title: Site-specific mutagenesis defines the intracellular role of the asparagine-linked oligosaccharides of chorionic gonadotropin beta subunit.
abstract: The beta subunit of human chorionic gonadotropin contains two asparagine (N)-linked oligosaccharides. To examine the structural and functional roles of these oligosaccharide units in vivo, we constructed mutant genes containing alterations in either the asparagine or threonine codons of the two glycosylation consensus sequences and inserted them into a eukaryotic expression vector. Wild-type and mutant CG beta proteins were expressed in Chinese hamster ovary cells alone or in the presence of native alpha subunit. Pulse-chase analysis of the beta-expressing clones showed that absence of the second N-linked sugar but not the first slows secretion 1.6-1.8-fold; absence of both N-linked units slows secretion 2-2.4-fold. Analysis of dimer clones reveals that greater than 80% of the native and glycosylation mutant CG beta subunits are secreted as dimer. However, pulse-chase analysis of these clones also reveals that the mutants completely devoid of N-linked sugars but not the single site mutants are slow to assemble with the alpha subunit. Thus, in vivo the two N-linked oligosaccharides of CG beta are critical for efficient secretion and assembly with the alpha subunit and are likely important for proper folding of the CG beta subunit.
authors: Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors: M M
authors: MM
authors: Matzuk
authors: I
authors: I
authors: Boime
mesh_terms: D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000502:physiology; D018997:Chorionic Gonadotropin, beta Subunit, Human; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D009154:Mutation; D009844:Oligosaccharides / Q000032:analysis; D010446:Peptide Fragments / Q000235:genetics* / Q000502:physiology
chemical_list: D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D009844:Oligosaccharides; D010446:Peptide Fragments; D001216:Asparagine