Mu Class Glutathione S-transferase Research Articles

Impact of the deletion glutathione S-transferase (class Mu) on lung cancer risk among smokers

BackgroundThe results of several studies assessing the effect of the glutathione S-transferase class Mu 1 (GSTM1) null variant on the genetic susceptibility of tobacco-related cancers have been conflicting. In this work, we aim to identify the impact of the deletion of GSTM1 on lung cancer risk among smokers.MethodsThis study was conducted on 20 patients diagnosed with primary lung cancer and 20 healthy individuals as a control group. They were subject to full medical history taking, complete clinical examination, and GSTM1 genotyping by PCR.ResultsBoth studied groups were matched for age, sex, and smoking status. No statistically significant difference was exhibited between the frequency of GSTM1 positive and GSTM1 null in the studied population. No risk of lung cancer associated with GSTM1 null genotype was demonstrated between the patients and control group (n = 14/20 cases) (p = 0.110, OR = 2.852, 95% CI 0.777–10.467). Additionally, there was no association between the risk of lung cancer and the presence of the gene either in smokers (p = 1, OR = 1.8 and 95% CI 0.124–26.196) or non-smokers; (p = 0.063, OR = 4.4 and 95% CI 0.889–21.78). No statistically significant risk was found between the frequencies of GSTM1 null and the various histopathological types of lung malignancy.ConclusionThe results of this work demonstrated no association between the occurrence of the GSTM1 null variant, even when stratified for smoking status, and the risk of lung cancer.

Prostaglandin synthase activity of sigma- and mu-class glutathione transferases in a parasitic trematode, Clonorchis sinensis.

Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-μ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-μ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 μm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 μm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1μm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.

The diverse functions of Mu-class Glutathione S-transferase HrGSTm1 during the development of Hyalomma rufipes with a focus on the detoxification metabolism of cyhalothrin

BackgroundGlutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes in living organisms with metabolic and detoxification functions, which can detoxify exogenous and endogenous compounds and thereby reduce the damage caused by toxic substances to the body. Ticks are obligate blood-sucking ectoparasites that can transmit various pathogens, and the characterization of tick-derived GSTs may help improve current understanding of the molecular mechanism of tick resistance to insecticides. In this study, a novel GST gene, named HrGSTm1, was identified from Hyalomma rufipes.MethodsSequence analysis was performed by using bioinformatics techniques. A prokaryotic expression system was used to obtain the recombinant expression protein rHrGSTm1. Detection of spatiotemporal expression patterns of target genes and their response to the toxicity of cyhalothrin on female H. rufipes was performed by using a quantitative PCR platform. The optimal enzymological parameters of rHrGSTm1 using glutathione as substrate were calculated. The antioxidant capacity of the recombinant protein was evaluated by DPPH• (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl). Knockdown of the HrGSTm1 genes through RNA interference was used to analyze their effects on the physiological parameters of ticks. The changes in HrGSTm1 messenger RNA expression patterns under cypermethrin stress were analyzed.ResultsThe complementary DNA sequence of HrGSTm1 contained a 672-bp open reading frame, which potentially encoded 223 amino acids. The predicted molecular weight was 25.62 kDa, and the isoelectric point 8.22. HrGSTm1 is a Mu-class GST, belonging to the cytoplasmic GSTs with no signal peptide observed. The Vmax and Km of rHrGSTm1 were 3.367 ± 0.81 uM and 2.208 ± 0.76 uM, respectively, and its activities were dependent on different temperatures and pH conditions; the scavenging rate of rHrGSTm1 to DPPH• reached 76.4% at 1.25 mg/ml. Variable expressions of HrGSTm1 were observed under various treatment periods and in different tissues, with the highest appearing in eggs (analysis of variance [ANOVA], F(2, 9) = 279.9, P < 0.0001) and Malpighian tubules (ANOVA, F(3, 12) = 290.5, P < 0.0001). After knockdown of HrGSTm1, compared with the control group, the mortality in the treatment group was increased by 16.7%, the average oviposition rate decreased by 33.9%, the average engorged body weight decreased by 287.38 mg and egg weight decreased by 127.46 mg, although only the engorged body weight was significantly different (t-test, t(44) = 2.886, P = 0.006). After exposure to three sublethal concentrations (LC05, LC10, LC50) of cyhalothrin, the expression level of HrGSTm1 in the midgut, ovary and salivary gland was upregulated, whereas in Malpighian tubules, it showed a trend of upregulation at first and then downregulation, implying different functions during the detoxification in different tissues.ConclusionsIn this study, a novel GST of the Mu-class was successfully isolated from H. rufipes and systematically subjected to bioinformatic analysis and recombination identification. The variation trend of HrGSTm1 expression level in different tissues suggests that the gene has different detoxification functions in different tissues. The potential function of this gene was analyzed to provide basic research for further investigation of its detoxification mechanism.Graphical

