Analysis of the Oligosaccharides on Rat Androgen-Binding Protein Using Serial Lectin Chromatography1

Abstract

The microheterogeneity seen when rat androgen-binding protein (rABP) is analyzed by two-dimensional polyacrylamide gel electrophoresis is attributable, at least in part, to the differential glycosylation of a single promoter. Further insight into the chemical nature of the oligosaccharide units on rABP was obtained by serial lectin chromatography. When rABP was chromatographed on immobilized Concanavalin A (Con-A), it was fractionated into three classes: (1) one that did not bind to the lectin (about 44% of the rABP), (2) one that was bound and could be eluted with 10 mM 1-O-methyl alpha-D-glucopyranoside (glucoside), about 34%, and (3) one that could be eluted with 0.5 M methyl alpha-D-mannopyranoside (mannoside), about 23%. Binding to Con-A indicates the presence of asparagine-linked oligosaccharides. Chromatography of the glucoside-eluted peak on lentil lectin (LcH) indicated that the rABP in that fraction contained a fucose residue on the chitobiose core. Chromatography of the mannoside-eluted peak on wheat germ agglutinin (WGA) indicated the presence of rABP with high mannose- (44%) and hybrid-type (56%) glycans attached. Chromatography on Ricinus communis I (RCA-I) lectin indicated a species containing galactosylated complex-type oligosaccharide chains. Treatment of rABP forms with exoglycosidases confirmed the presence of externally disposed fucose, sialic acid, mannose, and galactose residues. LcH chromatography indicated that about 30% of the rABP that did not bind to Con-A possessed triantennary oligosaccharides with fucose on the chitobiose core. About 28% of the rABP was retarded when it was chromatographed on Phaseolus vulgaris E lectin, suggesting the presence of bisected biantennary chains with terminal galactose residues. We were unable to detect rABP species with serine- or threonine-linked oligosaccharide chains in this fraction. Other forms of rABP in the nonretained fraction of Con-A were not resolved. Western blotting did not reveal major differences in relative molecular weight (Mr) among the rABP species; some differences in the ratio of the heavy to the light subunit of the molecule were detectable.