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

Abstract

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.

In Situ Visualization of PD-L1-Specific Glycosylation on Tissue Sections

“…Although the study of the structure and biological function of PD-L1-specific glycosylation has been facilitated by isolating the PD-L1 protein from cells or tissues for mass spectrometry, Western blotting analysis, or enzyme-linked immunosorbent assay, information about the location of PD-L1-specific glycosylation and its heterogeneous expression in different tumor regions has been missing. Recently, several methods combining the recognized molecules for protein labeling and metabolic oligosaccharide for glycosylation labeling to enable f luorescence r esonance e nergy t ransfer (FRET) have been developed for protein-specific glycosylation investigation. − However, these methods were confined to the analysis of living cells or model organisms, which were difficult to apply in clinical tissue samples due to the need for biological metabolism. Therefore, developing new strategies for in situ imaging of PD-L1-specific glycosylation on tissue sections is important for studying the effect of PD-L1 glycosylation on immune activity and discovering a reliable predictive biomarker for clinical immunotherapy response.…”

A living cell-based fluorescent reporter for high-throughput screening of anti-tumor drugs

“…More recently, a Forster resonance energy transfer (FRET) method was developed to monitor cellular O-GlcNAcylation levels [ 37 ]. The FRET method is based on the modification of catenin, which is labeled by using GFP and an O-GlcNAcylation-dependent fluorophore.…”

Metabolic glycoengineering – exploring glycosylation with bioorthogonal chemistry

“…Interestingly, Akt1-eGFP was found to be located in the cytosol as well as in the nucleus, but O-GlcNAcylation of the protein was increased in the nucleus as detected by a decreased fluorescence lifetime of eGFP in the nucleus compared to that in the cytosol. A similar approach was reported in fixed cells with Ac 4 GalNAz as an O-GlcNAc reporter in combination with CuAAC 156 or SPAAC labeling 157 to visualize the glycosylation of eGFP-tagged tau and b-catenin, respectively, by FLIM-FRET microscopy.…”