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

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 isofo...

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Autores principales: Jatooratthawichot, Peeranat, Talabnin, Chutima, Ngiwsara, Lukana, Rustam, Yepy Hardi, Svasti, Jisnuson, Reid, Gavin E., Ketudat Cairns, James R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761373/
https://www.ncbi.nlm.nih.gov/pubmed/33261081
http://dx.doi.org/10.3390/metabo10120488
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author Jatooratthawichot, Peeranat
Talabnin, Chutima
Ngiwsara, Lukana
Rustam, Yepy Hardi
Svasti, Jisnuson
Reid, Gavin E.
Ketudat Cairns, James R.
author_facet Jatooratthawichot, Peeranat
Talabnin, Chutima
Ngiwsara, Lukana
Rustam, Yepy Hardi
Svasti, Jisnuson
Reid, Gavin E.
Ketudat Cairns, James R.
author_sort Jatooratthawichot, Peeranat
collection PubMed
description 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.
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spelling pubmed-77613732020-12-26 Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells Jatooratthawichot, Peeranat Talabnin, Chutima Ngiwsara, Lukana Rustam, Yepy Hardi Svasti, Jisnuson Reid, Gavin E. Ketudat Cairns, James R. Metabolites Article 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. MDPI 2020-11-27 /pmc/articles/PMC7761373/ /pubmed/33261081 http://dx.doi.org/10.3390/metabo10120488 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Jatooratthawichot, Peeranat
Talabnin, Chutima
Ngiwsara, Lukana
Rustam, Yepy Hardi
Svasti, Jisnuson
Reid, Gavin E.
Ketudat Cairns, James R.
Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells
title Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells
title_full Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells
title_fullStr Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells
title_full_unstemmed Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells
title_short Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells
title_sort effect of expression of human glucosylceramidase 2 isoforms on lipid profiles in cos-7 cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761373/
https://www.ncbi.nlm.nih.gov/pubmed/33261081
http://dx.doi.org/10.3390/metabo10120488
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