| Summary: | 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.
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