Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells

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 next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di- O -acetylated GalNAz (1,3-Ac 2 GalNAz) and 1,3-di- O -propionylated GalNAz (1,3-Pr 2 GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr 2 GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG. Per- O -acetylated unnatural monosaccharides are popular tools for glycan labeling in live cells but can undergo unwanted side reactions with cysteines. Here, the authors develop unnatural sugars in a partially esterified form that are inert towards cysteines, and use them to probe O-GlcNAcylation in mESCs.