ABSTRACT
Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. GlcCer is mainly degraded by two enzymes, lysosomal acid ß-glucosidase (GBA) and nonlysosomal ß-glucosidase (GBA2), which may have different isoforms because of alternative splicing. To understand which GBA2 isoforms are active and how they affect glycosphingolipid levels in cells, we expressed nine human GBA2 isoforms in COS-7 cells, confirmed their expression by qRT-PCR and Western blotting, and assayed their activity to hydrolyze 4-methylumbelliferyl-ß-D-glucopyranoside (4MUG) in cell extracts. Human GBA2 isoform 1 showed high activity, while the other isoforms had activity similar to the background. Comparison of sphingolipid levels by ultra-high resolution/accurate mass spectrometry (UHRAMS) analysis showed that isoform 1 overexpression increased ceramide and decreased hexosylceramide levels. Comparison of ratios of glucosylceramides to the corresponding ceramides in the extracts indicated that GBA2 isoform 1 has broad specificity for the lipid component of glucosylceramide, suggesting that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks.
ABSTRACT
The number, position, and configuration of double bonds in lipids affect membrane fluidity and the recruitment of signaling proteins. Studies on mammalian sphingolipids have focused on those with a saturated sphinganine or mono-unsaturated sphingosine long chain base. Using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), we observed a marked accumulation of lipids containing a di-unsaturated sphingadiene base in the hippocampus of mice lacking the metabolic enzyme sphingosine kinase 2 (SphK2). The double bonds were localized to positions C4-C5 and C14-C15 of sphingadiene using ultraviolet photodissociation-tandem mass spectrometry (UVPD-MS/MS). Phosphorylation of sphingoid bases by sphingosine kinase 1 (SphK1) or SphK2 forms the penultimate step in the lysosomal catabolism of all sphingolipids. Both SphK1 and SphK2 phosphorylated sphinga-4,14-diene as efficiently as sphingosine, however deuterated tracer experiments in an oligodendrocyte cell line demonstrated that ceramides with a sphingosine base are more rapidly metabolized than those with a sphingadiene base. Since SphK2 is the dominant sphingosine kinase in brain, we propose that the accumulation of sphingadiene-based lipids in SphK2-deficient brains results from the slower catabolism of these lipids, combined with a bottleneck in the catabolic pathway created by the absence of SphK2. We have therefore uncovered a previously unappreciated role for SphK2 in lipid quality control.