Functional characterization of differentially expressed proteins coming from unisexual and bisexual infected Schistosoma japonicum female worms

Schistosomiasis is an important zoonotic disease affecting up to 40 kinds of animals and is responsible for ∼250 million human cases per year. Due to the extensive use of praziquantel for the treatment of parasitic diseases, drug resistance has been reported. Consequently, novel drugs and effective vaccines are urgently needed for sustained control of schistosomiasis. Targeting reproductive development of Schistosoma japonicum could contribute to the control of schistosomiasis. In this study, five highly expressed proteins (S. japonicum large subunit ribosomal protein L7e, S. japonicum glutathione S-transferase class-mu 26 kDa isozyme, S. japonicum UDP-galactose-4-epimerase and two hypothetical proteins SjCAX70849 and SjCAX72486) in 18, 21, 23, and 25-day mature female worms compared to single-sex infected female worms were selected based on our previous proteomic analysis. Quantitative real-time polymerase chain reaction analysis and long-term interference with small interfering RNA were performed to identify the biological functions of these five proteins. The transcriptional profiles suggested that all five proteins participated in the maturation of S. japonicum. RNA interference against these proteins resulted in morphological changes to S. japonicum. The results of an immunoprotection assay revealed that immunization of mice with recombinant SjUL-30 and SjCAX72486 upregulated production of immunoglobulin G-specific antibodies. Collectively, the results demonstrated that these five differentially expressed proteins were vital to reproduction of S. japonicum and, thus, are potential candidate antigens for immune protection against schistosomiasis.

Glutathione S-transferase gene diversity and their regulation by Nrf2 in Chinese mitten crab (Eriocheir sinensis) during nitrite stress

Eriocheir sinensis is one of the most important economic aquatic products in China. However, nitrite pollution has become a serious threat to the healthy culture of E. sinensis. Glutathione S-transferase (GST) is an important phase II detoxification enzyme, which plays a leading role in the cellular detoxification of exogenous substances. In this study, we obtained 15 GST genes (designated as EsGST1-15) from E. sinensis, and their expression and regulation in E. sinensis under nitrite stress were studied. EsGST1-15 belonged to different GST subclasses. EsGST1, EsGST2, EsGST3, EsGST4, and EsGST5 belonged to Delta-class GSTs; EsGST6 and EsGST7 are Theta-class GSTs; EsGST8 is a mGST-3-class GST; EsGST9 belonged to mGST-1-class GSTs; EsGST10 and EsGST11 belonged to Sigma-class GSTs; EsGST12, EsGST13, and EsGST14 are Mu-class GSTs; EsGST15 is a Kappa-class GST. Tissue distribution experiments showed that EsGSTs were widely distributed in all detected tissues. The expression level of EsGST1-15 was significantly increased in the hepatopancreas under nitrite stress, indicating that EsGSTs were involved in the detoxification of E. sinensis under nitrite stress. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a transcription factor that can activate the expression of detoxification enzyme. We detected the expression of EsGST1-15 after interfering with EsNrf2 in the hepatopancreas of E. sinensis with or without nitrite stress. Results showed that EsGST1-15 were all regulated by EsNrf2 with or without nitrite stress. Our study provides new information about the diversity, expression, and regulation of GSTs in E. sinensis under nitrite stress.

