Sigma Glutathione S-transferases Research Articles

Genome-wide identification glutathione-S-transferase gene superfamily in Daphnia pulex and its transcriptional response to nanoplastics

Glutathione S-transferases (GSTs) are key multifunctional phase II detoxification enzymes involved in the regulation of growth, development, and stress responses. However, the knowledge of GSTs in the model invertebrate organism Daphnia pulex at the genomic level remains limited. In the present study, 35 GST genes were identified in D. pulex (Dp-GST), belonging to eight subfamilies, with the sigma, mu, and delta/epsilon subfamilies constituting approximately 29 %, 20 %, and 20 % of the GST superfamily, respectively. Chromosome tandem duplication of genes within the same subfamily was observed, which may be the main force driving GST expansion in D. pulex. DpGST genes showed different expression patterns in response to nanoplastic exposure for 96 h and 21 days. Some homologous GST genes in D. pulex showed similar expression patterns in response to nanoplastic exposure, likely owing to their unique motifs. For example, motif 9 is found in all delta/epsilon GST genes, whereas motifs 1, 2, 3, 5, and 7 are highly conserved in sigma GST genes. The characterization of D. pulex GSTs extending the knowledge of GST-mediated environmental contaminants, especially nanoplastics.

Insecticide resistance associated overexpression of two sigma GST genes assists Nilaparvata lugens to remedy oxidative stress from feeding on resistant rice variety

Insect glutathione S-transferases (GSTs) participate in detoxifying insecticides and plant metabolites in two different ways, metabolizing toxic components and remedying oxidative stress. Here in Nilaparvata lugens, a major insect pest on rice, the roles of cytosolic GSTs in resistance to insecticides and to plant defences were evaluated. The over-expression in four resistant strains indicated that NlGSTs1 and NlGSTs2 were essential to resistances to four test insecticides and H2O2 through an antioxidation mechanism. RNAi verified the antioxidation function of NlGSTs1 and NlGSTs2 in the resistances as a common mechanism, regardless of the structural differences among insecticides and H2O2. NlGSTs1 and NlGSTs2 also provided protection for N. lugens against rice defense by the same mechanism, reducing H2O2 levels when N. lugens were fed on the resistant rice variety Mudogo. The antioxidation activity of recombinant NlGSTs1 and NlGSTs2 is higher than their direct detoxification, which supported the ability of these two GSTs to remedy oxidative stress. For oxidative stress remediation as a common mechanism of NlGSTs1 and NlGSTs2 in both insecticide resistance and host adaptability, the development of insecticide resistance might enhance the ability of insects to remedy oxidative stress from feeding on resistant rice variety and thus to lower the resistance level of rice variety to N. lugens. The results call for careful assessment on N. lugens control when both insecticides and resistant rice variety are applied.

Transcriptome and enzyme activity analyses of tolerance mechanisms in pearl oyster (Pinctada fucata) under high-temperature stress

Pearl oyster (Pinctada fucata) is an economically valuable shellfish for seawater pearl production. However, many pearl oysters are dying as the global climate warms and seawater temperatures rise. The breeding of high temperature-resistant strains is therefore urgent. We performed transcriptome analysis to reveal the response mechanism of P. fucata to long-term high-temperature stress. Nanopore sequencing identified 1787 loci and revealed high-quality full-length isoforms for 17,803 novel splice isoforms. The large-scale study suggested differential patterns of alternative splicing (AS) regulation and transcriptional regulation under heat stress. There were more AS events in the high-temperature group (HT) than the normal temperature (NT) control group. In total, 44 upregulated and 33 downregulated differentially expressed genes (DEGs) were evident between NT and HT. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that DEGs were mainly associated with defence mechanisms, phagosome signalling, cytoskeleton, posttranslational modification, protein turnover and chaperones under heat stress. Nine DEGs (c-fox, heat shock protein 60, heat shock protein 90, fucolectin-1, dermatopontin, caveolin-1, ETS homologous factor, glutathione S-transferase sigma 3 and fatty acyl-CoA hydrolase precursor) showed complex expression pattern changes from 1 h to 30 days of high-temperature induction. Furthermore, heat stress resulted in irregular shell microstructures and impacted enzyme activity, exemplified by continuous upregulation of lactate dehydrogenase (LDH), indicating that the energy needs of P. fucata needs were met in part by anaerobic metabolism. Integrated analyses demonstrated that long-term high-temperature stress can significantly impact internal bodily functions, and disrupt physiological and biochemical processes. High-temperature stress induces pearl oysters to activate gene expression and thereby alter immune responses, respiratory metabolism, antioxidant systems, biomineralisation, and secondary metabolism. These pathways likely coordinate to support P. fucata physiology under high-temperature stress. The findings deepen our understanding of the molecular mechanisms underlying the responses to long-term high-temperature stress, and provide valuable information for future breeding of thermotolerant P. fucata strains.

