γ‐Glutamyl Transpeptidase in Glutathione Biosynthesis

Abstract

Glutathione (GSH) is the most abundant nonprotein thiol in cells and has multiple biological functions. Glutathione biosynthesis by way of the γ‐glutamyl cycle is important for maintaining GSH homeostasis and normal redox status. As the only enzyme of the cycle located on the outer surface of plasma membrane, γ‐glutamyl transpeptidase (GGT) plays key roles in GSH homeostasis by breaking down extracellular GSH and providing cysteine, the rate‐limiting substrate, for intracellular de novo synthesis of GSH. GGT also initiates the metabolism of glutathione S‐conjugates to mercapturic acids by transferring the γ‐glutamyl moiety to an acceptor amino acid and releasing cysteinylglycine. GGT is expressed in a tissue‐, developmental phase‐, and cell‐specific manner that may be related to its complex gene structure. In rodents, there is a single GGT gene, and several promoters that generate different mRNA subtypes and regulate its expression. In contrast, several GGT genes have been found in humans. During oxidative stress, GGT gene expression is increased, and this is believed to constitute an adaptation to stress. Interestingly, only certain mRNA subtypes are increased, suggesting a specific mode of regulation of GGT gene expression by oxidants. Here, protocols to measure GGT activity, relative levels of total and specific GGT mRNA subtypes, and GSH concentration are described.

Introduction

GSH(γ‐glutamyl‐l‐cysteinyl‐l‐glycine) is the most abundant nonprotein thiol in cells, found ubiquitously at levels as high as 0.5–10 mM concentrations (Meister, 1988). GSH has multiple important biological functions, and these include conjugation of electrophiles, for example, in the detoxification of xenobiotic compounds and leukotriene metabolism, thiol‐disulfide exchange reactions in the maintenance of normal cellular redox status, and antioxidant functions in the removal of both endogenous and exogenous oxidants (Meister, 1992). GSH has also been implicated in cell signaling (Filomeni 2002, Sen 2000) and been suggested to act as a reservoir of cysteine in the transport of amino acids.

Consequently, maintenance of the reduced GSH pool is important for the general welfare of cells, especially in situations of severe oxidative stress; moreover, inability to do so may be detrimental to cells and organisms as a whole. Indeed, an elevated GSH content generally confers protection against oxidative damage, whereas depletion of GSH tends to enhance such damage. GSH deficiency has been associated with various diseases (Townsend 2003, Wu 2004) such as cardiovascular disease (Giugliano et al., 1995), neurodegenerative disorders (Cruz 2003, Dringen 2003, Halliwell 2001, Schulz 2000), aging (Richie, 1992), chronic lung disease (Rahman 1999, Rahman 1999, Rahman 1999), and viral infections (Droge, 1993).

The reactions of GSH with peroxides are catalyzed by members of GSH peroxidase (GPx) family or peroxiredoxin 6 (Prx6, also called 1‐cys Prx) and result in the production of GSH disulfide (GSSG):

$$2\mathit{GSH}+H_2{O}_2\underset{\mathit{Prx}6}{\overset{\mathit{GPx}}{\to }}\mathit{GSSG}+2H_{2}O$$

GSH's reactions with electrophiles are catalyzed by GSH S‐transferases (GST), resulting in the formation of glutathione conjugates. GSTs catalyze either reductive addition or exchange where X is usually a halide or hydroxyl moiety:

$$\begin{array}{l}\mathit{GSH}+\mathit{Electrophile}\stackrel{\mathit{GST}}{\to }\mathit{GS}-\mathit{Conjugate}\\ \mathit{GSH}+\mathit{Electrophile}-X\stackrel{\mathit{GST}}{\to }\mathit{GS}-\mathit{Conjugate}+\mathit{HX}\end{array}$$

Physiologically, most GSH is found in the reduced form; less than 1% of GSH exists as GSSG, so that the ratio of GSH/GSSG is maintained at approximately 100:1. Cells partly maintain this high intracellular reduced GSH pool by recycling GSSG back to GSH with GSSG reductase (Eq. 3) (Meister and Anderson, 1983):

$$\mathit{GSSG}+\mathit{NADPH}+H^{+}\stackrel{\mathit{GSSGReductase}}{\to }2\mathit{GSH}+{\mathit{NADP}}^{+}$$

Alternately, GSSG is extruded from cells by means of the multidrug resistance–associated protein that also transports GSH and GSH S conjugates out of cells (Lee et al., 1997).

