Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.
The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.
D000606:Amino Sugars / Q000378:metabolism; D001665:Binding Sites; D001894:Borohydrides; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005944:Glucosamine / Q000378:metabolism; D006020:Glycopeptides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D037102:Lectins / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D019158:N-Acetylneuraminic Acid; D012794:Sialic Acids / Q000032:analysis; D014909:Wheat Germ Agglutinins
D000606:Amino Sugars; D001894:Borohydrides; D006020:Glycopeptides; D037102:Lectins; D012794:Sialic Acids; D014909:Wheat Germ Agglutinins; C032233:lactosamine; C025364:sodium borohydride; D006026:Glycoside Hydrolases; D019158:N-Acetylneuraminic Acid; D005944:Glucosamine; D008358:Mannose