Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces

Abstract

A competitive binding assay has been developed to determine how modifications to the B subunit of cholera toxin affect the binding affinity of the subunit for an ileal brush border membrane surface. The Ricinus communis 120 agglutinin (RCA 120) specifically binds to terminal β- d-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside G m1 . Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA 120 agglutinin. Displacement of RCA 120 from brush border surfaces was proportional to the concentration of B subunit added. This assay was used to study the effect of modification of B subunit on competitive binding affinity for the ileal brush border surface. The B subunit of cholera toxin was modified by coupling an average of five sulfhydryl groups to each B subunit molecule and by reaction of the SH-modified B subunit with liposomes containing a surface maleimide group attached to phosphatidylethanolamine. SH-modified B subunit was approximately 200-fold more effective than native B subunit in displacing lectin from brush border surfaces in the competitive binding assay. The enhanced binding activity was retained on covalent attachment of the modified B subunit to the liposome surface. We conclude that the B subunit of cholera toxin may be a useful targeting agent for directing liposomes to cell surfaces that contain a ganglioside G m1 ligand.