College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
Nat Commun. 2019 Sep 6;10(1):4065. doi: 10.1038/s41467-019-11942-y.
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-AcGalNAz) and 1,3-di-O-propionylated GalNAz (1,3-PrGalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-PrGalNAz 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.
非天然单糖，如可代谢掺入细胞聚糖的叠氮糖，目前被用作聚糖成像和糖蛋白质组分析的主要工具。为了提高膜通透性和细胞摄取，通常对非天然糖进行全-O-乙酰化，但这会引起一个长期被忽视的副反应，即非酶 S-糖基化。在此，我们开发了 1,3-二酯化 N-叠氮乙酰半乳糖胺（GalNAz）作为代谢糖标记的下一代化学报告物。1,3-二-O-乙酰化 GalNAz（1,3-AcGalNAz）和 1,3-二-O-丙酰化 GalNAz（1,3-PrGalNAz）均表现出高效的蛋白 O-GlcNAcylation 标记，而没有人为的 S-糖基化。在小鼠胚胎干细胞（mESCs）中应用 1,3-PrGalNAz，我们鉴定出 ESRRB 是多能性的关键转录因子，是一种 O-GlcNAcylated 蛋白。我们表明，ESRRB 的 O-GlcNAcylation 对于 mESC 自我更新和多能性很重要。从机制上讲，ESRRB 由 O-GlcNAc 转移酶在丝氨酸 25 处 O-GlcNAc 化，这稳定了 ESRRB，促进了其转录活性，并促进了其与两个主要多能性调节因子 OCT4 和 NANOG 的相互作用。
