Preparation of VT1 and VT2 hybrid toxins from their purified dissociated subunits. Evidence for B subunit modulation of a subunit function.
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).
Head
S C
SC
Department of Microbiology, Hospital for Sick Children, Ontario, Canada.
Karmali
M A
MA
Lingwood
C A
CA
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification* / Q000633:toxicity; D001667:Binding, Competitive; D002470:Cell Survival / Q000187:drug effects; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000235:genetics / Q000302:isolation & purification*; D014176:Protein Biosynthesis / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000378:metabolism; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides / Q000378:metabolism; D014709:Vero Cells
D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; D004768:Enterotoxins; D011956:Receptors, Cell Surface; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides; C018549:globotriaosylceramide