Bisulfide bond mutations affect the folding of the human chorionic gonadotropin-β subunit in transfected Chinese hamster ovary cells

Previous kinetic studies have characterized the intracellular folding pathway of the human chorionic gonadotropin (hCG)-β subunit in which each of the folding intermediates can be biochemically identified based on the formation of disulfide (S-S) bonds: pβ1-early → pβ1-late → pβ2-free → pβ2-combined → native hCG-αβ. Based on these data, we postulated that hCG-β folding coincides with the formation of specific S-S bonds. We have now tested this hypothesis employing Chinese hamster ovary cells transfected with mutated hCG-β genes in which the Cys residues required for the formation of the final four (of six total) S-S bonds were replaced by Ala. When the Cys residues required for the third hCG-β S-S linkage to form (bond 9-90) were substituted, folding did not proceed beyond the earliest detectable folding intermediate (pβ1-early). In the absence of the subsequently formed S-S bond (bond 23-72), pβ1-early was converted into a second folding intermediate (pβ1-late), but conversion to the next intermediate (pβ2-free) was inhibited. When either of the final two S-S bonds (the carboxyl-terminal 93-100 or 26-110 bonds) were removed, conversion of pβ1-late to pβ2-free was detected, but conversion of pβ2-free to the last folding intermediate (pβ2-combined) was not observed. These data support the hypothesis that individual S-S bonds are involved in discrete steps in the hCG-β folding pathway.