Abstract

Over 15% of the cellular proteome is modified by O-linked N-acetyl glucosamine (O-GlcNAc), a post-translational modification (PTM) that consists of a single N-acetyl glucosamine sugar attached to serine or threonine residues of nuclear, cytosolic and mitochondrial proteins. Due to the ubiquitous nature of the modification, O-GlcNAc has been implicated in numerous biological processes, including immune response, cancer progression, neurodegenerative disease, and diabetes. In order to explore the role of O-GlcNAc on glycoproteins in human disease, we have now developed a cellular approach for target protein O-GlcNAcylation. The method uses a nanobody fused to an O-GlcNAc writer or eraser to control O-GlcNAc levels on the desired target protein in cells. Evaluation of the effect of O-GlcNAc on the targeted transcription factors revealed functions in protein stabilization and regulation of protein–protein interactions. Target protein O-GlcNAcylation will accelerate new insights to O-GlcNAc functions and reveal approaches to engineer these biological signals.