ASPARAGINE-LINKED OLIGOSACCHARIDES FACILITATE HUMAN CHORIONIC-GONADOTROPIN BETA-SUBUNIT FOLDING BUT NOT ASSEMBLY OF PREFOLDED BETA WITH ALPHA

To determine the role of asparagine (N)-linked oligosaccharide chains in protein folding and assembly, the well established hCG-beta in vitro folding and assembly assays were used to analyze how the human CG (hCG) beta-subunit devoid of one or two N-linked glycans folds and assembles under different conditions. Two approaches were used: 1) site-specific mutagenesis of hCG-beta synthesized in Chinese hamster ovary cells transfected with beta-mutants lacking the asparagine glycosylation sites; and 2) enzymatic deglycosylation of hCG-beta synthesized in JAR cells with peptide N-glycosidase F or endoglycosidase H. In both cases, [S-35]cysteine-labeled beta-subunits were used as substrates to measure the conversion of the hCG-beta folding intermediate p beta 1 into p beta 2 and assembly of p beta 2 with urinary alpha. Using the mutated substrates from Chinese hamster ovary cells, it was found that 60% of wild-type p beta 1 (two N-linked glycans), 60% of p beta 1 missing the Asn(13)-linked glycan, 40% of p beta 1 missing the Asn(30)-linked glycan, and 10% of p beta 1 missing two N-linked glycans were converted to the corresponding p beta 2, respectively. With the enzymatically deglycosylated substrate from JAR cells, 90% of p beta 1 (two N-linked glycans), 70% of p beta 1((1)) (one N-linked glycan), and 10% of p beta 1((0)) (without N-linked glycan) folded into p beta 2 under cysteamine and cystamine redox conditions with or without protein disulfide isomerase. These data demonstrate that at least one N-linked glycan is required for efficient folding of hCG-beta and that the Asn(30)-linked glycan is more important than Asn(13)-linked glycan for hCG-beta folding. It also was shown that the composition of N-linked glycans of hCG-p beta 1 did not change protein folding, since hCG-beta substrates with high mannose oligosacharides folded as efficiently as beta-substrates containing sialylated complex oligosaccharides. Moreover, assembly of the already folded, assembly-competent folding intermediate, p beta 2, was not affected by removal of one or both of the N-linked glycans of the beta-subunit. These data thus show that N-linked glycans play their most important role in the folding component-of the folding and assembly pathway for hCG-beta.