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 using Escherichia 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.
Hidaka
Y
Y
Division of Organic Chemistry and Division of Protein Metabolism, Institute for Protein Research, Osaka University, Suita, Japan. yuji@protein.osaka-u.ac.jp
Shimono
C
C
Ohno
M
M
Okumura
N
N
Adermann
K
K
Forssmann
W G
WG
Shimonishi
Y
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000502:physiology; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D021382:Protein Sorting Signals / Q000737:chemistry; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D004220:Disulfides; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; C097400:prouroguanylin; C044499:submandibular proteinase A; D012697:Serine Endopeptidases