In multiorgan systems, GSH is also transported from one organ to help maintain intracellular GSH content in other organs (Anderson 1980, Lauterburg 1984). Exogenous GSH may be transported in its intact form by certain epithelial cells to help maintain their intracellular GSH content (Hagen et al., 1988). However, extracellular GSH, GSSG, and glutathione S‐conjugates are usually broken down to their constituent amino acids by γ‐glutamyl transpeptidase (γ‐glutamyl transferase, GGT; EC2.3.2.2). These amino acids are then taken up and used intracellularly for de novo synthesis of GSH (Meister 1994, Suzuki 1993) through the γ‐glutamyl cycle (Fig. 1). The de novo synthesis of GSH consists of two consecutive ATP‐dependent reactions: in the first reaction, glutamate cysteine ligase (GCL, also known as γ‐glutamylcysteine synthase) uses glutamic acid and cysteine as the substrates to produce γ‐glutamylcysteine (γGC), which then reacts with glycine to form GSH in a reaction catalyzed by glutathione synthase. Detailed discussion of de novo synthesis of GSH and regulation of GCL can be found elsewhere (Deneke 1989, Dickinson 2004, Griffith 1999, Lu 2000). The remaining chapter will focus on GGT, its roles in the metabolism of GSH and GSH S‐conjugates, and the regulation of its gene expression.

GGT catalyzes the transfer of γ‐glutamyl moiety from GSH, GSH S‐conjugates, and other γ‐glutamyl compounds to acceptors such as amino acids, dipeptides, and H₂O. The catalytic mechanism of GGT is well known (Taniguchi 1998, Tate 1981). As discussed previously, GGT plays a key role in γ‐glutamyl cycle (Fig. 1) in the de novo synthesis of GSH (Meister 1974, Stark 2003). Here, GGT breaks down extracellular GSH to generate γ‐glutamyl compounds and cysteinylglycine, which is further cleaved by membrane‐bound dipeptidases. The constituent amino acids are then taken up and used by cells for intracellular resynthesis of GSH. One of the amino acids that are thus supplied by GGT is cysteine, the limiting substrate for GSH biosynthesis. Importantly, cysteine is a preferred acceptor for glutamate‐amino acid conjugation reaction by GGT, and the product, γ‐glutamylcystine, can be transported inside the cells and, after reduction to γ‐GC, used directly for GSH biosynthesis (Anderson 1983, Meister 1984). This GSH salvage pathway bypasses the rate‐limiting reaction catalyzed by GCL in de novo synthesis of GSH (Griffith et al., 1981) and may play an important role in the maintenance of GSH in cells.

Therefore, GGT is critical in maintaining GSH and cysteine homeostasis (Hanigan and Ricketts, 1993) and for cellular antioxidant function (Forman and Skelton, 1990). GGT^–/– mice showed glutathionemia, glutathionuria, cysteine deficiency, decreased tissue GSH levels, and increased susceptibility to oxidative stress, all of which could be reversed by N‐acetylcysteine (NAC), a pharmacological source of cysteine (Barrios 2001, Griffith 1980, Lieberman 1996, Jean 2002, Rojas 2000). These findings demonstrated the importance of GGT in GSH and cysteine homeostasis and suggested GGT as an integral component of endogenous antioxidant system in cells.

The cleavage of GSH S‐conjugates and other γ‐glutamyl compounds by GGT also plays an important role in the metabolism of natural biomolecules such as leukotriene C4, prostaglandins, and estrogen, as well as xenobiotic compounds such as carcinogens, mutagens, and drug compounds after their conjugation with GSH. In fact, the removal of γ‐glutamyl moiety from these conjugates often constitutes an initial step in the conversion of these compounds to mercapturic acids in the detoxification reactions.

Besides these roles in the metabolism of GSH and GSH S‐conjugates, other functions of GGT, such as the facilitation of amino acids transport (Hsu 1984, Vina 1983, Vina 1990) and pro‐oxidant effects, have also been described (del Bello 1999, Paolicchi 1997, Paolicchi 2002). The physiological significance of these findings, however, remains unclear.