In Vitro Disulfide-Coupled Folding of Guanylyl Cyclase-Activating Peptide and Its Precursor Protein

Hidaka, Yuji; Ohno, Megumu; Hemmasi, Bahram; Hill, O.; Forssmann, Wolf‐Georg; Shimonishi, Yasutsugu

doi:10.1021/bi9731246

Cited by 40 publications

(66 citation statements)

References 38 publications

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“…An essential contribution of the prosequences of guanylin and uroguanylin in the in vitro disulfide-coupled folding was previously shown (10,11). Furthermore, unusual elution behavior in size exclusion chromatography led to the suggestion of the homologous proteins proguanylin and prouroguanylin being dimers in solution (10,11). Moreover, it was suggested that dimer formation is involved in the folding pathway of prouroguanylin, with the critical protein concentration for dimerization ranging between 1 and 10 µM (13).…”

Section: Discussionmentioning

confidence: 99%

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Native and Recombinant Proguanylin Feature Identical Biophysical Properties and Are Monomeric in Solution

et al. 2002

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Guanylin, an intestinal peptide hormone and endogenous ligand of guanylyl cyclase C, is produced as the corresponding prohormone proguanylin. The mature hormone consists of 15 amino acid residues, representing the COOH-terminal part of the prohormone comprised of 94 amino acid residues. Here we report the recombinant expression and purification of proguanylin with its native disulfide connectivity, as well as the biophysical characterization of the recombinant and native protein. The comparison of recombinant and native proguanylin revealed identical biophysical and structural properties, as deduced from CZE, HPLC, and mass spectrometry, as well as NMR spectroscopy and CD spectroscopy at various temperatures and pH values. Exhaustive analytical ultracentrifugation studies were employed for protein concentrations up to the millimolar range to determine the association state of recombinant as well as native proguanylin, revealing both proteins to be monomeric at the applied solution conditions. As a result, a former identified close proximity between the termini of proguanylin is due to intramolecular interactions.The intestinal peptide hormone guanylin is an endogenous activator of guanylyl cyclase C (1). Activation of the cyclase markedly increases secretion of fluid and electrolytes into the intestinal lumen (2) by cGMP-mediated activation of the cystic fibrosis transmembrane conductance regulator (CFTR) (3, 4). For bioactivity, formation of the correct 1-3, 2-4 disulfide bonds of guanylin is necessary (1, 5). Even though guanylin possesses a unique cysteine connectivity, it is able to form two interconverting topological stereoisomers (6), with only one of them (A-form) showing significant biological effects (7).Guanylin is expressed as the corresponding prohormone containing 94 amino acid residues, with the mature hormone [guanylin-(80-94); numbering is according to the sequence of the prohormone] located at its COOH terminus, and is secreted into the intestinal mucosa and blood (8,9). It has been shown that an essential contribution of the prosequences of guanylin and the closely related uroguanylin is in the disulfide-coupled folding (10, 11). The prohormone, however, only shows negligible guanylyl cyclase C activating potency (10, 12). As an explanation for the inactivation of the bioactive COOH-terminal part of proguanylin, a shielding by its NH 2 terminus was suggested, since a close proximity of the NH 2 -and COOH-terminal regions of proguanylin was found (10). Since prouroguanylin and proguanylin show unusual elution behavior in size exclusion chromatography, they were assumed to be dimeric in solution (13). Nonetheless, no variation of the oligomerization state of native proguanylin was observed over the concentration range from 0.24 to 2.4 mM (10). As reversible association is concentration dependent, this result favors a monomeric state, but a very tight dimer cannot be excluded. An accurate determination of the oligomerization state, however, is a requirement for the correct interpretatio...

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Section: Discussionmentioning

confidence: 99%

“…It has been shown that an essential contribution of the prosequences of guanylin and the closely related uroguanylin is in the disulfide-coupled folding (10,11). The prohormone, however, only shows negligible guanylyl cyclase C activating potency (10,12).…”

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confidence: 99%

Native and Recombinant Proguanylin Feature Identical Biophysical Properties and Are Monomeric in Solution

et al. 2002

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“…A 41-amino-acid-residue peptide corresponding to the deduced mature Gal-6 peptide was synthesized by Genosphere Biotechnologies (Paris, France) using Fmoc solid-phase synthesis on a Symphony synthesizer (Protein Technology Inc., Tucson, AZ). Disulfide bridge formation was accomplished by the air-oxidation method as described by Hidaka et al (17). Synthesized Gal-6 was dissolved in 100 mM Tris-HCl (pH 8.0) in the presence of reduced and oxidized glutathione (2 mM glutathione, 1 mM glutathione disulfide) and incubated under an N 2 atmosphere at room temperature for 3 days.…”

Section: Methodsmentioning

confidence: 99%

The β-Defensin Gallinacin-6 Is Expressed in the Chicken Digestive Tract and Has Antimicrobial Activity against Food-Borne Pathogens

Dijk

Veldhuizen

Kalkhove

et al. 2007

Antimicrob Agents Chemother

124

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Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of ␤-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken ␤-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 g/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken ␤-defensin gallinacin-6 plays an important role in chicken innate host defense.

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“…24. The synthetic peptides were purified by RP-HPLC, and their identities were confirmed by MALDI-TOF MS and amino acid analysis (25,26).…”

Section: Structures Of Pad4mentioning

confidence: 99%

Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4

Arita

Shimizu

Hashimoto

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

125

130

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Histone arginine methylation is a posttranslational modification linked to the regulation of gene transcription. Unlike other posttranslational modifications, methylation has generally been regarded as stable, and enzymes that demethylate histone arginine residues have not been identified. However, it has recently been shown that human peptidylarginine deiminase 4 (PAD4), a Ca 2؉ -dependent enzyme previously known to convert arginine residues to citrulline in histones, can also convert monomethylated arginine residues to citrulline both in vivo and in vitro. Citrullination of histone arginine residues by the enzyme antagonizes methylation by histone arginine methyltransferases and is thus a novel posttranslational modification that regulates the level of histone arginine methylation and gene activity. Here we present the crystal structures of a Ca 2؉ -bound PAD4 mutant in complex with three histone N-terminal peptides, each consisting of 10 amino acid residues that include one target arginine residue for the enzyme (H3͞Arg-8, H3͞Arg-17, and H4͞Arg-3). To each histone N-terminal peptide, the enzyme induces a ␤-turn-like bent conformation composed of five successive residues at the molecular surface near the active site cleft. The remaining five residues are highly disordered. The enzyme recognizes each peptide through backbone atoms of the peptide with a possible consensus recognition motif. The sequence specificity of the peptide recognized by this enzyme is thought to be fairly broad. These observations provide structural insights into target protein recognition by histone modification enzymes and illustrate how PAD4 can target multiple arginine sites in the histone N-terminal tails.calcium binding ͉ histone modification ͉ rheumatoid arthritis ͉ protein deimination͞citrullination ͉ x-ray crystal structure

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In Vitro Disulfide-Coupled Folding of Guanylyl Cyclase-Activating Peptide and Its Precursor Protein

Cited by 40 publications

References 38 publications

Native and Recombinant Proguanylin Feature Identical Biophysical Properties and Are Monomeric in Solution

Native and Recombinant Proguanylin Feature Identical Biophysical Properties and Are Monomeric in Solution

The β-Defensin Gallinacin-6 Is Expressed in the Chicken Digestive Tract and Has Antimicrobial Activity against Food-Borne Pathogens

Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4

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