Agaricus bisporus agglutinin (ABA) isolated from edible mushroom has a potent anti-proliferative effect on malignant colon cells with considerable therapeutic potential as an anti-neoplastic agent. Since previous studies on the structural requirement for binding were limited to molecular or submolecular levels of Galβ1-3GalNAc (T; Thomsen–Friedenreich disaccharide glycotope; where Gal represents d-galactopyranose and GalNAc represents 2-acetamido-2-deoxy-d-galactopyranose) and its derivatives, the binding properties of ABA were further investigated using our collection of glycans by enzyme-linked lectinosorbent assay and lectin–glycan inhibition assay. The results indicate that polyvalent Galβ1-related glycotopes, GalNAcα1-Ser/Thr (Tn), and their cryptoforms, are the most potent factor for ABA binding. They were up to 5.5×105 and 4.7×106 times more active than monomeric T and GalNAc respectively. The affinity of ABA for ligands can be ranked as: multivalent Tα (Galβ1-3GalNAcα1-), Tn and I/II (Galβ1-3GlcNac/Galβ1-4GlcNAc, where GlcNAc represents 2-acetamido-2-deoxy-d-glucopyranose)>>>>monomeric Tα and Tn>I>>GalNAc>>>II, L (Galβ1-4Glc, where Glc represents d-glucopyranose) and Gal (inactive). These specific binding features of ABA establish the importance of affinity enhancement by high-density polyvalent (versus multiantennary I/II) glycotopes and facilitate our understanding of the lectin receptor recognition events relevant to its biological activities.
Abbreviations used: ABA, Agaricus bisporus agglutinin; ELLSA, enzyme-linked lectinosorbent assay; Gal, d-galactopyranose; GalNAc, 2-acetamido-2-deoxy-d-galactopyranose; Glc, d-glucopyranose; GlcNAc, 2-acetamido-2-deoxy-d-glucopyranose; HOC, human ovarian cyst fluid; OSM, ovine salivary glycoprotein; PNA, peanut (Arachis hypogaea) agglutinin; PSM, porcine salivary mucin; TBS, Tris/HCl-buffered saline; THGP, Tamm–Horsfall glycoprotein. The mammalian carbohydrate structural units in glycans used to define binding properties of ABA are: A, GalNAcα1-3Gal, human blood group A-specific disaccharide; Ah, GalNAcα1-3Gal(l-Fucα1-2), human blood group A-specific trisaccharide containing crypto H determinant; B, Galα1-3Gal, human blood group B-specific disaccharide; E, Galα1-4Gal, galabiose, the uropathogenic Escherichia coli receptor; F, GalNAcα1-3GalNAc; H, l-Fucα1-2Gal, human blood group H-specific disaccharide; I, Galβ1-3GlcNAc, human blood group type I precursor sequence; II, Galβ1-4GlcNAc, human blood group type II precursor sequence; L, Galβ1-4Glc; P, GalNAcβ1-3Gal; S, GalNAcβ1-4Gal; T, Galβ1-3GalNAc, Thomsen–Friedenreich disaccharide; Tα, Galβ1-3GalNAcα1-; Tn, GalNAcα1-Ser/Thr.