Knut Adermann

Dual Function of the Propeptide of Prouroguanylin in the Folding of the Mature Peptide DISULFIDE-COUPLED FOLDING AND DIMERIZATION

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498–8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced usingEscherichia coli or human kidney 293T cells,respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.

The mechanism of the propeptide-mediated folding of guanylyl cyclase activating peptides

Guanylyl cyclase activating peptides (GCAPs), endogenous ligands for guanylyl cyclase C, are initially secreted in the form of prohormones and then processed into mature forms (Fig. 1). In previous studies, we have shown that the mature forms, GCAPs, do not contain the requisite information to permit themselves to adopt the native disulfide pairing, and that the pro-leader peptides in the precursor proteins mediate the folding of the mature peptides, resulting in the correct disulfide pairing in the mature peptides [1,2].