Alteration of the carbohydrate binding specificity of verotoxins from Gal alpha 1-4Gal to GalNAc beta 1-3Gal alpha 1-4Gal and vice versa by site-directed mutagenesis of the binding subunit.

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RESUMO

Verotoxin 1 (VT-1) and Shiga-like toxin II (SLT-II) bind to the glycosphingolipid (GSL), globotriaosylceramide (Gb3), whereas pig edema disease toxin (VTE) binds to globotetraosylceramide (Gb4) and to a lesser degree Gb3. Amino acids important in the GSL binding specificity of VT-1 and VTE have been identified by site-directed mutagenesis. One mutation, Asp-18----Asn, in VT-1 resulted in binding to Gb4 in addition to Gb3 in a manner similar to VTE. Several mutations in VTE resulted in the complete loss of GSL binding; however, one mutation resulted in a change in the GSL binding specificity of the VTE B subunit. The double mutation Gln-64----Glu and Lys-66----Gln (designated GT3) caused a selective loss of Gb4 binding, effectively changing the binding phenotype from VTE to VT-1. Both wild-type VTE and GT3 were purified to homogeneity and binding kinetics in vitro were determined with purified GSLs from human kidney. The cell cytotoxicity spectrum of the mutant toxin was also found to be altered in comparison with VTE. These changes were consistent with the GSL content of the target cells.

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