Redox modulation of cell surface protein thiols in U937 lymphoma cells: the role of γ-glutamyl transpeptidase-dependent H₂O₂ production and S-thiolation

Abstract

The expression of gamma-glutamyl transpeptidase (GGT), a plasma membrane ectoenzyme involved in the metabolism of extracellular reduced glutathione (GSH), is a marker of neoplastic progression in several experimental models, and occurs in a number of human malignant neoplasms and their metastases. Because it favors the supply of precursors for the synthesis of GSH, GGT expression has been interpreted as a member in cellular antioxidant defense systems. However, thiol metabolites generated at the cell surface during GGT activity can induce prooxidant reactions, leading to production of free radical oxidant species. The present study was designed to characterize the prooxidant reactions occurring during GGT ectoactivity, and their possible effects on the thiol redox status of proteins of the cell surface. Results indicate that: (i) in U937 cells, expressing significant amounts of membrane-bound GGT, GGT-mediated metabolism of GSH is coupled with the extracellular production of hydrogen peroxide; (ii) GGT activity also results in decreased levels of protein thiols at the cell surface; (iii) GGT-dependent decrease in protein thiols is due to sulfhydryl oxidation and protein S-thiolation reactions; and (iv) GGT irreversible inhibition by acivicin is sufficient to produce an increase of protein thiols at the cell surface. Membrane receptors and transcription factors have been shown to possess critical thiols involved in the transduction of proliferative signals. Furthermore, it was suggested that S-thiolation of cellular proteins may represent a mechanism for protection of vulnerable thiols against irreversible damage by prooxidant agents. Thus, the findings reported here provide additional explanations for the envisaged role played by membrane-bound GGT activity in the proliferative attitude of malignant cells and their resistance to prooxidant drugs and radiation therapy.

Introduction

Adequate intracellular levels of the nucleophilic tripeptide GSH are crucial for the functioning of important cellular systems operating in the defense against prooxidant agents and for detoxification of electrophilic cytotoxic drugs. Despite the fact that uptake of intact GSH does occur in small intestine and kidney [1], [2], in most cell types, extracellular GSH cannot cross plasma membrane as such; thus, its intracellular levels depend on a balance between its consumption and its de novo synthesis; the latter in turn depends on an adequate supply of precursor amino acids. In a number of cell types, such a supply is warranted by γ-glutamyl transpeptidase (GGT; EC 2.3.2.2), an ectoenzyme with the active site oriented toward the outer cell surface, which is capable of cleaving extracellular GSH. Precursor amino acids are thus formed, which can cross the plasma membrane and are re-utilized for intracellular GSH synthesis [3]. Furthermore, because it has been documented that a continuous efflux of GSH occurs from a number of cell types through specific out-transporters [4], it appears that a major function of GGT ectoactivity is to expedite the salvage of extracellular GSH, which would otherwise be lost from the cell. γ-Glutamyl transpeptidase thus appears to participate in a “GSH cycling” in the plasma membrane [5].

The expression of significant levels of GGT has been reported to occur in a number of human malignant neoplasms, such as ovary [6], [7], colon [8], [9], lung [10], liver [11], sarcoma [12], melanoma [13], and leukemias [14], [15]; in many cases, levels of GGT detectable in metastases are higher than in corresponding primitive localizations. In a series of 60 human tumor cell lines, GGT was found to be significantly expressed in 70% of cases [16]. In addition, GGT expression is known to be a marker of neoplastic progression in several experimental models, such as rodent skin and liver chemical carcinogenesis [17]. Transfection of epithelial cells with the oncogene ras, while resulting in the appearance of metastatic behavior, is also accompanied by expression of GGT [18], [19]. Interestingly, in melanoma clones, the degree of GGT expression was found to be proportional to the invasive and migrating abilities [13]. Altogether, this and other evidence concur in highlighting GGT activity as a factor in human malignancy.

Because GGT plays a crucial role in the cellular supply of GSH, thus favoring in tumor cells the appearance of resistance against electrophilic chemotherapeutics, GGT has often been interpreted as a member in the cellular antioxidant enzyme systems [5], [6], [20]. However, the significance of GGT in the cellular redox equilibrium was made more complex by recent studies suggesting that oxidant compounds (superoxide anion, hydrogen peroxide, thiyl radicals) might be produced during GGT-mediated salvage of extracellular GSH, giving rise within the cell to oxidative processes such as lipid peroxidation [21], [22], [23], [24].

It is well established that several species originating from oxidative processes can interact with critical thiols of proteins causing noxious effects, such as the impairment of Ca²⁺-ATPases [25] or cytoskeletal alterations [26]. On the other hand, strong evidence has accumulated that the same oxidizing species can also play a nontoxic role as stimulants in transduction of proliferative signals, an effect likely due to their ability to interact with oxidizable regions of growth factor receptors, protein kinases, and transcription factors [27], [28], [29]. Previous studies showed that different pools of protein thiols can be differentially affected by oxidants, depending on the site where these originated [30]. In this respect, the peculiar location of GGT on cell plasma membrane raises the possibility that oxidants arising from its activity may primarily affect the thiol redox status of proteins located on the cell surface, possibly playing a physiological role in the modulation of their functions.

Our study was designed to investigate the nature of oxidants produced during GGT ectoactivity, and the effects possibly induced by modulation of GGT on the thiol redox status of cell surface proteins. Results indicate that an as yet unrecognized function of GGT might indeed be related to the modulation of protein thiols at the cell surface level, through the generation of hydrogen peroxide and reactive thiols capable of effecting S-thiolation of cellular proteins.