carbons. The manuscript presents the data relative to controls.