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.
Highlights
From the :j:The Eppley Institute for Research in Cancer and Allied Diseases and Departments of §Pharmacology and
The role of asparagine (N)-linked oligosaccharide chains in intracellular folding of the human chorionic gonadotropin-/J subunit was determined by examining the kinetics of folding in Chinese hamster ovary (CHO) cells transfected with wild-type or mutant hCG-IJ genes lacking one or both of the asparagine glycosylation sites
The terminology used here is: CHO aJ3WT or CHO J3WT for CHO cells transfected with wild-type hCG-J3 genes with or without co-transfection of the a gene, respectively; CHO af3Asn(1 +2) and CHO j3Asn(1 +2) for CHO cells transfected with hCG-J3 genes mutated at both N-linked glycosylation sites with or without co-transfection of the a gene, respectively; CHO J3Asn[1] or CHO J3Asn[2] for CHO cells transfected with hCG-J3 genes mutated at the Asn[13] or Asn[30] glycosylation sites, respectively
Summary
Depts. of Pathology, Cell Biology, and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030. Since proper disulfide bond formation is a critical event in the folding and maturation of functional hCG-{3 subunits (3, 5-7, 20, 27) and altered glycosylation can cause alteration of folding and disulfide bond formation (21, 22), we postulated thatN-linked oligosaccharides are involved in hCG-{3 folding by facilitating disulfide bond formation. To test this hypothesis, we analyzed the kinetics of intracellular folding of hCG-{3 in CHO cells transfected with either the wild-type hCG-{3 gene or hCG-{3 genes mutated atN-linked glycosylation sites. The slow folding process of unglycosylated hCG-!3 allowed us to detect molecular chaperone-hCG-13 complexes that may be involved in hCG-13 folding
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