Next-Generation Metabolic Glycosylation Reporters Enable Detection of Protein O-GlcNAcylation in Living Cells without S-Glyco Modification

Angew Chem Int Ed Engl. 2024 May 13;63(20):e202320247. doi: 10.1002/anie.202320247. Epub 2024 Apr 11.

Abstract

Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O-GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O-GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels-Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O-GlcNAcylation of several proteins and the protein-specific imaging of O-GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).

Keywords: FRET; bioorthogonal chemistry; carbohydrates; glycoproteins; metabolic engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosamine* / analogs & derivatives
  • Acetylglucosamine* / chemistry
  • Fluorescence Resonance Energy Transfer
  • Glycoproteins* / analysis
  • Glycosylation
  • HeLa Cells
  • Humans
  • Metabolic Engineering
  • Molecular Imaging*
  • Norbornanes* / chemistry
  • Protein Processing, Post-Translational*

Substances

  • 2-norbornene
  • Norbornanes
  • Acetylglucosamine
  • Glycoproteins