Head S C, Karmali M A, Lingwood C A
Department of Microbiology, Hospital for Sick Children, Ontario, Canada.
J Biol Chem. 1991 Feb 25;266(6):3617-21.
Verocytotoxins comprise a family of closely related subunit proteins. Two members of this group, VT1 and the immunologically distinct VT2, have been found to share similar physical properties, and yet several differences in their biological activities have been noted. The subunits of these toxins were separated using urea and isolated by high performance liquid chromatography gel filtration. Reconstituted VT1 and VT2 as well as VT1-A:VT2-B and VT2-A:VT1-B hybrid toxins were then prepared. The B subunit was found to determine cell culture specificity, cytotoxic titer, and antibody neutralizability as determined on Vero and MRC-5 cells. Cross-linking isolated B chains revealed 5 species upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis for both VT1-B and VT2-B. Using an in vitro translation system, both toxin A subunits inhibited protein synthesis at concentrations as low as 4 pM. In glycolipid binding assays, VT1 and VT1-B subunits competed equally on a molar basis with 125I-VT1 for the receptor, globotriaosylceramide, however, a 1000-fold excess of VT2 was required. Ligand analysis of direct VT1 and VT2 receptor binding assays revealed a difference in binding affinity constants (Kd of VT1 = 4.6 x 10(-8) M; VT2 = 3.7 x 10(-7) M).
志贺毒素由一组密切相关的亚基蛋白组成。已发现该组中的两个成员，即VT1和免疫上不同的VT2，具有相似的物理性质，但也注意到它们的生物活性存在一些差异。使用尿素分离这些毒素的亚基，并通过高效液相色谱凝胶过滤进行分离。然后制备了重组的VT1和VT2以及VT1-A:VT2-B和VT2-A:VT1-B杂合毒素。发现B亚基决定了在Vero和MRC-5细胞上测定的细胞培养特异性、细胞毒性滴度和抗体中和能力。对分离的B链进行交联后，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，VT1-B和VT2-B均显示出5种条带。使用体外翻译系统，两种毒素的A亚基在低至4 pM的浓度下均抑制蛋白质合成。在糖脂结合试验中，VT1和VT1-B亚基在摩尔基础上与125I-VT1对受体球三糖基神经酰胺的竞争能力相同，然而，需要1000倍过量的VT2。直接VT1和VT2受体结合试验的配体分析显示结合亲和常数存在差异（VT1的Kd = 4.6 x 10(-8) M；VT2的Kd = 3.7 x 10(-7) M）。
