The galactose-binding sites of the cytotoxic lectin ricin can be chemically blocked in high yield with reactive ligands prepared by chemical modification of glycopeptides containing triantennary N-linked oligosaccharides.

A glycopeptide containing a triantennary N-linked oligosaccharide from fetuin was modified by a series of chemical and enzymic reactions to afford a reagent that contained a terminal residue of 6-(N-methylamino)-6-deoxy-D-galactose on one branch of the triantennary structure and terminal galactose residues on the other two branches. Binding assays and gel filtration experiments showed that this modified glycopeptide could bind to the sugar-binding sites of ricin. The ligand was activated at the 6-(N-methylamino)-6-deoxy-D-galactose residue by reaction with cyanuric chloride. The resulting dichlorotriazine derivative of the ligand reacts with ricin, forming a stable covalent linkage. The reaction was confined to the B-chain and was inhibited by lactose. Bovine serum albumin and ovalbumin were not modified by the activated ligand under similar conditions, and we conclude, therefore, that the reaction of the ligand with ricin B-chain was dependent upon specific binding to sugar-binding sites. Ricin that had its galactose-binding sites blocked by the covalent reaction with the activated ligand was purified by affinity chromatography. The major species in this fraction was found to contain 2 covalently linked ligands per ricin B-chain, while a minor species contained 3 ligands per B-chain. The cytotoxicity of blocked ricin was at least 1000-fold less than that of native ricin for cultured cells in vitro, even though the activity of the A-chain in a cell-free system was equal to that from native ricin. Modified ricin that contained only 1 covalently linked ligand was also purified. This fraction retained an ability to bind to galactose affinity columns, although with a lower affinity than ricin, and was only 5- to 20-fold less cytotoxic than native ricin.