Binding of Escherichia coli verotoxins to cell surface protein on wild-type and globotriaosylceramide-deficient Vero cells.
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.
Devenish
J
J
Department of Pathobiology, University of Guelph, ON, Canada.
Gyles
C
C
LaMarre
J
J
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D016466:CHO Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003432:Cross-Linking Reagents; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli; D011956:Receptors, Cell Surface / Q000378:metabolism*; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; D014281:Trihexosylceramides / Q000172:deficiency*; 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