Evidence for the pro-oxidant effect of γ-glutamyltranspeptidase–related enzyme¹

Abstract

It has been previously reported that the metabolism of reduced glutathione (GSH) by γ-glutamyltranspeptidase (GGT) in the presence of chelated metals leads to free radical generation and lipid peroxidation (LPO). The present study demonstrates for the first time that an established cell line expressing GGT-rel, a human GGT-related enzyme, metabolizes extracellular GSH to cysteinylglycine (CysGly) in a time-dependent manner when cells were incubated in a medium containing 2.5 mM GSH and 25 mM glycylglycine. Supplementation with 150–165 μM Fe³⁺-EDTA resulted in a reactive oxygen species (ROS) generation process. The resulting data showed a significantly higher level (7.6-fold) of ROS production in the GGT-rel positive cells in comparison with the GGT-rel negative control cells. CysGly and Cys, but not GSH, were responsible for the observed ROS production, as we confirmed by measuring the same process in the presence of Fe³⁺-EDTA and different thiols. A higher iron reduction and an increased LPO level determined by malondialdehyde HPLC measurement were also found in GGT-rel–overexpressing cells compared to GGT-rel negative cells. Our data clearly indicate that in the presence of iron, not only GGT, but also GGT-rel has a pro-oxidant function by generation of a reactive metabolite (CysGly) and must be taken into account as a potential physiopathological oxidation system.

Introduction

Reduced glutathione (GSH), the major intracellular nonprotein thiol is mainly known as an important protector against free radical damages by providing reducing equivalents for several key antioxidant enzymes and also by scavenging hydroxyl radicals and singlet oxygen. The plasma membrane–bound enzyme γ-glutamyl transpeptidase [GGT (5-glutamyl)-peptide: aminoacid 5-glutamyltransferase E.C. 2.3.2.2] initiates the breakdown of GSH by removing the γ-glutamyl moiety and therefore providing amino acid precursors for the intracellular de novo synthesis of GSH. However, recent studies have shown that in the presence of chelated transition metals (iron or copper), pro-oxidant species can originate during the GGT-mediated metabolism of GSH [1], [2], [3], [4]. This process was explained by the autoxidation of the GGT-generated metabolite of GSH, cysteinylglycine (CysGly), which is able to form thiyl and oxygen radicals by reacting with Fe³⁺ and O₂ [1], [3].

In the last few years, it has been reported that the GGT/GSH/Fe³⁺ (Cu²⁺) oxidant system is able to induce lipid peroxidation (LPO) on different targets, e.g., polyunsaturated fatty acids [1], [5], human LDL [6], hepatocytes [7], liver microsomes [8], [9], HepG2 hepatoma cells [9], and also oxidative mutagenesis on DNA [2], [10]. Recent biochemical and histological findings suggest that the GGT-dependent oxidation of LDL may represent a potential mechanism in atherosclerosis and that the persistent increase of serum GGT is a possible risk of reinfarction in patients with ischemic heart disease [6]. In situ colocalization of GGT and LPO showed an evident association between GGT activity and oxidative damage in preneoplastic lesions from carcinogen-treated rats [7]. High levels of GGT expression were also reported in several human and mammalian cancers (liver, colon, ovary, prostate) suggesting that GGT is a marker of tumor progression, which could be mediated also by the oxidative damage linked to GGT activity [11].

On the other hand, it was shown that GGT-mediated metabolism of GSH leads to production of low levels of H₂O₂, which is essential to prevent apoptosis and maintain cell proliferation in U937 cells [4]. Furthermore, GGT-generated H₂O₂ and reactive thiol metabolites of GSH are involved in redox modulation of cell surface protein thiols by sulfhydryl oxidation and protein S-thiolation reactions, with possible consequences on cell resistance to pro-oxidant drugs and radiation therapy, as recently demonstrated by Dominici et al. [12].

In addition to GGT, another human GGT-related (GGT-rel) enzyme able to catalyze the cleavage of GSH has been identified [13], [14]. This enzyme, initially cloned from a human placental cDNA library, exhibited an overall similarity of 40% with human GGT. Similar to GGT, GGT-rel is also a membrane-bound dimer; the molecular weights of glycosylated heavy and light chains in HepG2 are 55 and 42 kDa, respectively [13]. Relatively little is known about the expression of GGT-rel in humans; most of the examined mouse tissues and human cell lines such as lymphoblast cell lines, pulmonary fibroblasts, kidney carcinoma (A498), lung carcinoma (ChaGo), and glioblastoma (A172) lacked abundant expression of GGT-rel. In contrast, GGT-rel expression was found in K562 and HepG2 cells and was inducible by dexamethasone [13].

Recently, a homologue of GGT-rel with 79% identity to human GGT-rel was identified in rat tracheal epithelial cells. Its expression was found to be induced by epidermal growth factor and increased in lung adenomas and carcinomas from rats chronically exposed to isobutylnitrite [15]. GGT-rel shows a substrate specificity different from GGT; it cannot cleave the common synthetic chromogenic substrates of GGT, e.g., L-γ-glutamyl-3-carboxy-4-nitroanilide or γ-glutamyl-4-methoxy-2-naphthylamide. The GGT-rel activity was determined in the GGT-rel transfected NIH3T3 cell line by degradation of extracellular GSH and was further confirmed by conversion of leukotriene C4 to leukotriene D4 [13]. However, the products of the enzymatic degradation of GSH (such as CysGly) have not been identified yet.

Although GGT is apparently much more abundant than GGT-rel, an important and particular physiological role of GGT-rel cannot be excluded. It was suggested that GGT-rel may be involved during particular stages of development, such as pregnancy or in specialized cell types [13], as well as a protective mechanism against oxidative stress or other toxic injuries [15]. One of the hypotheses proposed by Laouar et al. [16], [17] is devoted to the proteolytic function of GGT-related activities. In fact, these authors postulated that GGT-related proteins participate in the regulation of IL-6 and IGF1 metabolism. IL-6 dimerization was shown to be due to the membrane-bound GGT-related activity present in hematopoietic cells, as well as IGF-1, which was found to be a specific substrate for cathepsin D and GGT-related enzymes [16], [17]. Recent findings describe also the presence of GGT-rel genes in low-copy repeats, which mediate the common 3-MB deletion in patients with velo-cardio-facial syndrome [18].

In analogy with GGT, we assumed that GGT-rel might have also pro-oxidant properties resulting from the generation of a reactive metabolite of GSH (CysGly) in the presence of chelated iron. Our present work clearly demonstrates that a GGT-rel–transfected cell line metabolizes the extracellular GSH to CysGly and that this reaction is involved in a free radical generation process and consecutive induction of LPO, in the presence of chelated iron.