Improving reactivity of naphthalimide-based GST probe by imparting TPP cation: Development and application for live cell imaging

Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes comprising multiple classes and subtypes. This paper describes the synthesis and characterization of TPPBN-1, a naphthalimide derivative conjugated with a triphenylphosphonium (TPP) cation. When 4-bromonaphthalimide (BrNaph), a previously characterized GST substrate, was conjugated to a TPP cation, the conjugate showed increased reactivity towards most alpha- and mu-class GSTs, particularly the GSTA2 subtype, compared to the parent compound, but hardly towards Pi-class GSTs. Using this probe with enhanced reactivity, the enzymatic activity of endogenous GSTA1/2 in HepG2 cells was visualized by confocal fluorescence microscopy. The results demonstrated that modification with TPP cations, which are often used as tags for targeting mitochondria, can be used to enhance the reactivity of probes for specific GST subtypes.

Enhanced soluble expression of glutathione S-transferase Mu from Rutilus kutum by co-expression with Hsp70 and introducing a novel inhibitor for its activity

One of the most critical challenges in recombinant protein production is the increment of protein solubility. The overexpression of glutathione S-transferases (GSTs) in cancers leads to the fast detoxification of drug substrates that is subsequently followed by increased drug resistance. In this study, the co-expression of Rutilus kutum Hsp70 (RkHsp70) with the mu-class GST (RkGST-μ) from the same source was analyzed to increase the solubility of aggregate-prone RkGST-μ. Based on the results, the co-expressed RkGST-μ cells showed substantially higher solubility and specific activity compared to control. The kinetic properties of the enzyme were measured and its optimum pH and temperature were observed at pH 8 and 35 °C. Furthermore, this study introduces ZM-093, a sulfamethoxazole-based azo with IC50 values of 24.45 μM (p < 0.0001), as a novel RkGST-μ inhibitor. Overall, the present study provides clear findings in support of using molecular chaperones to enhance the solubility of recombinant proteins.

Activity of Noni Fruit (Morinda citrifolia L.) Ethanolic Extract on Class mu Glutation S-Transferase of Lung Rat

One of the main modalities of cancer treatment is chemotherapy, which uses chemicals that are generally electrophilic. These xenobiotic compounds sometimes does not produce effective response due to activity of glutathione S-transferase (GST) which inactivate the xenobiotics. Several natural phenolic compounds were reported to inhibit GST activity in vitro. Noni fruit (Morinda citrifolia L.) which contains flavonoids and other phenolic compounds such as scopoletin and morindon is proposed to interfere GST activity. This study aimed to analyze the effect of ethanolic extract of Noni fruit in vivo on GST activity in lung rat using 1,2-dichloro-4-nitrobenzene (DCNB). This substrate is a specific for class mu GST. First, rats were administered with ethanolic extract of Noni and dimethylbenz(α)anthracene (DMBA) for two weeks. The cytosolic fraction of lung was isolated then the GST activity was determined by simple kinetic program which was automatically calculated using spectrophotometer. The results showed that ethanolic extract of Noni in 1 and 5% (w/v) of concentration induced class mu GST activity, whereas 10% (w/v) of concentration inhibited class mu GST activity. After a treatment with DMBA, all tested concentrations of ethanolic extract of Noni inhibited class mu GST activity of lung rat significantly. These results indicated that Noni fruit extract can be further developed as a supportive agent of a chemotherapy drug.Keywords: DMBA, GST, Morinda citrifolia L., spectrophotometer.

