Mu Class Glutathione S-transferase Research Articles

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (kcat/Km) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50–180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix α4 region of rGSTM4-4 account for this pKa difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6–10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Molecular cloning and characterization of a mu-class glutathione S-transferase from Clonorchis sinensis

In biliary passages, Clonorchis sinensis causes epithelial hyperplasia and is assumed to promote carcinogenesis. Glutathione S-transferase (GST) is an antioxidant enzyme involved in phase II defense in trematodes. A clone (pcsGSTM1) encoding a GST was identified by screening a C. sinensis cDNA library with a PCR-synthesized cDNA probe. The predicted amino acid sequence encoded by pcsGSTM1 cDNA had a high degree of sequence identity and folding topology similar to the mu-class GSTs. The estimated molecular mass of the protein, 26 kDa, was consistent with an expression by pcsGSTM1 cDNA. The bacterially expressed recombinant csGSTM1 protein possessed an enzymatic GST activity and conjugated GSH to reactive carbonyls of lipid peroxidation. The recombinant csGSTM1 protein did not share antigenic epitope(s) with GSTs of Fasciola hepatica, Paragonimus westermani and Schistosoma japonicum. The csGSTM1 was identified to a mu-class GST in C. sinensis.

Pyruvate-induced long-term maintenance of glutathione s-transferase in rat hepatocyte cultures.

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.

GSTM1 null polymorphism and susceptibility to endometriosis and ovarian cancer.

It is likely that heritable genetic factors contribute to the development of endometriosis, which is a putative precursor of the endometrioid and clear cell histological subtypes of ovarian cancer. The phase II glutathione S-transferases (GSTs) are a family of enzymes responsible for metabolism of a broad range of xenobiotics and carcinogens. Allelic variants of GSTs that have impaired detoxification function may increase the rate of genetic damage and thereby increase the susceptibility to cancer. The null genetic polymorphism in the gene encoding the GST class mu (GSTM1) enzyme has been reported to be significantly elevated in endometriosis patients and may represent an endometriosis susceptibility allele. In this study the frequency of the GSTM1 null genotype was investigated in 84 cases of endometriosis, 293 cases of ovarian cancer and 219 controls. All cases and controls were derived from women resident in the south east of England. The frequency of the GSTM1 null allele was not over-represented in the endometriosis patients (47.6%) compared with the controls (48.9%) (P = 0.898). In the ovarian cancer group the GSTM1 null genotype was significantly elevated compared with controls (59.0 versus 48.9%, P = 0.025). When stratified according to histological subtype a significantly increased GSTM1 null genotype was only observed for the endometrioid (65.4%, P = 0.013) and the combined endometrioid/clear cell ovarian cancers (67.0%, P = 0.004). We conclude that the GSTM1 null allele is not an endometriosis susceptibility allele, however, it may predispose endometriotic lesions to malignant transformation to endometrioid and clear cell ovarian cancer.

Equilibrium folding of dimeric class mu glutathione transferases involves a stable monomeric intermediate.

The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.

Tyr115, Gln165 and Trp209 contribute to the 1,2-epoxy-3-(p-nitrophenoxy)propane-conjugating activity of glutathione S-transferase cGSTM1-1

