The effects of the carboxyl-terminus amino acids of the Shiga toxin B-subunit on retrograde transport.

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亚单位（STxB）来自肠道病原体痢疾志贺菌，负责与其受体球三糖神经酰胺（Gb3）结合，并沿着从质膜到内质网（ER）的逆行途径运输。在本研究中，为了阐明B片段的羧基末端（C末端/α1）氨基酸在逆行运输速度和逆行运输途径中的作用，通过定点突变和添加氨基酸尾对STxB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℃进入内质网的运输速度变慢。当酸性氨基酸尾“谷氨酰胺（Glu） - 丝氨酸（Ser）”（ES）添加到C末端氨基酸“R”上时，B片段的运输速度减慢并停留在高尔基体中。进一步的实验表明，氨基酸尾突变所产生的影响表现为STxB - EEEES≥ - EEES > - EES > - ES，这表明对尾的阻滞作用增强且酸性氨基酸的长度增加。这种影响可能是由酸性氨基酸尾产生的，而不仅仅是由氨基酸“E”产生。只有当酸性氨基酸尾的突变靠近C末端时，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将STxB系列蛋白开发为药物递送载体提供重要的见解。