g-Glutamyl transferase (GGT) deficiency in the GGT enu1 mouse results from a single point mutation that leads to a stop codon in the first coding exon of GGT mRNA (original) (raw)
Related papers
Mutagenesis, 1999
GGT enu1 , a recently described genetic murine model of γglutamyl transferase (GGT) deficiency, was induced by the point mutagen N-ethyl-N-nitrosourea and is inherited as an autosomal recessive trait. The phenotype of systemic GGT deficiency suggested a mutation site within the cDNA coding region which is common in all GGT transcripts. To identify this site, total lung and kidney RNA was isolated from normal and mutant mice, amplified by RT-PCR using GGT-specific primers, cloned as two overlapping~1 kb GGT cDNA fragments, sequenced and compared with that in the literature. A single base pair substitution was identified in the coding region at position 237, where thymidine became adenine, and this mutation replaced a leucine codon, TTG, with a termination codon, TAG. This mutation site was confirmed in mutant genomic DNA by PCR using primers that flanked the predicted site and spanned the intron between the common GGT non-coding exon and the first GGT coding exon. This PCR product was sequenced directly with the secondary 3Ј PCR primer, the mutation site identified and the protocol then utilized to genotype animals. In addition to this mutation, the steady-state level of GGT mRNA in mutant kidney is reduced 3-fold compared with the control. Heterodimeric GGT protein is not detectable by western blot in either whole kidney homogenate or a microsomal membrane fraction. The steady-state mRNA level of γ-glutatmyl cysteinyl synthetase was unchanged in mutant mice compared with normal, but that of heme oxygenase-1 and Cu,Zn-SOD was induced 4-and 3-fold, respectively. Hence, the GGT enu1 mouse model of GGT deficiency results from a single point mutation in the first coding exon of GGT mRNA and the resulting impairment in glutathione turnover induces oxidative stress in the kidney.
Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney
Biochemical and Biophysical Research Communications, 1989
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.
Glutamyl transferase deficiency results in lung oxidant stress in normoxia
2000
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.
Antioxidants & Redox Signaling, 2013
Aims: Human c-glutamyltranspeptidase 1 (hGGT1) is a cell-surface enzyme that is a regulator of redox adaptation and drug resistance due to its glutathionase activity. The human GGT2 gene encodes a protein that is 94% identical to the amino-acid sequence of hGGT1. Transcriptional profiling analyses in a series of recent publications have implicated the hGGT2 enzyme as a modulator of disease processes. However, hGGT2 has never been shown to encode a protein with enzymatic activity. The aim of this study was to express the protein encoded by hGGT2 and each of its known variants and to assess their stability, cellular localization, and enzymatic activity. Results: We discovered that the proteins encoded by hGGT2 and its variants are inactive propeptides. We show that hGGT2 cDNAs are transcribed with a similar efficiency to hGGT1, and the expressed propeptides are N-glycosylated. However, they do not autocleave into heterodimers, fail to localize to the plasma membrane, and do not metabolize c-glutamyl substrates. Substituting the coding sequence of hGGT1 to conform to alterations in a CX 3 C motif encoded by hGGT2 mRNAs disrupted autocleavage of the hGGT1 propeptide into a heterodimer, resulting in loss of plasma membrane localization and catalytic activity. Innovation and Conclusions: This is the first study to evaluate hGGT2 protein. The data show that hGGT2 does not encode a functional enzyme. Microarray data which have reported induction of hGGT2 mRNA should not be interpreted as induction of a protein that has a role in the metabolism of extracellular glutathione and in maintaining the redox status of the cell.
Gamma-glutamyl transferase deficiency results in lung oxidant stress in normoxia
American journal of physiology. Lung cellular and molecular physiology, 2002
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 express...
Gamma-glutamyltransferase-friend or foe within?
Liver International, 2016
Gamma-glutamyltransferase, is a liver enzyme which is located on the plasma membranes of most cells and organ tissues, but more commonly in hepatocytes and is routinely used in clinical practice to help indicate liver injury and as a marker of excessive alcohol consumption. Among the liver enzymes, important advances have especially been made in understanding the physiological functions of GGT. The primary role of GGT is the extracellular catabolism of glutathione, the major thiol antioxidant in mammalian cells, which plays a relevant role in protecting cells against oxidants produced during normal metabolism; GGT, therefore plays an important role in cellular defence. Beyond its physiological functions, circulating serum GGT has been linked to a remarkable array of chronic conditions and diseases, which include nonalcoholic fatty liver disease, vascular and non-vascular diseases, as well as mortality outcomes. This review summarizes the available epidemiological and genetic evidence for the associations between GGT and these adverse outcomes; the postulated biologic mechanisms underlying these associations; outlines areas of outstanding uncertainty; and the implications for clinical practice.
Hepatology
ssay of γ-glutamyltransferase (GGT) activity is a widely used test to indicate and monitor liver and biliary tract injury. We observed dominant inheritance of highly elevated plasma GGT levels, designated GGTemia, in two unrelated families. Neither clinical symptoms nor alterations of GGT substrates were associated with GGTemia. A plasma GGT fraction pattern distinguishes this trait from common liver diseases. Heterozygous γ-glutamyltransferase 1 (GGT1) mutations that
Cloning and analysis of the rat γ-glutamyltransferase gene
Journal of Biological Chemistry
We have isolated and characterized a complete structural gene encoding the enzyme gamma-glutamyltransferase ((5-glutamyl)-peptide:amino acid 5-glutamyltransferase; EC 2.3.2.2). The gene contains 8 exons and spans approximately 12 kilobases. Ras-transformed rat liver epithelial cells and rat kidney express RNAs which differ in length by approximately 0.3 kilobase pair. Comparison of the genomic sequence with kidney gamma GT cDNA sequence indicates that the first exon is noncoding, and nuclease protection and primer extension data have identified a potential kidney transcription start site (defined as +1) for this exon. The site is not associated with a TATA box, but there are two CCAAT boxes (-136 and -599) and two sites (-101 and -746) containing the consensus sequences to which the transcription factor SP1 is known to bind. There is also a sequence at -453 (TGTGGTTG) that is highly homologous to the core sequence (TGTGG(T)3-5G) of SV40 and polyoma viral enhancers.