Guanylin is a guanylyl cyclase (GC)-activating
peptide that is mainly secreted as the corresponding prohormone
of 94 amino acid residues. In this study, we show that
the originally isolated 15-residue guanylin, representing
the COOH-terminal part of the prohormone, is released from
the prohormone by cleavage of an Asp-Pro amide bond under
conditions applied during the isolation procedures. Thus,
the 15-residue guanylin is probably a non-native, chemically
induced GC-activating peptide. This guanylin molecule contains
two disulfide bonds that are absolutely necessary for receptor
activation. We demonstrate that the folding of the reduced
15-residue guanylin results almost completely in the formation
of the two inactive disulfide isomers. In contrast, the
reduced form of proguanylin containing the entire prosequence
folds to a product with the native cysteine connectivity.
Because proguanylin lacking the 31 NH2-terminal
residues of the prosequence folds only to a minor extent
to guanylin with the native disulfide bonds, it is evident
that this NH2-terminal region contributes significantly
to the correct disulfide-coupled folding. Structural studies
using CD and NMR spectroscopy show that native proguanylin
contains a considerable amount of α-helical and, to
a lesser extent, β-sheet structural elements. In addition,
a close proximity of the NH2- and the COOH-terminal
regions was found by NOESY. It appears that this interaction
is important for the constitution of the correct conformation
and provides an explanation of the minor guanylyl cyclase
activity of proguanylin by shielding the bioactive COOH-terminal
domain from the receptor.