In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein.
Guanylyl cyclase-activating peptide II (GCAP-II), an endogenous ligand of particulate guanylyl cyclase C (GC-C), is processed from the precursor protein and circulates in human blood. GCAP-II consists of 24 amino acid residues and contains two disulfide bridges. The correct disulfide paring of GCAP-II is an absolute requirement for its biological activity. This study shows that the folding of the peptide from the reduced form yields a peptide with the native disulfide paring as a minor product and with non-native ones as major products, regardless of the presence or absence of reduced and oxidized glutathione. The results suggest that GCAP-II does not possess sufficient information to permit the adoption of the native conformation and to effectively form the correct disulfide pairing and, as a result, that GCAP-II is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone. We studied whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient information to facilitate the folding of GCAP-II. For this purpose, we prepared proGCAP-II in Escherichia coli by a recombinant technique and examined the disulfide-coupled folding of proGCAP-II from the reduced form. proGCAP-II was quantitatively recovered with the correctly folded structure from the reduced form both in the presence and in the absence of reduced and oxidized glutathione. The protein contains only disulfide linkages at the same positions as the mature form of proGCAP-II, GCAP-II, and the biologically active isomer of GCAP-II in the molecule. These results provide evidence that the propeptide of proGCAP-II is a critical factor in the formation of the correct disulfide paring in the folding of the protein.
Hidaka
Y
Y
Institute for Protein Research, Osaka University, Japan. yuji@protein.osaka-u.ac.jp
Ohno
M
M
Hemmasi
B
B
Hill
O
O
Forssmann
W G
WG
Shimonishi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000096:biosynthesis; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D007536:Isomerism; D007668:Kidney; D008969:Molecular Sequence Data; D045265:Natriuretic Peptides; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D002135:Calcium-Binding Proteins; D004220:Disulfides; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D045265:Natriuretic Peptides; D010455:Peptides; D011498:Protein Precursors; C084362:uroguanylin; D006162:Guanylate Cyclase