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

Lauber, Thomas; Nourse, Amanda; Schulz, Axel; Marx, Ute C.

doi:10.1021/bi026434j

Cited by 7 publications

(14 citation statements)

References 26 publications

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“…Sample Preparation-Recombinant proguanylin was expressed as thioredoxin fusion protein in Escherichia coli AD494 (DE3) (Novagen, Madison, WI) and purified as described (10 C-glucose using the same purification protocol. As the expression strain lacks the capability to synthesize leucine, 15 N-labeled leucine was added to the medium according to the manufacturer's instructions (Novagen).…”

Section: Methodsmentioning

confidence: 99%

Solution Structure of Human Proguanylin

Lauber

Neudecker

Rösch

et al. 2003

Journal of Biological Chemistry

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The endogenous ligand of guanylyl cyclase C, guanylin, is produced as the 94-amino-acid prohormone proguanylin, with the hormone guanylin located at the COOH terminus of the prohormone. The solution structure of proguanylin adopts a new protein fold and consists of a three-helix bundle, a small three-stranded ␤-sheet of two NH 2 -terminal strands and one COOHterminal strand, and an unstructured linker region. The sequence corresponding to guanylin is fixed in its bioactive topology and is involved in interactions with the NH 2 -terminal ␤-hairpin: the hormone region (residues 80 -94) partly wraps around the first 4 NH 2 -terminal residues that thereby shield parts of the hormone surface. These interactions provide an explanation for the negligible bioactivity of the prohormone as well as the important role of the NH 2 -terminal residues in the disulfide-coupled folding of proguanylin. Since the ligand binding region of guanylyl cyclase C is predicted to be located around an exposed ␤-strand, the intramolecular interactions observed between guanylin and its prosequence may be comparable with the guanylin/receptor interaction.

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Section: Methodsmentioning

confidence: 99%

Solution Structure of Human Proguanylin

Lauber

Neudecker

Rösch

et al. 2003

Journal of Biological Chemistry

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“…For construction of a pET-32a derivative for production of recombinant proguanylin-C86S/C94S, the QuickChange Multi Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used according to the manufacturer's instructions with a single phosphorylated mutagenesis primer (CCA TGG 1 H) shift correlation double-resonance NMR experiment; HPLC, highperformance liquid chromatography; HOAc, acetic acid; HSQC, heteronuclear single-quantum coherence NMR experiment; NOE, nuclear Overhauser effect, also used for NOESY cross-peak; NOESY, NOE spectroscopy; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; NMR, nuclear magnetic resonance; SDS, sodium dodecyl sulfate; TOCSY, total correlation spectroscopy; Trx, E. coli thioredoxin A. TTA GCT TCC GGT ACA GGC AGC GTA GGC GCT GAT TTC ACA TG) and the previously published vector pET-32a-pres-proguanylin as a template (18).…”

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

“…Recombinant proguanylin, proguanylin-C48S/C61S, and proguanylin-C86S/C94S were expressed as thioredoxin fusion proteins in E. coli AD494-(DE3) cells (Novagen) and purified as described previously (18). For uniform (>95%) 15 N labeling, the recombinant proteins were isolated and purified from E. coli cultures grown in M9 minimal medium enriched with 15 NH 4 Cl using the same purification protocol.…”

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

“…To investigate whether proguanylin-C48S/C61S is still able to form the correct disulfide bonds in the hormone region despite the severe loss of tertiary structure in this protein, disulfide analysis was carried out by analytical HPLC and mass spectrometry after the protein had been treated with acetic acid (see Materials and Methods and refs 14 and 18) ( Figure 6). Biologically active guanylin with the native 1-3/2-4 disulfide connectivity is able to form two interconvertable topological isomers that give rise to a characteristic double peak in the chromatogram of acetic acid-treated proguanylin ( Figure 6a, double peak 1) (14,18). This characteristic double peak is also found for acetic acid-treated proguanylin-C48S/ C61S (Figure 6b, double peak 1), in addition to two peaks of identical molecular masses corresponding to the inactive disulfide isomers with 1-4/2-3 and 1-2/3-4 connectivities (Figure 6b, peaks 2 and 3, respectively), as deduced from comparison with reoxidized synthetic guanylin peptides (14).…”

