Over 15% of the cellular proteome is modified by O-linked <i>N</i>-acetyl glucosamine (O-GlcNAc), a post-translational modification (PTM) that consists of a single N-acetyl glucosamine monosaccharide 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. However, delineation of the function of O-GlcNAc modification on particular glycoproteins are hindered by the inability to control O-GlcNAc stoichiometry on specific proteins of interest in cells. A general method to control glycosylation on specific target proteins would enable the systematic evaluation of O-GlcNAc function in cells. To advance insight into the role of O-GlcNAc on specific proteins, we developed fusions of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) to nanobodies as proximity-directing agents for the chemical control of O-GlcNAc levels on a target protein in live cells. Fusion of the nanobody to a series of OGT and OGA constructs enabled the identification of the optimal fusion enzymes for selective control of O-GlcNAc stoichiometry on the target protein. Proximity-induced glycosylation or deglycosylation was demonstrated on a series of nucleocytoplasmic proteins representing the broad array of substrates for OGT and OGA. Evaluation of the effect of O-GlcNAc on some target proteins revealed altered subcellular localization or protein stabilization. Manipulation of O-GlcNAc stoichiometry using the proximity-directed enzymes reported here will catalyze additional discoveries of functions for O-GlcNAc and the enzymes that control it.

Chemical control of the O-GlcNAc proteome in cells

2019 Fall National Meeting

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