The carbohydrate chains of the β subunit of human chorionic gonadotropin produced by the choriocarcinoma cell line BeWo. Novel O-linked and novel bisecting-GlcNAc-containing N-linked carbohydrates

The N-linked carbohydrate chains of the β subunit of human chorionic gonadotropin (hCG-β) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F. Subsequently, the O-linked oligosaccharides were split off from the N-deglycosylated protein by mild alkaline borohydride treatment. The carbohydrate chains were purified in their intact sialylated forms by FPLC anion-exchange chromatography on Mono Q, HPLC on Lichrosorb-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. 1H-NMR spectroscopic analysis of the major fractions demonstrates the occurrence of the following sialylated diantennary and triantennary N-linked oligosaccharides. The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG-β (26%vs 2%). The same holds for the α1-6-fucosylation of the asparagine-bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of α2-6-linked Neu5Ac in the most abundant N-glycans, are new features for hCG-β. The major O-linked carbohydrate chains identified are the tetrasaccharide Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc-ol and the hexasaccharide Neu5Acα2-3Galβ1-4GlcNAcβ1-6(Neu5Acα2-3Galβ1-3)GalNAc-ol, both also found in normal urinary hCG. In addition, two novel O-glycans were characterized.