School of Life Sciences, Northeast Normal University, Changchun, Jilin 130024, PR China.
Mol Med Rep. 2012 Jul;6(1):220-6. doi: 10.3892/mmr.2012.885. Epub 2012 Apr 23.
The Shiga toxin B-subunit (STxB), from the enteric pathogen, Shigella dysenteriae, is responsible for the attachment of its receptor, globotriaosylceramide (Gb3), and navigates the retrograde pathway from the plasma membrane to the endoplasmic reticulum (ER). In this study, in order to demonstrate the role of carboxyl-terminus (C-terminus/al) amino acids of the B-fragment on the retrograde transport speed and the retrograde transport pathway, STxB was modified by site-directed mutagenesis and by the addition of an amino acid tail. The results showed that when the C-terminal amino acid, arginine [Arg (R)], was mutated to serine [Ser (S)], the speed of the B-fragment transportation into the ER at 37 ˚C was slower. When an acidic amino acid tail 'glutamine (Glu)-Ser' (ES) was added to the C-terminal amino acid 'R', the B-fragment transporting speed slowed down and remained in the Golgi apparatus. Further experiments showed that the effects induced by mutations of the amino acid tail resulted in STxB-EEEES ≥-EEES>-EES>-ES, demonstrating that the retardation effect on the tail was increased and the length of the acidic amino acid was augmented. The effect was possibly produced by an acidic amino acid tail, not only by the amino acid 'E'. The significant inhibitory effect on the speed of B-fragment retrograde transport was observed only when the mutations of the acidic amino acid tail were linked near to the C-terminus. These results may provide important insights for the study of transport mechanisms and for the development of STxB serial proteins as vectors for drug delivery.
志贺毒素 B 亚基（STxB）来源于肠道病原体志贺氏痢疾杆菌，负责其受体神经节苷脂 GM1 的附着，并将其导航到从质膜到内质网（ER）的逆行途径。在这项研究中，为了证明 B 片段羧基末端（C 末端/α）氨基酸在逆行运输速度和逆行运输途径上的作用，STxB 通过定点突变和添加氨基酸尾巴进行了修饰。结果表明，当 C 末端氨基酸精氨酸[Arg（R）]突变为丝氨酸[Ser（S）]时，B 片段在 37°C 下进入 ER 的运输速度较慢。当 C 末端氨基酸“精氨酸[Arg（R）]”添加酸性氨基酸尾巴“谷氨酰胺（Glu）-丝氨酸（Ser）”（ES）时，B 片段的运输速度减慢并留在高尔基体内。进一步的实验表明，氨基酸尾巴突变引起的效应导致 STxB-EEEES≥-EEES>-EES>-ES，表明对尾部的延迟效应增加，酸性氨基酸的长度增加。这种效应可能是由酸性氨基酸尾巴引起的，而不仅仅是由氨基酸“E”引起的。只有当酸性氨基酸尾巴的突变与 C 末端附近连接时，才观察到对 B 片段逆行运输速度的显著抑制作用。这些结果可能为运输机制的研究以及 STxB 系列蛋白作为药物输送载体的开发提供重要的见解。
