The glycerophospho metabolome and its influence on amino acid homeostasis revealed by brain metabolomics of GDE1(-/-) mice

Abstract

GDE1 is a mammalian glycerophosphodiesterase (GDE) implicated by in vitro studies in the regulation of glycerophophoinositol (GroPIns) and possibly other glycerophospho (GroP) metabolites. Here, we show using untargeted metabolomics that GroPIns is profoundly (>20-fold) elevated in brain tissue from GDE1(-/-) mice. Furthermore, two additional GroP metabolites not previously identified in eukaryotic cells, glycerophosphoserine (GroPSer) and glycerophosphoglycerate (GroPGate), were also highly elevated in GDE1(-/-) brains. Enzyme assays with synthetic GroP metabolites confirmed that GroPSer and GroPGate are direct substrates of GDE1. Interestingly, our metabolomic profiles also revealed that serine (both L-and D-) levels were significantly reduced in brains of GDE1(-/-) mice. These findings designate GroPSer as a previously unappreciated reservoir for free serine in the nervous system and suggest that GDE1, through recycling serine from GroPSer, may impact D-serine-dependent neural signaling processes in vivo.

Figure 1

GDE1(-/-) brains contain markedly elevated levels of multiple GroP-metabolites. (A) Mass ion peak intensity measurements from GDE1(+/+) versus (-/-) brains. Data are derived from untargeted metabolite profiling experiments performed in the negative ionization mode. Red spots represent m/z ions that are significantly elevated in GDE1(-/-) brains (in each case, fold-change > 10) and their m/z values are displayed. Isotopologues, adducts, and ions produced by in-source fragmentation were manually removed for clarity. (B) Extracted ion chromatograms (EICs) from individual LC-MS runs of GDE1(+/+) and (-/-) brain metabolomes. The retention times for the relevant peaks are shown in minutes. See also Figure S1, and Tables S1 and S3.

Figure 2

Structural characterization of GDE1-regulated brain metabolites. (A, C, E) Chromatographic behaviour on normal-phase LC of synthetic GroPIns, GroPSer, and GroPGate compared to the natural metabolites elevated in brains of GDE1(-/-) mice. Insets show observed and calculated masses (o.m. and c.m., respectively) for natural metabolites, as measured by quadrupole time-of-flight (QTOF) mass spectrometry (for higher-resolution FT-ICR mass-measurements see Table S4). (B, D, F) MS/MS spectra showing daughter ions that correspond to the glycerophosphate backbone (m/z 78.95, 96.97, 152.99, 171.00). Endogenous daughter ions matched their synthetic counterparts within 20 ppm which is typical for MS/MS ions obtained via QTOF mass spectrometry. The existence of the inositol (B) and glycerate (C) head-groups was confirmed by diagnostic peaks representing inositol- and dehydrated glycerate-phosphate, respectively. MS/MS profiles of synthetic (blue) and natural (red) metabolites are shown. See also Figure S2 and Tables S2 and S4.

Figure 3

GroPSer, GroPIns, and GroPGate are direct substrates of GDE1. Membrane preparations from GDE1-transfected COS-7 cells (A) or mouse brain samples (C) were tested for activity against synthetic GroP-metabolites with different head-groups – serine (GroPSer), glycerate (GroPGate), or inositol (GroPIns). Enzyme activity was determined by quantification of the release of free head group (serine, glycerate, and inositol from GroPSer, GroPGate, and GroPIns, respectively) by LC-MS in recombinant GDE1 assays. For mouse brain samples, enzyme activity against GroPSer, GroPGate was measured in the same way, and, for GroPIns, was determined by measuring reductions in consumed substrate (due to the presence of high background isobaric mass signals for inositol in brain samples.) All assays were performed with n=3, **, p < 0.01; * p < 0.05, student's t-test.

Figure 4

GDE1 regulates free serine levels in mouse brain. (A) Levels of serine are reduced by 1.6-fold in GDE1(-/-) brains; Data represent the average of six independent experiments per group ± standard error. *,p < 0.05. (B) Derivitization with BocPheOSu enables resolution of L- and D-serine via reverse-phase chromatography. (C) BocPheOSu derivitization of brain metabolomes reveals that both L- and D- serine levels are reduced by a similar magnitude in GDE1(-/-) brains. *, p < 0.05 for both enantiomers. (D) Working model for the role of GDE1 in serine metabolism. Phosphatidylserine (PtdS) which is exclusively located on the inner leaflet of the plasma membrane is de-acylated by A- or B-type phospholipases (PLA/B) whereupon the resulting GroPSer is released into the cytosol and is transported across the plasma membrane by a putative GroPSer transporter. Subsequently, extra-cellular GroPSer is exposed to the catalytic domain of GDE1 resulting in the release of serine and glycerol-3-phosphate. In the absence of GDE1, GroPSer accumulates, resulting in corresponding reductions in serine. See also Figure S3.