We investigated the epoxidase activity of a class mu glutathione S-transferase (cGSTM1-1), using 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as substrate. Trp209 on the C-terminal tail, Arg107 on the α4 helix, Asp161 and Gln165 on the α6 helix of cGSTM1-1 were selected for mutagenesis and kinetic studies. A hydrophobic side-chain at residue 209 is needed for the epoxidase activity of cGSTM1-1. Replacing Trp209 with histidine, isoleucine or proline resulted in a fivefold to 28-fold decrease in the kcatapp of the enzyme, while a modest 25 % decrease in the kcatapp was observed for the W209F mutant. The rGSTM1-1 enzyme has serine at the correponding position. The kcatapp of the S209W mutant is 2.5-fold higher than that of the wild-type rGSTM1-1. A charged residue is needed at position 107 of cGSTM1-1. The KmGSHapp of the R107L mutant is 38-fold lower than that of the wild-type enzyme. On the contrary, the R107E mutant has a KmGSHapp and a kcatapp that are 11-fold and 35 % lower than those of the wild-type cGSTM1-1. The substitutions of Gln165 with Glu or Leu have minimal effect on the affinity of the mutants towards GSH or EPNP. However, a discernible reduction in kcatapp was observed. Asp161 is involved in maintaining the structural integrity of the enzyme. The KmGSHapp of the D161L mutant is 616-fold higher than that of the wild-type enzyme. In the hydrogen/deuterium exchange experiments, this mutant has the highest level of deuteration among all the proteins tested.We also elucidated the structure of cGSTM1-1 co-crystallized with the glutathionyl-conjugated 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) at 2.8 Å resolution. The product found in the active site was 1-hydroxy-2-(S-glutathionyl)-3-(p-nitrophenoxy)propane, instead of the conventional 2-hydroxy isomer. The EPNP moiety orients towards Arg107 and Gln165 in dimer AB, and protrudes into a hydrophobic region formed by the loop connecting β1 and α1 and part of the C-terminal tail in dimer CD. The phenoxyl ring forms strong ring stacking with the Trp209 side-chain in dimer CD. We hypothesize that these two conformations represent the EPNP moiety close to the initial and final stages of the reaction mechanism, respectively.

Ligand-induced changes in the structure and dynamics of a human class Mu glutathione S-transferase.

Glutathione transferases are detoxification enzymes that catalyze the addition of glutathione (GSH) to a wide variety of hydrophobic compounds. Although this group of enzymes has been extensively characterized by crystallographic studies, little is known about their dynamic properties. This study investigates the role of protein dynamics in the mechanism of a human class mu enzyme (GSTM2-2) by characterizing the motional properties of the unliganded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these ligands are 10-20-fold lower than the diffusion limit, suggesting dynamic conformational heterogeneity of the active site. The off-rate of GSDNB is similar to the turnover number for its enzymatic formation, suggesting that product release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substrate. The dynamic properties of GSTM2-2 were investigated over a wide range of time scales using (15)N nuclear spin relaxation, residual dipolar couplings, and amide hydrogen-deuterium exchange rates. These data show that the majority of the protein backbone is rigid on the nanosecond to picosecond time scale for all forms of the enzyme. The presence of motion on the millisecond to microsecond time scale was detected for a small number of residues within the active site. These motions are likely to play a role in facilitating substrate binding and product release. The residual dipolar couplings also show that the conformation of the active site region is more open in solution than in the crystalline environment, further enhancing ligand accessibility to the active site. Amide hydrogen-deuterium exchange rates indicate a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate of a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site region. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 complex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic residues in the active site, including Met108. Replacement of Met108 with Ala increases the turnover number of the enzyme by a factor of 1.7.

The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin.

An overview is provided of the cancer chemoprevention actions of phenolic antioxidants and 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin). These agents principally appear to exert their beneficial effects through induction of phase II drug-metabolizing enzymes such as glutathione S-transferase (GST). The requirement for oxidative metabolism of the synthetic antioxidants to carbonyl-containing compounds, including quinones, in order that they can induce gene expression is discussed. Previous work has shown that the basic leucine zipper transcription factor Nrf2 is involved in induction of GST by the phenolic antioxidant butylated hydroxyanisole (BHA). Evidence is provided from a mouse possessing a targeted disruption of the Nrf2 gene that, in murine liver, the transcription factor regulates basal expression of several class Alpha and class Mu GST subunits, but not class Pi GST. In the Nrf2 knock-out mouse, hepatic induction of class Alpha and class Mu GST by BHA and the synthetic antioxidant ethoxyquin is similarly impaired, suggesting that these agents affect gene activation by a related mechanism. Significantly, residual induction of GST by antioxidants is apparent in the Nrf2 mutant mouse, indicating the existence of an alternative mechanism of gene activation.

