The asparagine-linked oligosaccharides of the human chorionic gonadotropin beta subunit facilitate correct disulfide bond pairing.
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.
Feng
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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha 68198-6805, USA.
Matzuk
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Mountjoy
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Bedows
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Ruddon
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Boime
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D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry*; D016466:CHO Cells; D002240:Carbohydrate Sequence; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D006224:Cricetinae; D003553:Cystine / Q000378:metabolism*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D046911:Macromolecular Substances; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D015292:Glycoprotein Hormones, alpha Subunit; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D009844:Oligosaccharides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D003553:Cystine; D001216:Asparagine