Localization of gamma-glutamyl transferase activity and protein in Zea mays organs and tissues

Gamma-glutamyl transferase/transpeptidase (GGT, (5-l-glutamyl)-peptide:amino-acid 5-glutamyltransferase; EC 2.3.2.2.) is an ectoenzyme promoting the cleavage of the gamma-glutamyl moiety of glutathione (GSH) and gamma-glutamyl related compounds. In this work, we describe the localization of GGT by enzymehistochemical and immunohistochemical analysis in maize plants. Our results show that the tissue spatial distribution of GGT activity closely correlates with the localization of the GGT protein. We also demonstrate that GGT activity and protein are unevenly distributed in tissues, being higher in the epidermis and stomata, parenchyma of conductive elements and root meristem. These results can contribute to our understanding of GGT function and regulation as well as its role in glutathione metabolism. To date, these are largely unknown in plants.     

A high performance gel filtration chromatography method for gamma-glutamyltransferase fraction analysis

The clinical relevance of serum gamma-glutamyltransferase (GGT) activity, in areas other than hepatic function, has recently been increased by several epidemiological associations. Still, GGT remains a nonspecific test because of the influence of various pathophysiological factors. We devised a procedure based on gel filtration chromatography, followed by postcolumn injection of fluorescent GGT substrate (gamma-glutamyl-7-amido-4-methylcoumarin), permitting the quantification of GGT fractions in serum or plasma. Plasma GGT molecular weight distribution was analyzed in healthy volunteers (20 males; mean+/-SD age 38+/-10 years; 20 females; age 44+/-13; total GGT 21+/-11 for males vs 13+/-7 for females; P<0.01). The method is highly sensitive (determination limit: 0.5 U GGT/L), with a linear dynamic range between 0.5 and 150 U/L for each fraction. Four GGT fractions of different molecular weight were detected in all subjects of both genders: b-GGT, m-GGT, s-GGT (likely lipoprotein-bound, molecular masses >2000, 940, and 140kDa, respectively), and a free fraction (f-GGT, 70kDa). f-GGT and s-GGT were the main fractions in subjects with lower and higher total GGT activity, respectively. Higher total GGT activity in males is related mainly to f-GGT (P<0.01). GGT fraction analysis may increase the sensitivity and specificity of the GGT activity test.     

Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.

The expression of gamma-glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood-brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary-specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT-protein-A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in porcine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary-adjacent pericytes in addition to the GGT-positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.     

Glutathione conjugates in the vacuole are degraded by gamma-glutamyl transpeptidase GGT3 in Arabidopsis

gamma-Glutamyl transpeptidase (GGT) is the enzyme responsible for breaking the gamma-glutamyl bond between Glu and Cys in glutathione (GSH). We are using this gene family to study GSH degradation in plants. There are four putative GGT genes in Arabidopsis, and one of them, GGT3 (At4g29210), is analyzed in this study. GGT3 is localized to the vacuole based on organelle-targeting programs, subcellular distribution of GFP fusion proteins during transient expression in onion (Allium cepa) epidermal tissues, and its ability to metabolize vacuolar substrates in Arabidopsis plants. While Northern blots and promoter:GUS expression patterns have suggested that GGT3 is transcribed at relatively high levels in all parts of the plant, a comparison of enzyme activities in different organs of wild-type and a ggt3 knockout mutant showed that GGT3 was a major contributor to total GGT activity in roots, but a relatively minor contributor in other tissues. Wild-type Arabidopsis plants treated with monobromobimane (mBB) form a fluorescent GSH-mBB conjugate that is moved into the vacuole and then metabolized to Cys-Gly-mBB and Cys-mBB in that order. The first step is catalyzed by GGT3, and GSH-mBB metabolism is completely blocked in the roots of ggt3 knockout plants. In ggt3 leaves, some GSH-mBB metabolism still proceeds using the apoplastic GGT1. This identifies GGT3 as catalyzing the obligate initial step in GSH conjugate metabolism, and suggests that it has an important role in protecting plants from some xenobiotic chemicals.     

