Cloning,characterization and tissue distribution of a pi-class glutathione S-transferase from clam (Venerupis philippinarum): Response to benzo[α]pyrene exposure

Glutathione S-transferases (GSTs) are phase II enzymes involved in major detoxification reactions of xenobiotics in many organisms. In this study, a full-length cDNA of GST-pi was cloned from the gill of Venerupis philippinarum by rapid amplification of cDNA ends (RACE) method for the first time. The full-length cDNA of V. philippinarum GST-pi (denoted as VpGSTp) was 1142 bp, with a 5′ untranslated region (UTR) of 87 bp, a 3′ UTR of 438 bp, and an open reading frame (ORF) of 618 bp encoding a protein of 205 amino acid residues with an estimated molecular mass of 23.9 kDa and an predicted isoelectric point (pI) of 7.9. The comparison of the deduced amino acid sequences with GSTs from other species showed that the enzyme belongs to the pi-class, and the amino acids defining the binding sites of glutathione (G-site) and for xenobiotic substrates (H-site) were highly conserved. Tissue distribution analysis of the VpGSTp mRNA revealed that the GST-pi expression level was observed higher in gill, adductor muscle, mantle and foot while lower in digestive gland. Using quantitative real-time PCR, the dose- and time-related effects of benzo[α]pyrene (B[α]P) on VpGSTp mRNA expression were investigated in gills and digestive gland. The results showed that a time-dependant increase in the expression of VpGSTp was induced by B[α]P and appeared a good linear relationship with B[α]P concentrations. All these results suggested that GST-pi in bivalve had an antioxidant role and VpGSTp expression may be a useful biomarker candidate for monitoring environmental contaminants such as PAHs.     

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) glutathione S-transferase

A cDNA of glutathione S-transferase (GST) was isolated from a cDNA library of salivary glands of Boophilus microplus. The recombinant protein was purified by glutathione affinity chromatography and assayed upon the chromogenic substrate CDNB. The 864 bp cloned fragment was sequenced and showed an open reading frame coding for a protein of 220 amino acids. Expression of the GST gene was tested by RT-PCR in tick tissues and larvae mRNA. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to the mammalian class mu GSTs.     

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.     

Hepatic and branchial glutathione S-transferases of two fish species: Substrate specificity and biotransformation of microcystin-LR

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min^? 1 mL^? 1 hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.     

Investigation of the role of conserved residues Ser13, Asn48 and Pro49 in the catalytic mechanism of the tau class glutathione transferase from Glycine max

Plant glutathione transferases (GSTs) play a key role in the metabolism of various xenobiotics. In this report, the catalytic mechanism of the tau class GSTU4-4 isoenzyme from Glycine max (GmGSTU4-4) was investigated by site-directed mutagenesis and steady-state kinetic analysis. The catalytic properties of the wild-type enzyme and three mutants of strictly conserved residues (Ser13Ala, Asn48Ala and Pro49Ala) were studied in 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction. The results showed that the mutations significantly affect substrate binding and specificity. The effect of Ser13Ala mutation on the catalytic efficiency of the enzyme could be explained by assuming the direct involvement of Ser13 to the reaction chemistry and the correct positioning of GSH and CDNB in the ternary catalytic complex. Asn48 and Pro49 were found to have a direct role on the structural integrity of the GSH-binding site (G-site). Moreover, mutation of Asn48 and Pro49 residues may bring about secondary effects altering the thermal stability and the catalytic activity (k_cat) of the enzyme without affecting the nature of the rate-limiting step of the catalytic reaction.     

Heterologous expression and functional characterization of avian mu-class glutathione S-transferases

