Immunochemical studies on the carbohydrate specificity of Maclura pomifera lectin

Maclura pomifera lectin was purified from crude extracts of seeds by adsorption onto soluble polyleucyl hog A + H blood group substance and elution with either melibiose or N-actyl-d-galactosamine. The purified lectin formed a single band in immunodiffusion and immunoelectrophoresis against rabbit antiserum to the crude extract. It migrated as a broad band of five very closely packed fine bands on polyacrylamide electrophoresis at both acidic and alkaline pH. A broad band was seen on isoelectrofocusing of the purified lectin in acrylamide gels. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, three bands with molecular weights of 14,500, 13,500, and 12,000 were seen. The carbohydrate specificity of the M. pomifera lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated well with blood group substances which had been subjected to mild acid hydrolysis or to Smith degradation and with blood group precursor substances. The active and inactive antifreeze glycoproteins, composed of repreating units of dGalβ1 → 3dGalNAcαl → OThrAlaAla, were the best, precipitating 90% of the lectin with less than 3.0 μg of glycoprotein being required for precipitation of 50% of 6.0 μg lectin N. Precipitability of this lectin with various native and blood group substances and glycoproteins is attributable to the terminal nonreducing DGalNAc and dGalβ1 → 3dGalNAc on the carbohydrate moiety of those compounds. Among the monosaccharides and glycosides tested for inhibition of precipitation, the nitrophenyl α-glycosides of dGalNAc and dGal were most active and were more than 130 times as active as dGalNAc and dGal, respectively. dGalNAc was 3.3 times as active as dGal. Nitrophenyl α- and methyl α-glycosides of dGalNAc and dGal were much more active than the corresponding β-anomers. Nitrophenyl α-glycosides were substantially better than the corresponding methyl α-glycosides, indicating a hydrophobic contribution to the site subterminal to the sugar moiety as has been found for Wistaria floribunda and Sophora japonica lectins. dGalβ1 → 3dGalNAc and its β-tosyl derivative were the best of the oligosaccharides tested. They were as active as methyl αdGalNAc, about 5.6 times less active than nitrophenyl α-glycosides of DGalNAc, and 5 and 18.5 times as active as dGalNAcα1 → 6dGal and melibiose, respectively. dGalβ1 → 3dGlcNAc, lactose, lacto-N-tetraose, N-acetyllactosamine as well as blood-group-specific oligosaccharides gave no inhibition in amounts comparable to those used with dGalβ → 3dGalNAc. From these results, it can be concluded that hydrophobic interaction is important for binding and that the combining size of the lectin is at least as large as a disaccharide, and of the compounds studied is most complementary to a mucintype disaccharide on a human erythrocyte membrane, DGalβ1 → 3dGalNAc.