志贺毒素B亚基C-末端氨基酸序列对其胞内逆向运输的影响.docVIP

 志贺毒素 B 亚基 C-末端氨基酸序列对其胞 内逆向运输的影响# 刘丹，范玉莹，李杰，高晓鸽，郝苗，薛慧婷** 5 10 15 20 25 30 35 （东北师范大学生命科学学院，长春 130024） 摘要：志贺毒素是由志贺杆菌分泌的蛋白毒素，B 亚基（STxB）负责毒素蛋白与它的受体糖 鞘脂 Gb3 结合，引导毒素蛋白从质膜到内质网的逆向途径。为了阐明 STxB 的 COOH 基端（C- 端）在逆向运输中的作用，我们通过氨基酸突变和修饰氨基酸尾巴的方法制备了一系列 STxB 融合蛋白并比较它们在细胞内的运输。结果显示，B 亚基 C-末端的 Arg（R）被 Ser（S）替 换后，B 亚基转运到内质网的速度变慢。如果在尾部修饰酸性氨基酸“Glu-Ser”（ES）， 同样会减慢蛋白转运的速度，更多蛋白停留在高尔基复合体内，随着氨基酸“E”的增加， 作用效果趋于明显，作用效果 STxB-EEEES≥-EEES-EES-ES，同时酸性氨基酸 Asp（D）具 有同样的抑制效果。并且只有当酸性氨基酸尾部与蛋白毒素直接相连时才具有影响转运的效 果，而突变掉末端氨基酸“R”再接连酸性氨基酸序列时，减缓蛋白运输的现象最为明显。 此研究的结果有利于更好地了解 STxB 在细胞内的逆向运输以及对 STxB 更好地开发和利用。 关键词：逆向运输；志贺毒素 B 亚基；Ｃ-末端；定点突变 The Effects of the End Amino Acids at Carboxyl-Terminus of Shiga Toxin B-Subunit on Its Retrograde Transport LIU Dan, FAN Yuying, LI Jie, GAO Xiaoge, HAO Miao, XUE Huiting (School of Life Sciences, Northeast Normal University, ChangChun 130024) Abstract: The B subunit of Shiga toxin (STxB) from enteric pathogens Shigella dysenteriae, is responsible for the attachment of its receptor globotriaosyl ceramide (Gb3), navigate the retrograde pathway from the plasma membrane to the endoplasmic reticulum. In order to demonstrate the role of end amino acid at carboxyl-terminus (C-terminus) of B-fragment on its retrograde transport speed and pathway, Shiga toxin B-subunit was modified by site-directed mutagenesis and addition of amino acid tail. The results showed that, when the end amino acid Arg (R) was mutated to be Ser (S), the speed of B-fragment transported into endoplasmic reticulum (ER) at 37 ℃ was slower. When adding acidic amino acid tail “Glu-Ser” (ES) behind the end amino acid “R”, B-fragment transporting speed slowed down and tended to remain in Golgi apparatus. Further studies showed that the effects induced by the mutations of amino acid tail were STxB-EEEES≥-EEES-EES-ES, suggesting that the retarded effect of the tail was increased along with the length of acidic amino acid augmented. The effect was probably produced by acidic amino acid tail, not only by amino acid “E”. The