Hepatic glutathione S-transferases (GSTs: EC2.5.1.1.8) catalyze the detoxification of reactive electrophilic compounds, many of which are toxic and carcinogenic intermediates, via conjugation with the endogenous tripeptide glutathione (GSH). Glutathione S-transferase (GST)-mediated detoxification is a critical determinant of species susceptibility to the toxic and carcinogenic mycotoxin aflatoxin B₁ (AFB₁), which in resistant animals efficiently detoxifies the toxic intermediate produced by hepatic cytochrome P450 bioactivation, the exo-AFB₁-8,9-epoxide (AFBO). Domestic turkeys (Meleagris gallopavo) are one of the most sensitive animals known to AFB₁, a condition associated with a deficiency of hepatic GST-mediated detoxification of AFBO. We have recently shown that unlike their domestic counterparts, wild turkeys (Meleagris gallopavo silvestris), which are relatively resistant, express hepatic GST-mediated detoxification activity toward AFBO. Because of the importance of GSTs in species susceptibility, and to explore possible GST classes involved in AFB₁ detoxification, we amplified, cloned, expressed and functionally characterized the hepatic mu-class GSTs tGSTM3 (GenBank accession no. JF340152), tGSTM4 (JF340153) from domestic turkeys, and a GSTM4 variant (ewGSTM4, JF340154) from Eastern wild turkeys. Predicted molecular masses of tGSTM3 and two tGSTM4 variants were 25.6 and 25.8 kDa, respectively. Multiple sequence comparisons revealed four GSTM motifs and the mu-loop in both proteins. tGSTM4 has 89% amino acid sequence identity to chicken GSTM2, while tGSTM3 has 73% sequence identity to human GSTM3 (hGSTM3). Specific activities of Escherichia coli-expressed tGSTM3 toward 1-chloro-2,4-dinitrobenzene (CDNB) and peroxidase activity toward cumene hydroperoxide were five-fold greater than tGSTM4 while tGSTM4 possessed more than three-fold greater activity toward 1,2-dichloro-4-nitrobenzene (DCNB). The two enzymes displayed equal activity toward ethacrynic acid (ECA). However, none of the GSTM proteins had AFBO detoxification capability, in contrast to recombinant alpha-class GSTs shown in our recent study to possess this important activity. In total, our data indicate that although turkey hepatic GSTMs may contribute to xenobiotic detoxification, they probably play no role in detoxification of AFBO in the liver.     

Structure-guided activity restoration of the silkworm glutathione transferase Omega GSTO3-3

Glutathione transferases (GSTs) are ubiquitous detoxification enzymes that conjugate hydrophobic xenobiotics with reduced glutathione. The silkworm Bombyx mori encodes four isoforms of GST Omega (GSTO), featured with a catalytic cysteine, except that bmGSTO3-3 has an asparagine substitution of this catalytic residue. Here, we determined the 2.20-Å crystal structure of bmGSTO3-3, which shares a typical GST overall structure. However, the extended C-terminal segment that exists in all the four bmGSTOs occupies the G-site of bmGSTO3-3 and makes it unworkable, as shown by the activity assays. Upon mutation of Asn29 to Cys and truncation of the C-terminal segment, the in vitro GST activity of bmGSTO3-3 could be restored. These findings provided structural insights into the activity regulation of GSTOs.     

Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Glutathione S-transferases (GST) catalyzing the conjugation of reduced glutathione to a vast range of xenobiotics including insecticides were characterized in the whitefly Bemisia tabaci. GST activities were determined in susceptible and resistant strains of B. tabaci towards artificial substrates, i.e. 1-chloro-2,4-dinitrobenzene (CDNB) in a photometric microplate assay and monochlorobimane (MCB) in a fluoroemtric microplate assay and characterized by their Michaelis-Menten kinetics. The inhibitory potential of ethacrynic acid was very effective with IC50-values between 0.9 and 5.8 microM depending on substrate and strain. The inhibitory effect of dicumarol was 10 times lower. Glutathione-affinity chromatography purified GST enzymes of two different B. tabaci strains appeared as a single band on SDS-PAGE and had a molecular mass of 23.5 kDa determined by MALDI mass spectrometry. The N-terminus of the purified enzyme was sequenced by Edman degradation. The nearly full-length cDNA of the enzyme was isolated by RT-PCR using a degenerate primer derived from the N-terminal amino acid sequence and contained an open reading frame encoding a 194-amino-acid protein. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to insect class sigma GSTs.     

