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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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Inhibition pattern of GSTP1-1 by salivary samples or authentic HOSCN.(A) Purified GSTP1-1 (20 pmoles) incubated with authentic HOSCN (10, 1, and 0.5 µM). (B) Purified GSTP1-1 (20 pmoles) was at 25°C incubated with 70 µl of saliva (full circle) (estimated inhibitor concentration 10 µM) or at different dilutions (full square 1∶10; full diamond 1∶20). 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-g008: Inhibition pattern of GSTP1-1 by salivary samples or authentic HOSCN.(A) Purified GSTP1-1 (20 pmoles) incubated with authentic HOSCN (10, 1, and 0.5 µM). (B) Purified GSTP1-1 (20 pmoles) was at 25°C incubated with 70 µl of saliva (full circle) (estimated inhibitor concentration 10 µM) or at different dilutions (full square 1∶10; full diamond 1∶20). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.

Mentions: Hypothiocyanite (OSCN−) is another oxidizing salivary component that displays a molecular mass compatible to that predicted by the exclusion chromatography on Biogel P2 (molecular mass = 74.1 Da). This compound represents one of the most active salivary antimicrobial agent formed in virtue of large amounts of salivary thiocyanate, H2O2 and salivary lactoperoxidase. Hypothiocyanite is not stable but its level in saliva is stabilized at about 10–20 µM concentration by the presence of a large excess of thiocyanate and a continuous generation of H2O2 by salivary bacteria and by epithelial Nox/Duox family NADPH oxidases [32], [35], [36]. A first indication that this compound may be the real inhibitor for GSTP1-1 has been obtained by using authentic OSCN−. Kinetics and the peculiar pH dependence of GSTP1-1 inactivation (Figure 8 and 9) are almost identical to those found using salivary samples. Moreover, using TNB as specific OSCN− titrant [22], we found that the concentration of this compound in saliva is almost identical to that calculated on the basis of GSTP1-1 inhibition (not shown). Furthermore, after titration of salivary OSCN− with stoichiometric amount of TNB and subsequent removal of DTNB with GSH (DTNB is a strong inhibitor of GSTP1-1 [23]), the salivary sample becomes unable to inactivate GSTP1-1 (Figure 10). A more accurate analysis allowed us to calculate the second order kinetic constants for the inactivation of GSTP1-1 in the presence of OSCN− or H2O2 (kOSCN¯ = 2.5×105 M−1 min−1; kH2O2 = 3×102 M−1 min−1). The ratio kOSCN¯/kH2O2 is about 800. In other words, hypothiocyanite is about 800 times more efficient than H2O2 in the GSTP1-1 inactivation.


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 pattern of GSTP1-1 by salivary samples or authentic HOSCN.(A) Purified GSTP1-1 (20 pmoles) incubated with authentic HOSCN (10, 1, and 0.5 µM). (B) Purified GSTP1-1 (20 pmoles) was at 25°C incubated with 70 µl of saliva (full circle) (estimated inhibitor concentration 10 µM) or at different dilutions (full square 1∶10; full diamond 1∶20). 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-g008: Inhibition pattern of GSTP1-1 by salivary samples or authentic HOSCN.(A) Purified GSTP1-1 (20 pmoles) incubated with authentic HOSCN (10, 1, and 0.5 µM). (B) Purified GSTP1-1 (20 pmoles) was at 25°C incubated with 70 µl of saliva (full circle) (estimated inhibitor concentration 10 µM) or at different dilutions (full square 1∶10; full diamond 1∶20). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.
Mentions: Hypothiocyanite (OSCN−) is another oxidizing salivary component that displays a molecular mass compatible to that predicted by the exclusion chromatography on Biogel P2 (molecular mass = 74.1 Da). This compound represents one of the most active salivary antimicrobial agent formed in virtue of large amounts of salivary thiocyanate, H2O2 and salivary lactoperoxidase. Hypothiocyanite is not stable but its level in saliva is stabilized at about 10–20 µM concentration by the presence of a large excess of thiocyanate and a continuous generation of H2O2 by salivary bacteria and by epithelial Nox/Duox family NADPH oxidases [32], [35], [36]. A first indication that this compound may be the real inhibitor for GSTP1-1 has been obtained by using authentic OSCN−. Kinetics and the peculiar pH dependence of GSTP1-1 inactivation (Figure 8 and 9) are almost identical to those found using salivary samples. Moreover, using TNB as specific OSCN− titrant [22], we found that the concentration of this compound in saliva is almost identical to that calculated on the basis of GSTP1-1 inhibition (not shown). Furthermore, after titration of salivary OSCN− with stoichiometric amount of TNB and subsequent removal of DTNB with GSH (DTNB is a strong inhibitor of GSTP1-1 [23]), the salivary sample becomes unable to inactivate GSTP1-1 (Figure 10). A more accurate analysis allowed us to calculate the second order kinetic constants for the inactivation of GSTP1-1 in the presence of OSCN− or H2O2 (kOSCN¯ = 2.5×105 M−1 min−1; kH2O2 = 3×102 M−1 min−1). The ratio kOSCN¯/kH2O2 is about 800. In other words, hypothiocyanite is about 800 times more efficient than H2O2 in the GSTP1-1 inactivation.

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