The carbohydrate chains of the beta 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 beta subunit of human chorionic gonadotropin (hCG-beta) isolated from the culture fluid of the choriocarcinoma cell line BeWo were released enzymatically by peptide-N4-(N-acetyl-beta-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. Residues not written in bold letters are variably present. [formula: see text] The incidence of triantennary carbohydrate chains is much higher than in normal urinary hCG-beta (26% vs 2%). The same holds for the alpha 1-6-fucosylation of the asparagine-bound GlcNAc (95% vs 42%). The presence of a bisecting GlcNAc and the occurrence of alpha 2-6-linked Neu5Ac in the most abundant N-glycans, are new features for hCG-beta. The major O-linked carbohydrate chains identified are the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-3(Neu5Ac alpha 2-6)GalNAc-ol and the hexasaccharide Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6(Neu5Ac alpha 2-3Gal beta 1-3)GalNAc-ol, both also found in normal urinary hCG. In addition, two novel O-glycans were characterized: [formula: see text]
Hård
K
K
Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Damm
J B
JB
Spruijt
M P
MP
Bergwerff
A A
AA
Kamerling
J P
JP
Van Dedem
G W
GW
Vliegenthart
J F
JF
D000117:Acetylglucosamine / Q000032:analysis*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D002822:Choriocarcinoma; D006063:Chorionic Gonadotropin / Q000096:biosynthesis / Q000737:chemistry* / Q000652:urine; D018997:Chorionic Gonadotropin, beta Subunit, Human; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000652:urine
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D009844:Oligosaccharides; D010446:Peptide Fragments; D000117:Acetylglucosamine