PDF(Pubmed)
Abstract：
Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.
摘要：
细菌毒素或效应蛋白对靶蛋白的单-O-糖基化是细菌干扰宿主细胞基本功能的公知机制。相应的糖基转移酶是重要的毒力因子，例如艰难梭菌毒素A和B。我们描述了耶尔森氏菌的两种具有高度序列同一性的糖基转移酶：来自人畜共患病原体小肠结肠炎耶尔森氏菌的YeGT和来自鼠病原体耶尔森氏菌的YkGT。我们表明，两者都通过在酪氨酸残基（RhoA中的Tyr-34）上连接N-乙酰葡糖胺（GlcNAc）来修饰Rho家族蛋白。值得注意的是,这些酶的靶蛋白特异性不同。虽然YeGT修改了RhoA，B和C,YkGT具有更宽的底物谱，不仅可以糖基化Rho，还可以糖基化Rac和Cdc42亚家族蛋白。诱变研究表明,残基177对于该更宽的目标谱是重要的。我们确定了YeGT在无配体状态下缩短了16个N末端残基（sYeGT）并与UDP结合的晶体结构，底物水解的产物。该结构将sYeGT分配给GT-A家族。它与来自毒素的糖基转移酶结构域具有高度的结构相似性。我们还证明，YeGT和YkGT的16个最N末端残基对于使用炭疽毒素的成孔保护性抗原介导的易位到宿主细胞中很重要。介导的引入Hela细胞或YeGT和YkGT的异位表达引起肌动蛋白细胞骨架的形态变化和重新分布。数据表明YeGT和YkGT可能是属于酪氨酸糖基化细菌糖基转移酶家族的细菌效应物。
