Targeted Control of Protein O-GlcNAcylation in Living Cells

O-linked N-acetylglucosamine glycosylation (O-GlcNAcylation) regulates many intracellular proteins, but linking a cellular phenotype to O-GlcNAcylation of a specific substrate remains difficult. Global perturbation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) changes many proteins at once and therefore provides limited information about individual substrates. Protein-selective approaches address this problem by directing OGT or OGA activity toward chosen proteins in living cells. Here, we review small-molecule recruiters, nanobody-based systems, inducible recruitment strategies, RNA aptamers, and complementary site-level approaches. We compare what each method can establish and the controls needed for interpretation, with particular attention to target engagement, target-protein O-GlcNAcylation, site assignment, selectivity, and functional validation. Together, these approaches provide increasingly direct ways to define how O-GlcNAcylation of individual proteins contributes to cellular function.