Galactose-induced dimerization of blocked ricin at acidic pH: Evidence for a third galactose-binding site in ricin B-chain

Blocked ricin is a glycoconjugate formed by covalent modification of each of the two galactose-binding sites of ricin with affinity ligands derived by modification of glycopeptides containing galactose-terminated, triantennary, N-linked oligosaccharides. Blocked ricin undergoes a pH-dependent reversible self-association, being predominantly dimeric at neutral pH and monomeric at acidic pH. The shift in the monomer-dimer equilibrium towards the monomeric form at acidic pH (pH 4) is inhibited by lactose, as shown by size-exclusion chromatography. This behavior of blocked ricin can be reproduced in studies with isolated blocked B-chain. The effect, which is dependent on the concentration of the sugar, is specific for sugars having terminal galactose moieties, or sugars having the same orientation of hydroxyl groups at C2 and C4 as galactose. These results are interpreted as providing further support for the notion that ricin B-chain has a third galactose-binding site, which may be important for the intracellular trafficking of ricin during intoxication of cells.