Immunochemical studies on the combining site of the d-galactopyranose and 2-acetamido-2-deoxy-d-galactopyranose specific lectin isolated from Bauhinia purpurea alba seeds

Abstract The combining site of the Bauhinia purpurea alba lectin was studied by quantitative precipitin and precipitin inhibition assays. Of 45 blood group substances, glycoproteins, and polysaccharides tested, 35 precipitated over 75% of the lectin. Precursor blood group substances with I activity (Cyst OG 10% from 20% and Cyst OG 20% from 10%), desialized fetuin, and desialized ovine salivary glycoprotein, in which more than 75% of the carbohydrate side chains have d GalN Ac linked through α1 → to the OH group of Ser or Thr of a protein core, completely precipitated the lectin. The poorly reactive blood group substances after mild acid hydrolysis or Smith degradation, as well as sialic acid-containing glycoproteins after removal of sialic acid, had substantially increased activity so that more than 80% of the lectin was precipitated. Precipitability with various blood group substances and glycoproteins is ascribable to the terminal nonreducing d GalNAc, d Galβ1 → 3 d GalNAc, d Galβ1 → 3 or 4 d GlcNAc, and d Galβ1 → 3 or 4 d GlcNAcβ1 → 3 d Gal determinants on the carbohydrate moiety. Of the monosaccharides tested for inhibition of precipitation, d GalNAc and its p -nitrophenyl and methyl α-glycosides were best. These compounds were four to five times better than the corresponding d Gal compounds but methyl β DGalNAc p was only about 40% more active than methyl β d Galp. The α-anomers of p -nitrophenyl DGalNAc p and d Galp, were twice as active as the corresponding β-anomers. Methyl αDGalNAc p was four times as active as the β-anomer but the inhibitory power of the methyl α- and β-anomers of d Gal were about equal. Among the oligosaccharides tested, d Galβ1 → 3 d GalNAc and its tosyl derivatives were most active, the tosyl glycosides being about twice as active as d Galβ1 → 3 d GalNAc, which was somewhat more active than d GalNAcα1 → 6 d Gal and d GalNAc, and 2.5 and 5 times as active as d GalNAcα1 → 3 d Galβ1 → 3 d GlcNAc and d GalNAcαl → 3 d Ga1, respectively (blood group A specific). These findings suggest that a subterminal d GalNAc β-linked and substituted on carbon 3 plays an important role in binding. Consistent with this inference are the findings that d Galβ1 → 3 d GlcNAc and d Galβ1 → 6 d Gal were poorer inhibitors although d Galβ1 → 3 d GlcNAc was two to three times as active as glycosides of d Gal. Oligosaccharides with terminal nonreducing d Gal and subterminal α-linked d Gal were as active or less active than d Gal. d Galβ1 → 3 d GlcNAcβ1 → 3 d Galβ1 → 4 d Glc (lacto- N -tetraose) and d Galβ1 → 3 d GlcNAcβ1 → 3 d Gal-β1-O-(CH 2 ) 8 COOCH 3 were equally active and 1.5 times as potent as d Galβ1 → 3 d GlcNAc whereas d Galβ1 → 3 d GlcNAcβ1 → 6 d Gal was only 40% as potent as d Galβ1 → 3 d GlcNAc suggesting that a third sugar may be part of the determinant. Substitution of d Galβ1 → 3 d GlcNAcβ1 → 3 d Galβ1 → 4 d Glc on the subterminal d GlcNAc by l Fucα1 → 4 in lacto- N -fucopentaose II reduced activity fourfold; if the nonreducing d Gal is substituted by l Fucα1 → 3 as in lacto- N -fucopentaose I its activity is almost completely abolished. This suggests that a terminal nonreducing d Gal as well as subterminal d GlcNAc are contributing to binding. The β → 3 linkage of the terminal d Gal to the subterminal amino sugar is significant since d Galβ1 → 4 d GlcNAc is a poorer inhibitor. Although the available data suggest that the combining site of the lectin Bauhinia purpurea alba may be most complementary to the structure d Galβ1 → 3 d GalNAcβ1 → 3 d Gal, several other possibilities remain to be tested when suitable oligosaccharides become available.