Intracellular Folding Pathway of the Cystine Knot-Containing Glycoprotein Hormone #alpha#-Subunit

摘要

Three of the five disulfide bonds in the glycoprotein hormone a-subunit (GPH-a) form a cystine knot motif that stabilizes a three-Ioop antiparallel structure. Previously, we described a mutant ( ak) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-a folding and secretion. In this study, we used ak as a model to study the intracellular GPH-a folding pathway. Cystine knot formation proceeded through a I-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-a has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-a 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-a and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.