Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
PLoS One. 2012;7(10):e46964. doi: 10.1371/journal.pone.0046964. Epub 2012 Oct 8.
It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB(7/8)-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His(6)-tag to different proteins increased their binding affinity to the protective antigen (PA) PA(63)-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB(7/8)-family of toxins. His(6)-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to -70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA(63). Cell-based experiments demonstrated that addition of an N-terminal His(6)-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA(63). Our results revealed that addition of His(6)-tags to several factors increase their binding properties to PA(63) and enhance the property to intoxicate cells.
定义细菌毒素的生化特性以将它们用作分子注射器装置，以便将酶活性递送到宿主细胞中是很有意义的。AB(7/8)-型二元毒素是最有效和最特异的细菌蛋白毒素之一。B 亚基寡聚化形成一个孔，该孔以高亲和力结合宿主细胞受体和酶 A 亚基。这允许复合物的内吞作用，随后将 A 亚基注入宿主细胞的细胞质中。在这里，我们报告说，在不同的蛋白质中添加 N 端 His(6)-标签会增加它们与保护性抗原 (PA) PA(63)-通道的结合亲和力，无论它们是否与 AB(7/8)-型毒素家族有关（C2I）或无关（gpJ、EDIN）。当跨膜侧设置为-70 mV 对应于细胞内部时，His(6)-EDIN 表现出结合稳定性常数的电压依赖性增加，约增加 25 倍。令人惊讶的是，C. botulinum 毒素 C2II-通道没有表现出这种 PA(63)的特征。基于细胞的实验表明，添加 N 端 His(6)-标签也会促进 C2I 或 EDIN 通过 PA(63)感染内皮细胞。我们的结果表明，向几种因子添加 His(6)-标签会增加它们与 PA(63)的结合特性，并增强感染细胞的特性。
