Gamma-Glutamyl Transferase: The Silent Partner?

ABSTRACT

Glutathione is one of the most abundant proteins in vivo involved in maintaining cellular homeostasis and is essential for the regulation of oxidant stress. Gamma-Glutamyl transferase (GGT) is the first enzyme of the gamma glutamyl cycle that regulates the antioxidant glutathione, hence it is a critical enzyme in glutathione homeostasis. Recent findings have indicated upregulation of GGT in inflammation, increasing antioxidant defence whilst potentially driving leukotriene-induced inflammation. GGT is a marker of future comorbid diseases consistent with inflammation (and oxidative stress) as a key central pathophysiological process. COPD reflects several distinct pathological phenotypes. Inflammation (and hence oxidative stress) is influenced by other factors such as bacterial colonisation and exacerbations. The increased incidence of other co-morbid conditions with systemic inflammation suggests that common pathophysiological processes are responsible. Active oxidant stress and hence the role of GGT may play a role in these processes. Future studies of systemic and local GGT function and genotypes in well characterised patients may lead to a better understanding of the processes involved and hence the development of new treatment strategies.

Keywords: :

• Gamma-glutamyl transferase
• Glutathione
• COPD
• Biomarkers
• Inflammation

INTRODUCTION

Gamma-glutamyl transferase (GGT) is commonly recognized as the enzyme, which indicates liver disease and alcohol misuse (1, 2). Since the 1960s, the measurement of GGT enzyme activity has become an important routine diagnostic test for liver damage (3).

GGT is a heterodimeric protein, which is bound on the extracellular surface of membranes of secretory cells. It is critical to glutathione homeostasis (4) as it is the first major enzyme of the gamma-glutamyl cycle that regulates the metabolism of the antioxidant glutathione (GSH, γ-glutamyl-cysteinyl-glycine) (Figure 1). Glutathione is one of the most abundant proteins in vivo involved in maintaining cellular homeostasis. It is essential for regulating oxidant stress and in detoxification of drugs, pollutants, and carcinogens. In addition, glutathione is involved in cell cycle regulation, apoptosis, and cell signalling (5). GGT catalyzes the hydrolysis and transfer of the γ-glutamyl residue of glutathione and other γ-glutamyl compounds to acceptor molecules such as dipeptides and amino acids. The process regenerates the pool of amino acids available for re-use (6) and thus plays a role in all processes in which oxidative stress is increased at the vascular endothelial level. Oxidative stress occurs when oxidants and free radicals reach cytotoxic levels, resulting in damage to cellular components and producing pathological changes leading to organ damage and disease in vivo. Oxidants and reactive oxygen species are increased following exposure to various environmental factors such as infections, pollution, excessive alcohol, and smoking, as well as being a feature of ageing and general inflammatory responses. The oxidant burden is modulated by several antioxidants such as glutathione, catalase, and vitamins A, C, and E.

GGT also has a key role in the interconversion of the inflammatory mediator leukotriene C4 (LTC4), which is composed of leukotriene A4 and glutathione into leukotriene D4 (LTD4). Leukotrienes are important in asthma, allergic reactions, and inflammation. They are a family of lipids derived from arachidonic acid, which produce inflammation in asthma and bronchitis. LTC4 and LTD4 are cysteinyl leukotrienes and are responsible for the spasmogenic activity in anaphylaxsis. Thus, leukotrienes are important in inflammatory responses and GGT may therefore play a role in leukotriene-mediated inflammation (7).

The general importance of GGT has been demonstrated in a mouse model of GGT deficiency, an autosomal recessive trait, which was induced with N-ethyl-N-nitrosourea. The resulting mutant mouse (GGTenu1) failed to grow to normal size, had a reduced life span, was infertile and had glutathionuria (8, 9).

GGT deficiency in humans is rare, occurring as an autosomal recessive trait described in seven patients from five families identified worldwide (10). The deficiency causes glutathionuria, and the levels of glutathione are higher in plasma and urine compared to normal. Although three patients had total leukotriene D4 deficiency (11–15), other effects and particularly clinical abnormalities were not observed, possibly due to the small number of individuals studied and the potential lack of epigenetic factors that amplify predisposition to clinical phenotypes.

Figure 1.  A diagrammatic representation of the gamma-glutamyl cycle.

MOLECULAR STUDIES

GGT is a glycosylated heterodimer that consists of a heavy chain subunit (Mr 50,000–62,000) and a light chain subunit (Mr 22,000–30,000) (molecular weight variations are due to glycosylation). The two subunits are associated non-covalently to produce the enzyme activity, and the protein is anchored in the plasma membrane by an N-terminal membrane-spanning fragment of the heavy chain (Figure 2; 16–18).