Differential regulation of gamma-glutamyltransferase mRNAs in four human tumour cell lines.

Human gamma-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-alpha (TNF-alpha), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-alpha and TPA did not alter these parameters. In LNCap cells, TNF-alpha, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-alpha, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.     

Differential regulation of γ-glutamyltransferase mRNAs in four human tumour cell lines

Human γ-glutamyltransferase (GGT) belongs to a multigenic family and at least three mRNAs are transcribed from the gene that codes for an active enzyme. Four human tumour cell lines (HepG2, LNCap, HeLa and U937) with different GGT levels were used to investigate how GGT activity, total GGT mRNA and each individual GGT mRNA subtype responded to tumour necrosis factor-α (TNF-α), 12-O-tetradecanoylphorbol 13-acetate (TPA) or sodium butyrate treatment. Butyrate reduced the GGT activity in HepG2 cells, and the level of total GGT mRNA accordingly, whereas TNF-α and TPA did not alter these parameters. In LNCap cells, TNF-α, TPA, and butyrate reduced the activity as well as the level of GGT total mRNA. In HeLa cells no significant changes were observed either in activity or in mRNA level whereas TPA induced both GGT activity and mRNA levels in U937 cells. The distribution of each GGT mRNA subtype (A, B and C) was found to be cell specific: type B mRNA was the major form in HepG2 cells, while type A was the major form in LNCap and HeLa, type A and type C were expressed almost at the same level in U937 cells. The GGT mRNA subtypes were also differently modulated in these cells after TNF-α, TPA or butyrate treatment, suggesting that they are regulated by distinct and cell type specific mechanisms.     

Helicobacter pylori γ-glutamyltranspeptidase induces cell cycle arrest at the G1-S phase transition

In our previous study, we showed that Helicobacter pylori γ-glutamyltranspeptidase (GGT) is associated with H. pylori-induced apoptosis through a mitochondrial pathway. To better understand the role of GGT in apoptosis, we examined the effect of GGT on cell cycle regulation in AGS cells. To determine the effect of recombinant GGT (rGGT) on cell cycle distribution and apoptosis, rGGT-treated and untreated AGS cells were analyzed in parallel by flow cytometry using propidium iodide (PI). We found that rGGT inhibited the growth of AGS cells in a time-dependent manner, and that the pre-exposure of cells to a caspase-3 inhibitor (z-DEVD-fmk) effectively blocked GGT-induced apoptosis. Cell cycle analysis showed G1 phase arrest and apoptosis in AGS cells following rGGT treatment. The rGGT-mediated G1 phase arrest was found to be associated with down-regulation of cyclin E, cyclin A, Cdk 4, and Cdk 6, and the up-regulation of the cyclindependent kinase (Cdk) inhibitors p27 and p21. Our results suggest that H. pylori GGT induces cell cycle arrest at the G1-S phase transition.     

Gamma-glutamyl transpeptidase activity in isolated cells and in various regions of the mouse brain during early development and aging

Differences in the distribution of gamma-glutamyl transpeptidase (GGT) activity during early and late postnatal development of the mouse brain were studied at the cellular and regional level. From the 7th to the 25th day of brain development, GGT activity rose evenly in the neuronal perikarya, glial cells and in the neuropil. At 25 days and 5 weeks of age, a 1.5-fold enrichment was observed in nerve cell bodies and in neuroglia as compared with the neuropil and the original cell suspension. During the same period, an abrupt increase in GGT activity was observed in brain capillaries, and between the 10th day and the 5th week, this was 1.5-5 times higher than the enzyme activity in cellular elements and in the neuropil. This increase in enzyme activity in brain capillaries continued up to the age of 12 months, when it was succeeded by a decrease in GGT activity to the level found at 3 months. GGT activity in the choroid plexi rose significantly (more than double) between the age of 5 weeks and 18 months and was a whole order higher than GGT activity in 10 other regions of the mouse brain, whose development displayed no pronounced changes in the activity of the enzyme. A single dose of dexamethasone, or adrenalectomy, were used to demonstrate any participation by steroid hormones in the regulation of GGT activity in the brain tissue. Of the two techniques, only adrenalectomy led to a statistically significant decrease in activity in brain and liver tissue, while the apparent increase in GGT activity after a single dose of dexamethasone was not statistically significant.     

Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 μM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.     

Post-translational processing of Neisseria meningitidis gamma-glutamyl aminopeptidase and its association with inner membrane facing to the cytoplasmic space

We previously demonstrated that gamma-glutamyl aminopeptidase (also called gamma-glutamyl transpeptidase) (GGT) of Neisseria meningitidis is involved in the bacterial multiplication in cerebrospinal fluid. To further understand the function of meningococcal GGT, the biochemical properties were investigated in this study. The deduced amino acid sequence in N. meningitidis GGT was 37% identical to that of Escherichia coli GGT and that of Helicobacter pylori GGT, respectively, while a typical signal sequence was not found at the N-terminus of meningococcal GGT. Western blotting using rabbit antiserum against recombinant meningococcal GGT protein demonstrated that the meningococcal GGT is processed into two subunits in N. meningitidis at the conserved amino acid, threonine 427. The experiments on subcellular fractionation suggested that the majority of meningococcal GGT is associated with inner membrane facing to the cytoplasmic side. This cell localization might be unique for N. meningitidis compared to other GGTs.     

Localization of members of the gamma-glutamyl transpeptidase family identifies sites of glutathione and glutathione S-conjugate hydrolysis

gamma-Glutamyl transpeptidases (GGTs) are essential for hydrolysis of the tripeptide glutathione (gamma-glutamate-cysteine-glycine) and glutathione S-conjugates since they are the only enzymes known to cleave the amide bond linking the gamma-carboxylate of glutamate to cysteine. In Arabidopsis thaliana, four GGT genes have been identified based on homology with animal GGTs. They are designated GGT1 (At4g39640), GGT2 (At4g39650), GGT3 (At1g69820), and GGT4 (At4g29210). By analyzing the expression of each GGT in plants containing GGT:beta-glucuronidase fusions, the temporal and spatial pattern of degradation of glutathione and its metabolites was established, revealing appreciable overlap among GGTs. GGT2 exhibited narrow temporal and spatial expression primarily in immature trichomes, developing seeds, and pollen. GGT1 and GGT3 were coexpressed in most organs/tissues. Their expression was highest at sites of rapid growth including the rosette apex, floral stem apex, and seeds and might pinpoint locations where glutathione is delivered to sink tissues to supplement high demand for cysteine. In mature tissues, they were expressed only in vascular tissue. Knockout mutants of GGT2 and GGT4 showed no phenotype. The rosettes of GGT1 knockouts showed premature senescence after flowering. Knockouts of GGT3 showed reduced number of siliques and reduced seed yield. Knockouts were used to localize and assign catalytic activity to each GGT. In the standard GGT assay with gamma-glutamyl p-nitroanilide as substrate, GGT1 accounted for 80% to 99% of the activity in all tissues except seeds where GGT2 was 50% of the activity. Protoplasting experiments indicated that both GGT1 and GGT2 are localized extracellularly but have different physical or chemical associations.     

Gamma-glutamyl compounds: substrate specificity of gamma-glutamyl transpeptidase enzymes