The Enhanced Affinity for Thiolate Anion and Activation of Enzyme-bound Glutathione Is Governed by an Arginine Residue of Human Mu Class Glutathione S-Transferases

A series of chimeric human Mu class glutathione S-transferases were designed to determine mechanisms by which they activate enzyme-bound glutathione (GSH) for reaction with electrophilic substrates. In view of evidence that the His(107) residue of hGSTM1a-1a is important for catalysis (Patskovsky, Y. V., Patskovska, L. N., and Listowsky, I. (1999) Biochemistry 38, 1193-1202), the cognate Arg(107) residue of the hGSTM2 subunit was replaced (R107N or R107H) and arginine residues were also incorporated into position 107 of hGSTM1 (H107R) and hGSTM4 (S107R) subunits. The major distinguishing kinetic properties invariably associated with enzymes containing an Arg(107) residue include an inverse dependence of k(cat) on viscosity and lower K(m(GSH values relative to enzymes with other residues at that position. Moreover, affinities for GSH thiolate anion binding are greater for enzymes containing Arg(107))), with K(d) values of 20-50 microM that are consistent with the K(m(GSH values (10-25 microM) obtained by steady-state kinetic analyses. Both thermodynamic and kinetic and data indicate that the Arg(107))) residue is specifically involved in enhancing the binding affinity of GSH thiolate anion relative to that of the protonated form. These enzymes therefore, can be more effective at lower GSH concentrations. Combined mutations indicate that both Arg(107) and Tyr(6) residues are required for thiolate anion formation and stabilization. The three-dimensional structure of ligand-free hGSTM2-2 determined by x-ray crystallography suggests that Arg(107) maintains an electrostatic interaction with the Asp(161) side chain (3 A apart), but is distant from the GSH-binding site. However, an alternative energetically favorable model places the guanidino group 4 A from the sulfur atom of bound GSH. It is suggested therefore, that in solution, motion of the positively charged arginine into the catalytic pocket could provide a counter ion to promote ionization of the sulfhydryl group of GSH, thereby accounting for the observed greater affinity of enzymes containing Arg(107) for binding of thiolate anion.

Identification of mu-class glutathione transferases M2-2 and M3-3 as cytosolic prostaglandin E synthases in the human brain.

Cytosolic prostaglandin (PG) E synthase was purified from human brain cortex. The N-terminal amino acid sequence, PMTLGYXNIRGL, was identical to that of the human mu-class glutathione transferase (GST) M2 subunit. Complementary DNAs for human GSTM2, GSTM3, and GSTM4 subunits were cloned, and recombinant proteins were expressed as homodimers in Escherichia coli. The recombinant GSTM2-2 and 3-3 catalyzed the conversion of PGH2 to PGE2 at the rates of 282 and 923 nmol/min/mg of protein, respectively, at the optimal pH of 8, whereas GSTM4-4 was inactive; although all three enzymes showed GST activity. The PGE synthase activity depended on thiols, such as glutathione, dithiothreitol, 2-mercaptoethanol, or L-cysteine. Michaelis-Menten constants and turnover numbers for PGH2 were 141 microM and 10.8 min(-1) for GSTM2-2 and 1.5 mM and 130 min(-1) for GSTM3-3, respectively. GSTM2-2 and 3-3 may play crucial roles in temperature regulation, nociception, and sleep-wake regulation by producing PGE2 in the brain.

The effect of thioacetamide on the activity and expression of cytosolic rat liver glutathione-S-transferase.

The effect of thioacetamide (TA), an hepatotoxic and hepatocarcinogenic compound, on the expression and activity of the cytosolic enzyme glutathione-S-transferase (GST) was studied in rat liver. Four h following the administration of 14C-labeled thioacetamide (50 mg/Kg), several subunits of GST were found to be radioactively labeled. A single sublethal dose of TA (250 mg/Kg) decreased by three-fold the expression of class alpha GST at 24-48 h of treatment, but did not significantly affect the transcription of class mu GST. The activity of the enzyme toward 1-chloro-2,4-dinitrobenzene was mildly inhibited (66% of the control) by a 24 h TA treatment and gradually increased thereafter. It is proposed that the covalent binding of TA or its derivative to the GST subunits does not affect the activity of the enzyme. Nevertheless, GST activity inhibition is due to the deleterious effect of TA on GST transcription.

Glutathione S-transferase M1 null genotype: lack of association with tumour characteristics and survival in advanced breast cancer.

