Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin beta-subunit by disruption of disulfide bonds.
The human chorionic gonadotropin beta-subunit (hCGbeta) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGbeta produced in Chinese hamster ovary (CHO) cells: wild-type hCGbeta (betaWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of betaWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled betaWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGbeta molecule.
Moriwaki
T
T
Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Aichi, Japan.
Suganuma
N
N
Furuhashi
M
M
Kikkawa
F
F
Tomoda
Y
Y
Boime
I
I
Nakata
M
M
Mizuochi
T
T
D000117:Acetylglucosamine / Q000737:chemistry; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005690:Galactose / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D009154:Mutation; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D014316:Tritium
D018997:Chorionic Gonadotropin, beta Subunit, Human; D004220:Disulfides; D009844:Oligosaccharides; D014316:Tritium; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D003545:Cysteine; D000409:Alanine; D000117:Acetylglucosamine; D005690:Galactose