Human Gamma-glutamyl Transpeptidase Research Articles

Design and evaluation of novel analogs of 2-amino-4-boronobutanoic acid (ABBA) as inhibitors of human gamma-glutamyl transpeptidase

Inhibitors of gamma-glutamyl transpeptidase (GGT1, aka gamma-glutamyl transferase) are needed for the treatment of cancer, cardiovascular illness and other diseases. Compounds that inhibit GGT1 have been evaluated in the clinic, but no inhibitor has successfully demonstrated specific and systemic GGT1 inhibition. All have severe side effects. L-2-amino-4‑boronobutanoic acid (l-ABBA), a glutamate analog, is the most potent GGT1 inhibitor in vitro. In this study, we have solved the crystal structure of human GGT1 (hGGT1) with ABBA bound in the active site. The structure was interrogated to identify interactions between the enzyme and the inhibitor. Based on these data, a series of novel ABBA analogs were designed and synthesized. Their inhibitory activity against the hydrolysis and transpeptidation activities of hGGT1 were determined. The lead compounds were crystalized with hGGT1 and the structures solved. The kinetic data and structures of the complexes provide new insights into the critical role of protein structure dynamics in developing compounds for inhibition of hGGT1.

Structure of 6-diazo-5-oxo-norleucine-bound human gamma-glutamyl transpeptidase 1, a novel mechanism of inactivation.

Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM-1 min-1 and the Ki was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.

Abstract 4546: Depleting cysteine: A novel approach to tumor therapy

Abstract A cure remains elusive for many types of tumors that initially respond to chemotherapy but recur with drug-resistant disease. Cysteine, (or its oxidized form cystine) is an essential amino acid for tumors. Enhanced uptake of cysteine and/or cystine is required for tumors to maintain their intracellular glutathione (GSH) levels and redox balance. In addition, some stem cells within epithelial tumors express CD44v8-10, which stabilizes the cystine transporter, increasing cystine uptake into the cell and enhancing resistance to pro-oxidant therapy. Depleting cysteine during treatment has a profound effect on sensitizing the tumor to therapy. We have a novel strategy that reduces the cysteine (and cystine) concentration in serum and extracellular fluid. The strategy exploits the finding that a severe cysteine deficiency develops in mice lacking the enzyme gamma-glutamyl transpeptidase (GGT). This enzyme reclaims the cysteine from GSH as the blood is filtered through the kidney. Inhibiting GGT activity results in excretion of GSH in the urine and a precipitous drop in the concentration of cysteine and cystine in serum. The inhibitors of GGT that have been evaluated in the clinic are glutamate analogs and are too toxic for clinical use. We have discovered and are developing a new class of GGT inhibitors that are not glutamate analogs. These inhibitors are orders of magnitude less toxic than the glutamate analogs. We have determined the crystal structure of human GGT. In the current study we have co-crystallized human GGT with a series of inhibitors. We report the identification of residues within the active site that are critical for inhibitor binding. We have identified changes in the conformation of the active site upon substrate binding. We have also delineated movement within the active site during substrate cleavage. Site-directed mutagenesis and functional assays have confirmed the role of the key residues identified in the structural studies. These data provide new insights for continued development of a potent, specific, non-toxic inhibitor of GGT that can be used clinically in cancer therapy. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640. Citation Format: Marie H. Hanigan, Anthony W. G. Burgett, Nancy Wakeham, Simon S. Terzyan. Depleting cysteine: A novel approach to tumor therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4546. doi:10.1158/1538-7445.AM2015-4546

