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Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle.

Hnia K, Notarnicola C, de Santa Barbara P, Hugon G, Rivier F, Laoudj-Chenivesse D, Mornet D - PLoS ONE (2008)

Bottom Line: From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions.Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells.A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

View Article: PubMed Central - PubMed

Affiliation: INSERM ERI 25 Muscle et Pathologies, Université Montpellier 1, EA 4202, CHU Arnaud de Villeneuve, Montpellier, France.

ABSTRACT

Unlabelled: The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties.

Background: GNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells.

Methodology/principal findings: From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods.

Conclusion/significance: Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

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Related in: MedlinePlus

Interaction of the N-terminal region of actin with purified chicken GNK-1.Part (A): H-NMR spectrum of actin (200 mM) residues 1–28 containing 250 mM DTT, pH 6.9. Part (B): H-NMR spectrum after gastrokine-1 addition (25 mM) in similar conditions. Part (C): Difference spectrum (A–B) showing identification of the proton side chain belonging to specific actin residues that were perturbed upon gastrokine-1 addition and which are indicated in the corresponding area of the 1H-NMR spectrum. The identity of the protons involved, as well as the amino acid to which they belong, are given in a box over the corresponding peaks. The summary of this particular experiment is stated in the “Table 2” summarizes the results of this experiment along with other results obtained with different actin peptides.
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pone-0003854-g007: Interaction of the N-terminal region of actin with purified chicken GNK-1.Part (A): H-NMR spectrum of actin (200 mM) residues 1–28 containing 250 mM DTT, pH 6.9. Part (B): H-NMR spectrum after gastrokine-1 addition (25 mM) in similar conditions. Part (C): Difference spectrum (A–B) showing identification of the proton side chain belonging to specific actin residues that were perturbed upon gastrokine-1 addition and which are indicated in the corresponding area of the 1H-NMR spectrum. The identity of the protons involved, as well as the amino acid to which they belong, are given in a box over the corresponding peaks. The summary of this particular experiment is stated in the “Table 2” summarizes the results of this experiment along with other results obtained with different actin peptides.

Mentions: To shed further light on the actin residues involved in the gastrokine-1 association, we used few actin peptides and tested interactions by the proton NMR technique. Each actin synthetic peptide was recorded in relation to its amino acid sequence (“Table 2”). Proton NMR spectroscopy was performed as detailed in a previous report [22]. Spectral perturbations resulting from complex formation between the synthetic actin peptides and gastrokine-1 were monitored by difference spectroscopy and by application of the two-pulse spin echo technique [22]. The most evident association was found between N-terminal peptide (residues 1 to 28) and gastrokine-1. “Figure 7” shows the respective records corresponding to the H-NMR spectra of N-terminal actin peptide alone (A), of the mixture of N-terminal peptide and gastrokine-1 (B) and the complex formation (C) which corresponds to the differential spectrum (A–B). Our data showed that the gastrokine-1/actin interface involves a specific proton belonging to well identified side chains of a few N-terminal actin residues. Although the proton mapping could not be precisely done for each residue, Glu-2 and -4 (E) as well as Thr- 5 and -6 or Phe-21 were clearly involved in the gastrokine-1 interaction (shown in bold in the N-terminal sequence of the 28 first actin amino acid residues). The other protons implicated in the gastrokine-1 association were in the side chain CH3 of Val/Leu and Ala or CH2 of Asp residues. According to these results, the underlined residues, mapped into the following actin sequence : DEDETTALVCDNGSGLVKAGFAGDDAPR, could correspond to the GNK1 actin binding site. This is in complete agreement with the EDC-induced covalent association between actin and gastrokine-1 which promotes the activation of actin acid residues for direct crosslink with gastrokine-1, as previously reported for myosin S1 and/or other actin binding proteins [23], [24], [25], [26].


Biochemical properties of gastrokine-1 purified from chicken gizzard smooth muscle.

Hnia K, Notarnicola C, de Santa Barbara P, Hugon G, Rivier F, Laoudj-Chenivesse D, Mornet D - PLoS ONE (2008)

Interaction of the N-terminal region of actin with purified chicken GNK-1.Part (A): H-NMR spectrum of actin (200 mM) residues 1–28 containing 250 mM DTT, pH 6.9. Part (B): H-NMR spectrum after gastrokine-1 addition (25 mM) in similar conditions. Part (C): Difference spectrum (A–B) showing identification of the proton side chain belonging to specific actin residues that were perturbed upon gastrokine-1 addition and which are indicated in the corresponding area of the 1H-NMR spectrum. The identity of the protons involved, as well as the amino acid to which they belong, are given in a box over the corresponding peaks. The summary of this particular experiment is stated in the “Table 2” summarizes the results of this experiment along with other results obtained with different actin peptides.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2588339&req=5

pone-0003854-g007: Interaction of the N-terminal region of actin with purified chicken GNK-1.Part (A): H-NMR spectrum of actin (200 mM) residues 1–28 containing 250 mM DTT, pH 6.9. Part (B): H-NMR spectrum after gastrokine-1 addition (25 mM) in similar conditions. Part (C): Difference spectrum (A–B) showing identification of the proton side chain belonging to specific actin residues that were perturbed upon gastrokine-1 addition and which are indicated in the corresponding area of the 1H-NMR spectrum. The identity of the protons involved, as well as the amino acid to which they belong, are given in a box over the corresponding peaks. The summary of this particular experiment is stated in the “Table 2” summarizes the results of this experiment along with other results obtained with different actin peptides.
Mentions: To shed further light on the actin residues involved in the gastrokine-1 association, we used few actin peptides and tested interactions by the proton NMR technique. Each actin synthetic peptide was recorded in relation to its amino acid sequence (“Table 2”). Proton NMR spectroscopy was performed as detailed in a previous report [22]. Spectral perturbations resulting from complex formation between the synthetic actin peptides and gastrokine-1 were monitored by difference spectroscopy and by application of the two-pulse spin echo technique [22]. The most evident association was found between N-terminal peptide (residues 1 to 28) and gastrokine-1. “Figure 7” shows the respective records corresponding to the H-NMR spectra of N-terminal actin peptide alone (A), of the mixture of N-terminal peptide and gastrokine-1 (B) and the complex formation (C) which corresponds to the differential spectrum (A–B). Our data showed that the gastrokine-1/actin interface involves a specific proton belonging to well identified side chains of a few N-terminal actin residues. Although the proton mapping could not be precisely done for each residue, Glu-2 and -4 (E) as well as Thr- 5 and -6 or Phe-21 were clearly involved in the gastrokine-1 interaction (shown in bold in the N-terminal sequence of the 28 first actin amino acid residues). The other protons implicated in the gastrokine-1 association were in the side chain CH3 of Val/Leu and Ala or CH2 of Asp residues. According to these results, the underlined residues, mapped into the following actin sequence : DEDETTALVCDNGSGLVKAGFAGDDAPR, could correspond to the GNK1 actin binding site. This is in complete agreement with the EDC-induced covalent association between actin and gastrokine-1 which promotes the activation of actin acid residues for direct crosslink with gastrokine-1, as previously reported for myosin S1 and/or other actin binding proteins [23], [24], [25], [26].

Bottom Line: From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions.Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells.A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

View Article: PubMed Central - PubMed

Affiliation: INSERM ERI 25 Muscle et Pathologies, Université Montpellier 1, EA 4202, CHU Arnaud de Villeneuve, Montpellier, France.

ABSTRACT

Unlabelled: The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties.

Background: GNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells.

Methodology/principal findings: From fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods.

Conclusion/significance: Altogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

Show MeSH
Related in: MedlinePlus