Cystine knot mutations affect the folding of the glycoprotein hormone alpha-subunit. Differential secretion and assembly of partially folded intermediates.
The common glycoprotein hormone alpha-subunit (GPH-alpha) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10-60, 28-82, and 32-84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-alpha folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin beta-subunit (hCG-beta). Mutation of non-cystine knot disulfide bond 7-31, bond 59-87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-beta. From these studies we conclude that: (i) the cystine knot of GPH-alpha is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-alpha folding, its ability to be secreted, and its ability to heterodimerize with hCG-beta.
Darling
R J
RJ
Eppley Institute for Research in Cancer and Allied Diseases and the Department of Pharmacology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Ruddon
R W
RW
Perini
F
F
Bedows
E
E
D000409:Alanine; D019943:Amino Acid Substitution; D002460:Cell Line; D003545:Cysteine; D003553:Cystine; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D004220:Disulfides; D015292:Glycoprotein Hormones, alpha Subunit; D011994:Recombinant Proteins; D003553:Cystine; D003545:Cysteine; D000409:Alanine; D004229:Dithiothreitol