Abstract 683: Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase

Abstract We have reported that 73% of ovarian tumors express the cell surface enzyme gamma-glutamyl transpeptidase (GGT). Clinical studies have shown a poorer prognosis for ovarian tumors that are GGT-positive compared to GGT-negative tumors. GGT-positive tumors are able to use the glutathione in the interstitial fluid as an additional source of cysteine, the limiting component of intracellular glutathione synthesis in tumors. The concentration of cysteine/cystine in standard tissue culture media is more than 3 times the concentration in interstitial fluid. In this study we show that in studies of cultured cells, the high concentration of cysteine in tissue culture media obscured the role of GGT expression in drug resistance. However, culturing human ovarian tumor cells in tissue culture media containing physiological concentrations of cystine and glutathione demonstrated that the supply of cysteine/cystine regulates intracellular glutathione concentrations. In these studies, inhibition of GGT blocked the use of extracellular glutathione as a source of cysteine and sensitized the tumors to cisplatin, carboplatin and melphalan. Inhibiting GGT in patients would also inhibit GGT in the kidney resulting in the excretion of glutathione in the urine and depletion of cysteine from serum and interstitial fluid. As a result, inhibition of GGT in patients would sensitize both GGT-positive and GGT-negative tumors to pro-oxidant chemotherapy. Clinical application of this therapeutic strategy is limited by the fact that most inhibitors of GGT are glutamate analogs and too toxic for use in humans. We discovered a non-glutamate analog that is an uncompetitive inhibitor of GGT, and are developing more potent, non-toxic GGT inhibitors. We recently published the crystal structure of human GGT. We have now also solved the structure of the substrate-bound enzyme, the form of the enzyme to which our uncompetitive inhibitors bind. Using structure-based drug design and our physiologically relevant tissue culture system we are designing new GGT inhibitors and evaluating them for their ability to sensitize tumors to chemotherapy regimens used in the clinic. Supported by an Institutional Development Award (IDeA) from the National Institutes of Health under grant P20GM103640. Citation Format: Marie H. Hanigan, Nancy Wakeham, Stephanie Wickham, Simon S. Terzyan. Sensitizing tumors to pro-oxidant therapy by inhibiting gamma-glutamyl transpeptidase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 683. doi:10.1158/1538-7445.AM2014-683

Mechanistic study of BNP7787-mediated cisplatin nephroprotection: modulation of gamma-glutamyl transpeptidase

The mechanisms for cisplatin-induced renal cell injury have been the focus of intense investigation for many years with a view to provide a more effective and convenient form of nephroprotection. BNP7787 (disodium 2,2'-dithio-bis ethane sulfonate; dimesna, Tavocept), is a water-soluble disulfide investigational new drug that is undergoing clinical development for the prevention and mitigation of clinically important chemotherapy-induced toxicities associated with platinum-type chemotherapeutic agents. We hypothesized that part of BNP7787's mechanism of action (MOA) pertaining to the potential prevention of cisplatin-induced nephrotoxicity involves the inhibition of gamma-glutamyl transpeptidase (GGT) activity, mediated by BNP7787-derived mesna-disulfide heteroconjugates that contain a terminal gamma-glutamate moiety [e.g., mesna-glutathione (MSSGlutathione) and mesna-cysteinyl-glutamate (MSSCE)]. Inhibition studies were conducted on human and porcine GGT to determine the effect of mesna-disulfide heteroconjugates on the enzyme's activity in vitro. These studies utilized a fluorimetric assay that monitored the hydrolysis of L-gamma-glutamyl-7-amino-4-trifluoromethylcoumarin (GG-AFC) to AFC. Mesna-disulfide heteroconjugates that contained gamma-glutamyl moieties were potent inhibitors of human and porcine GGT. An in situ-generated mesna-cisplatin conjugate was not a substrate for GGT. The GGT xenobiotic metabolism pathway is postulated to be a major toxification pathway for cisplatin nephrotoxicity, and BNP7787 may play a novel and critical therapeutic role in the modulation of GGT activity. We further postulate that there are two general mechanisms for BNP7787-mediated nephroprotection against cisplatin-induced nephrotoxicity involving this pathway. First, the active BNP7787 pharmacophore, mesna, produces an inactive mesna-cisplatin conjugate that is not a substrate for the GGT toxification pathway (GGT xenobiotic metabolism pathway) and, second, BNP7787-derived mesna-disulfide heteroconjugates may serve as selective, potent inhibitors of GGT, possibly resulting in nephroprotection by a novel means.

A Novel, Species-specific Class of Uncompetitive Inhibitors of γ-Glutamyl Transpeptidase

Expression of gamma-glutamyl transpeptidase (GGT) in tumors contributes to resistance to radiation and chemotherapy. GGT is inhibited by glutamine analogues that compete with the substrate for the gamma-glutamyl binding site. However, the glutamine analogues that have been evaluated in clinical trials are too toxic for use in humans. We have used high throughput screening to evaluate small molecules for their ability to inhibit GGT and have identified a novel class of inhibitors that are not glutamine analogues. These compounds are uncompetitive inhibitors, binding the gamma-glutamyl enzyme complex. OU749, the lead compound, has an intrinsic K(i) of 17.6 microm. It is a competitive inhibitor of the acceptor glycyl-glycine, which indicates that OU749 occupies the acceptor site while binding to the gamma-glutamyl substrate complex. OU749 is more than 150-fold less toxic than the GGT inhibitor acivicin toward dividing cells. Inhibition of GGT by OU749 is species-specific, inhibiting GGT isolated from human kidney with 7-10-fold greater potency than GGT isolated from rat or mouse kidney. OU749 does not inhibit GGT from pig cells. Human GGT expressed in mouse fibroblasts is inhibited by OU749 similarly to GGT from human cells, which indicates that the species specificity is determined by differences in the primary structure of the protein rather than species-specific, post-translational modifications. These studies have identified a novel class of inhibitors of GGT, providing the basis for further development of a new group of therapeutics that inhibit GGT by a mechanism distinct from the toxic glutamine analogues.