Metal binding ability of glutathione transferases conserved between two animal species,the vanadium-rich ascidian Ascidia sydneiensis samea and the schistosome Schistosoma japonicum

Glutathione transferases (GSTs) are multifunctional enzymes found in many organisms. We recently identified vanadium-binding GSTs, designated AsGSTs, from the vanadium-rich ascidian, Ascidia sydneiensis samea. In this study, the metal-selectivity of AsGST-I was investigated. Immobilized metal ion affinity chromatography (IMAC) analysis revealed that AsGST-I binds to V(IV), Fe(III), and Cu(II) with high affinity in the following order Cu(II) > V(IV) > Fe(III), and to Co(II), Ni(II), and Zn(II) with low affinity. The GST activity of AsGST-I was inhibited dose-dependently by not V(IV) but Cu(II). A competition experiment demonstrated that the binding of V(IV) to AsGST-I was not inhibited by Cu(II). These results suggest that AsGST-I has high V(IV)-selectivity, which can confer the specific vanadium accumulation of ascidians. Because there are few reports on the metal-binding ability of GSTs, we performed the same analysis on SjGST (GST from the schistosome, Schistosoma japonicum). SjGST also demonstrated metal-binding ability although the binding pattern differed from that of AsGST-I. The GST activity of SjGST was inhibited by Cu(II) only, as that of AsGST-I. Our results indicate a possibility that metal-binding abilities of GSTs are conserved among organisms, at least animals, which is suggestive of a new role for these enzymes in metal homeostasis or detoxification.     

Engineering GST M2-2 for high activity with indene 1,2-oxide and indication of an H-site residue sustaining catalytic promiscuity

The substrate-binding H-site of human glutathione transferase (GST) M2-2 was subjected to iterative saturation mutagenesis in order to obtain an efficient enzyme with the novel epoxide substrate indene 1,2-oxide. Residues 10, 116, and 210 were targeted, and the activities with the alternative substrates, benzyl isothiocyanate and the prodrug azathioprine, undergoing divergent chemical reactions were monitored for comparison. In general, increased activities were found when the smaller residues Gly, Ser, and Ala replaced the original Thr210. The most active mutant T210G was further mutated at position 116, but no mutant showed enhanced catalytic activity. However, saturation mutagenesis of position 10 identified one double mutant T210G/I10C with 100-fold higher specific activity with indene 1,2-oxide than wild-type GST M2-2. This enhanced epoxide activity of 50 μmol min^− 1 mg^− 1 resulted primarily from an increased k_cat value (70 s^− 1). The specific activity is 24-fold higher than that of wild-type GST M1-1, which is otherwise the most proficient GST enzyme with epoxide substrates. A second double mutant T210G/I10W displayed 30-fold increased activity with azathioprine, 0.56 μmol min^− 1 mg^− 1. In both double mutants, the replacement of Ile10 led to narrowed acceptance of alternative substrates. Ile10 is evolutionarily conserved in related class Mu GSTs. Conservation usually indicates preservation of a particular function, and in the Mu class, it would appear that the conserved Ile10 is not necessary to maintain catalytic functions but to prevent loss of broad substrate acceptance. In summary, our data underscore the facile transition between alternative substrate selectivity profiles in GSTs by a few mutations.     

Glutathione transferases,regulators of cellular metabolism and physiology

Background

The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions.

Scope of review

The review covers the genetics, structure and function of the human cytosolic GSTs with particular attention to their emerging roles in cellular metabolism.

Major conclusions

All the catalytically active GSTs contribute to the glutathione conjugation or glutathione dependant-biotransformation of xenobiotics and many catalyze glutathione peroxidase or thiol transferase reactions. GSTs also catalyze glutathione dependent isomerization reactions required for the synthesis of several prostaglandins and steroid hormones and the catabolism of tyrosine. An increasing body of work has implicated several GSTs in the regulation of cell signaling pathways mediated by stress-activated kinases like Jun N-terminal kinase. In addition, some members of the cytosolic GST family have been shown to form ion channels in intracellular membranes and to modulate ryanodine receptor Ca^2 + channels in skeletal and cardiac muscle.

