Molecular cloning and oxidative stress response of a sigma-class glutathione S-transferase of the bumblebee Bombus ignitus

Glutathione S-transferases (EC 2.5.1.18; GSTs) are multifunctional enzymes that are mainly involved in xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTS gene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H(2)O(2) overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H(2)O(2) overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3h after H(2)O(2) exposure, whereas BiGSTS and BiGSTD are highly induced at 9h after H(2)O(2) exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitus are differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.     

Purification and characterization of a novel sigma-class glutathione S-transferase of the fall webworm, Hyphantria cunea

Abstract:  An enzyme that possesses glutathione S -transferase (GST) activity was found in the fall webworm, Hyphantria cunea . The enzyme was purified to homogeneity for the first time by ammonium sulphate fractionation and affinity chromatography. The N-terminal sequence of the purified protein was similar to those of Sigma-class GSTs. The purified GST retained more than 75% of its original GST activity after incubation at pH 5–8. Incubation for 30 min at temperatures below 50°C scarcely affected the activity. The enzyme was able to catalyse the reaction of glutathione with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation.     

A second stress-inducible glutathione S-transferase gene from Schizosaccharomyces pombe

A second glutathione S-transferase gene (GST II) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The nucleotide sequence determined contains 1908 bp including an open reading frame of 230 amino acids that would encode a protein of a molecular mass of 26843.4 Da. The amino acid sequence of the putative GST II is very homologous with that of the previously isolated GST gene (GST I) located in the same chromosome III of S. pombe. The cloned GST II gene produces the functional GST in S. pombe, and it gives much higher GST in the stationary phase than in the exponential phase. Regulation of the GST II gene was studied using the GST II-lacZ fusion. The synthesis of beta-galactosidase from the fusion plasmid is greatly enhanced by the treatments with oxidative stresses such as menadione and mercuric chloride. It is also induced by o-dinitrobenzene, one of the GST substrates. NO-generating S-nitroso-N-acetylpenicillamine has a weak induction effect on the expression of GST II gene. These results indicate that the S. pombe GST II gene is involved in the oxidative stress response and detoxification. However, physiological meaning on the existence of the two similar GST genes in S. pombe remains unknown yet.     

Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].     

Thermodynamics of glutathione binding to the tyrosine 7 to phenylalanine mutant of glutathione S-transferase from Schistosoma japonicum

The binding of glutathione (GSH) to the tyrosine 7 to phenylalanine mutant of Schistosoma japonicum glutathione S-transferase (SjGST-Y7F) has been studied by isothermal titration calorimetry (ITC). At pH 6.5 and 25 °C this mutant shows a higher affinity for glutathione than wild type enzyme despite an almost complete loss of activity in the presence of 1-chloro-2,4-dinitrobenzene (CDNB) as second substrate. The enthalpy change upon binding of GSH is more negative for the mutant than for the wild type GST (SjGST). Changes in accessible solvent areas (ASA) have been calculated based on enthalpy and heat capacity changes. ASA values indicated the burial of apolar surfaces of protein and ligand upon binding. A more negative ΔC_p value has been obtained for the mutant enzyme, suggesting a more hydrophobic interaction, as may be expected from the change of a tyrosine residue to phenylalanine.     

A comparison of erythrocyte glutathione S-transferase activity from human foetuses and adults.

Glutathione S-transferase activity was measured in partially purified haemolysates of erythrocytes from human foetuses and adults. Enzyme activity was present in erythrocytes obtained between 12 and 40 weeks of gestation. The catalytic properties of the enzyme from foetal cells were similar to those of the enzyme from adult erythrocytes, indicating that probably only one form of the erythrocytes enzyme exists throughout foetal and adult life.     

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

A soluble auxin-binding protein from Hyoscyamus muticus is a glutathione S-transferase.