Identification and characterization of the novel genes encoding glutathione S-transferases in Mytilus galloprovincialis

The superfamily of glutathione S-transferases (GST) plays an essential role in the xenobiotic metabolism, binding compounds to the glutathione, and is like a cell protector during the influence of various negative external factors. Nevertheless, there are very few works devoted to the investigation of these genes in marine invertebrates. Up to this time, only three classes of cytosolic GSTs for one of the leading commercial molluscs Mytilus galloprovincialis were described. We sequenced the whole transcriptome from the gill tissues and, using bioinformatic analysis, detected ten classes of glutathione S-transferases, which are expressed in the mussel M. galloprovincialis. For the first time, two subfamilies were described: mitochondrial GST (kappa class) and microsomal (MAPEG), as well as five classes of the family of cytosolic GSTs (mu, omega, rho, tau, theta). Omega and sigma GST classes might be rapidly regulated genes due to the lack of introns and this assumption was confirmed by the investigation of short-term hypoxia on M. galloprovincialis. Seven new classes of GST revealed a greater gene variety of this detoxifying enzyme in mussels than expected. The obtained nucleotide sequences are necessary for future investigations of GSTs expression in response to various external factors (pollution, oxygen starvation, infection, etc.).

Bioaccumulation and function analysis of glutathione S-transferase isoforms in Manila clam Ruditapes philippinarum exposed to different kinds of PAHs

This study analyzed the function of different glutathione S-transferase (GST) isoforms and detoxification metabolism responses in Manila clam, Ruditapes philippinarum, exposed to 4 kinds of polycyclic aromatic hydrocarbons (PAHs) single, and their mixtures for 15 days under laboratory conditions. 13 kinds of GSTs in R. philippinarum were classified, and the results of tissue distribution indicated that 12 kinds of GSTs (except GST sigma 3) expressed most in digestive glands. We detected the mRNA expression levels of aryl hydrocarbon receptor signaling pathway, and detoxification system in digestive glands of clams exposed to benzo[a]pyrene (BaP), chrysene (CHR), benzo[a]anthracene (BaA), benzo[b]fluoranthene (BbF), and BaP + CHR + BaA + BbF, respectively. Among these genes, we selected GST-sigma, GST-omega and GST-pi as potential indicators to BaP; GST-sigma, GST-A and GST-rho to CHR; GST-pi, GST-sigma, GST-A, GST-rho and GST-microsomal to BaA; GST-theta and GST-mu to BbF; while GST-pi and GST-mu to the mixture of BaP, CHR, BaA and BbF. Additionally, the bioaccumulation of PAHs in tissues increased remarkably over time, and showed an obvious dose-effect. Under the same concentration, the bioaccumulation in single exposure group was higher than that in mixture group, and the bioaccumulation of PAHs in tissues with different concentrations of stress was irregular. The results revealed the metabolic differences and bioaccumulation rules in clams exposed to four kinds of PAHs, and provided more valuable information for the PAHs risk assessment.

Characterization of a sigma class GST (GSTS6) required for cellular detoxification and embryogenesis in Tribolium castaneum

The sigma glutathione S-transferases (GSTSs) are a class of cytosolic glutathione S transferases (GSTs) that play important roles in antioxidant defense in insects, but the mechanisms by which GSTSs contribute to antioxidant activity remain unclear. Here, we isolated a GSTS (GSTS6) from Tribolium castaneum and explored its function. Homology and phylogenetic analysis revealed that TcGSTS6 shared high identity with other evolutionarily conserved GSTSs. The recombinant TcGSTS6 protein had strong activity toward cumene hydroperoxide and 4-hydroxynonenal but low activity toward the universal substrate 1-chloro-2,4-dinitrobenzene. Exposure to various types of oxidative stress, including heat, cold, UV and pathogenic microbes, significantly induced TcGSTs6 expression, which indicates that it is involved in antioxidant defense. Knockdown TcGSTs6 by using RNA interference (RNAi) caused reduced antioxidant capacity, which was accomplished by cooperating with other antioxidant genes. Moreover, treatment with various insecticides such as phoxim, lambda-cyhalothrin, dichlorvos and carbofuran revealed that TcGSTS6 plays an important role in insecticide detoxification. The RNAi results showed that TcGSTS6 is essential for embryogenesis in T. castaneum. Our study elucidates the mechanism by which a GSTS contributes to antioxidant activity and enhances our understanding of the functional diversity of GSTSs in insects.

