CARBOHYDRATE-COMPOSITION OF THE ALPHA-SUBUNIT OF HUMAN CHORIOGONADOTROPIN (HCG-ALPHA) AND THE FREE-ALPHA MOLECULES PRODUCED IN PREGNANCY - MOST FREE-ALPHA AND SOME COMBINED HCG-ALPHA MOLECULES ARE FUCOSYLATED

The carbohydrate compositions of pregnancy-derived hCGα (dissociated from intact hCG) and free α-subunit were analyzed using a combination of chemical analysis, lectin affinity chromatography, and glycosidase sensitivity. For direct compositional analysis, parallel samples were hydrolyzed in tri-fluoroacetic acid and analyzed for sialic acid and neutral sugars without prior derivitization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at high pH, and the resolved monosaccharides were quantified by pulsed amperometric detection. The amounts of sugar that were found relative to peptide indicated the presence of two N-linked oligosaccharides per molecule on both hCGα and free α. Free α contained 2.5-fold higher amounts of sialic acid and galactose as well as a higher amount of iV-acetylglucosamine than did hCG α. Free α also contained a 6-fold higher amount of fucose than did hCG α (1.2 vs. 0.2 residues of fucose/molecule) Serial fractionation of intact hCGα and free α molecules by lectin affinity chromatography indicated that virtually all of the hCG α -subunits contained at least one Concanavalin-A (Con-A)-binding site, whereas as many as 32% of the free a molecules could not bind to Con-A. Chromatography on Lens culinaris (Lch) resulted in 12% binding of hCG α and approximately 72% binding of free α (80-85% of the Con-A-bound free a and 47-48% of the Con-A-nonbound free α bound to Lch)Endoglycosidase-H (endo-H) treatment of hCG α released a portion of the oligosaccharides. The endo-H-released material appeared to be a monoantennary hybrid based on DEAE-binding properties and carbohydrate composition. In contrast to hCG α, free a was completely resistant to endo-H treatment. Incubation of endo-H-resistant hCGα with glycopeptidase-A resulted in the release of two components, which could be separated into monoantennary and biantennary fractions on the basis of size and chargeThe collective data suggest that hCGα contains primarily monoantennary hybrid oligosaccharide structures and relatively little fucose. In contrast, free a contains primarily multiantennary oligosaccharide structures, and most of the free a molecules contain at least one oligosaccharide with fucose attached to the asparagine-linked N-acetylglucosamine residue. © 1990 by The Endocrine Society.