Misfolded human chorionic gonadotropin β subunits are secreted from transfected Chinese hamster ovary cells

There are six intramolecular disulfide (S-S) bonds that form during intracellular folding of the human chorionic gonadotropin (hCG)-β subunit. Site-directed mutagenesis of every pair of Cys residues involved in the formation of each S-S bond was used to examine the roles that S-S bonds play in β subunit folding and secretion. Tryptic maps of secreted hCG-β showed that only one S-S bond formed in all S-S bond mutants that failed to fold from the earliest detectable β folding intermediate, pβ1, into a second major intermediate, pβ2 (C34A-C88A, C38A-C57A or C9A-C90A mutants), whereas all 5 remaining S-S bonds formed in mutants when pβ1 → pβ2 conversion occurred (C23A-C72A, C93A-C100A, or C26A-C110A mutants). Nonreducing SDS- polyacrylamide gel electrophoresis showed that β multimers were secreted from cells expressing S-S bond mutations where the folding of pβ1 → pβ2 was blocked. However, for mutations where pβ1 → pβ2 conversion was efficient, β monomers rather than multimers were secreted. For all cell lines studied, secreted hCG-β migrated as monomeric β during reducing SDS- polyacrylamide gel electrophoresis, indicating that hCG-β multimers formed via intermolecular cross-linking of unpaired thiols. Tryptic maps of hCG-β isolated from mutants lacking the 34-88 bond, where >80% turnover occurs, showed that only the 38-57 S-S bond formed. β Subunits lacking the 9-90 linkage also have only S-S bond 38-57 formed, but <10% turnover of C9A-C90A, hCG-β occurs. Thus, subtle conformational differences between partially folded or misfolded β subunits may determine whether hCG-β is degraded, or undergoes intracellular translocation and secretion.