Li Wei, Gao Ling, Cui Shiyong, Wei Tiantian, Sun Jiayu, Zhou Xinyue, Liang Shuyu, Pan Xiaoqing, Pan Xuanzhen, Gao Chuanping, Wang Yingze, Chang Junhan, Wang Chunting, Lv Pinou, Xiao Junyu, Dai Peng, Chen Xing
College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China.
Nat Chem Biol. 2025 Jul 14. doi: 10.1038/s41589-025-01968-3.
Unlike N-glycosylation, protein O-glycosylation often lacks a strict consensus sequon, making synthesis of homogeneous O-glycoproteins and site-specific engineering of O-glucosylation challenging. Here we identify Legionella effector LtpM as a versatile protein β-O-glucosyltransferase recognizing extremely short two-residue sequons G-T/S and S-G. X-ray crystallography, molecular simulation and biochemical studies together reveal a unique catalytic mechanism: four residues of LtpM (F166, Q167, W228 and K225) serve as 'gatekeepers' above the binding pocket of the uridine diphosphate (UDP)-glucose sugar donor to form a narrow clamp for the substrate proteins, limiting the residue adjacent to serine or threonine to be exclusively glycine. By exploiting the short sequons, LtpM is developed as a powerful tool for site-specifically O-glucosylating various eukaryotic proteins of interest. In particular, O-glucose serves as a functional surrogate for O-linked N-acetylglucosamine in a synaptic Ras GTPase-activating protein. Additionally, LtpM accepts 6-azido analog of UDP-glucose and enables site-specific bioorthogonal conjugation of proteins.
与N-糖基化不同，蛋白质O-糖基化通常缺乏严格的共有序列，这使得合成同质O-糖蛋白以及对O-葡萄糖基化进行位点特异性工程改造具有挑战性。在这里，我们鉴定出嗜肺军团菌效应蛋白LtpM是一种多功能蛋白质β-O-葡萄糖基转移酶，它能识别极短的双残基序列G-T/S和S-G。X射线晶体学、分子模拟和生化研究共同揭示了一种独特的催化机制：LtpM的四个残基（F166、Q167、W228和K225）在尿苷二磷酸（UDP）-葡萄糖糖供体的结合口袋上方充当“守门人”，为底物蛋白形成一个狭窄的夹子，限制丝氨酸或苏氨酸相邻的残基只能是甘氨酸。通过利用这些短序列，LtpM被开发成为一种强大的工具，用于对各种感兴趣的真核蛋白进行位点特异性O-葡萄糖基化。特别是，O-葡萄糖在突触Ras GTP酶激活蛋白中作为O-连接的N-乙酰葡糖胺的功能替代物。此外，LtpM接受UDP-葡萄糖的6-叠氮类似物，并能够实现蛋白质的位点特异性生物正交共轭。
