The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the beta subunit of human chorionic gonadotropin (hCG- beta), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG-beta were investigated: the rate-limiting events in the folding of this protein, and the assembly of hCG-beta with, hCG-alpha. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate-limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG-beta folding were attained in a 2 mM glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10muM cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half-time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the beta subunit under these conditions yields a functional protein, based on its ability to assemble with the alpha subunit. The rates of assembly of hCG-beta with hCG-alpha in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t(1/2/sb>= 9 to 12 min)). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine.(c) 1994 John Wiley & Sons, Inc.

Three of the five disulfide bonds in the glycoprotein hormone alpha-subunit (GPH-alpha) form a cystine knot motif that stabilizes a three-loop antiparallel structure. Previously, we described a mutant (alpha(k)) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-alpha folding and secretion. In this study, we used alpha(k) as a model to study the intracellular GPH-alpha folding pathway. Cystine knot formation proceeded through a 1-disulfide intermediate that contained the 28-82 disulfide bond. Formation of disulfide bond 10-60, then disulfide bond 32-84, followed the formation of 28-82. Whether the two non-cystine knot bonds 7-31 and 59-87 could form independent of the knot was also tested. Disulfide bond 7-31 formed rapidly, whereas 59-87 did not form when all cysteine residues of the cystine knot were converted to alanine, suggesting that 7-31 forms early in the folding pathway and that 59-87 forms during or after cystine knot formation. Finally, loop 2 of GPH-alpha has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-alpha folding. To test this, we replaced residues 36-55 in the flexible loop 2 with an artificially flexible glycine chain. Consistent with our hypothesis, folding and secretion were unaffected when loop 2 was replaced with the glycine chain. Based on these findings, we describe a model for the intracellular folding pathway of GPH-alpha and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X(1)-Gly-X(2)-Cys and Cys-X(3)-Cys. Previously, we characterized the contributions that cystine residues of the hCG subunit cystine knots make in folding, assembly, and bioactivity. Here, we determined the contributions that noncysteine residues make in hCG folding, secretion, and assembly. When the X(1), X(2), and X(3) residues of hCG-alpha and -beta were substituted by swapping their respective cystine knot motifs, the resulting chimeras appeared to fold correctly and were efficiently secreted. However, assembly of the chimeras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X(2) residue made the greatest individual contribution. Analysis by tryptic mapping, high performance liquid chromatography, and SDS-polyacrylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X(1)-Gly-X(2)-Cys sequence of either the alpha- or beta-subunit cystine knot resulted in non-native disulfide bond formation and subunit misfolding. This occurred even when the most conservative change possible (Gly --> Ala) was made. From these studies we conclude that all three "X" residues within the hCG cystine knots are collectively, but not individually, required for the formation of assembly-competent hCG subunits and that the invariant Gly residue is required for efficient cystine knot formation and subunit folding.

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.

The hCGbeta gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGbeta gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGbeta gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGbeta gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in beta gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether betaV79M hCG had biological properties that differed from those of wild-type hCG, a beta-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone alpha-subunit in Chinese hamster ovary cells and 293T cells. When we examined betaV79M hCG biosynthesis, we detected atypical betaV79M hCG folding intermediates, including a betaV79M conformational variant that resulted in a beta-subunit with impaired ability to assemble with the alpha-subunit. The inefficient assembly of betaV79M hCG appeared to be independent of beta-subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the betaV79M subunit to fold correctly. The majority of the V79M beta-subunit synthesized was secreted as unassembled free beta. Although the amount of alphabeta hCG heterodimer formed and secreted by betaV79M-producing cells was less than that by wild-type beta-producing cells, the hCG that was secreted as alphabeta V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.