Structure of Asparagine-Linked Oligosaccharides on Human and Rabbit Testosterone-Binding Globulin1

Abstract

We have previously demonstrated, using two-dimensional polyacrylamide gel electrophoresis, that much of the microheterogeneity of human (h) and rabbit (rb) testosterone-binding globulin (TeBG) is due to differential glycosylation of a single protomer. Since glycosylation has been shown to be a physiologically important modification of proteins, we have examined the structure of the oligosaccharide chains attached to hTeBG and rbTeBG to facilitate future studies on the mechanisms of action of the proteins. The structures of the oligosaccharides attached to TeBG were determined by using serial lectin chromatography. About 10% of the TeBG from castrated male rabbits and about 20% of the TeBG from pregnant rabbits and from a human sample were not retained on a column of immobilized concanavalin-A (Con-A). This fraction would consist of TeBG with attached asparagine (Asn)-linked tri- and tetraantennary complex and serine/threonine (O)-linked oligosaccharides as well as non-glycosylated forms. None of the lectins used to subfractionate these species was effective. Forty to 50% of the TeBG applied to Con-A possessed biantennary complex oligosaccharides as indicated by the fact that it could be eluted with 10 mM 1-O-methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin (WGA). About 8% of the biantennary complex oligosaccharides on hTeBG and none of those on rbTeBG were fucosylated on the chitobiose core, as determined by chromatography on Lens culinaris lectin (LcH). Galactosylated oligosaccharides were also present on the TeBG in this fraction as indicated by its interaction with Ricinus communis-I (RCA-I). Thirty to 40% of the TeBG applied to Con-A was retained and could be eluted with 0.5 M methyl-alpha-D-mannopyranoside. This fraction contains TeBG possessing high mannose-type, hybrid-type, and complex galactosylated glycans as determined by chromatography on Con-A, WGA, and RCA-I. Evidence based on the binding of mannoside-eluted TeBG to Con-A, WGA, and RCA-I indicated that at least the TeBG in this fraction contained two glycosylation sites and that the sites were differentially glycosylated.