The genome of the freshwater monogonont rotifer Brachionus angularis: Identification of phase I, II, and III detoxification genes and their roles in molecular ecotoxicology

Brachionus spp. rotifers, which are widely distributed in aquatic environments, have been proposed as model organisms for ecotoxicological studies. Although the genomes of several rotifers belonging to the genus Brachionus have been assembled, the genome for the freshwater rotifer Brachionus angularis remains unknown. In this study, we analyzed the whole-genome sequence of B. angularis, which revealed a total length of 56.5 Mb and 21 contigs. The N50 and the GC content were 5.42 Mb and 23.66%, respectively. A total of 13,952 genes were predicted. Of them, we identified the main detoxification-related gene families, including those for cytochrome P450, glutathione S-transferase (GST), and the ATP-binding cassette transporter. In comparison with other Brachionus rotifers, massive species-specific expansion in GST sigma genes was found in B. angularis. This whole-genome analysis of B. angularis provides a basis for molecular ecotoxicological studies and provides useful biological tools for comparative studies of the evolution of detoxification mechanisms in Brachionus spp.

Modulation of glutathione S-transferase and superoxide dismutase in response to heavy metals in brackish water flea Diaphanosoma celebensis

Superoxide dismutase (SOD) and glutathione S-transferase (GST) serve as biomarkers of oxidative stress in aquatic organisms. However, the modulatory effects of heavy metals on the expression of small crustaceans are yet unknown. The acute toxicity of inorganic arsenic (As), cadmium (Cd), and lead (Pb) to the brackish water flea Diaphanosoma celebensis (4 days old) was evaluated to determine their sublethal concentrations. Modulations in the activities and transcription of SODs (Cu/Zn-SOD and Mn-SOD) and GSTs (mu, kappa, theta, and sigma) in response to sublethal concentrations of heavy metals were investigated for 48 h. The 48-h median lethal concentration (LC50) was determined to be 2.62 mg/L, 2.60 mg/L, and 0.37 mg/L for Cd, As, and Pb, respectively, and the order of toxicity in this species was Pb > Cd = As. SOD and GST activities were induced after exposure to all metals; in particular, GST activity was sensitively enhanced upon As exposure. Real-time reverse-transcriptase polymerase chain reaction showed that GST mu and GST sigma were specific for As, and GST kappa and GST theta for Cd. Mn-SOD mRNA expression was more sensitive to As. These findings suggest that D. celebensis SOD and GST may be involved in the antioxidant defense mechanisms to heavy metals. These genes may serve as useful biomarkers for risk assessment of heavy metal pollution in aquatic ecosystems. This study may be helpful for the better understanding of the molecular mode of action of heavy metals in small crustaceans and provide preliminary data for future studies evaluating the combined effects of heavy metal mixture.

Generation of oxidative stress and induction of apoptotic like events in curcumin and thymoquinone treated adult Fasciola gigantica worms