Treatment With Anti-γ-Glutamyl Transpeptidase Antibody Attenuates Osteolysis in Collagen-Induced Arthritis Mice

The effectiveness of a new antibody treatment on arthritis-associated osteolysis was studied by using CIA mice. GGT, a newly identified bone-resorbing factor, was upregulated in arthritic joints. We generated monoclonal antibodies against GGT and injected them into CIA mice. Mice treated with antibodies showed a reduction in osteoclast number and bone erosion. Gamma-glutamyl transpeptidase (GGT) acts as a bone-resorbing factor that stimulates osteoclast formation. GGT expression has been detected in active lymphocytes that accumulate at inflammation sites, such as rheumatoid arthritis (RA). We hypothesize that GGT is an effective target for suppression of arthritis-related osteoclastogenesis and joint destruction. Here, we describe the therapeutic effect of neutralizing antibodies against GGT on joint destruction using a collagen-induced arthritis (CIA) mouse model. GGT expression in the synovium of RA patients and CIA mice was determined by immunohistochemistry and RT-PCR. Monoclonal antibodies were generated against recombinant human GGT (GGT-mAbs) using BALB/c mice. Antibody treatment was performed by intraperitoneal injections of GGT-mAbs into CIA mice. Effects of antibody treatment on arthritis and bone erosion were evaluated by incidence score, arthritis score, and histopathological observations. The role of GGT in osteoclast development was examined by using the established osteoclastogenic culture system. GGT expression was significantly upregulated in inflamed synovium. Immunohistochemistry revealed that GGT was present in lymphocytes, plasma cells, and macrophages, as well as capillaries. Injection of GGT-mAbs significantly decreased the number of osteoclasts and attenuated the severity of joint destruction in CIA mice. In vitro examination showed that GGT enhanced RANKL-dependent osteoclast formation. GGT stimulated the expression of RANKL in osteoblasts and its receptor RANK in osteoclast precursors, respectively. This study indicates that inflamed synovial tissue-derived GGT acts as a risk factor for joint destruction and that the antibody-mediated inhibition of GGT significantly decreases osteoclast number and bone erosion in CIA mice. GGT antagonists might be novel therapeutic agents for attenuating joint destruction in RA patients.

Kinetic Characterization and Identification of the Acylation and Glycosylation Sites of Recombinant Human γ-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathione homeostasis control. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase, and confirmed four of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization.

Gamma-glutamyl transpeptidase is up-regulated on memory T lymphocytes.

The ectoenzyme gamma-glutamyl transpeptidase (GGT) hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH levels and modifies the activity of GSH-containing adducts. Previous data suggested that this enzyme was present on mitogen-activated T lymphocytes. However, the level of GGT protein expression on human mononuclear cell subsets has not been determined. A novel mAb to human GGT, 3A8, was developed. 3A8 was used to show that the expression of GGT is, in fact, highest on resting T cells that express markers of the memory phenotype, specifically CD45RO and decreased expression of CD45RB. The peripheral blood of patients with rheumatoid arthritis was found to have expanded numbers of T cells expressing levels of GGT up to 10-fold higher than controls. In addition, the CD4(+) T cell subset with the capacity to migrate across a human endothelial cell monolayer expresses high GGT levels. GGT expression was up-regulated on peripheral blood T cells following activation in vitro by either superantigen, phorbol ester, or IL-15, a stimulatory cytokine synthesized in rheumatoid synovium. Resting peripheral blood T cells that express GGT have higher levels of intracellular thiols than those that do not. These observations suggest that GGT may play an important role in the regulation of lymphocytes that are at a particular developmental stage.

