Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (β-D-Gal(1→3)D-GalNAc)

Abstract

The binding of Artocarpus integrifolia lectin to Ndansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene- 1-sulfonyl) leads to a 100%i ncrease in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74×l0⁴ M⁻¹ at 20°C. The lectin has two binding sites for N-dansylgalactosamine. The values of -ΔH and -ΔS for the bindinogf N-dansylgalactosamine are in the rangeof values reported for severale ctin-monosaccharide interactions, indicating an absence of nonpolar interactiono f the dansylm oiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and tchoen sequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that theO H groups at C-2, C- 3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-α-Dgalactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Galβ1→3GalNAc binds very strongly to the lectinw hen compared with methyl-β-D-galactopyranoside, the β(1→3)-linked disaccharides such as Galβ1→3GlcNAc, and the β(1→4)-linked disaccharides, N-acetyllactosamine andla ctose. The major stabilizing force for the avid bindingof Tantigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore,e xtended. These data takent ogether suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overaltlo pography of its combining site when compared with that of peanut (Arachis hypogaea) agglutininThe results of stopped flow spectrometry for the binding of N-dansylgalactosamine to the Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of k₊₁ and k₋₁, at 21°C are 8.1×10⁵ M⁻¹ s^−l and 50 s⁻¹, respectively. The activation parameters indicate eant halpy-controlled association process.