Fasciolosis is a neglected tropical disease caused by the liver fluke Fasciola gigantica. The absence of successful vaccine and emerging resistance in flukes against the drug of choice, triclabendazole, has necessitated the search for alternatives including phyto-therapeutic approaches. Curcumin and thymoquinone, the active ingredients of Curcuma longa and Nigella sativa plants respectively, were first screened for their binding affinity with Glutathione-S-transferase (GST) molecule through in silico molecular docking followed by in vitro treatment of worms with varying concentrations of the test compounds.The in silico molecular docking of curcumin and thymoquinone with sigma GST revealed strong hydrogen bonding as well as hydrophobic interactions with high fitness scores but showing inter-specific differences. The in vitro treatment of F. gigantica worms with both curcumin and thymoquinone resulted in a significant increase in the generation of reactive oxygen species (ROS) whereas the level of reduced glutathione, a primary redox regulator, was found to be significantly decreased (p < 0.05). The two compounds not only inhibited the GST activity, which is an important detoxification enzyme and also a key drug/vaccine target for the control of fasciolosis but also significantly inhibited the activity of antioxidant enzymes glutathione peroxidase and glutathione reductase that are vital in maintenance of redox homeostasis. The immunohistochemistry performed using anti sigma GST polyclonal antibodies revealed that both the compounds used in the present study significantly reduced immunofluorescence in the vitellaria, developing eggs present in the ovary and the intestinal caecae indicating inhibition of GST enzyme in these regions of the worms. Further, following treatment with curcumin and thymoquinone, chromatin condensation and DNA fragmentation was also observed in F. gigantica worms.In conclusion, both curcumin and thymoquinone generated oxidative stress in the worms by production of ROS and significantly inhibiting their antioxidant and detoxification ability. The oxidative stress along with induction of apoptotic like events would compromise the survival ability of worms within the host. However, further studies are required to establish their anthelmintic potential alone and in combination with the commonly used anthelmintic drugs under in vivo conditions.

Genome-wide identification of the entire 90 glutathione S-transferase (GST) subfamily genes in four rotifer Brachionus species and transcriptional modulation in response to endocrine disrupting chemicals

Genome-wide identification of glutathione S-transferase (GST), a major phase II detoxification enzyme, was investigated in four different aquatic model rotifer species Brachionus koreanus, B. plicatilis, B. rotundiformis, and B. calyciflorus. GSTs are ubiquitous antioxidant enzymes that play versatile function including cellular detoxification, stress alleviation, and production of the radical conjugates. Among the four rotifers, B. rotundiformis was found with the least number of GST genes (total 19 GST genes), whereas the other three species shared 23 to 24 GST genes. Among the identified GST genes, belonging to the cytosolic GST superfamily, the expansion of GST sigma classes mainly occurs through tandem duplication, resulting in tandem-arrayed gene clusters on the chromosomes. Overall, the number of genes discovered in this study was highest in the sigma class, zeta, alpha, and omega in descending order. With integration of phylogenetic analysis and xenobiotic-mediated GST mRNA expression patterns along with previous enzymatic activities, the functional divergence among species-specific GST genes was clearly observed. This study covers full identification of GST classes in three marine rotifer and one fresh-water rotifer species and their important role in marine environmental ecotoxicology.

Glutathione S-Transferase Regulation in Calanus finmarchicus Feeding on the Toxic Dinoflagellate Alexandrium fundyense.

The effect of the dinoflagellate, Alexandrium fundyense, on relative expression of glutathione S-transferase (GST) transcripts was examined in the copepod Calanus finmarchicus. Adult females were fed for 5-days on one of three experimental diets: control (100% Rhodomonas spp.), low dose of A. fundyense (25% by volume, 75% Rhodomonas spp.), and high dose (100% A. fundyense). Relative expression of three GST genes was measured using RT-qPCR on days 0.5, 1, 2 and 5 in two independent experiments. Differential regulation was found for the Delta and the Sigma GSTs between 0.5 to 2 days, but not on day 5 in both experiments. The third GST, a microsomal, was not differentially expressed in either treatment or day. RT-qPCR results from the two experiments were similar, even though experimental females were collected from the Gulf of Maine on different dates and their reproductive output differed. In the second experiment, expression of 39 GSTs was determined on days 2 and 5 using RNA-Seq. Global gene expression analyses agreed with the RT-qPCR results. Furthermore, the RNA-Seq measurements indicated that only four GSTs were differentially expressed under the experimental conditions, and the response was small in amplitude. In summary, the A. fundyense diet led to a rapid and transient response in C. finmarchicus in three cytosolic GSTs, while a fourth GST (Omega I) was significantly up-regulated on day 5. Although there was some regulation of GSTs in response the toxic dinoflagellate, the tolerance to A. fundyense by C. finmarchicus is not dependent on the long-term up-regulation of specific GSTs.

