Human chorionic gonadotropin β-subunit affects the folding and glycosylation of α-Cys mutants

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha -subunit but differ in their hormone-specific beta -subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of beta -subunits is rigid, whereas that of alpha -subunit is flexible and can be molded by a alpha -subunit. Previously, we reported that secreted forms of alpha mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCG beta -subunit affects the conformations of alpha mutants, or-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCG beta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha -subunit were assessed by continuous labeling with [S-35]methionine/cysteine, immunoprecipitation with anti-alpha or -hCG beta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that alpha homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of beta -subunit. This finding indicated that hCG beta -subunit rescued the alpha half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the cu mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive alpha -subunits was less than 5% of total alpha -subunits. In contrast to free alpha -subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCG beta -subunit affects the folding and glycosylation of the alpha -subunit mutants.