Caracterization of glutathione S-transferase of Dermacantor marginatus and effect of the recombinant antigen as a potential anti-tick vaccine

Dermacentor marginatus is one of the main tick species in northwestern China, and is a vector of various tick-borne pathogens. Tick control method largely depends on chemical agents, but the disadvantages of using such approach would cause environmental damage and the risk of developing tick resistance to acaricides. Vaccination of tick protective antigen is an eco-friendly approach which is an alternative and promising method to mitigate tick infestation in livestock. In the study, a mu-class glutathione S-transferase (GST) sequence of D. marginatus was cloned and the recombinant protein (rDmGST) was expressed. Transcriptional level of the GST was measured together with native GST activity of the tick. Finally, A vaccine trial on rabbits against D. marginatus was proceeded to evaluate the anti-tick effect of rDmGST. Results reveled that the CDs of the D. margiantus glutathione S-transferase mu 1 gene has 669 base pair nucleotide sequence encoding a 223 amino acid. The deduced GST protein sequence had over 95 % similarity with that of D. variabilis. The rDmGST was efficiently expressed soluble and purified by His trap affinity chromatography. Enzyme activity of native GST and transcriptional profiles of the GST showed up-regulation in different stages and organs of D. marginaus during blood feeding. Polyclonal antibody reacted with rDmGST in Western blotting. Tick challenge on rDmGST inoculated rabbits showed reductions in adult female engorgement rate, total egg mass and egg hatching rate with an overall vaccine efficacy of 43.69 %. The results of the experiment indicated the GST has potential value to be an effective protective antigen of D. marginatus.

Gene silencing by RNA interference in the ectoparasitic mite, Psoroptes ovis

The presence of components of the RNA interference (RNAi) pathway in Psoroptes ovis, an ectoparasitic mite responsible for psoroptic mange, was investigated through interrogation of the P. ovis genome. Homologues of transcripts representing critical elements for achieving effective RNAi in the mite, Tetranychus urticae and the model organisms Caenorhabditis elegans and Drosophila melanogaster were identified and, following the development of a non-invasive immersion method of double stranded RNA delivery, gene silencing by RNAi was successfully demonstrated in P. ovis. Significant reductions in transcript levels were achieved for three target genes which encode the Group 2 allergen (Pso o 2), mu-class glutathione S-transferase (PoGST-mu1) and beta-tubulin (Poβtub). This is the first demonstration of RNAi in P. ovis and provides a mechanism for mining transcriptomic and genomic datasets for novel control targets against this economically important ectoparasite.

Monomeric Camelus dromedarius GSTM1 at low pH is structurally more thermostable than its native dimeric form.

Glutathione S‒transferases (GSTs) are multifunctional enzymes that play an important role in detoxification, cellular signalling, and the stress response. Camelus dromedarius is well-adapted to survive in extreme desert climate and it has GSTs, for which limited information is available. This study investigated the structure-function and thermodynamic properties of a mu-class camel GST (CdGSTM1) at different pH. Recombinant CdGSTM1 (25.7 kDa) was expressed in E. coli and purified to homogeneity. Dimeric CdGSTM1 dissociated into stable but inactive monomeric subunits at low pH. Conformational and thermodynamic changes during the thermal unfolding pathway of dimeric and monomeric CdGSTM1 were characterised via a thermal shift assay and dynamic multimode spectroscopy (DMS). The thermal shift assay based on intrinsic tryptophan fluorescence revealed that CdGSTM1 underwent a two-state unfolding pathway at pH 1.0–10.0. Its Tm value varied with varying pH. Another orthogonal technique based on far-UV CD also exhibited two-state unfolding in the dimeric and monomeric states. Generally, proteins tend to lose structural integrity and stability at low pH; however, monomeric CdGSTM1 at pH 2.0 was thermally more stable and unfolded with lower van't Hoff enthalpy. The present findings provide essential information regarding the structural, functional, and thermodynamic properties of CdGSTM1 at pH 1.0–10.0.

