Identification of three oligosaccharide binding sites in ricin.
The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests 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 blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred 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 results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.
Steeves
R M
RM
ImmunoGen, Inc., 148 Sidney Street, Cambridge, Massachusetts 02139, USA.
Denton
M E
ME
Barnard
F C
FC
Henry
A
A
Lambert
J M
JM
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D012276:Ricin / Q000737:chemistry / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D009844:Oligosaccharides; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D012276:Ricin