Altered expression of gamma-glutamyl transpeptidase in human tumors

Elevated levels of gamma-glutamyl transpeptidase (GGT) activity and intracellular glutathione in tumor cells have been correlated with resistance to several classes of chemotherapeutic drugs. In this study, the first comprehensive analysis of GGT expression in human malignant neoplasms, 451 tumors were immunostained with an antibody directed against a c-terminus peptide of the human GGT protein. Analysis of the immunostaining revealed that GGT was expressed in 22 of 44 lung carcinomas and 16 of 22 ovarian surface epithelial carcinomas, although normal pulmonary and ovarian epithelium are GGT-negative. The tumor samples were obtained from patients before the start of therapy; therefore, GGT was not induced by radiation or chemotherapy. There was no GGT expression in mesotheliomas, Hodgkin's disease, non-Hodgkin's lymphomas, melanomas, basal cell carcinomas, and most soft tissue sarcomas, all of which are derived from GGT-negative cells. Carcinomas arising from some GGT-positive epithelium retained their GGT-positive phenotype. These included renal cell carcinomas, hepatocellular and cholangiocarcinomas, and carcinomas of the prostate and thyroid whereas both pancreatic adenocarcinomas and infiltrating carcinomas of the breast showed a wide range of GGT expression. Further studies are underway to determine whether expression of GGT plays a role in the inherent resistance of some tumors to alkylating agents and other classes of chemotherapeutic drugs.

Expression of an active glycosylated human gamma-glutamyl transpeptidase mutant that lacks a membrane anchor domain.

A mutant of human gamma-glutamyl transpeptidase (EC 2.3.2.2, a membrane-bound enzyme of importance in glutathione metabolism) that differs from the wild type by deletion of the putative signal peptide/anchor domain (amino acid residues 1-27) was expressed in insect cells using a baculovirus system. In contrast to the wild-type enzyme--which, as expected, was mainly cell-associated--the mutant enzyme was secreted into the medium. The mutant and wild-type enzymes were purified and found to exhibit virtually identical catalytic properties. The mutant enzyme was glycosylated and processed into two subunits, as found for the wild-type enzyme. Brefeldin A inhibited secretion of the mutant enzyme and led to its accumulation in cells. The findings indicate that gamma-glutamyl transpeptidase can be targeted to the endoplasmic reticulum in a manner that does not involve function of an amino-terminal "signal/anchor" domain and that this domain is involved primarily in a membrane anchoring function. Another region of the enzyme may function as a signal domain.

Extracellular glutathione is a source of cysteine for cells that express .gamma.-glutamyl transpeptidase

We show that gamma-glutamyl transpeptidase (GGT) is a glutathionase that enables cells to use extracellular glutathione as a source of cysteine. We transfected NIH/3T3 mouse fibroblasts with a plasmid containing cDNA for human GGT, and obtained stably transformed cell lines that expressed GGT in its proper orientation on the outer surface of the cell. NIH/3T3 fibroblasts require cysteine for growth and are unable to use extracellular glutathione as a source of cysteine. We demonstrate GGT-positive fibroblasts are able to grow in cysteine-free medium supplemented with glutathione. Cysteine derived from the cleavage of extracellular glutathione can be used to maintain intracellular levels of glutathione. GGT-positive NIH/3T3 cells were able to replenish intracellular glutathione when incubated in cysteine-free medium containing glutathione. GGT-negative cells could not. Therefore, GGT is a glutathionase that provides the cell with access to a secondary source of cysteine.

Significance of Arg-107 and Glu-108 in the catalytic mechanism of human gamma-glutamyl transpeptidase. Identification by site-directed mutagenesis.

The catalytic properties of wild-type and mutant enzymes produced by expression of cDNA (for gamma-glutamyl transpeptidase) in COS-1 cells were analyzed. Substitutions of Lys-100 to Asn, Glu-102 to Gln, and ARg-112, -139, -147, and -150 to Gln resulted in expression of fully active enzymes. Replacement of Arg-107 with Gln or His led to complete loss of enzyme activity, whereas substitution to Lys resulted in partial loss of activity, indicating that the positive charge at position 107 is essential for enzyme activity. In the Arg-107-to-Lys mutant, the apparent Km for the donor L-gamma-glutamyl-3-carboxy-4-nitroanilide was increased 8.4-fold compared to that of the wild-type enzyme. Although 32% of the wild-type hydrolysis activity was retained in this mutant, the activity could not be enhanced by addition of an acceptor substrate, glycylglycine, indicating that the transpeptidation reaction was specifically prevented. Substitution of Glu-108 to Gln increased the apparent Km for glycylglycine 8.5-fold while increasing the apparent Km for the donor only 1.6-fold. The apparent Vmax was decreased to 4-5% of wild-type for both substrates. These data suggest that Arg-107 plays a significant role in substrate binding rather than catalysis, and that Glu-108 is an important, although not essential, participant in both acceptor binding and catalysis.