General significance

In addition to their well established roles in the conjugation and biotransformation of xenobiotics, GSTs have emerged as significant regulators of pathways determining cell proliferation and survival and as regulators of ryanodine receptors that are essential for muscle function. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Plasmodium vivax: molecular cloning, expression and characterization of glutathione S-transferase

Malaria parasite glutathione S-transferases (GSTs) are postulated to be essential for parasite survival by protecting the parasite against oxidative stress and buffering the detoxification of heme-binding compounds; therefore, GSTs are considered potential targets for drug development. In this study, we identified a Plasmodium vivax gene encoding GST (PvGST) and characterized the biochemical properties of the recombinant enzyme. The PvGST contained 618 bp that encoded 205 amino acids and shared a significant degree of sequence identity with GSTs from other Plasmodium species. The recombinant homodimeric enzyme had an approximate molecular mass of 50kDa and exhibited GSH-conjugating and GSH-peroxidase activities towards various model substrates. The optimal pH for recombinant PvGST (rPvGST) activity was pH 8.0, and the enzyme was moderately unstable at 37 degrees C. The K(m) values of rPvGST with respect to GSH and CDNB were 0.17+/-0.09 and 2.1+/-0.4mM, respectively. The significant sequence homology and similar biochemical properties of PvGST and Plasmodium falciparum GST (PfGST) indicate that they may have similar molecular structures. This information may be useful for the design of specific inhibitors for plasmodial GSTs as potential antimalarial drugs.     

Roles for glutathione transferases in plant secondary metabolism

Plant glutathione transferases (GSTs) are classified as enzymes of secondary metabolism, but while their roles in catalysing the conjugation and detoxification of herbicides are well known, their endogenous functions are largely obscure. Thus, while the presence of GST-derived S-glutathionylated xenobiotics have been described in many plants, there is little direct evidence for the accumulation of similarly conjugated natural products, despite the presence of a complex and dichotomous metabolic pathway which processes these reaction products. The conservation in glutathione conjugating and processing pathways, the co-regulation of GSTs with inducible plant secondary metabolism and biochemical studies showing the potential of these enzymes to conjugate reactive natural products are all suggestive of important endogenous functions. As a framework for addressing these enigmatic functions we postulate that either: (a) the natural reaction products of GSTs are unstable and undergo reversible S-glutathionylation; (b) the conjugation products of GSTs are very rapidly processed to derived metabolites; (c) GSTs do not catalyse conventional conjugation reactions but instead use glutathione as a cofactor rather than co-substrate; or (d) GSTs are non-catalytic and function as transporter proteins for secondary metabolites and their unstable intermediates. In this review, we describe how enzyme biochemistry and informatics are providing clues as to GST function allowing for the critical evaluation of each of these hypotheses. We also present evidence for the involvement of GSTs in the synthesis of sulfur-containing secondary metabolites such as volatiles and glucosinolates, and the conjugation, transport and storage of reactive oxylipins, phenolics and flavonoids.     

Glutathione transferases with vanadium-binding activity isolated from the vanadium-rich ascidian Ascidia sydneiensis samea

Some ascidians accumulate vanadium in vanadocytes, which are vanadium-containing blood cells, at high levels and with high selectivity. However, the mechanism and physiological significance of vanadium accumulation remain unknown. In this study, we isolated novel proteins with a striking homology to glutathione transferases (GSTs), designated AsGST-I and AsGST-II, from the digestive system of the vanadium-accumulating ascidian Ascidia sydneiensis samea, in which the digestive system is thought to be involved in vanadium uptake. Analysis of recombinant AsGST-I confirmed that AsGST-I has GST activity and forms a dimer, as do other GSTs. In addition, AsGST-I was revealed to have vanadium-binding activity, which has never been reported for GSTs isolated from other organisms. AsGST-I bound about 16 vanadium atoms as either V(IV) or V(V) per dimer, and the apparent dissociation constants for V(IV) and V(V) were 1.8 × 10⁻⁴ M and 1.2 × 10⁻⁴ M, respectively. Western blot analysis revealed that AsGSTs were expressed in the digestive system at exceptionally high levels, although they were localized in almost all organs and tissues examined. Considering these results, we postulate that AsGSTs play important roles in vanadium accumulation in the ascidian digestive system.     