Glutathione S-transferase (GST)M1, a member of the mu class GST gene family, has been shown to be polymorphic because of a partial gene deletion. This results in a failure to express the GSTM1 gene in 50-60% of individuals. Several studies have demonstrated a possible link with the GSTM1-null genotype and susceptibility to cancer. Furthermore, a GSTM1 isoenzyme has been positively associated with protective effect against mutagenic drugs, such as alkylating agents and anthracyclines. To determine whether GSTM1 polymorphisms are associated with tumour characteristics and survival in advanced breast cancer patients, and whether it may constitute a prognostic factor. We genotyped 92 patients receiving primary chemotherapy, which included cyclophosphamide, doxorubicine and 5-fluorouracil. The relationships between allelism at GSTM1 and clinicopathological parameters including age, menopausal status, tumour size, grade hormone receptors, involved nodes and p53 gene mutations were analysed. Of the patients with GSTM1-positive genotype, tissue samples obtained before and after treatment were available from 28 cases, allowing RNA extraction and GSTM1 expression by reverse transcription polymerase chain reaction. Relationships with clinical response to chemotherapy, and disease-free and overall survival were also evaluated. The data obtained was analysed using logistic regression to estimate the odds ratio and 95% confidence interval. Of 92 patients, 57.6% (n = 53) were classified as heritably GSTM1-deficient, and 42.4% (n = 39) were of the GSTM1-positive genotype. There were no statistically significant relationships between GSTM1-null genotype and the clinicopathological parameters analysed. No relationship was observed between GSTM1 RNA expression and objective clinical response to chemotherapy. Objective clinical response to chemotherapy was related only to clinical tumour size (P = 0.0177) and to the absence of intraductal carcinoma (P = 0.0013). GSTM1-null genotype had no effect on disease-free or overall survival. The absence of hormone receptors (P = 0.002), the presence of a mutated p53 gene (P = 0.0098) and lack of response to primary chemotherapy (P = 0.0086) were the only factors associated with reduced disease-free or overall survival. GSTM1-null genotype alone had no effect on tumour characteristics and outcome of patients with advanced breast cancers. The lack of correlation of GSTM1 genotype with clinical tumour features, clinical response to chemotherapy and survival exclude a role for GSTM1 polymorphism as a prognostic factor in advanced breast cancer.

Haemopoietic Lineage Sensitivity and Individual Susceptibility to PCB126: Relation to Glutathione S-transferase μ

The effect of PCB126 on the in vitro growth potential of human myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells derived from human cord blood haemopoietic cells was examined. The possible link between individual variability in susceptibility and the presence of the xenobiotic metabolizing enzyme glutathione S-transferase class mu (GST μ) was studied. After density centrifugation separation, mononuclear cord blood cells were cultured in the presence of PCB126 (10 −6 m and 10 −8 m). Several cord blood samples were simultaneously tested for: (1) colony formation and (2) the presence of the GST μ gene measured by PCR. An interindividual variability in the response to PCB126 was present. At 10 −8 m and 10 −6 m PCB126, respectively 8 (40%) and 10 (50%) out of 20 cord blood samples showed a significant dose-dependent decrease in CFU-GM numbers. Erythroid progenitors were less affected by PCB126. At 10 −8 m and 10 −6 m PCB126, respectively only two (12%) and three (18%) out of 17 cord blood samples showed a significant decrease in BFU-E numbers. The presence or absence of the GST μ gene was determined using PCR. The GST μ gene was present in 52% (14 out of 27 samples tested) of the cord blood samples. Damage to the myeloid and erythroid haemopoietic progenitor cells at either PCB126 concentrations was not correlated with the presence of the GST μ gene.

Characterization and molecular cloning of a glutathione S-transferase gene from the tick, Boophilus microplus (Acari: Ixodidae)

A glutathione S-transferase (GST) was purified from the larval cattle tick, Boophilus microplus (Acari: Ixodidae), by glutathione-affinity chromatography. The purified enzyme appeared as a single band on SDS-PAGE and has a molecular mass of 25.8 kDa determined by mass spectrometry. The N-terminus of the purified enzyme was sequenced. The full-length cDNA of the enzyme was isolated by RT-PCR using degenerate oligonucleotides derived from the N-terminal amino acid sequence. The cDNA contains an open reading frame encoding a 223-amino-acid protein with the N-terminus identical to the purified GST. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian mu class GST.

