Effect of expression of human glucosylceramidase 2 isoforms on lipid profiles in COS-7 cells

Abstract

Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. It is continuously formed and degraded in glycosphingolipid metabolism. GlcCer is mainly degraded by two enzymes, lysosomal acid β-glucosidase (GBA) and nonlysosomal β-glucosidase (GBA2). Deficiencies of GBA and GBA2 affect glycosphingolipid metabolism, resulting in neurological diseases, such as Gaucher Disease and Hereditary Spastic Paraplegia. 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-β-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 compared to control and other isoforms. 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. These studies suggest that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell, acting on glucosylceramides with a wide range of lipid components. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks.AbbreviationsCDaseceramidaseCerSceramide synthaseCerceramideCBEConduritol-β-epoxideDAG or DGdiacylglycerolERendoplasmic reticulumGSLglycosphingolipidGBA2glucosylceramidase 2GBAglucosylceramidaseGlcCerglucosylceramideGalCergalactosylceramideHexCerhexosylceramideHexCer(d18: 1/16: 0)hexosylceramide with a C18 sphingosine (d18: 1) and N-acyl group (16: 0)MGmonoacylglycerolLPCLysophosphatidylcholineLPELysophosphatidylethanolaminePAPhosphatidic acidPCphosphatidylcholinePEphosphatidylethanolaminePGPhosphatidylglycerolPIphosphatidylinositolPSphosphatidylserineSLsphingolipidSMsphingomyelinSMasesphingomyelinaseSMSsphingomyelin synthaseSphsphingosineSPTserine palmitoyltransferaseS1Psphingosine-1-phosphate4MUG4-Methylumbelliferyl-β-D-glucopyranosideTGtriacylglycerolView Full Text