Glutathione S-transferases in the Japanese quail: Tissue distribution and purification of the liver isozymes

Cytosolic glutathione S-transferase (GST) activities toward 1-chloro-2,4-dinitro-benzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EA), 1,2-epoxy-3-(p-nitrophenoxyl)propane (EPNP), trans-4-phenyl-3-buten-2-one (t-PBO), δ³-androstene-3,17-dione (ASD) and trans-stilbene oxide (t-SO); cytosolic glutathione peroxidase activity toward cumene hydroperoxide (CuOOH); and microsomal GST activity toward CDNB were examined in liver, kidney, brain, and lung of adult male and female Japanese quail. In all cases, the renal specific activity per milligram protein was higher than the hepatic activity and was the highest among the four tissues examined. No consistent sex differences in GST activity were observed. The GSTs were purified from quail liver cytosol by S-hexylglutathione and glutathione affinity chromatography. Total GSTs eluted from the S-hexylglutathione affinity column were further separated by chromatofocusing, and the microheterogeneity of the GST isozymes was shown by high-resolution native isoelectrofocusing (IEF) in polyacrylamide slab gels and by SDS-PAGE. Five subunits were identified: QL1 (30.5 kDa), QL2 (27.2 kDa), QL3a (26.8 kDa), QL3b (26.5 kDa), and QL4 (25.5 kDa). Western blot analysis revealed that QL1 and QL2 reacted with antibodies raised against the rat Mu class GSTs (Yb1 and Yb2), and QL3a and QL3b reacted with those raised against the Alpha class (rat Ya and mouse a). Substrate specific activity of each isoform was determined with CDNB, DCNB, CuOOH, EA, t-PBO, ASD, and t-SO. QL3a and QL3b have high reactivity toward CuOOH, while QL1 and QL2 showed high activity toward t-SO. The N-terminal amino acid sequence of QL2 was identical to that of the chicken Mu class GST subunit CL2. However, no sequence was obtained with QL1 due to possible N-terminal blockage. © 1996 John Wiley & Sons, Inc.     

Plants for waste water treatment--effects of heavy metals on the detoxification system of Typha latifolia

Upon treatment with Cd and As cattail (Typha latifolia) showed induced catalase, monodehydroascorbate reductase and ascorbate peroxidase activities in leaves but strong inhibition in rhizomes. Peroxidase activity in leaves of the same plants was inhibited whereas linear increase was detected after Cd treatment in rhizomes.Glutathione S-transferase measurements resulted in identical effects of the trace elements on the substrates CDNB, DCNB, NBC, NBoC, fluorodifen. When GST was assayed with the model substrate DCNB, a different pattern of activity was observed, with strongly increasing activities at increasing HM concentrations. Consequently, to improve the success rates, future phytoremediation plans need to preselect plant species with high antioxidative enzyme activities and an alert GST pattern capable of detoxifying an array of organic xenobiotics.     

Glutathione S-transferase and S-crystallins of cephalopods: Evolution from active enzyme to lens-refractive proteins

Our previous studies have shown that the S-crystallins of cephalopod (Ommastrephes sloani pacificus) eye lenses comprise a family of at least ten members which are evolutionarily related to glutathione S-transferase (GST, EC 2.5.1.18). Here we show by cDNA cloning that there are at least 24 different S-crystallins that are 46–99% identical to each other by amino acid sequence in the squid Loligo opalescens. In each species, all but one S-crystallin (SL11 in O. pacificus and Lops4 in L. opalescens) examined has an inserted central peptide of variable length and sequence. cDNA expression studies conducted in Escherichia coli showed that squid GST (which is expressed little in the lens) has very high enzymatic activity using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate; by contrast, SL20-1 of O. pacificus and Lops 12 of L. opalescens (which are encoded by abundant lens mRNAs) have no GST activity. Interestingly, SL11 and Lops4 have some enzymatic activity with the CDNB substrate. Site-specific mutations at Y7 or W38, both residues essential for activity of vertebrate GSTs, or insertion of the central peptide present in the inactive SL20-1, reduced the specific activity of squid GST by 30- to 100-fold. These data indicate that the S-crystallins consist of a family of enzymatically inactive proteins (when using CDNB as a substrate) which is considerably larger than previously believed and that GST activity was lost by gradual drift in sequence as well as by insertion of an extra peptide by exon shuffling. The results are also consistent with the idea that SL11 and Lops4 are orthologous crystallins representing the first descendants of the ancestral GST gene in the pathway which gave rise to the extensive S-crystallin family of lens proteins. Correspondence to: S.I. Tomarev