CHEMICAL SYNTHESIS OF GLOBOTRIOSE AND GALABIOSE - RELATIVE STABILITIES OF THEIR COMPLEXES WITH ESCHERICHIA-COLI SHIGA-LIKE TOXIN-1 AS DETERMINED BY DENATURATION-TITRATION WITH GUANIDINIUM CHLORIDE

Globotriose [alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc] is the carb ohydrate moiety of the globotriosyl ceramide (Gb(3)), also known as th e germinal centre B-cell differentiation antigen CD77, a glycolipid pr esent on the plasma membrane of certain mammalian cells. In Gb(3), glo botriose functions as the cell-surface receptor for Shiga toxin and fo r the Shiga-like toxins (verocytotoxins). Here we report the chemical synthesis of globotriose and the corresponding terminal disaccharide, galabiose [alpha-D-Gal(1-->4)-beta-D-Gal]. Globotriose and galabiose a re attached via a linker to CNBr-activated Sepharose to generate affin ity matrices that permit the one-step purification of recombinant Shig a-like toxin-l from crude E. coli homogenates. Toxin is released from either of the immobilised saccharides by elution with 6 M guanidinium chloride. After dilution of the denaturant, the released toxin had ful l catalytic activity. Denaturation-titration experiments show that the bound toxin is released from galabiose-Sepharose at 2.3 M guanidinium chloride, while its release from globotriose-Sepharose requires a hig her concentration of 4.8 M. These results indicate that the glucose co mponent of globotriose contributes similar to 2.6 kcal mol(-1) to the binding;energy relative to galabiose.