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

Molecules

MatthewG. Alteen¹  Christine Terryn²  Tony Lefebvre³  Christophe Biot³  Corentin Spriet³  Stephan Hardiville³  Angelina Kasprowicz³  Vincent Rigolot³ 

[1] Department of Chemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;PICT Platform, University of Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51100 Reims, France;Univ. Lille, CNRS, UMR 8576–UGSF–Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France;

关键词: bioorthogonal chemistry;    fluorescence;    glycosylation;    metabolic incorporation;    GFP;    β-catenin;   

DOI  :  10.3390/molecules25194501

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown