GGT Gene Research Articles

Increases in the mRNA levels of γ-Glutamyltransferase and heme oxygenase-1 in the rat lung after ozone exposure

γ-Glutamyltransferase (GGT) and heme oxygenase-1 (HO-1) are induced by chemical and physical stresses producing an oxidative burden on tissues and cells. Both enzymes are proposed to have an antioxidant role in protecting cells and tissues from oxidative burden. To explore the effects of ozone (O 3), the major oxidant in photochemical smog, on the expression of GGT and HO-1 genes in the lung, we exposed rats to 0.4 ppm O 3 for up to 7 days. After exposures, mRNA levels of GGT and HO-1 in the lung were measured by RNA blot analysis. Although a 1-day exposure did not change either GGT or HO-1 mRNA levels in the lung, both genes responded to prolonged exposure to O 3. GGT mRNA was increased to 149% (P < 0.01) and 158% (P < 0.01) of the control by 3- and 7-day exposures, respectively. HO-1 mRNA was also elevated to 174% (P < 0.01) and 184% (P < 0.001) of the control after 3- and 7-day exposures, respectively. The elevation of GGT and HO-1 mRNA after prolonged exposure to O 3 suggests that expression of these genes is not involved in the acute respiratory response, but in the recovery process from lung damage induced by O 3.

Nitrogen Dioxide Exposure Activates γ-Glutamyl Transferase Gene Expression in Rat Lung

Exposure to nitrogen dioxide (NO2) has been shown to activate glutathione metabolism in lung and lung lavage. Since GGT is a key enzyme in glutathione metabolism and we have previously characterized GGT expression in distal lung epithelium and in lung surfactant, we examined the NO2exposed lung for induction of γ-glutamyl transferase (GGT) mRNA, protein, and enzyme activity. We found that the GGT gene product is induced in lung by NO2. The GGT mRNA level in lung increases 2-fold within 6 hr and 3-fold after 24 hr of exposure to this oxidant gas, and this 3-fold elevation persists even after 14 days of exposure. The pattern of GGT mRNA expression switches from the single GGT mRNA III transcript in the normal lung to the dual expression of GGT mRNA I and mRNA III. Enzyme activity in whole lung increases 1.6- to 2.5-fold while extracellular surfactant-associated GGT activity accumulates 5.5-fold and GGT protein accumulates in lung surfactant. Induction of GGT mRNA and protein is evident in cells of the bronchioles byin situhybridization and immunolocalization, respectively. In contrast, alveolar type 2 cells lack anin situhybridization signal and exhibit a reduction in the intensity of immunostaining with prolonged exposure. Our studies show that NO2induces GGT mRNA expression, including GGT mRNA1, in lung and GGT protein and enzyme activity in lung and lung lavage in response to the oxidative stress of NO2inhalation.

Analysis of low temperature inducible mechanism of gamma-glutamyltranspeptidase of Escherichia coli K-12.

Escherichia coli K-12 cultured at 20 degrees C has more gamma-glutamyltranspeptidase (GGT: EC 2.3.2.2) activity than that cultured at 37 or 42 degrees C. On Western blot analysis, E. coli K-12 cells cultured at 20 degrees C produced more GGT protein than those cultured at 37 degrees C. mRNA of the GGT gene (ggt) in the cells was also measured and it was found that the level of ggt mRNA at 20 degrees C was 10-fold higher than that at 37 degrees C. When the ggt promoter was replaced by a tac promoter, GGT activity at 37 degrees C from the tac promoter was 5-fold higher than that at 37 degrees C from the ggt promoter, though there was less difference in GGT activity between both grown at 20 degrees C. The ggt mRNA at 20 degrees C was found to be more stable than that at 37 degrees C. These results suggested that the higher GGT activity in E. coli K-12 cells grown at 20 degrees C was due to a higher level of GGT protein at 20 degrees C caused by higher level of ggt mRNA at 20 degrees C because of a low-temperature dependent ggt promoter as well as the stability of ggt mRNA at 20 degrees C.

Organization of the 5′-End of the Porcine γ-Glutamyl Transpeptidase Gene and Identification of Three Different mRNAs in the Kidney

Three different mRNAs coding for the porcine γ-glutamyl transpeptidase (GGT) in the kidney were identified by 5′-RACE-PCR. These differ in their 5′-noncoding region. Genomic Southern blot analysis has demonstrated the existence of a single GGT gene in the porcine genome. Thus, the existence of multiple mRNAs can only be explained by the use of different promoters or alternative splicing. Four GGT-specific genomic clones containing the complete 5′-end of the gene were isolated and characterized, revealing six exons common to all three mRNAs. Four of these exons were located in the coding region comprising the codons for amino acids 1 to 138. Two exons and an intervening sequence were identified upstream from these six common exons representing the unique 5′-ends of the three mRNAs. The coding exons show a significant sequence homology to mouse, rat, and human GGT cDNA, whereas exons 1 and 3 display no homology.

