Immunochemical studies on the combining site of the d-galactose/ N-acetyl- d-galactosamine specific lectin from Erythrina cristagalli seeds

Abstract

The combining site of the Erythrina cristagalli lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated best with two fractions of a precursor human ovarian cyst blood group substance with I and i activities. A 1, A 2, B, H, Le a, and Le b blood group substances precipitated poorly to moderately and substances of the same blood group activity precipitated to varying extents. These differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. Specific precipitates with the poorly reactive blood group substances were found to be more soluble than those reacting strongly. Precipitation was minimally affected by EDTA or divalent cations. Among the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl β dGal was most active and was 10 times more active than methyl β dGal, indicating involvement of hydrophobic contacts in site specificity. Methyl α dGalNAc, p-nitrophenyl α dGalNAc, methyl α dGal, N-acetyl- d-galactosamine, p-nitrophenyl α dGal, methyl β dGal, and p-nitrophenyl β dGalNAc were progressively less active than p-nitrophenyl β dGal. The best disaccharide inhibitor dGalβ1 → 4 dGlcNAc was 7.5 times more potent than p-nitrophenyl β dGal. A tetraantennary and triantennary oligosaccharide containing four and three dGalβ1 → 4 dGlcNAcβ1 → branches, respectively, were, because of cooperative binding effects, 1.6 and 2.5 times more active than the bi- and monoantennary oligosaccharides, respectively. dGalβ1 → 4 dGlcNAcβ1 → 6 dGal and dGalβ1 → 4 dGlcNAcβ1 → 2 dMan had the same activity, being 1.5 times more active than dGalβ1 → 4 dGlcNAc, which was 2.6 and 8.5 times more active than dGalβ1 → 3 dGlcNAc and dGalβ1 → 3 dGlc, respectively. Substitutions by N-acetyl- d-galactos-amine or l-fucose on the d-galactose of inhibitory compounds blocked activity. These results suggest that a hydrophobic interaction with the subterminal sugar is important in the binding and that the specificity of the lectin combining site involves a terminal dGalβ1 → 4 dGlcNAc and the β linkage of a third sugar.