We have used the photoaffinity label azido-[3H]IAA (5-N3-[7-3H]indole-3-acetic acid), a biologically active analog of indole-3-acetic acid, to identify auxin-binding proteins (ABPs) in the soluble fraction of Hyoscyamus muticus. A 25-kD polypeptide previously described (H. Macdonald, A. M. Jones, P. J. King [1991] J Biol Chem 266: 7393-7399) has now been purified to homogeneity by conventional methods. Binding of azido-[3H]IAA to the purified protein was reduced by active auxins but not by inactive indoles. Partial amino acid sequences of the purified protein showed high homology to glutathione S-transferase (GST) from tobacco (ParB) and from maize (GT32). The conclusion that the 25-kD ABP is a GST is further supported by high GST activity in fractions highly enriched in the 25-kD polypeptide and recognition of the ABP by antibodies against GST from wheat and maize. Furthermore, purification of a protein from a soluble protein extract from H. muticus by affinity chromatography on glutathione-agarose also yielded a 25-kD polypeptide that was indistinguishable in its N-terminal amino acid sequence and biochemical characteristics from the protein purified by conventional methods. Possible functions of GST in auxin action are discussed.     

Characterization and regulation of glutathione S-transferase gene from Schizosaccharomyces pombe

A glutathione S-transferase (GST) gene has been cloned from Schizosaccharomyces pombe for the first time. The nucleotide sequence determined was found to contain 2030 base pairs including an open reading frame of 229 amino acids that would encode a protein of a molecular mass of 27017 Da. The cloned GST gene was expressed and was found to function in S. pombe, Saccharomyces cerevisiae, and Escherichia coli. The plasmid pGT207 encoding the S. pombe GST gene appeared to be able to accelerate the growth of a wild type S. pombe culture. In a culture of S. pombe containing plasmid pGT207, the growth was inhibited less by mercuric chloride than in a culture with vector alone. The 1088 bp region upstream from the GST gene as well as the region encoding the N-terminal 14 amino acids was transferred into the promoterless beta-galactosidase gene of plasmid YEp357R to yield the fusion plasmid pYSH2000. beta-Galactosidase synthesis was induced by cadmium chloride, mercuric chloride, hydrogen peroxide, and menadione. It was also induced by high temperature. These results suggest that the cloned S. pombe GST gene is involved in the oxidative stress response.     

Induction of glutathione S-transferase activity and glutathione level in plants exposed to glyphosate

Treatment with the herbicide glyphosate led to significantly increased activities of the enzyme gluiathione S-transferase (GST, EC 2.5.1.18) in wheat ( Triticum aestivum L. cv. Kadett and cv. Satu), pea ( Pisum sativum L. ev. Debreceni Világoszöld) and in maize ( Zea mays. L. Pioneer 3839 hybrid) tissues. GST activities in wheat seedlings (cv. Kadett) exposed to 960 μM glyphosate for 4 days were ca 6-fold and 3-fold higher in shoots and roots, respectively, than in the controls. Glyphosate increased the GST activity to a lesser extent in pea and maize than in wheat. In wheat seedlings (cv. Satu) exposed let 120 μM glyphosate gradual increases in the content of non-protein thiols were observed. After 7 days exposure to glyphosate the thiol levels rose to about 360% and 220% of the controls in wheal shoots and roots, respectively. The elevation of thiol content in glyphosate-treated plants was shown to be primarily due increases of glutathione level. These results suggest that the enhanced glutathione metabolism may have a role in the mode of action or degradation of this herbicide.     

Purification and biochemical properties of glutathione S-transferase from Lactuca sativa

A glutathione S-transferase (GST) from Lactuca sativa was purified to electrophoretic homogeneity approximately 403-fold with a 9.6% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure. The activity of the enzyme was significantly inhibited by ShexylGSH and S-(2,4-dinitrophenyl) glutathione. The enzyme displayed activity towards 1-chloro-2,4-dinitrobenzene, a general GST substrate and high activities towards ethacrynic acid. It also exhibited glutathione peroxidase activity toward cumene hydroperoxide.     

