concanavalin-a and acetic-anhydride

The effect of a "bisecting" 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, linked (1----4) to the beta-D-mannopyranosyl group of asparagine-linked complex and hybrid oligosaccharides, on the binding of [14C]acetylated glycopeptides to columns of immobilized concanavalin A (Con A), Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin-120 (RCA-120) was investigated. The presence of this "bisecting" GlcNAc group caused significant inhibition of the binding to ConA-agarose of biantennary complex glycopeptides in which the two branches are terminated at their nonreducing ends by two GlcNAc groups, or by a Gal and a GlcNAc group, or by two Gal groups, or by a Man and a GlcNAc group. Binding of biantennary, complex glycopeptides to E-PHA-agarose required a "bisecting" GlcNAc group, a Gal group at the nonreducing terminus of the alpha-D-Man-p-(1----6) branch, and a terminal or internal GlcNAc residue linked beta-(1----2) to the alpha-D-Manp-(1----3) branch. Binding to RCA-120-agarose occurred only when at least one nonreducing terminal Gal group was present, and increased as the proportion of terminal Gal groups increased; the presence of a "bisecting" GlcNAc group caused either enhancement or inhibition of these binding patterns. It is concluded that a "bisecting" GlcNAc group affects the binding of glycopeptides to all three lectin columns.

Topics: Acetic Anhydrides; Acetylation; Acetylglucosamine; Carbohydrate Conformation; Carbohydrate Sequence; Carbon Radioisotopes; Concanavalin A; Glucosamine; Glycopeptides; Lectins; Oligosaccharides; Phytohemagglutinins; Plant Lectins; Plants, Toxic; Ricin; Ricinus communis; Sepharose

Oligosaccharides were prepared from alpha 1-antitrypsin (PiM) and human serum transferrin by hydrazinolysis and then reacetylated with radiolabelled acetic anhydride. Gel permeation chromatography revealed similarities between the two preparations. The antitrypsin oligosaccharides were resolved into 76% concanavalin A binding and 24% nonbinding sugar chains by lectin affinity chromatography; both fractions were further resolved by preparative paper electrophoresis. Small amounts of neutral oligosaccharides, as well as approximately 30% "monosialo" and 67% "disialo" biantennary oligosaccharides, were identified in the lectin-binding pool. The disialo oligosaccharide fraction was susceptible to sequential degradation with exoglycosidases. Upon incubation of the monosialo biantennary oligosaccharide fraction with CMP-[14C]sialic acid and a rat liver Golgi preparation as a source of transferase, a new doubly labelled peak was formed that eluted from the gel permeation chromatography column in the same volume as the disialo biantennary oligosaccharide fraction. The paper electrophoretogram of the lectin-nonbinding oligosaccharides revealed the presence of limited amounts of neutral oligosaccharides and oligosaccharides carrying two and three negative charges. The lectin-nonbinding oligosaccharide fraction with two charges was tentatively identified as disialo biantennary oligosaccharide containing a bisecting N-acetylglucosamine residue, and the fraction with three negative charges was tentatively identified as trisialo triantennary oligosaccharide.

Topics: Acetic Anhydrides; Acetylation; alpha 1-Antitrypsin; Carbohydrate Sequence; Carbon Radioisotopes; Chromatography, Affinity; Concanavalin A; Humans; Oligosaccharides; Transferrin; Tritium