The Asparagine-linked Oligosaccharides of the Human Chorionic Gonadotropin β Subunit Facilitate Correct Disulfide Bond Pairing

Abstract

The role of asparagine (N)-linked oligosaccharide chains in intracellular folding of the human chorionic gonadotropin (hCG)-beta subunit was determined by examining the kinetics of folding in Chinese hamster ovary (CHO) cells transfected with wild-type or mutant hCG-beta genes lacking one or both of the asparagine glycosylation sites. The half-time for folding of p beta 1 into p beta 2, the rate-determining step in beta folding, was 7 min for wild-type beta but 33 min for beta lacking both N-linked glycans. The p beta 1-->p beta 2 half-time was 7.5 min in CHO cells expressing the beta subunit missing the Asn13-linked glycan and 10 min for the beta subunit missing the Asn30-linked glycan. The inefficient folding of hCG-beta lacking both N-linked glycans correlated with the slow formation of the last three disulfide bonds (i.e. disulfides 23-72, 93-100, and 26-110) to form in the hCG-beta-folding pathway. Unglycosylated hCG-beta was slowly secreted from CHO cells, and beta subunit-folding intermediates retained in cells for more than 5 h were degraded into a hCG-beta core fragment-like protein. However, coexpression of the hCG-alpha gene enhanced folding and formation of disulfide bonds 23-72, 93-100, and 26-110 of hCG-beta lacking N-linked glycans. In addition, the molecular chaperones BiP, ERp72, and ERp94, but not calnexin, were found in a complex with unglycosylated, unfolded hCG-beta and may be involved in the folding of this beta form. These data indicate that N-linked oligosaccharides assist hCG-beta subunit folding by facilitating disulfide bond formation.

Characterization of a recombinant proteinase 3, the autoantigen in Wegener's granulomatosis and its reactivity with anti‐neutrophil cytoplasmic autoantibodies

“…Our analysis of the importance of the carbohydrate moieties in the binding of PR3 to Wegener's autoantibodies clearly shows that autoantibody recognition of PR3 epitope(s) is mainly oligosaccharide-independent in PMN-derived PR3, which already has a proper folding. Glycosylation, however, may also be involved in the folding process of PR3 as suggested by other reports showing that the N-linked oligosaccharides and their processing are critical for proper glycoprotein folding and assembly [27].…”

Specific N-linked glycosylation sites modulate synthesis and secretion of von Willebrand factor

“…23 It is also consistent with the role of glycosylation of the CK domains in Muc2 and human chorionic gonadotrophin which similarly affect dimer formation. 33,34 It is not known precisely how glycosylation promotes dimer formation but in the case of the Muc2, dimers were shown to form at an increased rate, but to be less stable and to rapidly revert to monomers. 33 It has been demonstrated that core glycan residues can interact with neighbouring amino acids and decrease flexibility of the local peptide region, stabilizing the protein structure.…”

Expression of a bioactive, single‐chain choriogonadotropin in Dictyostelium discoideum

“…The crystal structure of hCG elucidates that both the A and the β subunits contain unusual cystine-knot structures, involving three disulphide bridges each [26]. In addition, single-chain hCG contains four Nlinked sugars that are essential for intracellular folding and transport, as well as for biological activity [4]. Since functional single-chain hCG can be produced by Dictyostelium,we conclude that this organism contains a large variety of chaperones and folding enzymes, which are able to perform all post-translational modifications necessary for biological activity of glycoprotein hormones.…”

“…Since both the A and the β subunits contain a so-called cystine knot, it can be anticipated that protein disulphide isomerase plays a key role in the facilitation of the folding process [3]. In addition, it has been shown that the N-linked oligosaccharide side chains are required for proper folding, disulphide formation and secretion of hCG [4].The gonadotropins have been expressed in Chinese Hamster Ovary (CHO) cells, and their recombinant derivatives have bioCorrespondence to P. D. J. …”