Properties of human vasopressin precursor constructs: Inefficient monomer folding in the absence of copeptin as a potential contributor to diabetes insipidus
These studies were aimed at an initial characterization of the human vasopressin precursor and the evaluation of factors leading to misfolding by the pathological 87STOP mutation. This mutation deletes the precursor's glycosylated copeptin segment, which has been considered unnecessary for folding, and the last seven neurophysin residues. We investigated the role in folding of the last seven neurophysin residues by comparing the properties of the 87STOP precursor and its derivative neurophysin with those of the corresponding wild-type proteins from which copeptin had been deleted, leading to the following conclusions. First, despite modulating effects on several protein properties, the last seven neurophysin residues do not make a significant net thermodynamic contribution to precursor folding; stabilities of the mutant and wild-type precursors to both guanidine denaturation and redox buffer unfolding are similar, as are in vitro folding rates. Second, the monomeric forms of both precursors are unstable and predicted to fold inefficiently at physiological pH and temperature, as evidenced by precursor behavior in redox buffers and by thermodynamic calculations. Third, both precursors are significantly less stable than the bovine oxytocin precursor. These results, together with earlier studies elsewhere of vasopressin precursor behavior within rat neurons, are shown to represent a self-consistent argument for a role for glycosylated copeptin in vasopressin precursor folding in vivo, copeptin most probably assisting refolding by facilitating interaction of misfolded monomers with the calnexin/calreticulin system. This hypothesis provides an explanation for the absence of copeptin in the more stable oxytocin precursor and suggests that the loss of copeptin contributes to 87STOP pathogenicity. Reported cell culture studies of rat precursor folding are also discussed in this context. Most generally, the results emphasize the significance of monomer stability in the folding pathways of oligomeric proteins.