Figure 2.  A diagrammatic illustration of the expression of the gamma-glutamyltransferase gene.

Thus far, 13 genes and pseudogenes of the GGT family have been identified, and of these, 6 are active (19) and 7 are located on chromosome 22 (20). The human GGT sequence was first cloned from fetal liver using a cDNA sequence of rat GGT (21, 22) and has subsequently been cloned from human placenta (22), a human hepatoma cell line (23), fetal liver (21), and human lung (24).

Tissue-specific differences in GGT are due to an “unusual untranslated region” (UTR) in the 5' region of the sequences and are attributed to alternative splicing (25), although the GGT protein product remains the same. Many single-base mutations have been identified in the GGT protein, resulting in different amino acid residues in the translation product; however, the putative protein coding regions show a high degree of homology (26). Mutations in the protein have not occurred in the known light-chain active site residue or the putative glutamine-binding site, suggesting these are most critical for function and health.

The tissue-specific inactive form of the GGT enzyme is a truncated protein consisting of only the heavy chain portion of GGT (26). The mRNA coding for this truncated protein has been detected in human liver, kidney, brain, intestine, stomach, placenta, and mammary gland. The clinical importance, if any, of this inactive form remains unknown.

GAMMA-GLUTAMYL TRANSFERASE AND OTHER DISEASES

Although GGT is most commonly recognized as the marker of liver damage, recently it has been shown to be associated with several other organs and their diseases. Serum GGT activity has been identified as a predictor of complications of atherosclerosis and has similar activity to that found in the tissues (27). The buildup of GGT in atherosclerotic plaques has been implicated in the triggering or amplifying of oxidative stress, thus leading to clinically important cardiovascular disease (28). The relationship between the inflammation seen in COPD and the increased incidence of cardiovascular disease is complex. Whether it reflects overspill of inflammation in the lung affecting systemic organs or vice versa remains unknown (29). Alternatively, it may reflect predisposing genotypes that affect more than one organ through the same pathophysiological processes. The association of GGT in the plasma and atheromatous plaques may reflect any or all of these explanations.

Similarly, plasma GGT is elevated in type-II diabetes (30) and gestational diabetes mellitus (31) and is an early biomarker for this condition (32). GGT has also been found to act as a bone-reabsorbing factor in rheumatoid arthritis (33), and it may be involved in oxidative stress in the pathogenesis of myotonic dystrophy type I (34). GGT has also been implicated in the progression of various cancers (35, 36) and their drug-resistance mechanisms (37). In addition, GGT has been found to be associated with obesity, correlating with visceral fat content (38). GGT levels could therefore serve as an indicator of the visceral fat content in the treatment of obesity. This common link between many metabolic conditions associated with COPD and features of inflammation such as oxidative stress and GGT may suggest common therapeutic paradigms.

GAMMA-GLUTAMYL TRANSFERASE AS A BIOMARKER

Recent studies have explored the use of GGT as a biomarker. GGT has been shown to be an independent marker for future acute coronary events (39), myocardial infarctions (40), and cardiac death (41). GGT has also been found to be an independent predictor of type-2 diabetes mellitus for both men and women in the general population (42) and used as an early predictor for the development of diabetes (32). GGT1 has also been highlighted as a biomarker for clear cell ovarian cancer (43).

More recent studies have begun to explore the role of GGT in an inflammatory environment. Daubeuf et al. (44) demonstrated that the expression of GGT mRNA was induced by three cytokines, IFNα, IFNβ, and TNFα. Subsequently, Reuter et al. (45) identified that the expression and activity of GGT was stimulated by TNFα via the NF-κB signalling pathway by recruiting Sp1 and RNA polymerase II to the GGT promotor. These findings indicate that inflammatory conditions can increase GGT synthesis. The implication of these findings could be that TNFα induces GGT expression either as a protective mechanism in conditions where oxidative stress is increased or in order to induce leukotriene synthesis and thus amplify inflammation (46). Further studies are necessary to explore these possibilities.

GAMMA-GLUTAMYL TRANSFERASE AND THE LUNG

Glutathione is essential for antioxidant defence in the lung (47) with 140-fold higher levels in epithelial lining fluid compared to plasma (48). This suggests a significant degree of local production and compartmentalization in the lung rather than simple diffusion from the blood (at least in health). However, GGT levels in the lung are low, and therefore have not been thought of as central to the development of lung disease. When there are insufficient levels of glutathione (for example, due to GGT deficiency), the oxidant/antioxidant balance is disrupted. Oxidants and free radicals can cause direct damage in the lung, leading to inactivation of antiproteases, damage to epithelial cells, and upregulation of proinflammatory cytokine gene expression via transcription factors such as nuclear factor κB. A deficiency of GGT would therefore predispose the lung to oxidant stress. The importance of GGT has been demonstrated in a GGT-deficient mouse which developed oxidative stress, pulmonary oedema, and bronchiolar cellular injury. This provides evidence that GGT is essential to control oxidative stress which influences the pathophysiology of many distinctive lung diseases (49). GGT is likely to be protective and therefore critical in maintaining lung glutathione homeostasis.

