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 dGalN 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 dGalNAc, dGalβ1 → 3 dGalNAc, dGalβ1 → 3 or 4 dGlcNAc, and dGalβ1 → 3 or 4 dGlcNAcβ1 → 3 dGal determinants on the carbohydrate moiety. Of the monosaccharides tested for inhibition of precipitation, dGalNAc and its p-nitrophenyl and methyl α-glycosides were best. These compounds were four to five times better than the corresponding dGal compounds but methyl βDGalNAc p was only about 40% more active than methyl β dGalp. The α-anomers of p-nitrophenyl DGalNAc p and dGalp, 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 dGal were about equal. Among the oligosaccharides tested, dGalβ1 → 3 dGalNAc and its tosyl derivatives were most active, the tosyl glycosides being about twice as active as dGalβ1 → 3 dGalNAc, which was somewhat more active than dGalNAcα1 → 6 dGal and dGalNAc, and 2.5 and 5 times as active as dGalNAcα1 → 3 dGalβ1 → 3 dGlcNAc and dGalNAcαl → 3 dGa1, respectively (blood group A specific). These findings suggest that a subterminal dGalNAc β-linked and substituted on carbon 3 plays an important role in binding. Consistent with this inference are the findings that dGalβ1 → 3 dGlcNAc and dGalβ1 → 6 dGal were poorer inhibitors although dGalβ1 → 3 dGlcNAc was two to three times as active as glycosides of dGal. Oligosaccharides with terminal nonreducing dGal and subterminal α-linked dGal were as active or less active than dGal. dGalβ1 → 3 dGlcNAcβ1 → 3 dGalβ1 → 4 dGlc (lacto- N-tetraose) and dGalβ1 → 3 dGlcNAcβ1 → 3 dGal-β1-O-(CH 2) 8COOCH 3 were equally active and 1.5 times as potent as dGalβ1 → 3 dGlcNAc whereas dGalβ1 → 3 dGlcNAcβ1 → 6 dGal was only 40% as potent as dGalβ1 → 3 dGlcNAc suggesting that a third sugar may be part of the determinant. Substitution of dGalβ1 → 3 dGlcNAcβ1 → 3 dGalβ1 → 4 dGlc on the subterminal dGlcNAc by lFucα1 → 4 in lacto- N-fucopentaose II reduced activity fourfold; if the nonreducing dGal is substituted by lFucα1 → 3 as in lacto- N-fucopentaose I its activity is almost completely abolished. This suggests that a terminal nonreducing dGal as well as subterminal dGlcNAc are contributing to binding. The β → 3 linkage of the terminal dGal to the subterminal amino sugar is significant since dGalβ1 → 4 dGlcNAc 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 dGalβ1 → 3 dGalNAcβ1 → 3 dGal, several other possibilities remain to be tested when suitable oligosaccharides become available.