Site-specific mutagenesis defines the intracellular role of the asparagine-linked oligosaccharides of chorionic gonadotropin β subunit

The β 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 CGβ proteins were expressed in Chinese hamster ovary cells alone or in the presence of native α subunit. Pulse-chase analysis of the β-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 >80% of the native and glycosylation mutant CGβ 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 α-subunit. Thus, in vivo the two N-linked oligosaccharides of CGβ are critical for efficient secretion and assembly with the α subunit and are likely important for proper folding of the CGβ subunit.