Gamma-glutamyl compounds include antioxidants, inflammatory molecules, drug metabolites, and neuroactive compounds. Two cell surface enzymes that metabolize gamma-glutamyl compounds have been identified: gamma-glutamyl transpeptidase (GGT1) and gamma-glutamyl leukotrienase (GGT5). There is controversy in the literature regarding the substrate specificity of these enzymes. To address this issue, we have developed a method for comprehensive kinetic analysis of compounds as substrates for GGT enzymes. Our assay is sensitive, quantitative, and conducted at physiological pH. We evaluated a series of gamma-glutamyl compounds as substrates for human GGT1 and human GGT5. The K_m value for reduced glutathione was 11 μM for both GGT1 and GGT5. However, the K_m values for oxidized glutathione were 9 μM for GGT1 and 43 μM for GGT5. Our data show that the K_m values for leukotriene C₄ are equivalent for GGT1 and GGT5 at 10.8 and 10.2 μM, respectively. This assay was also used to evaluate serine–borate, a well-known inhibitor of GGT1, which was 8-fold more potent in inhibiting GGT1 than in inhibiting GGT5. These data provide essential information regarding the target enzymes for developing treatments for inflammatory diseases such as asthma and cardiovascular disease in humans. This assay is invaluable for studies of oxidative stress, drug metabolism, and other pathways that involve gamma-glutamyl compounds.     

Geranylgeranyltransferase I mediates BDNF‐induced synaptogenesis

Geranylgeranyltransferase I (GGT) is a prenyltransferase that mediates lipid modification of Rho small GTPases, such as Rho, Rac, and Cdc42, which are important for neuronal synaptogenesis. Although GGT is expressed in brain extensively, the function of GGT in central nerves system is largely unknown so far. We have previously demonstrated that GGT promotes the basal and neuronal activity and brain‐derived neurotrophic factor (BDNF)‐induced dendritic morphogenesis of cultured hippocampal neurons and cerebellar slices. This study is to explore the function and mechanism of GGT in neuronal synaptogenesis. We found that the protein level and activity of GGT gradually increased in rat hippocampus from P7 to P28 and subcellular located at synapse of neurons. The linear density of Synapsin 1 and post‐synaptic density protein 95 increased by over‐expression of GGT β, while reduced by inhibition or down‐regulation of GGT. In addition, GGT and its known substrate Rac was activated by BDNF, which promotes synaptogenesis in cultured hippocampal neurons. Furthermore, BDNF‐induced synaptogenesis was eliminated by GGT inhibition or down‐regulation, as well as by non‐prenylated Rac1 over‐expression. Together, our data suggested that GGT mediates BDNF‐induced neuronal synaptogenesis through Rac1 activation.     

Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney

In human, the two subunits of gamma-glutamyl transpeptidase (GGT) arise from a common precursor encoded by a multigene family. Until now, a single specific coding sequence for this precursor (type I) has been identified in human placenta and liver. In the present study, we have isolated from a human kidney cDNA library, a GGT specific clone (0.8 Kb). The sequence of which (type II) i) covers the carboxy terminal part of the GGT precursor, ii) exhibits 22 point mutations and a 30 bp deletion as compared to the type I GGT sequence. The sequencing of a human genomic clone reveals that this type II GGT mRNA is encoded by a different gene than the type I GGT mRNA. Both type I and type II GGT mRNAs are expressed in human liver, while almost exclusively type II GGT mRNA is detected in human kidney.     

Gamma-glutamyl transferase deficiency results in lung oxidant stress in normoxia

gamma-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGT(enu1) mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGT(enu1) lung. Glutathione content was barely decreased in GGT(enu1) lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGT(enu1) alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGT(enu1) lung. When GGT(enu1) mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.     

Characterization of the extracellular gamma-glutamyl transpeptidases, GGT1 and GGT2, in Arabidopsis

gamma-Glutamyl transpeptidase (GGT) is the only enzyme known that can cleave the gamma-peptide bond between glutamate and cysteine in glutathione, and is therefore a key step in glutathione degradation. There are three functional GGT genes in Arabidopsis, two of which are considered here. GGT1 and GGT2 are apoplastic, associated with the plasma membrane and/or cell wall. RNA blots and analysis of enzyme activity in knockout mutants suggest that GGT1 is expressed most strongly in leaves but is found throughout the plant. A GGT1::GUS fusion construct showed expression only in vascular tissue, specifically the phloem of the mid-rib and minor veins of leaves, roots and flowers. This localization was confirmed in leaves by laser microdissection. GGT2 expression is limited to embryo, endosperm, outer integument, and a small portion of the funiculus in developing siliques. The ggt2 mutants had no detectable phenotype, while the ggt1 knockouts were smaller and flowered sooner than wild-type. In ggt1 plants, the cotyledons and older leaves yellowed early, and GSSG, the oxidized form of glutathione, accumulated in the apoplastic space. These observations suggest that GGT1 is important in preventing oxidative stress by metabolizing extracellular GSSG, while GGT2 might be important in transporting glutathione into developing seeds.     

