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<p>Unlike <i>N</i>-glycosylation, protein <i>O</i>-glycosylation often lacks a strict consensus sequon, making synthesis of homogeneous <i>O</i>-glycoproteins and site-specific engineering of <i>O</i>-glucosylation challenging. Here we identify <i>Legionella</i> effector LtpM as a versatile protein β-<i>O</i>-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 <i>O</i>-glucosylating various eukaryotic proteins of interest. In particular, <i>O</i>-glucose serves as a functional surrogate for <i>O</i>-linked <i>N</i>-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.</p><p></p>

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Protein β-O-glucosylation by Legionella LtpM through short consensus sequons G-T/S and S-G

  • 期刊论文

  • 发表日期：

    2025年7月14日

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  • Wei Li,
  • Ling Gao,
  • Shiyong Cui,
  • Tiantian Wei,
  • Jiayu Sun,
  • Xinyue Zhou,
  • Shuyu Liang,
  • Xiaoqing Pan,
  • Xuanzhen Pan,
  • Chuanping Gao,
  • Yingze Wang,
  • Junhan Chang,
  • Chunting Wang,
  • Pinou Lv,
  • Junyu Xiao,
  • Peng Dai,
  • Xing Chen

摘要

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.