THE ASPARGINE-LINKED OLIGOSACCHARIDES OF THE HUMAN CHORIONIC-GONADOTROPIN BETA-SUBUNIT FACILITATE CORRECT DISULFIDE BOND PAIRING

The role of asparagine (N)-linked oligosaccharide chains in intracellu lar folding of the human chorionic gonadotropin (hCG)-beta subunit was determined by examining the kinetics of folding in Chinese hamster ov ary (CHO) cells transfected with wild-type or mutant hCG-beta genes la cking 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 b eta folding, was 7 min for wildtype beta but 33 min for beta lacking b oth 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 Asn(13)-linked gl ycan and 10 min for the beta subunit missing the Asn(30)-linked glycan . The inefficient folding of hCG-beta lacking both N linked glycans co rrelated 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-foldi ng pathway. Unglycosylated hCG-beta was slowly secreted from CHO cells , and beta subunit-folding intermediates retained in cells for more th an 5 h were degraded into a hCG-beta core fragment-like protein, Howev er, 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-lin ked glycans, In addition, the molecular chaperones BiP, ERp72, and ERp 94, but not calnexin, were found in a complex with unglycosylated, unf olded hCG-beta and may be involved in the folding of this beta form, T hese data indicate that N-linked oligosaccharides assist hCG-beta subu nit folding by facilitating disulfide bond formation.