Identification of three oligosaccharide binding sites in ricin

The galactoside-binding sites of ricin B chain can be blocked by affinity-d irected chemical modification using a reactive ligand derived from asialogl ycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is mo dified to contain a reactive dichlorotriazine moiety. Two separate galactos ide-binding sites have been clearly established in the ricin B chain by X-r ay crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 2 60-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A me thod was developed using thiol-specific labeling of the ligand combined wit h subsequent immunoaffinity chromatography which allowed the isolation of r icin B chain peptides covalently linked to the Ligand from proteolytic dige sts of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of bloc ked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferr ed that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These res ults are interpreted as providing support for the notion that the ricin B c hain has three oligosaccharide binding sites.