A microRNA-1 gene, tci-miR-1-3p, is involved in cyflumetofen resistance by targeting a glutathione S-transferase gene, TCGSTM4, in Tetranychus cinnabarinus.

microRNA-1 (miR-1) is a well-studied conservative microRNA (miRNA) involved in immune responses in mammals and insects. However, little is known about its role in pesticide resistance in arthropods. In this study, we found that a microRNA belong to miR-1 family (tci-miR-1-3p) was significantly down-regulated in a cyflumetofen-resistant strain (CYR) of Tetranychus cinnabarinus compared with its homologous susceptible strain (SS), indicating an involvement of miR-1 in cyflumetofen resistance inmites. One glutathione S-transferase (GST) gene (TCGSTM4, a mu class GST gene), a candidate target gene of tci-miR-1-3p, was found to be significantly down-regulated when tci-miR-1-3p was over-expressed. The specific interaction between tci-miR-1-3p and the target sequence in the 3' untranslated region of TCGSTM4 was confirmed. A decrease or increase in tci-miR-1-3p abundance through feeding miRNA inhibitors or mimics significantly increased or decreased TCGSTM4 expressions at the mRNA and protein levels, respectively. In addition, an over-expression of tci-miR-1-3p resulted in a decrease in the tolerance of T. cinnabarinus to cyflumetofen in both SS and CYR strains, and vice versa. After decreasing TCGSTM4 transcription via RNA interference, T. cinnabarinus became more sensitive to cyflumetofen in both resistant and susceptible mites, and the change in mortality was greater in CYR than that in SS. Moreover, the recombinant TCGSTM4 could significantly decompose cyflumetofen, indicating that TCGSTM4 is a functional gene responsible for cyflumetofen resistance in mites.

Comprehensive analysis of the catalytic and structural properties of a mu-class glutathione s-transferase from Fasciola gigantica

Glutathione S‒transferases (GSTs) play an important role in the detoxification of xenobiotics. They catalyze the nucleophilic addition of glutathione (GSH) to nonpolar compounds, rendering the products water-soluble. In the present study, we investigated the catalytic and structural properties of a mu-class GST from Fasciola gigantica (FgGST1). The purified recombinant FgGST1 formed a homodimer composed of 25 kDa subunit. Kinetic analysis revealed that FgGST1 displays broad substrate specificity and shows high GSH conjugation activity toward 1-chloro-2,4-dinitrobenzene, 4-nitroquinoline-1-oxide, and trans-4-phenyl-3-butene-2-one and peroxidase activity towards trans-2-nonenal and hexa-2,4-dienal. The FgGST1 was highly sensitive to inhibition by cibacron blue. The cofactor (GSH) and inhibitor (cibacron blue) were docked, and binding sites were identified. The molecular dynamics studies and principal component analysis indicated the stability of the systems and the collective motions, respectively. Unfolding studies suggest that FgGST1 is a highly cooperative molecule because, during GdnHCl-induced denaturation, a simultaneous unfolding of the protein without stabilization of any partially folded intermediate is observed. The protein is stabilized with a conformational free energy of about 10 ± 0.3 kcal mol−1. Additionally, the presence of conserved Pro-53 and structural motifs such as N-capping box and hydrophobic staple, further aided in the stability and proper folding of FgGST1.

Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets

BackgroundScabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools.MethodsWe performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase (SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi).ResultsWe provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme (SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls.ConclusionsA series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

Crystal structure of a class-mu glutathione S-transferase from whiteleg shrimp Litopenaeus vannamei: structural changes in the xenobiotic binding H-site may alter the spectra of molecules bound.