However, the effects of airway inflammation on glutathione metabolism in bronchial asthma using the GGT(enu1) mouse (GGT-deficient mutant) produced different results (50). Asthma is also thought to be an inflammatory condition exacerbated by an oxidant/antioxidant imbalance. Asthma induced with IL-13 was associated with 10-fold higher levels of glutathione in the lung lining fluid than the equivalent wild-type mouse due to lack of turnover of glutathione. However, despite this the mutant mouse did not develop mucous cell hyperplasia, upregulation of mucin related genes, epidermal growth factor regulation or hyperreactivity of the airways. The results suggest that the glutathione levels in the mutant mouse had a major protective effect and that the role of GGT may differ between diseases. The model suggests therefore that the inhibition of GGT may even be a potential therapy for asthma, whereas enhancement may be of importance in COPD. Clearly, further work is necessary to address these apparent disparities.

COPD is an inflammatory condition (51) with evidence of oxidant stress (52). Smoking is a major component in the pathogenesis of COPD as cigarette smoke contains high concentrations of free radicals and oxidants and increases recruitment of neutrophils and macrophages to the lungs of smokers, which results in inflammation. The presence of oxidants due to cigarette smoking has produced an increase in antioxidants such as glutathione, as a result of upregulation of antioxidant gene expression in chronic smokers. These results were obtained in vitro by exposing epithelial cells to cigarette smoke condensate as well as in vivo in rats exposed to cigarette smoke (53, 54).

However, epidemiological studies have shown that reduced lung function is associated with a decrease in antioxidants (55). This is consistent with oxidant stress playing a direct or indirect role in the pathophysiology of COPD. Whether this reflects specific phenotypes causing or being associated with reduced FEV1 remains to be determined.

COPD patients have elevated levels of C-Reactive Protein (CRP). Serum CRP has a positive correlation with GGT (57), indicating there may be an underlying relationship between GGT, general inflammation and oxidative stress in COPD.

Recent work from our group has found that GGT levels are more likely to be elevated in alpha-1-antitrypsin patients (25.7%) compared to healthy volunteers (11%). In addition, the plasma levels of GGT increased with the severity of COPD and chronic bronchitis, suggesting it reflects airflow obstruction and bronchial inflammation (58).

The role of leukotrienes in COPD has been investigated with particular emphasis on leukotriene B4, a known potent inflammatory mediator (59). However, the contribution of the cysteinyl leukotrienes (LTC4 and LTD4), which are the byproducts of GGT catabolism, is also known to cause mucus secretion, bronchoconstriction, and infiltration of inflammatory cells. Cysteinyl leukotriene inhibitiors used to treat asthma have been administered to COPD patients and reports showed some positive effects on treatment. Thus, changes to GGT levels will affect these cysteinyl leukotrienes and may have downstream consequences in the lung that are features of COPD (60).

COPD is associated with other comorbidities that have elevated levels of GGT, such as diabetes (61), cardiovascular disease (62), and osteoporosis (63), suggesting there may be a pathophysiological connection between GGT and these conditions.

Despite the involvement of the oxidant/antioxidant imbalance and the recognized genetic component to COPD, gene association studies involving GGT have yet to be conducted (64).

Finally, in addition to the 2.4 kb normal length GGT, the human lung expresses a specific shorter (1.2 kb) transcript of GGT (6, 23). This produces a truncated protein composed of a small portion of the heavy chain subunit and all of the light chain subunit and is referred to as type-II GGT. The significance of the truncated protein in the lung is currently unknown although some components for enzyme activity are present and this requires further investigation.

In summary, GGT is an important modulator of oxidant stress, as it is the first enzyme of the gamma-glutamyl cycle that regulates the antioxidant glutathione. Recent findings have indicated upregulation of GGT in inflammation, increasing antioxidant defence whilst potentially driving leukotriene-induced inflammation. GGT is a marker of future comorbid diseases consistent with inflammation (and oxidative stress) as a key central pathophysiological process. It is now accepted that COPD reflects several distinct pathological phenotypes. Inflammation (and hence oxidative stress) is influenced by other factors such as bacterial colonization and exacerbations. The increased incidence of other comorbid conditions with systemic inflammation suggests that common pathophysiological processes are responsible. Active oxidant stress and hence the role of GGT may play a role in these processes. Future studies of systemic and local GGT function and genotypes in well-characterized patients may lead to a better understanding of the processes involved and hence the development of new treatment strategies.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.