Gene expression analysis of R1 mESCs stably expressing WT ESRRB and Ser 25A (the O-GlcNAc residue mutant) ESRRB

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per O-acetylated, which, however, can induce a long-overlooked side reaction, non enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N azidoacetylgalactosamine (GalNAz) as the next generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O propionylated GalNAz (1,3-Pr2GalNAz) exhibited high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identified ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We showed that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 25 (Ser 25), which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG. Three biological replicates of R1 embryonic stem cells stably expressing WT ESRRB and Ser 25A ESRRB were analysed.