Glutathione S-transferases (GSTs) are dimeric proteins that play a key role in phase II cellular detoxification. Here, the first crystal structure of a GST class-mu from marine crustacean shrimp Litopenaeus vannamei is reported at a resolution of 2.0 Å. The coordinates reported here have the lowest sequence identity with previously reported GSTs class-mu deposited at the Protein Data Bank (PDB), although they have subtle conformational differences. One key feature of GST class-mu from L. vannamei is the active site crevice markedly reduced when it is compared with other GSTs class-mu. This finding together with the chemical change of residues into the cavity (F112 and Y210) points to a particular specialization in which smallest xenobiotics with nonstandard chemical characteristics can be bound to the H-site. This suggests that marine organisms have evolved structural strategies to provide efficient selectivity toward xenobiotics to be disposed of by the phase II detoxification process.

The Glutathione-S-Transferase, Cytochrome P450 and Carboxyl/Cholinesterase Gene Superfamilies in Predatory Mite Metaseiulus occidentalis

Pesticide-resistant populations of the predatory mite Metaseiulus (= Typhlodromus or Galendromus) occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) have been used in the biological control of pest mites such as phytophagous Tetranychus urticae. However, the pesticide resistance mechanisms in M. occidentalis remain largely unknown. In other arthropods, members of the glutathione-S-transferase (GST), cytochrome P450 (CYP) and carboxyl/cholinesterase (CCE) gene superfamilies are involved in the diverse biological pathways such as the metabolism of xenobiotics (e.g. pesticides) in addition to hormonal and chemosensory processes. In the current study, we report the identification and initial characterization of 123 genes in the GST, CYP and CCE superfamilies in the recently sequenced M. occidentalis genome. The gene count represents a reduction of 35% compared to T. urticae. The distribution of genes in the GST and CCE superfamilies in M. occidentalis differs significantly from those of insects and resembles that of T. urticae. Specifically, we report the presence of the Mu class GSTs, and the J’ and J” clade CCEs that, within the Arthropoda, appear unique to Acari. Interestingly, the majority of CCEs in the J’ and J” clades contain a catalytic triad, suggesting that they are catalytically active. They likely represent two Acari-specific CCE clades that may participate in detoxification of xenobiotics. The current study of genes in these superfamilies provides preliminary insights into the potential molecular components that may be involved in pesticide metabolism as well as hormonal/chemosensory processes in the agriculturally important M. occidentalis.

Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure.

Glutathione S-transferases (GSTs) are a family of multifunctional phase II enzymes that are involved in the detoxification of exogenous and endogenous compounds. In this study, a full-length cDNA of Mu-class GST (PmMuGST) was isolated from the hepatopancreas of Penaeus monodon using rapid amplification of cDNA ends method. The full length cDNA of PmMuGST is 867 bp, contains an open read frame of 660 bp, and encodes a polypeptide of 219 amino acids with a molecular mass of 25.61 kDa and pI of 6.15. Sequence analysis indicated that the predicted protein sequence of PmMuGST was very similar to (86 %) that of Litopenaeus vannamei. A conserved domain of GST_N_Mu_like (PSSM: cd03075) and GST_C_family_superfamily_like (PSSM: cl02776) was indentified in PmMuGST. Real time quantitative RT-PCR analysis indicated that PmMuGST was present in all of the tested tissues. PmMuGST transcripts both in the hepatopancreas and in the muscle were significantly induced after 14 days of treatment with a low dosage of AFB1 (50 μg/kg) exposure and were significantly inhibited after 42 and 56 days of a high dosage of AFB1 (1000, 2500 μg/kg AFB1) exposure. Taken together, the Mu-class GST from P. monodon was inducible and was involved in the response to AFB1 exposure.

Characterisation of Dermanyssus gallinae glutathione S-transferases and their potential as acaricide detoxification proteins.