Characterization of Glutathione S-Transferase of Taenia solium

A Taenia solium glutathione-S-transferase fraction (SGSTF) was isolated from a metacestode crude extract by affinity chromatography on reduced glutathione (GSH)-sepharose. The purified fraction displayed a specific glutathione S-transferase (GST) activity of 2.8 micromol/min/mg and glutathione peroxidase selenium-independent activity of 0.22 micromol/min/mg. Enzymatic characterization of the fraction suggested that the activity was closer to the mammalian mu-class GSTs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and enzyme activity analysis showed that the fraction was composed of a major band of Mr = 26 kd and that the active enzyme was dimeric. Immunohistochemical studies using specific antibodies against the major 26-kd band of the SGSTF indicated that GST protein was present in the tegument, parenchyma, protonephridial, and tegumentary cytons of the T. solium metacestode. Antibodies generated against the SGSTF tested in western blot showed cross-reactivity against GSTs purified from Taenia saginata, T. taeniaeformis, and T. crassiceps, but did not react with GSTs from Schistosoma mansoni, or mice, rabbit, and pig liver tissue. Furthermore, immunization of mice with SGSTF reduced the metacestode burden up to 74.2%. Our findings argue in favor of GST having an important role in the survival of T. solium in its hosts.

Evolution of differential substrate specificities in Mu class glutathione transferases probed by DNA shuffling

A library of variant enzymes was created by combined shuffling of the DNA encoding the human Mu class glutathione transferases GST M1-1 and GST M2-2. The parental GSTs are 84 % sequence identical at the protein level, but their specific activities with the substrates aminochrome and 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG) differ by more than 100-fold. Aminochrome is of particular interest as an oxidation product of dopamine and of possible significance in the etiology of Parkinson’s disease, and cyanoDMNG is a model for genotoxic and potentially carcinogenic nitroso compounds. GST M2-2 has at least two orders of magnitude higher catalytic activity with both of the substrates than any of the other known GSTs, including GST M1-1. The DNA library of variant Mu class GST sequences contained “mosaic” structures composed of alternating segments of both parental sequences. All clones contained the 5′-end of a GST M1-1 clone optimized for high-level expression in Escherichia coli. The remainder of the sequences derived from segments of GST M2-2 and GST M1-1 DNA. All of the clones analyzed contained between two and seven distinct DNA segments. In addition, each clone contained an average of approximately one point mutation. None of the library clones analyzed was identical with either of the two parental structures. Variant GST sequences were expressed in E. coli, and their enzymatic activities with aminochrome, cyanoDMNG, and 1-chloro-2,4-dinitrobenzene (CDNB) were determined in bacterial lysates. Such screening of more than 70 clones demonstrated a continuous range of activities covering at least two orders of magnitude for each of the substrates. For a given clone, the activities with aminochrome and cyanoDMNG, in spite of their different chemistries, were clearly correlated, whereas no strong correlation was found with CDNB. This functional correlation suggests a common structural basis for the enzymatic mechanisms for conjugation of aminochrome and denitrosation of cyanoDMNG. From an evolutionary perspective, the results show that recombination of segments from homologous proteins gives rise to a large proportion of functionally competent proteins with a range of activities. The data support the proposal that natural evolution of protein functions may involve recombination of DNA segments followed by selection for advantageous functional properties of the resulting proteins. Clearly, the same approach can be utilized in the engineering of proteins displaying novel functions by in vitro evolution.

Different expression of glutathione S-transferase alpha, mu and pi in childhood acute lymphoblastic and myeloid leukaemia.

