Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging
Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its &l...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-10-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/25/19/4501 |
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| author | Angelina Kasprowicz Corentin Spriet Christine Terryn Vincent Rigolot Stephan Hardiville Matthew G. Alteen Tony Lefebvre Christophe Biot |
| author_facet | Angelina Kasprowicz Corentin Spriet Christine Terryn Vincent Rigolot Stephan Hardiville Matthew G. Alteen Tony Lefebvre Christophe Biot |
| author_sort | Angelina Kasprowicz |
| collection | DOAJ |
| description | Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its <i>O</i>-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of β-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the <i>O</i>-GlcNAcylation status of β-catenin in HeLa cells. The changes in <i>O</i>-GlcNAcylation of β-catenin were varied by perturbing global cellular <i>O</i>-GlcNAc levels with the inhibitors of <i>O</i>-GlcNAc transferase (OGT) and <i>O</i>-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation. |
| first_indexed | 2024-03-10T15:55:14Z |
| format | Article |
| id | doaj.art-b65e1cab4d9548c3a9113cd1fcd53b6f |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-10T15:55:14Z |
| publishDate | 2020-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-b65e1cab4d9548c3a9113cd1fcd53b6f2023-11-20T15:46:15ZengMDPI AGMolecules1420-30492020-10-012519450110.3390/molecules25194501Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and ImagingAngelina Kasprowicz0Corentin Spriet1Christine Terryn2Vincent Rigolot3Stephan Hardiville4Matthew G. Alteen5Tony Lefebvre6Christophe Biot7Univ. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, FranceUniv. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, FrancePICT Platform, University of Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51100 Reims, FranceUniv. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, FranceUniv. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, FranceDepartment of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, CanadaUniv. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, FranceUniv. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, FranceMonitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its <i>O</i>-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of β-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the <i>O</i>-GlcNAcylation status of β-catenin in HeLa cells. The changes in <i>O</i>-GlcNAcylation of β-catenin were varied by perturbing global cellular <i>O</i>-GlcNAc levels with the inhibitors of <i>O</i>-GlcNAc transferase (OGT) and <i>O</i>-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.https://www.mdpi.com/1420-3049/25/19/4501bioorthogonal chemistryfluorescenceglycosylationmetabolic incorporationGFPβ-catenin |
| spellingShingle | Angelina Kasprowicz Corentin Spriet Christine Terryn Vincent Rigolot Stephan Hardiville Matthew G. Alteen Tony Lefebvre Christophe Biot Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging Molecules bioorthogonal chemistry fluorescence glycosylation metabolic incorporation GFP β-catenin |
| title | Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging |
| title_full | Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging |
| title_fullStr | Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging |
| title_full_unstemmed | Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging |
| title_short | Exploring the Potential of β-Catenin <i>O</i>-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging |
| title_sort | exploring the potential of β catenin i o i glcnacylation by using fluorescence based engineering and imaging |
| topic | bioorthogonal chemistry fluorescence glycosylation metabolic incorporation GFP β-catenin |
| url | https://www.mdpi.com/1420-3049/25/19/4501 |
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