Exploring the Potential of β-Catenin -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 -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 -GlcNAcylation status of β-catenin in HeLa cells. The changes in -GlcNAcylation of β-catenin were varied by perturbing global cellular -GlcNAc levels with the inhibitors of -GlcNAc transferase (OGT) and -GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.