Inactivation of Human Salivary Glutathione Transferase P1-1 by Hypothiocyanite: A Post-Translational Control System in Search of a Role

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.

Erythrocyte glutathione transferase: a general probe for chemical contaminations in mammals

“…The presence of a detectable inactive oxidized form of GSTP1-1 has been recently found in salivary samples of healthy human subjects. 11 The possible presence of such oxidized forms in the blood of different mammals will be explored below.…”

“…9 The resulting oxidized enzyme is fully inactive. 9 The presence of oxidized GSTP1-1 has recently been discovered in human saliva, 11 but it has never been observed in mammalian blood. After incubation of hemolyzed total blood with DTT, we observed a conjugation rate of GSH with CDNB higher than that found in the absence of reducing treatment, suggesting the presence of a significant, not spurious amount of oxidized e-GST ( Figure 2a ).…”

The cysteine proteome

“…Support for this possibility is limited, but HOSCN, and not HOCl or HOBr, induces cell signaling by reaction with serum factor(s) [202, 203]. HOSCN selectively targets salivary GSTP, but not GSTA or GSTM [204]. Altered redox signaling of HOSCN may be reflected in altered cellular responses to this oxidant instead of others, e.g., removal of H 2 O 2 and HOCl by SCN − protects against mammalian cell death and decreases inflammation [179, 205].…”

Biochemical mechanisms and therapeutic potential of pseudohalide thiocyanate in human health

“…HOSCN can inhibit GSH S -transferase pi (GSTP1), GAPDH and ATPase activity in erythrocytes suspended at 2% hematocrit and may be a more potent ATPase inhibitor than HOCl or HOBr, though this result might have differed with intact cells instead of isolated membranes [126]. The cysteine residues of constitutively inactive GSTP1-1 in human saliva are kept oxidized by HOSCN [143]. NF-κB signaling was strongly implicated in the eCAM and tissue factor (TF)-inducing effects of HOSCN in serum-treated HUVECs.…”

“…As complexity increases in biological systems from isolated proteins to subcellular fractions, intact cells, tissues, organs and ultimately whole organisms it is increasingly less likely that HOSCN generated in situ will reach any one of these particular targets, as demonstrated in multiple studies wherein whole cells or tissues were not as sensitive to HOSCN as isolated proteins. However, the observations that HOSCN transduces eCAM and TF expression in whole cells submerged in 10% serum are particularly compelling [116,146] and it is now known that HOSCN is an important negative regulator of at least one salivary enzyme [143]. …”