title: Human chorionic gonadotropin beta-subunit affects the folding and glycosylation of alpha-cys mutants.
abstract: 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 beta-subunit. Previously, we reported that secreted forms of a 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 hCGbeta-subunit affects the conformations of alpha mutants, alpha-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGbeta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha-subunit were assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or -hCGbeta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that a 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 hCGbeta-subunit rescued the a half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the alpha 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 hCGbeta-subunit affects the folding and glycosylation of the alpha-subunit mutants.
authors.affiliation: Department of Obstetrics and Gynecology, Handa City Hospital, Handa, Aichi, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Furuhashi
authors.forename: N
authors.initials: N
authors.lastname: Suganuma
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry*; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006596:Hexosaminidases / Q000737:chemistry; D006801:Humans; D009154:Mutation; D017510:Protein Folding; D014162:Transfection
chemical_list: D018997:Chorionic Gonadotropin, beta Subunit, Human; D015292:Glycoprotein Hormones, alpha Subunit; D006596:Hexosaminidases; D003545:Cysteine