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Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells

1
School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
2
Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 3000, Thailand
3
Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
4
Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC 3010, Australia
5
School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia
*
Authors to whom correspondence should be addressed.
Metabolites 2020, 10(12), 488; https://doi.org/10.3390/metabo10120488
Received: 29 September 2020 / Revised: 20 November 2020 / Accepted: 25 November 2020 / Published: 27 November 2020
(This article belongs to the Section Lipid Metabolism)
Glucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. GlcCer is mainly degraded by two enzymes, lysosomal acid β-glucosidase (GBA) and nonlysosomal β-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-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. 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. View Full-Text
Keywords: glycolipids; sphingolipids; cerebrosides; ceramides; lipidomics; enzymology glycolipids; sphingolipids; cerebrosides; ceramides; lipidomics; enzymology
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MDPI and ACS Style

Jatooratthawichot, P.; Talabnin, C.; Ngiwsara, L.; Rustam, Y.H.; Svasti, J.; Reid, G.E.; Ketudat Cairns, J.R. Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells. Metabolites 2020, 10, 488. https://doi.org/10.3390/metabo10120488

AMA Style

Jatooratthawichot P, Talabnin C, Ngiwsara L, Rustam YH, Svasti J, Reid GE, Ketudat Cairns JR. Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells. Metabolites. 2020; 10(12):488. https://doi.org/10.3390/metabo10120488

Chicago/Turabian Style

Jatooratthawichot, Peeranat, Chutima Talabnin, Lukana Ngiwsara, Yepy H. Rustam, Jisnuson Svasti, Gavin E. Reid, and James R. Ketudat Cairns 2020. "Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells" Metabolites 10, no. 12: 488. https://doi.org/10.3390/metabo10120488

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