Synthesis of human gamma-glutamyl transpeptidase (GGT) during the fetal development of liver

We have determined expression of human GGT gene encoding γ-glutamyl transpeptidase (GGT) during fetal development of liver using the Northern-blot analysis with a cloned human GGT cDNA and immunohistochemistry with a monoclonal antibody. GGT mRNA could be detected as early as the 12th week of gestation. It then increased gradually to a peak of approx. threefold the amount at week 12, at week 40, just before birth. The size of the mRNA in the fetal liver was 2.7 kb and mRNA of the same size was detected both in the human fetal kidney and human hepatocellular carcinoma as well as normal adult liver. Immunohistochemical analyses show that GGT increased as the fetal liver developed in parallel with the increase in mRNA. Histochemically, GGT was shown to be located in the wall of bile canaliculi when synthesis was low in early development, but to be distributed, in addition, all over the cell membrane of the fetal hepatocytes when synthesis was high at the later stage of development.

An alternatively processed mRNA specific for gamma-glutamyl transpeptidase in human tissues.

Human gamma-glutamyl transpeptidase (GGT)1 is composed of two subunits derived from a single precursor (Nash, B., and Tate, S.S. (1984) J. Biol. Chem. 259, 678-685; Finidori, J., Laperche, Y., Tsapis, R., Barouki, R., Guellaën, G., and Hanoune, J. (1984) J. Biol. Chem. 259, 4687-4690) consisting of 569 amino acids (Laperche, Y., Bulle, F., Aissani, T., Chobert, M.N., Aggerbeck, M., Hanoune, J., and Guellaën, G. (1986) Proc Natl. Acad. Sci. U.S.A. 83, 937-941). In the present study we report the cloning of an altered form of this precursor from human liver. We have isolated two clones, one 2,632 base pairs (bp) long from a fetal liver cDNA library and one 926 bp long from an adult liver cDNA library, each containing a 22-bp insertion that introduces a premature stop codon and shortens the open reading frame to 1,098 bp when compared with known human cDNA sequences specific for GGT. Sequence analysis of a human genomic GGT clone shows that this insertion of 22 bp is generated by a splicing event involving an alternative 3'-acceptor site. By polymerase chain reaction experiments we demonstrate that the alternatively spliced mRNA is present in polysomes from the microsomal fraction of a human hepatoma cell line (Hep G2) and thus could encode an altered GGT molecule of 39,300 Da (366 amino acids) encompassing most of the heavy subunit which is normally 41,500 Da (380 amino acids). The altered mRNA is detected in various human tissues including liver, kidney, brain, intestine, stomach, placenta, and mammary gland. This report is the first demonstration of an alternative primary sequence in the mRNA coding for GGT, a finding that could be related to the presence of some inactive forms of GGT detected in human tissues.

Cloning and nucleotide sequence of human gamma-glutamyl transpeptidase.

We have identified the gene for human gamma-glutamyl transpeptidase [GGT; glutamine:D-glutamyl-peptide 5-glutamyltransferase (also called gamma-glutamyltransferase), EC 2.3.2.2] in a BCR gene-related region located in band q11----qter of chromosome 22. Two cDNAs complementary to the GGT mRNA have been isolated from a human placental library constructed in phage lambda gt11. The largest cDNA has a size of 2535 base pairs (bp) and an open reading frame of 1707 nucleotides encoding 569 amino acids. By using a probe corresponding to this cDNA, a mRNA of approximately 2.4 kilobases was detected by RNA blot-hybridization analysis in mouse kidney RNA. The GGT precursor encoded by the coding sequence would have an estimated Mr of 61,400. We compared our nucleotide and deduced amino acid sequences with the published results of rat kidney cDNAs. The human and rat amino acid sequences are similar; however, a considerable discrepancy in nucleotide sequence was found within a 180-bp fragment of the heavy chain, resulting in a completely different amino acid sequence for this region. In addition, the 5' untranslated sequence of the human cDNA (669 bp) is substantially larger than that determined in the rat cDNA (227 bp). Our results may be valuable for further studies on the protein structure of human GGT as well as studies on the regulation of the enzyme.

The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat γ-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa ( M r 38336) and of 189 aa ( M r 20000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.

Gamma-Glutamyl transpeptidase: a single copy gene in the rat and a multigene family in the human genome.

gamma-Glutamyl transpeptidase (GGT) genomic sequences were isolated from rat and human libraries using a rat GGT cDNA as a cross-species hybridization probe. Characterization of the human GGT clones by restriction mapping clearly establishes that at least four different GGT genes or pseudogenes are present in the human genome. All the rat genomic clones cover a 12.5-kilobase sequence and exhibit a unique restriction pattern. A precise quantitation of the rat GGT gene copy number by Southern blot analysis demonstrates that this sequence is present as a single copy/rat haploid genome. Therefore, the GGT gene organization is different between rat and human species; this raises the possibility of different regulatory mechanisms in the two species.