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.
Highlights
The complete set of glycans, i.e., the glycome, represents a large diversity of structures and functions and plays a central role in biology and health [1]
O-GlcNAcylation is much simpler than glycosylation occurring within the Molecules 2020, 25, 4501; doi:10.3390/molecules25194501
We opted for a bioorthogonal chemical reporter strategy to detect the O-GlcNAc modification state of β-catenin
Summary
The complete set of glycans, i.e., the glycome, represents a large diversity of structures and functions and plays a central role in biology and health [1]. Glycans are specific markers for numerous pathologies, including cancers [2]. This functional diversity is linked to a great structural diversity of glycans. The various forms of glycosylation are essential for cell homeostasis. The most complex forms of glycosylation, namely N-linked protein, O-linked protein, and O-linked mucin-type glycosylation, take place mainly in the endoplasmic reticulum and the Golgi apparatus where the requisite biosynthetic enzymes reside. Another widespread form of glycosylation is O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) [4]. O-GlcNAcylation is much simpler than glycosylation occurring within the Molecules 2020, 25, 4501; doi:10.3390/molecules25194501 www.mdpi.com/journal/molecules
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