Section: Spectroscopic Analysis Of Proguanylin-c86s/c94s Andmentioning

confidence: 99%

“…To further investigate if the structure of the prosequence is indeed essential for and involved in the correct assembly of the three-dimensional structure of proguanylin, we recombinantly expressed two disulfide mutant proteins ( Figure 1a) in addition to the wild-type protein (18). The substitutions of cysteine residues 86 and 94 with serine were expected to enhance the flexibility of the hormone region and thereby disturb the stabilizing interactions between the termini.…”

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Role of Disulfide Bonds for the Structure and Folding of Proguanylin

et al. 2004

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The intestinal peptide hormone guanylin circulates mainly as its corresponding prohormone of 94 amino acids and is the first identified endogenous ligand of intestinal guanylyl cyclase C. While the prohormone is biologically inactive, it is processed to the fully functional form with 15 amino acid residues corresponding to the COOH terminus of the precursor protein. In addition to this inactivation of the hormone region, the prosequence makes an essential contribution to the disulfide-coupled folding of the hormone. On the basis of the recently determined solution structure of proguanylin, explanations for these functions of the prosequence were found, indicating that interstrand contacts between the NH 2 -terminal -hairpin of the prosequence and the COOH-terminal hormone region are crucial for formation of the correct disulfide bonds of guanylin. To further investigate the role of individual disulfide bonds upon stabilization of the overall three-dimensional structure of proguanylin and to test the assumption of a direct effect of the prosequence on the structure of the hormone region, we studied the cysteine double mutant proteins proguanylin-C48S/C61S and proguanylin-C86S/C94S. Disulfide determination as well as CD and NMR spectroscopy of proguanylin-C48S/C61S reveals an essential function of the Cys48-Cys61 disulfide bond for the stability of the hydrophobic core and thereby for the stability of the overall three-dimensional structure of proguanylin. Furthermore, sequence specific resonance assignment of the second disulfide deletion mutant, proguanylin-C86S/C94S, and comparison of the NMR spectra of this protein with those of the wild-type protein demonstrate that the rigid helical core structure of proguanylin is not affected by the mutation. Additionally, analysis of the interstrand contacts between the termini reveals a direct effect of the prosequence on the conformation of the hormone region. On the basis of these results, we propose a cooperative mechanism that leads to formation of the correct disulfide pattern of guanylin.The peptide hormone guanylin is the first identified endogenous ligand of intestinal guanylyl cyclase C (GC-C) (1) and is part of a newly discovered entero-endocrine axis connecting intestine and kidney by regulating electrolyte and fluid secretion in the intestine (2) as well as kaliuresis and diuresis in the kidney (3-6). In the intestine, binding of guanylin to GC-C leads to an increased level of intracellular cGMP that subsequently activates the cystic fibrosis transmembrane conductance regulator chloride channel, resulting in an increased level of secretion of Cl -, HCO 3 -, and water into the intestinal lumen, and simultaneously inhibits sodium absorption (7,8). For GC-C binding and activation, formation of the correct 1-3/2-4 disulfide bond pattern of guanylin is required (1, 9), allowing formation of two interconvertable topological stereoisomers (10), with only one of them (Aisomer) showing biological activity (11).The main circulating form of guanylin is the cor...

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Solution Structure, Dynamics, and Hydrodynamics of the Calcium-bound Cross-reactive Birch Pollen Allergen Bet v 4 Reveal a Canonical Monomeric Two EF-Hand Assembly with a Regulatory Function

Neudecker

Nerkamp

Eisenmann

et al. 2004

Journal of Molecular Biology

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Birch pollinosis is one of the prevailing allergic diseases. In all, 5 -20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca 2þ -binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal a-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca 2þ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4.

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

Cited by 7 publications

References 26 publications

Solution Structure of Human Proguanylin

Solution Structure of Human Proguanylin

Role of Disulfide Bonds for the Structure and Folding of Proguanylin

Solution Structure, Dynamics, and Hydrodynamics of the Calcium-bound Cross-reactive Birch Pollen Allergen Bet v 4 Reveal a Canonical Monomeric Two EF-Hand Assembly with a Regulatory Function

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