Abstract
We have examined the carbohydrate binding specificity of the B4 lectin from Vicia villosa seeds. The B4 lectin agglutinates Tn-exposed erythrocytes specifically and binds to these erythrocytes (1.4 X 10(6) sites/cell) with an association constant of 4.2 X 10(7) M-1. The concentrations of saccharides and glycopeptides of defined structure which cause 50% inhibition of B4 lectin binding to Tn-exposed erythrocytes were determined. N-Acetylgalactosamine is the best monosaccharide inhibitor, causing 50% inhibition of binding at a concentration of 0.04 mM. Other monosaccharides inhibit lectin binding in the following order of decreasing potency: N-acetylgalactosamine greater than methyl-alpha-galactopyranoside greater than p-nitrophenyl-alpha- or beta-galactopyranoside greater than methyl-beta-galactopyranoside, galactose greater than galactosamine greater than mannose, N-acetylglucosamine. The disaccharide Gal beta 1,3GalNAc causes 50% inhibition of binding at a concentration of 2.8 mM, a concentration similar to that of the p-nitrophenyl-alpha- or beta-galactopyranosides. Glycopeptides containing O-glycosidically linked oligosaccharide units are significantly more potent inhibitors of lectin binding than the oligosaccharide units alone. The most potent glycopeptide inhibitor is a fetuin glycopeptide containing two alpha-linked N-acetylgalactosamine units. This glycopeptide causes 50% inhibition of lectin binding at a concentration of 0.00034 mM and probably closely resembles the B4 lectin binding site on Tn-exposed erythrocytes.
Highlights
The disaccharide GalPl,3GalNAc was as potent as the p-nitrophenylgalactopyranosides, causing 50% inhibition of lectin binding at a concentration of 2.8 mM
We have shown that the B4 lectin from V. villosa seeds agglutinates Tn-exposed erythrocytes but not, AI, B, or 0 erythrocytes
Our studies show that there areapproximately 1.4 X lo6binding sites for the B4lectin on Tn-exposed erythrocytes
Summary
N-Acetylgalactosaniine Residues a-Linked to Serinoer Threonine Residuesin Cell Surface Glycoproteins”. Mouse cytotoxic T lymphocytes (2). recent reports have questioned these findings, our preliminary studies with cell) with an association constantof 4.2 x107~ - l T. he the purified B4 lectin have been consistent with this observam ~. Tin binding in the following order of decreasing poserine or threonine residues in their surface glycoproteins(Tntency:N-acetylgalactosamine > methyl-a-galactopy- exposed erythrocytes). We have examined the carbohydrate ranoside > p-nitrophenyl-a-or 8-galactopyranoside> binding specificityof the B, lectin by utilizingsaccharides and methyl+-galactopyranoside, galactose > galactosa- glycopeptidesof defiied structureto inhibit the binding of the mine > mannose, N-acetylglucosamine. The studies suggest possiride Gal/31,3GalNAcauses 60%inhibitionof binding at ble oligosaccharidestructures to which the Bq lectin may bind a concentration of 2.8 m ~ a,concentration similar to on mouse cytotoxic T lymphocytes.
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