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.     

Distribution and properties of glutathione S-transferase from T. infestans

The glutathione transferase from T. infestans is able to render aqueous metabolites when incubated in vitro with malathion, parathion and fenitrothion. It is a soluble enzyme present in every developmental stage and widely distributed in all insect organs. The purification procedure applied, consisting of fractionation with ammonium sulfate and Bio-Gel P-60 chromatography, gives an unique molecular form catalytically active using methyl iodide as substrate in polyacrylamide gel electrophoresis (PAGE). One of the most active substrates is the 1-chloro-2,4-dinitrobenzene (CDNB), with an activity maximum at pH 7.5 and at 45 degrees C temperature. Its activation energy calculated from an Arrhenius plot is 14,846 cal mol-1. The enzyme susceptibility to inhibition by thiol reagents shows three degrees of responses; slight, moderate or high, depending on the compounds used. The kinetics of the enzyme catalysed reaction with the purified fraction is complex, and resembles that reported for glutathione S-transferase A from rat liver, showing a biphasic kinetic mechanism in which the reaction pathway depends on the concentration of GSH. In general, the properties of this insect enzyme are similar to those enzymes isolated from vertebrate organisms.     

Structure and chromosomal localization of human and mouse genes for hematopoietic prostaglandin D synthase. Conservation of the ancestral genomic structure of sigma-class glutathione S-transferase.

Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST). We isolated the genes and cDNAs for human and mouse H-PGDSs. The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively. Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme. Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner. Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin. By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively. The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns. The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS. These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family.     

Cloning and transcriptional analysis of two sialic acid-binding lectins (SABLs) from razor clam Solen grandis

Sialic acid-binding lectin (SABL) plays crucial role in both innate and adaptive immune responses benefiting from its predominant affinity toward glycan. In the present study, two SABLs from razor clam Solen grandis (designated as SgSABL-1 and SgSABL-2) were identified, and their expression patterns, both in tissues and towards microorganism glycan stimulation, were then characterized. The cDNA of SgSABL-1 and SgSABL-2 was 988 and 1281 bp, containing an open reading frame (ORF) of 744 and 570 bp, respectively, and deduced amino acid sequences showed high similarity to other invertebrates SABLs. Both SgSABL-1 and SgSABL-2 encoded a C1q domain. SgSABL-1 and SgSABL-2 were found to be constitutively expressed in a wide range of tissues with different levels, including mantle, gill, gonad, hemocyte, muscle, and hepatopancreas, and both of them were highly expressed in hepatopancreas. SgSABL-1 and SgSABL-2 could be significantly induced after razor clams were stimulated by acetylated subunits-containing glycan LPS and PGN, suggesting the two SgSABLs might perform potential function of glycan recognition. In addition, SgSABL-2 could also be induced by β-1,3-glucan. All these results indicated that SgSABL-1 and SgSABL-2 might be involved in the immune response against microbe infection and contributed to the pathogens recognition.     

Short communication direct effect of Cd on glutathione S-transferase and glutathione reductase from Calystegia sepium

Interactions between heavy metals, glutathione, glutathione S-transferase (GST), and glutathione reductase (GR) are being investigated by many working groups, but evaluation of the direct effect of Cd+ on these enzymes in vitro is lacking. We report here the effect of cadmium (10, 50, 100, 250 microM CdSO4) on partially purified enzymes from Calystegia sepium. Plants were grown under normal field conditions without metals and the enzymes were extracted by Tris buffer and partially purified by ammonium sulphate fractionation and gel filtration. Glutathione S-transferase activity was measured with different substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), p-nitrobenzylchloride (NBC), and the herbicide Fluorodifen. GST activity was significantly lower in leaf compared to stem, flower, and rhizome and the inhibitory effect of Cd was obtained with NBC and Fluorodifen substrates at 250 microM. There was no effect of Cd on GR activity up to 250 microM.