Devenish J, Gyles C, LaMarre J
Department of Pathobiology, University of Guelph, ON, Canada.
Can J Microbiol. 1998 Jan;44(1):28-34.
We have examined verotoxin (VT) binding to cell surface proteins. When Vero or globotriaosylceramide (Gb3) deficient Vero (VRP) cells were incubated with 125I-labelled verotoxin 2(VT2) and disuccinimidyl suberate cross-linker, SDS-PAGE of cell lysates showed radiolabelled bands at 44, 50, 60, 86, 102, and 138 kDa. When 125I-labelled verotoxin 1 (VT1) was cross-linked, radioactive bands occurred at 51, 67, 101, 160, 188, and 232 kDa. In contrast, 125I-labelled VT1 B subunit produced a single radioactive band migrating at 50 kDa. CHO cells did not bind labelled VT. VT2 binding to VRP cells fit a rectangular hyperbola suggesting a single class of binding sites. In contrast, VT1 and VT1 B subunit binding to VRP cells was best fit by sigmoidal curves suggesting the presence of positive cooperativity between at least two binding sites. Scatchard analysis of VT2 binding data yielded 3.5 x 10(9) molecules bound/microgram of cell protein with an equilibrium dissociation constant (KD) of 13 nM. The apparent KD was 9.7nM for VT1 and 73.2 nM for VT1 B subunit. These results indicate that VT binds to a protein, or proteins, on the surface of susceptible cells and that there appear to be differences between VT1 and VT2 binding. Interactions between VT1 or VT2 and the proteins demonstrated here may be important in the biological activity of VT.
我们检测了志贺毒素（VT）与细胞表面蛋白的结合情况。当将Vero细胞或缺乏球三糖神经酰胺（Gb3）的Vero细胞（VRP细胞）与125I标记的志贺毒素2（VT2）及辛二酸二琥珀酰亚胺酯交联剂一起孵育时，细胞裂解物的SDS - PAGE显示在44、50、60、86、102和138 kDa处有放射性标记条带。当125I标记的志贺毒素1（VT1）进行交联时，在51、67、101、160、188和232 kDa处出现放射性条带。相比之下，125I标记的VT1 B亚基产生了一条迁移至50 kDa的单一放射性条带。CHO细胞不结合标记的VT。VT2与VRP细胞的结合符合矩形双曲线，表明存在单一类别的结合位点。相比之下，VT1和VT1 B亚基与VRP细胞的结合最适合用S形曲线拟合，表明至少两个结合位点之间存在正协同性。对VT2结合数据进行Scatchard分析，得出每微克细胞蛋白结合3.5×10⁹个分子，平衡解离常数（KD）为13 nM。VT1的表观KD为9.7 nM，VT1 B亚基的表观KD为73.2 nM。这些结果表明，VT与易感细胞表面的一种或多种蛋白质结合，并且VT1和VT2的结合似乎存在差异。此处展示的VT1或VT2与蛋白质之间的相互作用可能在VT的生物活性中起重要作用。
