Structures of N-glycosidic carbohydrate units of human chorionic gonadotropin.

The structures of the 4 asparagine-linked carbohydrate units of aand P-subunits of human chorionic gonadotropin (hCG-a and hCG+) have been determined by carrying out the structural investigations on the tryptic glycopeptides (uT-8, aT-lla, p-2, and pT-3 (Bellisario, R., Carlsen, R. B., and Bahl, 0. P. (1973) J. Biol. Chem. 248, 6796-6809; Carlsen, R. B., Bahl, 0. P., and Swaminathan, N. (1973) J. Biol. Chem. 248, 6810-6827). The glycopeptides were prepared from the tryptic digests of the reduced carboxamitimethyl-asialo hCG-cY and hCG-/I by gel filtration on Sephadex G-50, followed by further purification of the carbohydrate-containing fractions individually by high voltage paper electrophoresis at pH 1.8. The methods for the structural determination included techniques such as periodate oxidation, methylation with the identification of the methylated sugar by gas-liquid chromatography-mass spectrometry, and hydrolysis with glycosidases. The periodate oxidation resulted in the destruction of two out of three mannoses in the first cycle, destruction of one mannose in the second cycle, and one N-acetylglucosamine in the third cycle for each of the N-glycosidic carbohydrate units except in the case of hCG-fl glycopeptides, there was an additional loss of fucose residue in the first cycle. The precise intersugar linkages were determined by the methylation of each of the glycopeptides. The methylation studies indicated that all of the N-acetylglucosamine residues were 1,4-O-substituted with the exception of hCG-8 glycopeptides in which one of the N-acetylglucosamine residues was 1,4,6-linked; one mannose was 1,3,6-linked, one &a-linked, and one 1,6-linked, and the fucose was linked to N-acetylglucosamine by 1 + 6 linkage. The anomeric configuration of the glycosidic bonds was determined by sequential degradation of hCG subunits and the glycopeptides with specific glycosidases. All the N-glycosidic carbohydrate units appear to have the same basic structure except that fucose is present in only the glycopeptides from hCG+?.