Expression of three major classes of glutathione S-transferases (GSTs), i.e. alpha, mu and pi class, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) were studied in childhood acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and normal peripheral blood lymphocytes by flow cytometry. In vitro cytotoxicity of 4-hydroxy-ifosfamide (IFOS), daunorubicin (DNR) and prednisolone (PRED) was assessed by the MTT assay. Expression of alpha, mu and pi class GST did not significantly differ between leukaemic cells from 100 initial and 14 unrelated relapse ALL patients (GSTalpha P=026; GSTmu P=O009; GSTpi P=0.13). The expression of GSTalpha (1.4-fold, P=0.0004), GSTpi (13-fold, P = 0001) and to a lesser extent also GSTmu (1.1-fold, P=0.03) was higher in ALL compared with normal peripheral blood lymphocytes. Expression of GSTmu and GST7pi was significantly higher in 18 AML compared with 100 ALL patients at initial diagnosis (respectively 1.3-fold, P=0.0005 and 2-fold, P<0.0001). In contrast, GSTalpha was median 2-fold lower expressed in the AML samples (P< 0.0001). Expression levels of alpha, mu and pi class GSTs were not related to the degree of resistance to IFOS, DNR and PRED nor to immunophenotype, white blood cell count or age at presentation of childhood ALL. One exception was a remarkably low expression of GSTalpha in IFOS-sensitive samples compared with a heterogenous expression in IFOS-resistant samples (P= 0.02). Expression of GSTpi, but not of GSTalpha or GSTmu, weakly correlated with the expression of MRP (Rs 0.36, P = 0.002, n = 74) but not with P-gp. However, a high expression of both GSTpi and MRP was not associated with in vitro resistance to IFOS, DNR or PRED. The present data suggest that expression of GSTs is not linked to the degree of resistance to IFOS, DNR and PRED or clinical risk factors in childhood ALL. Whether the high expression of GSTmu and GSTpi in AML cells contributes to the relative resistance to IFOS, DNR and PRED compared with ALL samples (P < or = 0.0001) warrants further study.

Solution structure of the carboxyl terminus of a human class mu glutathione S-transferase: NMR assignment strategies in large proteins

Strategies to obtain the NMR assignments for the HN, N, CO, Cα and Cβ resonance frequencies for the human class mu glutathione-S-transferase GSTM2-2 are reported. These assignments were obtained with deuterated protein using a combination of scalar and dipolar connectivities and various specific labeling schemes. The large size of this protein (55 kDa, homodimer) necessitated the development of a novel pulse sequence and specific labeling strategies. These aided in the identification of residue type and were essential components in determining sequence specific assignments. These assignments were utilized in this study to characterize the structure and dynamics of the carboxy-terminal residues in the unliganded protein. Previous crystallographic studies of this enzyme in complex with glutathione suggested that this region may be disordered, and that this disorder may be essential for catalysis. Furthermore, in the related class alpha protein extensive changes in conformation of the C terminus are observed upon ligand binding. On the basis of the results presented here, the time-averaged conformation of the carboxyl terminus of unliganded GSTM2-2 is similar to that seen in the crystal structure. NOE patterns and 1H-15N heteronuclear nuclear Overhauser enhancements suggest that this region of the enzyme does not undergo motion on a rapid time scale.

Exogenous 17beta-estradiol blocks alpha and mu but not pi class glutathione S-transferase immunoreactivity in epithelium of Syrian hamster vas deferens.

Members of the glutathione S-transferase (GST) family of detoxification enzymes play a role in chemotherapy resistance in certain cancers but have not been directly implicated as agents whose absence may predispose tissues to hormonally induced tumorigenesis. Here we report the development of a polyclonal antiserum to a hamster mu class GST, and immunohistochemical analysis of alpha, mu, and pi class GSTs to study the effects of hormone treatment on their expression in reproductive tract tissues of male golden Syrian hamsters. These animals develop leiomyosarcomas in the vas deferens after treatment with testosterone propionate (TP) and 17beta-estradiol (E2). High levels of all three GST classes were detected throughout the reproductive tract epithelium of control animals. In 100% of the experimental animals, 4 weeks of treatment either with E2 alone, or with E2 plus TP promoted a complete loss of immunostaining for alpha and mu class GSTs, but not for pi class GSTs, only in the epithelial lining of the vas deferens. In contrast, treatment with TP alone resulted in moderate hyperplasia of smooth muscle in the proximal vas deferens, with no cellular atypia and no changes in immunoreactivity of any of the GST classes. The consistent and site-specific nature of these results strongly suggests a functional role for GSTs in hormonally induced carcinogenic process. (J Histochem Cytochem 47:91-98, 1999)