Abstract
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.
Highlights
The common glycoprotein hormone ␣-subunit (GPH-␣) 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-␣ folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin -subunit
GPH-␣ is a unique subunit in that it contains intrinsic structural characteristics that allow it to combine with four different  subunits, suggesting that GPH-␣ must contain a high degree of flexibility
Summary
To characterize the role of each disulfide bond, we have converted each pair of cysteine residues involved in a disulfide bond to alanine From these experiments, we have determined that removal of any of the three cystine knot disulfide bonds (10 – 60, 28 – 82, or 32– 84) significantly disrupted folding and resulted in multiple folding intermediates. The results reported here are the first to describe the construction and folding of a cystine knot-containing protein from which all other disulfide bonds have been removed, except those involved in the knot This has enabled us to study the role of the cystine knot in GPH-␣ secretion and heterodimerization with hCG- and should serve as a simplified model protein for future studies involving the cystine knot motif
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