Identification of glutathione S-transferase genes in Leptinotarsa decemlineata and their expression patterns under stress of three insecticides

Glutathione S-transferases (GSTs) is a family of multifunctional enzymes that are involved in detoxification of poisonous compounds. In the present paper, the Leptinotarsa decemlineata genome and transcriptome dataset were mined and 30 GST genes were identified. These GSTs belonged to cytosolic (29 genes) and microsomal (1 gene) classes. Among them 3 GSTs (LdGSTe2, LdGSTs4, and LdGSTo3) possessed splice variants. Of the 29 cytosolic LdGSTs, 3, 10, 5, 4, 4, and 1 members were classified as delta, epsilon, omega, sigma, theta, and zeta subclasses respectively, along with 2 unclassified genes. Phylogenetic analysis suggest that epsilon, omega and sigma subclasses appear to undergo species-specific bloom. Moreover, most epsilon, omega and sigma GSTs are tandemly arranged in three chromosome scaffolds. To find GST candidates involving in insecticide detoxification, we tested the mRNA levels of 20 GST transcripts under stress of cyhalothrin, fipronil or endosulfan. Out of them, LdGSTe2a, LdGSTe2b, LdGSTo5 and LdGSTt1 were significantly overexpressed after exposure to each of the three insecticides. Two other genes were respectively upregulated after cyhalothrin (LdGSTe10 and LdGSTu2) or endosulfan (LdGSTd1 and LdGSTu2) treatment. The diversified expression responses to insecticide exposure suggest that the LdGSTs may depend on a functionally complex system to detoxify different classes of insecticides. In addition, our findings provide a base for a better understanding of the evolution of insecticide resistance, and functional research on specific GST genes.

Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain.

Cloning and characterization of a novel sigma-like glutathione S-transferase from the giant panda parasitic nematode, Baylisascaris schroederi.

BackgroundBaylisascaris schroederi, an intestinal nematode of the giant panda, is the cause of the often fatal disease, baylisascariasis. Glutathione S-transferases (GSTs) are versatile enzymes that can affect parasite survival and parasite-host interactions and, are therefore, potential targets for the development of diagnostic tests and vaccines.MethodsIn this study, we identified a full-length cDNA that encoded a novel, secretory sigma-like GST (Bsc-GSTσ) from a B. schroederi-omic dataset. Following cloning and sequencing, sequence and structural analyses and comparative modeling were performed using online-bioinformatics and proteomics tools. The recombinant Bsc-GSTσ (rBsc-GSTσ) protein was prokaryotically expressed and then used to detect antigenicity and reactivity using immunoblotting assays. In addition, the native protein in female adult B. schroederi was located via immunofluorescence techniques, while the preliminary ELISA-based serodiagnostic potential of rBsc-GSTσ was assessed in native and infected mouse sera.ResultsBsc-GSTσ contained a 621-bp open reading frame that encoded a polypeptide of 206 amino acids with two typical sigma GST domain profiles, including a GST_N_Sigma_like at the N-terminus and a GST_C_Sigma_like at the C-terminus. The presence of an N-terminal signal sequence indicated that Bsc-GSTσ was a secretory protein. Sequence alignment and phylogenetic analyses showed that Bsc-GSTσ was a nematode-specific member of the Sigma class GSTs and shared the closest genetic distance with its homologue in Ascaris suum. Further comparative structure analyses indicated that Bsc-GSTσ possessed the essential structural motifs (e.g., βαβαββα) and the consensus secondary or tertiary structure that is typical for other characterized GSTσs. Immunolocalization revealed strong distributions of native Bsc-GSTσ in the body hypodermis, lateral chords, gut epithelium, gut microvilli, oviduct epithelium, and ovaries of adult female worms, similar to its homologue in A. suum. Building on good immunogenic properties, rBsc-GSTσ-based ELISA exhibited a sensitivity of 79.1% and a specificity of 82.0% to detect anti-B. schroederi IgG antibodies in the sera of experimentally infected mice.ConclusionThis study presents a comprehensive demonstration of sequence and structural-based analysis of a new, secretory sigma-like GST from a nematode, and its good serodiagnostic performance suggests that rBsc-GSTσ has the potential to detect B. schroederi and, therefore, could be used to develop an ELISA-based serological test to diagnose baylisascariasis in giant pandas.

Molecular screening of insecticides with sigma glutathione S-transferases (GST) in cotton aphid Aphis gossypii using docking.

