Intracellular folding, assembly, and secretion of the cystine knot-containing glycoprotein hormone alpha-subunit.

摘要

The common glycoprotein hormone α-subunit (GPH-α) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10-60, 28-82, and 32-84). Additionally, there are two non-cystine knot disulfides (7-31 and 59-87). By converting cysteine pairs to alanine, we have determined the role of individual disulfide bonds in GPH-a folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin β-subunit (hCG-β) Mutation of bond 7-31, bond 59-87, or both (leaving only the cystine knot, termed α_k) resulted in efficient folding and secretion. Conversely, cystine knot mutants were inefficiently secreted and formed multiple folding intermediates that display differential secretion and assembly properties. These results suggest that the GPH-α cystine knot is necessary and sufficient for folding and that the ability to fold correlates with secretion and assembly.; Using α_k as a model to study the intracellular folding pathway of a cystine knot protein, we determined that cystine knot formation proceeds through a 1-disulfide intermediate containing 28-82. Formation of disulfide 10-60, then 32-84, follows the formation of 28-82. Non-cystine knot bond 7-31 appears to form before cystine knot formation, while 59-87 appears to form after the cystine knot. The flexibility of loop 2 suggests that it does not actively drive folding. Indeed, replacement of loop 2 with a flexible glycine chain did not affect folding or secretion. A similar result was obtained for the hCG-β loop 2, suggesting that this finding may be applicable to the mechanism of folding for other cystine knot proteins.; hCG subunits contain the consensus sequences Cys-x₁-GIY-X ₂-Cys and CYS-X₃-CYS in their cystine knots. By swapping cystine knot motifs, we determined the contributions that non-cysteine residues make in folding, secretion, and assembly. Although the chimeras showed no alterations in folding, heterodimer formation was abrogated, suggesting that the ‘x’ residues function in a subunit-specific manner. Mutation of the Gly in either GPH-α or hCG-β resulted in subunit misfolding. Therefore, non-cysteine residues of the hCG cystine knots are functionally important for both folding and subunit assembly.