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Inactivation of human salivary glutathione transferase P1-1 by hypothiocyanite: a post-translational control system in search of a role.

Fabrini R, Bocedi A, Camerini S, Fusetti M, Ottaviani F, Passali FM, Topazio D, Iavarone F, Francia I, Castagnola M, Ricci G - PLoS ONE (2014)

Bottom Line: A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein.The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function.In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", 00133 Rome, Italy.

ABSTRACT
Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

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Related in: MedlinePlus

Inhibition of cysteine variants of GSTP1-1 by saliva.(A) C47S variant (20 pmoles) incubated at 25°C with 70 µl of saliva (open circle) or saliva 1∶20 diluted (open triangle). (B) C101S variant (20 pmoles) incubated with 70 µl saliva (open circle) or saliva diluted 1∶10 (cross). (C) C47S/C101S double variant (20 pmoles) incubated with 70 µl of saliva (open diamond). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.
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pone-0112797-g003: Inhibition of cysteine variants of GSTP1-1 by saliva.(A) C47S variant (20 pmoles) incubated at 25°C with 70 µl of saliva (open circle) or saliva 1∶20 diluted (open triangle). (B) C101S variant (20 pmoles) incubated with 70 µl saliva (open circle) or saliva diluted 1∶10 (cross). (C) C47S/C101S double variant (20 pmoles) incubated with 70 µl of saliva (open diamond). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.

Mentions: Experiments with specific Cys variants of GSTP1-1 further confirmed the involvement of Cys47 and Cys101 in the observed inactivation. As shown in Figure 3, replacement of both these residues by Ser fully prevented the inactivation due to the salivary inhibitor. Conversely, single point mutations involving only Cys47 or Cys101 allowed salivary inactivation but to a lower extent and with slower kinetics than that observed with native GSTP1-1. The fast and almost complete inactivation observed within the native enzyme confirms that its inactivation mainly involves the oxidation of only Cys47 and Cys101 (Figure 3). As above indicated, alternative intramolecular disulfides can be formed, but only in the absence of Cys47 or Cys101 and possibly involving Cys14.


Inactivation of human salivary glutathione transferase P1-1 by hypothiocyanite: a post-translational control system in search of a role.

Fabrini R, Bocedi A, Camerini S, Fusetti M, Ottaviani F, Passali FM, Topazio D, Iavarone F, Francia I, Castagnola M, Ricci G - PLoS ONE (2014)

Inhibition of cysteine variants of GSTP1-1 by saliva.(A) C47S variant (20 pmoles) incubated at 25°C with 70 µl of saliva (open circle) or saliva 1∶20 diluted (open triangle). (B) C101S variant (20 pmoles) incubated with 70 µl saliva (open circle) or saliva diluted 1∶10 (cross). (C) C47S/C101S double variant (20 pmoles) incubated with 70 µl of saliva (open diamond). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231102&req=5

pone-0112797-g003: Inhibition of cysteine variants of GSTP1-1 by saliva.(A) C47S variant (20 pmoles) incubated at 25°C with 70 µl of saliva (open circle) or saliva 1∶20 diluted (open triangle). (B) C101S variant (20 pmoles) incubated with 70 µl saliva (open circle) or saliva diluted 1∶10 (cross). (C) C47S/C101S double variant (20 pmoles) incubated with 70 µl of saliva (open diamond). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.
Mentions: Experiments with specific Cys variants of GSTP1-1 further confirmed the involvement of Cys47 and Cys101 in the observed inactivation. As shown in Figure 3, replacement of both these residues by Ser fully prevented the inactivation due to the salivary inhibitor. Conversely, single point mutations involving only Cys47 or Cys101 allowed salivary inactivation but to a lower extent and with slower kinetics than that observed with native GSTP1-1. The fast and almost complete inactivation observed within the native enzyme confirms that its inactivation mainly involves the oxidation of only Cys47 and Cys101 (Figure 3). As above indicated, alternative intramolecular disulfides can be formed, but only in the absence of Cys47 or Cys101 and possibly involving Cys14.

Bottom Line: A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein.The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function.In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Sciences and Technologies, University of Rome "Tor Vergata", 00133 Rome, Italy.

ABSTRACT
Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

Show MeSH
Related in: MedlinePlus