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Exploring the Potential of β-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging

1
Univ. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
2
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, US 41–UMS 2014–PLBS, F-59000 Lille, France
3
PICT Platform, University of Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51100 Reims, France
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Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada
*
Author to whom correspondence should be addressed.
Molecules 2020, 25(19), 4501; https://doi.org/10.3390/molecules25194501
Received: 16 July 2020 / Revised: 24 September 2020 / Accepted: 24 September 2020 / Published: 1 October 2020
(This article belongs to the Section Chemical Biology)
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 O-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 O-GlcNAcylation status of β-catenin in HeLa cells. The changes in O-GlcNAcylation of β-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation. View Full-Text
Keywords: bioorthogonal chemistry; fluorescence; glycosylation; metabolic incorporation; GFP; β-catenin bioorthogonal chemistry; fluorescence; glycosylation; metabolic incorporation; GFP; β-catenin
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MDPI and ACS Style

Kasprowicz, A.; Spriet, C.; Terryn, C.; Rigolot, V.; Hardiville, S.; Alteen, M.G.; Lefebvre, T.; Biot, C. Exploring the Potential of β-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging. Molecules 2020, 25, 4501.

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