Cloning of cDNA and genomic structure of the mouse γ-glutamyl transpeptidase-encoding gene

We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse γ-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exons and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.

Triiodothyronine decreases gammaglutamyltranspeptidase expression in cultured rat hepatocytes.

Recently, an increase in gammaglutamyltranspeptidase (GGT) activity and mRNA in liver of hypothyroid rats has been reported. The aim of this study was to verify if triiodothyronine (T3) can directly affect GGT expression in primary cultures of rat hepatocytes. Results obtained from adult rat hepatocytes cultured in serum-free medium demonstrate: 1) a rise in GGT mRNA level magnified by dexamethasone during the maintenance of hepatocytes in culture which parallels the stimulation of GGT activity; 2) a negative effect of T3 on GGT activity of cultured hepatocytes which reflects a specific inhibition of GGT gene expression. The T3 effect on GGT expression in cultured hepatocytes is in line with previous observations on hypothyroid rat liver suggesting an important role for thyroid hormone in maintaining the differentiated adult liver phenotype in the rat.

Expression of multiple γ-glutamyltransferase genes in man

In clinical and pharmacological laboratories, the assay for gamma-glutamyltransferase (GGT) activity is an important diagnostic test, but one with high biological variability. Although the human genome contains multiple GGT genomic sequences, the diagnostic tests generally assume that only a single GGT gene is active. In the current study, segments encompassing parts of seven different potential human GGT genes have been molecularly cloned. Based on sequence determination of exons within these distinct genomic clones, oligonucleotide primers were designed which would prime and PCR-amplify putative mRNA of all seven potential GGT genes, if expressed. Gene-specific oligonucleotide probes were then utilized to assay the transcriptional status of the seven possible GGT genes in a wide variety of human RNAs. Our results show that a single GGT gene exhibits ubiquitous expression in all RNAs tested, including those from fetal and adult liver. A surprisingly large number of four additional GGT genes is expressed in man. Interestingly, these novel GGT genes are expressed in a tissue-restricted manner, which suggests that their corresponding gene products exhibit distinct functions in these specific tissues.

Molecular cloning of the gamma-glutamyltranspeptidase gene from a Pseudomonas strain.

gamma-Glutamyltranspeptidase (GGT) was purified from a Pseudomonas sp. strain A14. The purified enzyme was found to be composed of two nonidentical subunits with molecular weights of 39,000 and 22,000 and had a pI of > 8.6. The partial N-terminal amino acid sequences of both subunits and some proteolytic fragments were determined. Using mixed oligonucleotides designed from the partial amino acid sequences as hybridization probes, one cosmid clone which contained the GGT gene was isolated from a Pseudomonas sp. strain A14 cosmid genome library, and the DNA sequence of the GGT gene was determined. The nucleotide sequence and the protein sequence analysis revealed that GGT was synthesized as a precursor protein of 575 amino acids and then processed to mature enzyme, presumably after removal of a signal peptide. Comparison of the predicted amino acid sequence of Pseudomonas GGT with published results for Escherichia coli K-12 and rat kidney GGTs shows that the protein sequence of Pseudomonas GGT is 51% and 33% identical to the E. coli and rat GGT sequences, respectively. Higher similarity is observed among the small subunits, which have been thought to have a binding site for the gamma-glutamyl residue. Expression of the cloned Pseudomonas GGT gene in E. coli was subjected to Western blot analysis using antibody raised against the purified GGT. This suggested that processing of the precursor protein to its subunits is temperature-dependent, because the amount of mature GGT protein was increased when the culture was performed at low temperature.

Organization of the 5' end of the rat gamma-glutamyl transpeptidase gene: structure of a promoter active in the kidney.

