The specificity of the combining site of the lectin from Vicia villosa seeds which reacts with cytotoxic T-lymphoblasts☆

The combining site of purified Vicia villosa lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin, which specifically hemagglutinated blood group A erythrocytes nevertheless precipitated to different extents with blood group A1, A2, H, B and precursor I substances from saliva and ovarian cysts, and with different blood group substances of the same specificity indicating an interaction of the lectin with a non-blood group determinant site. The agglutinating and precipitating activities of the lectin were not strongly affected by EDTA or bivalent cations. Precipitates of Vicia villosa lectin with certain blood group glycoproteins showed unusually high solubilities as compared to other lectin glycoprotein and antigen-antibody precipitates. Inhibition assays with various monosaccharides, glycosides and oligosaccharides indicate that the Vicia villosa lectin is specific for terminal, non-reducing, α-linked dGalNAc. Of the monosaceharides tested, methyl αdGalNAc, p-NO2-phenyl αdGalNAc and dGalNAc were best. They were about 100 times better inhibitors than the corresponding dGal compounds. The most potent inhibitor was methyl αdGalNAc which was 10 times more active than dGalNAc and 18 times more potent than the β-anomer. Among disaccharides tested, dGalNAcαl → 3dGal was most active, about as potent as methyl αdGalNAc, twice as active as dGalNAcαl → 6dGal and 8 times more potent than dGalNAcαl → 6dGalNAc, indicating the importance of a subterminal αl → 3-linkeddGal in the binding. dGalNAcαl → 3dGalβl → 3dGlcNAc was 7 times less active and the blood group A determinant was less than 140 as active as dGalNAcαl → 3dGal. These findings indicate that the combining site of the Vicia villosa lectin is at least as large as the disaccharide dGalNAcαl → 3dGal and that the αl → 3 linkage is important in the binding. The unique nature of this site is consistent with its high specificity for a glycoprotein on cytotoxic T-lymphocytes.