Inactivation of human salivary glutathione transferase P1-1 by hypothiocyanite: A Post-Translational control system in search of a role

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 a- 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 4264 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 disease

Reactivation rate of GSTP1-1.

<p>Purified GSTP1-1 (20 pmoles) was incubated with HOSCN (10 µM, final concentration) for 20 min, 25°C. Then the samples were incubated with DTT at different concentrations for 2.5 min, 37°C in potassium phosphate buffer 0.1 M, pH 8.4, and the activity was measured. Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p

Inactivation of GSTP1-1 by GSSG and CysSSCys.

<p>(A) GSTP1-1 (20 pmoles) incubated at 25°C with variable amounts of oxidized glutathione or oxidized cysteine in 70 µl of 0.1 M potassium phosphate buffer, pH 7.0. For comparison the same amount of GSTP1-1 was incubated with 70 µl saliva. (B) pH dependence of the inactivation by cystine (100 µM). The activity was evaluated after 5 min of incubation. Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p

Inhibition of purified GSTP1-1 by H₂O₂.

<p>GSTP1-1 (20 pmoles) was incubated at 25°C with two different concentrations of H₂O₂, 10 µM, corresponding to the physiological level (open triangle) and 100 µM (open square) in 70 µl potassium phosphate buffer, pH 7.0. For comparison GSTP1-1 (20 pmoles) was also incubated with 70 µl of saliva (open circle). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p

Inactivation of GSTP1-1 by human defensins (HNP-1 and HNP-2).

<p>GSTP1-1 (90 pmoles) was incubated at 25°C with HNP-1 (0.54 nmoles) (open triangle), HNP-2 (open diamond) (0.54 nmoles) and both HNP-1 0.27 nmoles and HNP-2 (0.27 nmoles) (open square) in 70 µl (final volume) of potassium phosphate buffer, pH 7.0 (these defensin levels reproduce the average concentration of these proteins in saliva). An identical amount of GSTP1-1 was also incubated with 70 µl saliva (open circle). Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same sample). Error bars represent SEM.</p

pH dependence of GSTP1-1 inhibition.

<p>(A) Purified GSTP1-1 (20 pmoles) incubated with saliva (estimated inhibitor concentration 5 µM) at different pH values (from 4.3 to 8.0) with suitable additions of 0.1 M potassium dihydrogen phosphate or 0.1 M potassium monohydrogen phosphate. After incubation (1 min, 25°C) the activity was measured. (B) GSTP1-1 incubated as in (A) with authentic HOSCN 5 µM. Each experiment was performed in triplicate (i.e. three different spectrophotometric determinations on the same salivary sample). Error bars represent SEM.</p

Inhibition pattern of GSTP1-1 by salivary samples or authentic HOSCN.

<p>(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.</p

Inter-day and intra-day levels of salivary GSTP1-1.

<p>(A) Inter-day activity of GSTP1-1 (early morning recovery -Men+women). (B) Sex distribution of the salivary GSTP1-1 as average of three days (early morning recovery). (C) Average of salivary GSTP1-1 (men+women) during three days at different time of saliva recovery (8 am, 11 am, 3 pm). Values expressed as mU/mg of salivery proteins. (D) as in (C) with GSTP1-1 activity expressed as mU/ml of saliva. Error bars represent SEM.</p