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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| Format: | Article |
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MDPI AG
2020-11-01
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| Series: | Metabolites |
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| Online Access: | https://www.mdpi.com/2218-1989/10/12/488 |
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| author | Peeranat Jatooratthawichot Chutima Talabnin Lukana Ngiwsara Yepy Hardi Rustam Jisnuson Svasti Gavin E. Reid James R. Ketudat Cairns |
| author_facet | Peeranat Jatooratthawichot Chutima Talabnin Lukana Ngiwsara Yepy Hardi Rustam Jisnuson Svasti Gavin E. Reid James R. Ketudat Cairns |
| author_sort | Peeranat Jatooratthawichot |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T14:30:37Z |
| format | Article |
| id | doaj.art-e1e49476268b435db538c526598d267e |
| institution | Directory Open Access Journal |
| issn | 2218-1989 |
| language | English |
| last_indexed | 2024-03-10T14:30:37Z |
| publishDate | 2020-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Metabolites |
| spelling | doaj.art-e1e49476268b435db538c526598d267e2023-11-20T22:38:57ZengMDPI AGMetabolites2218-19892020-11-01101248810.3390/metabo10120488Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 CellsPeeranat Jatooratthawichot0Chutima Talabnin1Lukana Ngiwsara2Yepy Hardi Rustam3Jisnuson Svasti4Gavin E. Reid5James R. Ketudat Cairns6School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandSchool of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandLaboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, ThailandDepartment of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC 3010, AustraliaLaboratory of Biochemistry, Chulabhorn Research Institute, Bangkok 10210, ThailandDepartment of Biochemistry and Molecular Biology, University of Melbourne, Parkville, VIC 3010, AustraliaSchool of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandGlucosylceramide (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.https://www.mdpi.com/2218-1989/10/12/488glycolipidssphingolipidscerebrosidesceramideslipidomicsenzymology |
| spellingShingle | Peeranat Jatooratthawichot Chutima Talabnin Lukana Ngiwsara Yepy Hardi Rustam Jisnuson Svasti Gavin E. Reid James R. Ketudat Cairns Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells Metabolites glycolipids sphingolipids cerebrosides ceramides lipidomics enzymology |
| 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 | glycolipids sphingolipids cerebrosides ceramides lipidomics enzymology |
| url | https://www.mdpi.com/2218-1989/10/12/488 |
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