BackgroundGlutathione S-transferases (GSTs) facilitate detoxification of drugs by catalysing the conjugation of the reduced glutathione (GSH) to electrophilic xenobiotic substrates and therefore have a function in multi-drug resistance. As a result, knowledge of GSTs can inform both drug resistance in, and novel interventions for, the control of endo- and ectoparasite species. Acaricide resistance and the need for novel control methods are both pressing needs for Dermanyssus gallinae, a highly economically important haematophagous ectoparasite of poultry.MethodsA transcriptomic database representing D. gallinae was examined and 11 contig sequences were identified with GST BlastX identities. The transcripts represented by 3 contigs, designated Deg-GST-1, −2 and −3, were fully sequenced and further characterized by phylogenetic analysis. Recombinant versions of Deg-GST-1, −2 and −3 (rDeg-GST) were enzymically active and acaricide-binding properties of the rDeg-GSTs were established by evaluating the ability of selected acaricides to inhibit the enzymatic activity of rDeg-GSTs.Results6 of the identified GSTs belonged to the mu class, followed by 3 kappa, 1 omega and 1 delta class molecules. Deg-GST-1 and −3 clearly partitioned with orthologous mu class GSTs and Deg-GST-2 partitioned with delta class GSTs. Phoxim, permethrin and abamectin significantly inhibited rDeg-GST-1 activity by 56, 35 and 17 % respectively. Phoxim also inhibited rDeg-2-GST (14.8 %) and rDeg-GST-3 (20.6 %) activities.ConclusionsDeg-GSTs may have important roles in the detoxification of pesticides and, with the increased occurrence of acaricide resistance in this species worldwide, Deg-GSTs are attractive targets for novel interventions.

Substrate profiling of glutathione S-transferase with engineered enzymes and matched glutathione analogues.

The identification of specific substrates of glutathione S-transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G-W40A-R41A mutant (GST-KWR) of the mu-class glutathione S-transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH-R1) as the cosubstrate. The GSH-R1 conjugation products can react with an azido-based biotin probe for ready enrichment and MS identification. Proof-of-principle studies were carried to detect the products of GSH-R1 conjugation to 1-chloro-2,4-dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.

A mu class glutathione S-transferase from Manila clam Ruditapes philippinarum (RpGSTμ): Cloning, mRNA expression, and conjugation assays

Glutathione S-transferases (GSTs) are enzymes that catalyze xenobiotic metabolism in the phase II detoxification process. GSTs have a potential for use as indicators or biomarkers to assess the presence of organic and inorganic contaminants in aquatic environments. In this study, a full-length cDNA of a mu (μ) class GST (RpGSTμ) was identified from Manila clam (Ruditapes philippinarum) and biochemically characterized. The 1356 bp of the cDNA included an open reading frame of 651 bp encoding a polypeptide of 217 amino acid residues with a molecular mass of 25.04kDa and an estimated pI of 6.34. Sequence analysis revealed that the RpGSTμ possessed several characteristic features of μ class GSTs, such as a thioredoxin-like N-terminal domain containing binding sites for glutathione (GSH), a C-terminal domain containing substrate binding sites, and a μ loop. The recombinant RpGSTμ (rRpGSTμ) protein exhibited GSH-conjugating catalytic activity towards several substrates, and significantly strong activity was detected against 4-nitrophenethyl bromide (5.77±0.55) and 1-chloro-2,4-dinitrobenzene (CDNB, 3.19±0.05). Kinetic analysis as a function of GSH and CDNB concentrations revealed relatively low Km values of 1.03±0.46mM and 0.56±0.20mM, respectively, thereby indicating a GSH-conjugation attributed with high rates. The optimum pH and temperature for the catalytic activity of the rRpGSTμ protein were 7.7 and 37°C, respectively. The effect of two inhibitors, Cibacron blue and hematin, on the activity of rRpGSTμ was evaluated and the IC50 values of 0.65μM and 9μM, respectively, were obtained. While RpGSTμ transcripts were highly expressed in gills and hemocytes, a significant elevation in mRNA levels was detected in these tissues after lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (poly I:C) and live bacterial (Vibrio tapetis) challenges. These findings collectively suggest that RpGSTμ functions as a potent detoxifier of xenobiotic toxicants present in the aquatic environment, and that its mRNA expression could be modulated by pathogenic stress signal(s).