Two gamma-glutamyl transpeptidase mRNAs (mRNAI and mRNAII), with alternate 5'-untranslated regions, are expressed in the rat kidney. Oligonucleotides were designed based upon these two alternate 5' sequences and used as primers to amplify GGT genomic DNA sequences. The genomic organization of the mRNAI and mRNAII 5'-untranslated sequences reveals that the mRNAs are encoded from two separate promoters which are 2.1 kbp apart on the single GGT gene. A 2775 base pair genomic sequence, which contains the proximal GGT promoter, was cloned from two overlapping amplified fragments. S1 mapping analysis shows that the kidney GGT mRNAI is transcribed from several start sites on this promoter which displays neither a classical TATA box nor Sp1 binding sites. Chimeric plasmids, including the GGT promoter region for mRNAI, associated with the chloramphenicol acetyltransferase (CAT) reporter gene, were transiently expressed in a kidney (LLCPK) and in a hepatoma (HTC) cell line. A sequence extending 308 bases upstream from the major GGT mRNAI start site drives a promoter activity which is 5-fold higher in LLCPK than in HTC cells and is sufficient to confer cell specificity to the GGT proximal promoter.

Excretion and rapid purification of γ-glutamyltranspeptidase from Escherichia coli K-12

tolA strains of Escherichia coli harboring plasmids carrying the ggt gene excreted about 70% of their γ-glutamyltranspeptidase (GGT) (EC 2.3.2.2) into the medium. The excreted GGT was purified to electrophoretic homogeneity by a simple two-step method with a yield of 51%.

Description and analysis of differential sensitivity to glucocorticoids in Fao cells

This study shows that the derived hepatoma cell line Fao displays different sensitivities for glucocorticoid induction of tyrosine aminotransferase (TAT), alanine aminotransferase (AAT) and gamma-glutamyltransferase (GGT). This was seen in the different behaviors of nine steroids with respect to these three effects: (1) in the presence of full agonists (dexamethasone or deacylcortivazol), half-maximal induction of GGT occurred at approx 5- to 6-fold higher agonist concentrations than those required for half-maximal induction of AAT and TAT; 1. (2) in the presence of full antagonists (RU 486, R5020, or progesterone) the GGT response induced by an equal agonist concentration was inhibited at concentrations approx 4- to 5-fold lower than those required for an equivalent inhibition of TAT response; 2. (3) in the presence of cortexolone, deoxycorticosterone, 11β-hydroxyprogesterone and dexamethasone-3'-oxetanone, there was a partial agonistic effect (30–50%) on TAT and AAT responses, whereas there was a mainly antagonistic effect (very weak agonistic effect: 0–10%) on GGT response; 3. (4) regardless of the steroid or its full or partial agonist activity, a given TAT induction level (50%, for example) always corresponded to the same AAT and GGT induction levels (50 and 10% respectively). We provide evidence showing that the three above-mentioned biological responses are mediated via the same type of glucocorticoid receptor binding site. Consequently, this differential behavior probably originates from a phenomenon occurring after the common steps (activation, translocation) that follow the formation of the steroid-receptor complex. This leads us to propose a model in which this phenomenon is assumed to originate from a difference in the affinities of the activated receptor for the nuclear acceptor sites of the TAT and GGT genes.

Induction of gamma-glutamyl transpeptidase in primary cultures of normal rat hepatocytes by liver tumor promoters and structurally related compounds.

Rat hepatocytes maintained for up to 6 days in primary culture were used to test a variety of xenobiotics and steroids for effects on the activity of gamma-glutamyltranspeptidase (GGT) in normal cells. In control cultures GGT activity was low and increased slowly with time. When added to cultures for 5 days, a variety of xenobiotics and steroids increased GGT activity to levels 2- to 6-times those of control cultures. Induction of GGT was potentiated for most test compounds by 20-30 nM dexamethasone and diminished by nicotinamide or adenosine-3',5'-monophosphate. Effective non-genotoxic inducers included phenobarbital and some structurally related compounds, p,p'-dichlorodiphenyltrichloroethane,alpha- and gamma-hexachlorocyclohexanes, Aroclor 1254, butyl hydroxytoluene, nafenopin, various estrogens, progesterone, pregnenolone-16 alpha-carbonitrile and cyproterone acetate. A number of compounds including barbituric acid, butyl hydroxyanisole, acetaminophen, saccharin, caffeine, clofibrate and some bile acids failed to induce GGT. Except for 2-acetylaminofluorene and diethylnitrosamine, genotoxic compounds tested did not increase GGT. The results establish that a structurally diverse group of xenobiotics and steroids, many of which are considered to be liver tumour promoters, may directly enhance GGT gene expression in normal hepatocytes. Thus, a variety of compounds used in experimental studies of liver cancer induction as promoters may elevate GGT by mechanism(s) not necessarily related to carcinogenesis.