ALTERATION OF THE GLYCOLIPID BINDING-SPECIFICITY OF THE PIG EDEMA TOXIN FROM GLOBOTETRAOSYL TO GLOBOTRIASOYL CERAMIDE ALTERS INVIVO TISSUE TARGETING AND RESULTS IN A VEROTOXIN-1 LIKE DISEASE IN PIGS

All members of the verotoxin (VT) family specifically recognize globo- series glycolipids on the surface of susceptible cells. Those toxins t hat are associated with human disease, VT1, VT2, and VT2c, bind to glo botriaosyl ceramide (Gb3) while VT2e, which is associated with edema d isease of swine, binds preferentially to globotetraosyl ceramide (Gb4) . We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell , G. J., K. Ramotar, B. Boyd, B. W Toye, C. A. Lingwood, and J. L. Bru nton. 1992. Proc. Natl. Acad. Sci. USA. 89:524). The concomitant mutat ion of Gln64 and Lys66 in the VT2e binding subunit to the correspondin g residues (Glu and Gln, respectively) found in VT2 effectively conver ted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro. We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared wi th classical VT2e-induced edema disease. The tissue localization of ra diolabeled GT3 after intravascular injection was elevated in neural ti ssues compared with VT2e accumulation, while localization of GT3 to th e gastrointestinal tract was relatively reduced. Accordingly, the path ological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemor rhage and edema of the cerebellum, and submucosa of the stomach and la rge intestine. In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease. VT2e, u nlike GT3 or VT1, bound extensively to red cells, which have high leve ls of Gb4. The overall tissue distribution of VT2e was thus found to b e influenced by regional blood flow to each organ and not solely by th e Gb4 levels of these tissues. Conversely, the distribution of GT3 (an d VT1), which cleared more rapidly from the circulation, correlated wi th respective tissue Gb3 levels rather than blood flow. These studies indicate the primary role of carbohydrate binding specificity in deter mining systemic pathology, suggest that the red cells act as a toxin c arrier in edema disease, and indicate that red cell binding does not p rotect against the pathology of systemic verotoxemia.