Glutathione S-transferases (GSTs) are one of the major families of detoxifying enzymes that detoxifies different chemical compounds including insecticides in different insect species. Among the GST subclasses, sigma GSTs are found to be the most abundant and conserved among different insect orders. These GSTs are found to play an important role in lipid peroxidation as well as detoxification. Cotton aphid, Aphis gossypii is the most damaging sucking pest with a wide range of hosts and vector of more than 50 plant viruses. Resistance to insecticides in A. gossypii is reported in India and in other countries. Glutathione S transferases (GSTs), an oxidative enzyme is understood to have a role in insecticide resistance and plant resistance breakdown. In relation to this, we have focused on the sigma 1 (GenBank Accession No: JN989964.1) and sigma 2 (GenBank Accession No: JN989965.1) GSTs of A. gossypii and their interaction with plant natural compounds and insecticides. Molecular screening of different insecticides (Chlorphinamidine, Mevinphos, Nitenpyrum, Piperonyl butoxide, Tetrachlorovinphos, Pyrethrins, Resmetrin, Pirimicarb and Dinotefuran) and known plant derived natural compounds (Catechin, Gossypol, Myrcene, Kaempferol, P-coumaric acid, Quercetin, Tannins, α-mangostin, Capsaicin, Cinnamic acid, Citronellal, Curcumin, Dicumarol, Ellagic acid, Eugenol, Geriniol, Isoeugenol, Juglone, Menadione, Methyl jasmonate, Morin, Myricetin, Myristicin, Piperine, Plumbagin, Tangitinin C, Thymol, Vanillin, Alpha pipene, α-terpineol Apigenin and β-Caryophyllene) with sigma 1 and sigma 2 GST protein models was completed using Maestro 9.3 (Schrodinger, USA). This exercise showed the binding of piperonyl butoxide with sigma 1 GST and tannin with sigma 2 GST for further consideration.

Interaction of Omega, Sigma, and Theta glutathione transferases with p38b mitogen-activated protein kinase from the fruit fly, Drosophila melanogaster.

Glutathione S-transferases (GSTs) are a diverse family of phase II detoxification enzymes found in almost all organisms. Besides playing a major role in the detoxification of xenobiotic and toxic compounds, GSTs are also involved in the regulation of mitogen activated protein (MAP) kinase signal transduction by interaction with proteins in the pathway. An in vitro study was performed for Theta, Omega, Sigma GSTs and their interaction with MAP kinase p38b protein from the fruit fly Drosophila melanogaster Meigen (Diptera: Drosophilidae). The study included the effects of all five Omega class GSTs (DmGSTO1, DmGSTO2a, DmGSTO2b, DmGSTO3, DmGSTO4), all five Theta class GSTs (DmGSTT1, DmGSTT2, DmGSTT3a, DmGSTT3b, DmGSTT4), and one Sigma class glutathione transferase on the activity of Drosophila p38b, including the reciprocal effect of this kinase protein on glutathione transferase activity. It was found that DmGSTT2, DmGSTT3b, DmGSTO1, and DmGSTO3 activated p38b significantly. Substrate specificities of GSTs were also altered after co-incubation with p38b. Although p38b activated DmGSTO1, DmGSTO2a, and DmGSTT2, it inhibited DmGSTT3b and DmGSTO3 activity toward xenobiotic and physiological substrates tested. These results suggest a novel link between Omega and Theta GSTs with the p38b MAP kinase pathway.

Characterization and functional analysis of four glutathione S-transferases from the migratory locust, Locusta migratoria.

Glutathione S-transferases (GSTs) play an important role in detoxification of xenobiotics in both prokaryotic and eukaryotic cells. In this study, four GSTs (LmGSTd1, LmGSTs5, LmGSTt1, and LmGSTu1) representing different classes were identified from the migratory locust, Locusta migratoria. These four proteins were heterologously expressed in Escherichia coli as soluble fusion proteins, purified by Ni2+-nitrilotriacetic acid agarose column and biochemically characterized. LmGSTd1, LmGSTs5, and LmGSTu1 showed high activities with 1-chloro-2, 4-dinitrobenzene (CDNB), detectable activity with p-nitro-benzyl chloride (p-NBC) and 1, 2-dichloro-4-nitrobenzene (DCNB), whereas LmGSTt1 showed high activity with p-NBC and detectable activity with CDNB. The optimal pH of the locust GSTs ranged between 7.0 to 9.0. Ethacrynic acid and reactive blue effectively inhibited all four GSTs. LmGSTs5 was most sensitive to heavy metals (Cu2+ and Cd2+). The maximum expression of the four GSTs was observed in Malpighian tubules and fat bodies as evaluated by western blot. The nymph mortalities after carbaryl treatment increased by 28 and 12% after LmGSTs5 and LmGSTu1 were silenced, respectively. The nymph mortalities after malathion and chlorpyrifos treatments increased by 26 and 18% after LmGSTs5 and LmGSTu1 were silenced, respectively. These results suggest that sigma GSTs in L. migratoria play a significant role in carbaryl detoxification, whereas some of other GSTs may also involve in the detoxification of carbaryl and chlorpyrifos.

