Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer

doi:10.1016/S0167-0115(97)00020-7

Regulatory Peptides

Volume 70, Issues 2–3, 18 June 1997, Pages 111-120

https://doi.org/10.1016/S0167-0115(97)00020-7 Get rights and content

Abstract

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4–12 and 7–15, and its disulfide isomer, with disulfides positions 4–15 and 7–12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by ¹H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.

Introduction

Guanylin (Gu) is a recently discovered endogenous hormone initially isolated from rat jejunum [1]as a peptide composed of 15 amino acid residues with two possible intramolecular disulfide bridges. It stimulates intestinal membrane guanylate cyclase and shows sequence homology with heat stable enterotoxin, STa, from pathogenic bacteria, which causes secretory diarrhoea by a cGMP/guanylate cyclase-dependent mechanism [2]. The sequence of rat and human Gu was deduced from the cDNA sequence as the C-terminal fragment, positions 101–115, of the 115 amino acid human prepro-Gu 3, 4, 5. Characterization of human pro-Gu expressed in mammalian cells indicated that it corresponds to the C-terminal 94 amino acids 6, 7. Additionally Kuhn et al. have isolated a protein from human hemofiltrate that equates with pro-Gu as it had a molecular weight of 10 336 corresponding to the same 94 residues [8]. Two disulfide bridges of the intact guanylin were predicted from the homology with STa, that is, Cys⁴–Cys¹² and Cys⁷–Cys¹⁵. More recently the structurally related peptide, uroguanylin 9, 10has been isolated and shown to also have four cysteine residues at similar positions. Conformational stabilization by disulfide-bond formation is important for the biological actions of these peptides. Hence, to clarify the physiological significance of this peptide family, we have prepared two types of Gu with different disulfide forms [11], that is Gu (SS: 4–12 and 7–15) and iso-Gu (SS: 4–15 and 7–12). The present paper describes the synthesis, disulfide-linkage characterization, solution structures of two of the possible disulfide isomers of Gu by nuclear magnetic resonance (NMR) spectroscopy, binding studies and cGMP production to unequivocally establish the form of the biologically relevant intact form.

Section snippets

Chemical synthesis

Chemicals for peptide assembly such as protected amino acids, resins and coupling reagents were SynProPep® products from Shimadzu (Kyoto, Japan). All other solvents and chemicals were of analytical grade and used as received. Parallel simultaneous assembly of peptide chains was performed on the Fmoc-Cys(Acm)-p-alkoxybenzyl alcohol resin (60 mg, 0.5 meq/g) by the solid-phase method using a synthesizer [12], Shimadzu PSSM-8 System (Kyoto, Japan), by the 9-fluorenylmethyloxycarbonyl (Fmoc)

Results

Assembly of the peptide chain was successfully and efficiently performed by the Fmoc-tBu strategy using a simultaneous multiple peptide synthesizer [12]. Protected peptide-resin was cleaved with TFA-scavenger cocktail to give quantitative yields of the tetra S-protected derivatives. Peptides were analyzed by RP-HPLC, sequencing, amino acid analysis to give peptide contents, and LSIMS to give exact molecular weights which allowed discrimination of sulfhydryl and disulfides. The two Acm moieties

Discussion

Each lane of the independent simultaneous multiple synthesizer provided rapid assembly of the same backbone peptides that are completely contamination free and have different Cys side-chain protection groups. As two of three intramolecular disulfide isomers, (SS: 4–12, 7–15 and 4–15, 7–12) were possible biologically active forms from molecular dynamics calculations, they were efficiently synthesized by the Fmoc-strategy using a simultaneous multiple peptide synthesizer. Multiple lanes were used

Acknowledgements

The authors wish to thank M. Yamaguchi, Shimadzu Corp. for his technical assistance. A part of the present work was supported by the Monbushou International Scientific Research Program from the Ministry of Education, Science and Culture of Japan.

References (26)

R.C Wiegand et al.
Rat guanylin cDNA: Characterization of the precursor of an endogenous activator of intestinal guanylate cyclase
Biochem Biophys Res Commun
(1992)
R.C Wiegand et al.
Human guanylin: cDNA isolation, structure, and activity
FEBS Lett
(1992)
K.C Garcia et al.
Processing and characterization of human proguanylin expressed in Escherichia coli
J Biol Chem
(1993)
M Kuhn et al.
The circulating bioactive form of human guanylin is a high molecular weight peptide (10.3 kDa)
FEBS Lett
(1993)
M.M Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)
H Ozaki et al.
Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli
J Biol Chem
(1991)
M Nakazato et al.
Identification of biologically active and inactive human uroguanylins in plasma and urine and their increases in renal insufficiency
Biochem. Biophys. Res. Commun.
(1996)
M.G Currie et al.
Guanylin: An endogenous activator of intestinal guanylate cyclase
Proc Natl Acad Sci USA
(1992)
M Field et al.
Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine
Proc Natl Acad Sci USA
(1978)
F.J De Sauvage et al.
Precursor structure, expression, and tissue distribution of human guanylin
Proc Natl Acad Sci USA
(1992)

S Schultz et al.

Cloning and expression of guanylin

J Biol Chem

(1992)

F.K Hamra et al.

Uroguanylin: Structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase

Proc Natl Acad Sci USA

(1993)

T Kita et al.

Characterization of human uroguanylin: a member of the guanylin peptide family

Am J Physiol

(1994)

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Human guanylin, coded by the GUCA2A gene, is a member of a peptide family that activates intestinal membrane guanylate cyclase, regulating electrolyte and water transport in intestinal and renal epithelia. Deregulation of guanylin peptide activity has been associated with colon adenocarcinoma, adenoma and intestinal polyps. Besides, it is known that mutations on guanylin receptors could be involved in meconium ileus. However, there are no previous works regarding the alterations driven by single nucleotide polymorphisms in guanylin peptides. A comprehensive in silico analysis of missense SNPs present in the GUCA2A gene was performed taking into account 16 prediction tools in order to select the deleterious variations for further evaluation by molecular dynamics simulations (50 ns). Molecular dynamics data suggest that the three out of five variants (Cys104Arg, Cys112Ser and Cys115Tyr) have undergone structural modifications in terms of flexibility, volume and/or solvation. In addition, two nonsense SNPs were identified, both preventing the formation of disulfide bonds and resulting in the synthesis of truncated proteins. In summary the structural analysis of missense SNPs is important to decrease the number of potential mutations to be in vitro evaluated for associating them with some genetic diseases. In addition, data reported here could lead to a better understanding of structural and functional aspects of guanylin peptides.
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Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer

Abstract

Introduction

Section snippets

Chemical synthesis

Results

Discussion

Acknowledgements

Biochem Biophys Res Commun

FEBS Lett

J Biol Chem

FEBS Lett

Anal Biochem

J Biol Chem

Biochem. Biophys. Res. Commun.

Guanylin: An endogenous activator of intestinal guanylate cyclase

Proc Natl Acad Sci USA

Heat-stable enterotoxin of Escherichia coli: in vitro effects on guanylate cyclase activity, cyclic GMP concentration, and ion transport in small intestine

Proc Natl Acad Sci USA

Precursor structure, expression, and tissue distribution of human guanylin

Proc Natl Acad Sci USA

Cloning and expression of guanylin

J Biol Chem

Uroguanylin: Structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase

Proc Natl Acad Sci USA

Characterization of human uroguanylin: a member of the guanylin peptide family

Am J Physiol