Ac34FGlcNAz is an effective metabolic chemical reporter for O-GlcNAcylated proteins with decreased S-glyco-modification

Abstract

O-GlcNAcylation is a ubiquitous post-translational modification governing vital biological processes in cancer, diabetes and neurodegeneration. Metabolic chemical reporters (MCRs) containing bio-orthogonal groups such as azido or alkyne, are widely used for labeling of interested proteins. However, most MCRs developed for O-GlcNAc modification are not specific and always lead to unexpected side reactions termed S-glyco-modification. Here, we attempt to develop a new MCR of Ac₃4FGlcNAz that replacing the 4-OH of Ac₄GlcNAz with fluorine, which is supposed to abolish the epimerization of GALE and enhance the selectivity. The discoveries demonstrate that Ac₃4FGlcNAz is a powerful MCR for O-GlcNAcylation with high efficiency and the process of this labeling is conducted by the two enzymes of OGT and OGA. Most importantly, Ac₃4FGlcNAz is predominantly incorporated intracellular proteins in the form of O-linkage and leads to negligible S-glyco-modification, indicating it is a selective MCR for O-GlcNAcylation. Therefore, we reason that Ac₃4FGlcNAz developed here is a well characterized MCR of O-GlcNAcylation, which provides more choice for label and enrichment of O-GlcNAc associated proteins.

Keywords: Metabolic labeling; O-GlcNAc; OGA; OGT; S-glyco-modification.