Molecular Characterization of the Sigma Class Gutathione S-Transferase From Chilo suppressalis and Expression Analysis Upon Bacterial and Insecticidal Challenge

The insect glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotic compounds and are related to insecticides resistance. The full-length cDNA sequences encoding the sigma class GST protein (CsGSTsigma) was cloned from the Asiatic rice borer, Chilo suppressalis (Walker), one of the most important rice pests in Asia. The comparison of amino acid sequences showed that CsGSTsigma is highly similar to the sigma GST isolated from the domestic silkworm, Bombyx mori (L.). A homology model of CsGSTsigma was constructed and its binding environment for GSH is identical to that in the equivalent site of sigma GST from the fruit fly, Drosophila melanogaster Meigen. The developmental changes of the relative mRNA expression levels of CsGSTsigma were examined in Asiatic rice borer, and the highest expression level of this gene is in adult followed by the third-instar larvae stage. Furthermore, one gram-positive bacterium and two chemical insecticides were found to be able to induce the increasing expression of CsGSTsigma, suggesting that CsGSTsigma might work as an antioxidant enzyme to against the negative effects caused by both pathogens and xenobiotics.

Heterologous expression and characterization of a sigma glutathione S-transferase involved in carbaryl detoxification from oriental migratory locust, Locusta migratoria manilensis (Meyen)

Glutathione S-transferases (GSTs) play a major role in detoxification of xenobiotics and resistance to insecticides in insects. In the present study, a sigma-class GST gene (LmGSTs3) was identified from the locust, Locusta migratoria manilensis. Its full-length cDNA sequence is 828bp containing an open reading frame (ORF) of 612bp that encodes 204 amino acid residues. The predicted protein molecular mass and pI are 23.4kDa and 7.62, respectively. Recombinant LmGSTs3 was heterologously expressed in Escherichia coli as a soluble fusion protein. Its optimal activity was observed at pH 8.0. Incubation for 30min at temperatures below 40°C scarcely affected activity. The LmGSTs3 at pH values between 4.0 and 11.0 retained more than 80% of its original activity. Ethacrynic acid and cibacron blue were very effective inhibitors of LmGSTs3 with I50-values 1.7 and 3.7μM, respectively. In response to heavy metal (CuSO4, CdCl2) exposure there was a concentration-dependent and time-dependent decrease in activity. The nymph mortalities after carbaryl treatment increased 38.7% after LmGSTs3 were silenced. These results suggest that LmGSTs3 may be involved in carbaryl detoxification in L. migratoria manilensis.

Molecular cloning and differential expression patterns of sigma and omega glutathione S-transferases from Venerupis philippinarum to heavy metals and benzo[a]pyrene exposure

Glutathione S-transferases (GSTs) are a class of enzymes that facilitate the detoxification of xenobiotics, and also play important roles in antioxidant defense. We identified two glutathione S-transferase isoforms (VpGSTS, sigma GST; VpGSTO, omega GST) from Venerupis philippinarum by RACE approaches. The open reading frames of VpGSTS and VpGSTO were of 612 bp and 729 bp, encoding 203 and 242 amino acids with an estimated molecular mass of 22.88 and 27.94 kDa, respectively. The expression profiles of VpGSTS and VpGSTO responded to heavy metals and benzo[a]pyrene (B[a]P) exposure were investigated by quantitative real-time RT-PCR. The expression of VpGSTS and VpGSTO were both rapidly up-regulated, however, they showed differential expression patterns to different toxicants. Cd displayed stronger induction of VpGSTS expression with an approximately 12-fold increase than that of VpGSTO with a maximum 6.4-fold rise. Cu exposure resulted in similar expression patterns for both VpGSTS and VpGSTO. For B[a]P exposure, the maximum induction of VpGSTO was approximately two times higher than that of VpGSTS. Altogether, these findings implied the involvement of VpGSTS and VpGSTO in host antioxidant responses, and highlighted their potential as a biomarker to Cd and B[a]P exposure.