Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin beta subunit.
Human chorionic gonadotropin (hCG) consists of two noncovalently joined alpha and beta subunits similar to the other glycoprotein hormones. To study the function of the individual disulfide bonds in subunit assembly and secretion, site-directed mutagenesis was used to convert the 12 cysteine (Cys) residues in the beta subunit of hCG to either alanine or serine. Both cysteines of proposed disulfide pairs were also mutated. These mutant hCG beta genes were transfected alone or together with the wild-type alpha gene into Chinese hamster ovary cells. Only 3-10% assembly could be achieved with derivatives containing single Cys mutations at positions 26, 110, 72, and 90, whereas no assembly was detected with the other 8 mutants. However, double mutations of pairs 26-110 or 23-72 showed increased dimer formation (11 and 36%, respectively). The secretion rate of individual mutants varied significantly. Whereas the Cys-23 and 72 mutants were secreted normally (t1/2 = 140-190 min), the Cys-26 mutant was secreted faster (t1/2 = 70 min), and the other 9 mutants were secreted slower (t1/2 = 280-440 min); mutations of both Cys at 26 and 110 caused much faster secretion (t1/2 = 34 min). Although the secretion rate of these mutants differed, they were quantitatively recovered in the medium except for mutant Cys-88, Cys-23-72, and Cys-34-88 (40, 55, and 10% secreted, respectively). Thus, interruption of any disulfide bond in the hCG beta subunit alters the structure sufficiently to block dimer formation and in some cases slow secretion, although the stability for most of the mutant hCG beta subunits is not greatly affected. The data indicate that interruption of any hCG beta disulfide bond generates different structural forms that are unable to assemble with the alpha subunit, and that the structural requirements for stability and assembly are different.
Suganuma
N
N
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Matzuk
M M
MM
Boime
I
I
D000818:Animals; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000096:biosynthesis / Q000235:genetics*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D003001:Cloning, Molecular; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism*; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D014162:Transfection
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D003545:Cysteine