Citation

  • Authors: Kasprowicz A. et al.
  • Year: 2020
  • Journal: Molecules 25 4501
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell type: HeLa
    Description: Human cervix epitheloid carcinoma cells

Method

For microscopy, cells were grown on glass coverslips and transfected at 70% confluency. Transfections were performed using 1 µL of Jetoptimus (Polyplus), 1 µg of the plasmid in 900 µL DMEM, according to the manufacturer’s instruction. The transfections mix was replaced 4 h later with fresh medium containing Ac4GAlNAz, Ac4GalNAl, or Ac4ManNAz (200 µM), with or without Ac45SGlcNAc (100 µM) or Thiamet G (1 µM), for 24 h.

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.

Pubmed