Exploring the Potential of β-Catenin -GlcNAcylation by Using Fluorescence-Based Engineering and Imaging.
Fiche publication
Date publication
octobre 2020
Journal
Molecules (Basel, Switzerland)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr TERRYN Christine
Tous les auteurs :
Kasprowicz A, Spriet C, Terryn C, Rigolot V, Hardiville S, Alteen MG, Lefebvre T, Biot C
Lien Pubmed
Résumé
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.
Mots clés
GFP, bioorthogonal chemistry, fluorescence, glycosylation, metabolic incorporation, β-catenin
Référence
Molecules. 2020 Oct 1;25(19):