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 have developed an original tool enabling labeling and visualization of the cell cycle key-regulator b-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 b-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 b-catenin in HeLa cells. Moreover, the changes in O-GlcNAcylation of b-catenin were varied by perturbing global cellular O-GlcNAc levels with 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.

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Subject: Chemistry and Materials Science - Applied Chemistry

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