Is serum gamma glutamyltransferase a marker of oxidative stress?

The primary role of cellular gamma glutamyltransferase (GGT) is to metabolize extracellular reduced glutathione (GSH), allowing for precursor amino acids to be assimilated and reutilized for intracellular GSH synthesis. Paradoxically, recent experimental studies indicate that cellular GGT may also be involved in the generation of reactive oxygen species in the presence of iron or other transition metals. Although the relationship between cellular GGT and serum GGT is not known and serum GGT activity has been commonly used as a marker for excessive alcohol consumption or liver diseases, our series of epidemiological studies consistently suggest that serum GGT within its normal range might be an early and sensitive enzyme related to oxidative stress. For example, serum and dietary antioxidant vitamins had inverse, dose-response relations to serum GGT level within its normal range, whereas dietary heme iron was positively related to serum GGT level. More importantly, serum GGT level within its normal range positively predicted F2-isoprostanes, an oxidative damage product of arachidonic acid, and fibrinogen and C-reactive protein, markers of inflammation, which were measured 5 or 15 years later, in dose-response manners. These findings suggest that strong associations of serum GGT with many cardiovascular risk factors and/or events might be explained by a mechanism related to oxidative stress. Even though studies on serum and/or cellular GGT is at a beginning stage, our epidemiological findings suggest that serum GGT might be useful in studying oxidative stress-related issues in both epidemiological and clinical settings.     

Studies on turnover rates of rat gamma-glutamyltranspeptidase after chronic ethanol administration in vivo

Chronic ethanol administration to rats was shown to result in a significant increase of hepatic and serum GGT activities, contrasting to the decreased levels observed in pancreas, intestine, brain, and kidney by the new alcock regimen method. The kinetics of rat GGT synthesis and degradation in vivo among the different sources after chronic ethanol administration has been studied by use of acivicin, which irreversibly inactivates GGT. The comparison of kinetics of GGT return after acivicin injection showed that the kidney and serum GGT exhibits biphasic half-lives in contrast to liver, pancreatic, intestinal, and brain GGT half-lives in chronic ethanol-administered rats. The present studies on kinetics of GGT synthesis (Ks) and degradation (Kd) in vivo would seem to indicate the existence of three types of systems. That is, Ks rather than Kd may be preferential in liver and serum whereas Kd is apparently increased in kidney and intestine without noticeable change in Ks. The reverse phenomenon is also observed for pancreas and brain. These findings suggest that the contributions of alterations in the rates of GGT synthesis and degradation to changing levels of GGT have been evaluated as a mechanism for enzyme adaptation in animal tissues as a change from the control diet to the ethanol diet.     

A mutant Bacillus subtilis gamma-glutamyltranspeptidase specialized in hydrolysis activity

gamma-Glutamyltranspeptidase (GGT) catalyzes the hydrolysis of gamma-glutamyl compounds and the transfer of their gamma-glutamyl moieties to amino acids and peptides. The transpeptidation activity of Bacillus subtilis GGT is about 10-fold higher than its hydrolysis activity. In B. subtilis GGT, substitution of Asp-445 with Ala abolished its transpeptidation activity. The specific activity for hydrolysis of D445A GGT was 40.2% of that of the wild-type GGT. The K(m) value for L-glutamine was 15.3 mM. D445A GGT was salt tolerant like the wild-type GGT. These results indicate that D445A GGT will be highly useful as a 'glutaminase' in food industry.