Effect of covalent modification on the binding of cholera toxin B subunit to ileal brush border surfaces.
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 communis120 agglutinin (RCA120) specifically binds to terminal beta-D-galactosyl residues such as those found in oligosaccharide side chains of glycoproteins and ganglioside GM1. Conditions were designed to produce binding competition between the B subunit of cholera toxin and the RCA120 agglutinin. Displacement of RCA120 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 GM1 ligand.
Uwiera
R E
RE
Veterinary Physiological Sciences, University of Saskatchewan, Saskatoon, Canada.
Romancyia
D A
DA
Wong
J P
JP
Forsyth
G W
GW
D000818:Animals; D001667:Binding, Competitive; D002772:Cholera Toxin / Q000737:chemistry* / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007082:Ileum / Q000378:metabolism*; D066298:In Vitro Techniques; D007583:Jejunum / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008081:Liposomes; D046911:Macromolecular Substances; D008871:Microvilli / Q000378:metabolism*; D037121:Plant Lectins; D013329:Structure-Activity Relationship; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013552:Swine
D037102:Lectins; D008081:Liposomes; D046911:Macromolecular Substances; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1; D013388:Succinimides; D013438:Sulfhydryl Compounds; D005677:G(M1) Ganglioside; D002772:Cholera Toxin