Glutathione S-transferase cDNA Research Articles

The Detection of Circulating Antigen Glutathione S-Transferase in Sheep Infected with Fasciola hepatica with Double-Antibody Sandwich Signal Amplification Enzyme-Linked Immunosorbent Assay

Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin–streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong’an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong’an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens.

Detoxification and antioxidant functions and regulatory mechanisms of two Delta-class GSTs in paddy crayfish (Procambarus clarkii) after imidacloprid stress

Glutathione S-transferases (GSTs) are phase II metabolic detoxification enzymes, which are widely found in organisms, and play an important role in helping organisms to resist toxic compounds. In this study, the two Delta-class GSTs cDNA sequences were cloned from Procambarus clarkii (designated as PcGSTD1 and PcGSTD2). Tissue specific expression profile showed that PcGST1,2 were expressed in all 6 tissues, with the highest expression in hepatopancreas. Subcellular localization assay showed that PcGSTD1, 2 were mainly expressed in the cytoplasm of HEK-293 T cells. Recombinant PcGSTD1, 2 showed the highest catalytic activity to the GST model substrate 1-chloro-2,4-dinitrobenzene (CDNB) at 20 and 30 °C, pH 8 and 7, respectively. The mRNA expression of PcGSTD1, 2 and the GSTs activity varied with the time of imidacloprid challenge. The BL21(DE3) expressing PcGSTD1, 2 proteins could more resistant to H2O2. The dsRNA experiments showed that PcKeap1b, PcNrf1, and PcMafK affected the transcription levels of PcGSTD1, 2. The GST-Pulldown results revealed that PcbZIP and PcMafK recombinant proteins could bind to each other in vitro. The gel mobility shift assay demonstrated that PcMafK recombinant protein had affinity with the promoter of PcGSTD2. The Dual luciferase assays analyzed the activity of the promoters after different truncations, the core region of PcGSTD1 promoter was at −440 bp to +54 bp, and that of PcGSTD2 promoter was between −1609∼−1125 bp. These results suggested that PcGSTD1, 2 respond positively to imidacloprid stress in P. clarkii, and the transcriptional expressions of PcGSTD1, 2 were influenced by the factors of PcKeap1b/PcNrf1/PcMafK.

Expression profile of a novel glutathione S-transferase gene in the marine polychaete Perinereis aibuhitensis in short-term responses to phenanthrene, fluoranthene, and benzo[α]pyrene

Polychaete worms can eliminate polycyclic aromatic hydrocarbons (PAHs) in environments through a mechanism that increases their water solubility. This detoxification starts with cytochrome P450 enzymes (CYPs) and then with glutathione S-transferases (GSTs). Here, a novel GST gene was identified and characterized from the widespread polychaete Perinereis aibuhitensis. The full-length cDNA of GST is 1544 bp and encodes 256 amino acids, belonging to the omega class. Gene expression patterns in P. aibuhitensis showed that its transcriptional level was positively correlated with the concentration of benzo[α]pyrene (0.5, 2, 4, and 8 μg/L) exposure but was negatively correlated with a PAH benzene ring after it was exposed to the same mass concentrations of fluoranthene (3.2 μg/L), phenanthrene (2.9 μg/L), and benzo[α]pyrene (4.0 μg/L) during the 14-day experimentation. These findings indicate that omega GST may play an important role in the phase II detoxification of PAHs in polychaete worms, and the persistence and bioavailability of PAHs may depend on benzene rings.

Effect of polyphenols extracted from Punica granatum and Acacia saligna plants on glutathione S-transferase of the cattle tick Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae).

Ticks are hematophageal ectoparasites that transport major pathogens around the world. Glutathione S-transferases (GST) are involved in resistance to acaricide and redox balancing during the life cycle of the tick. The inhibition of tick GST enzymes by certain phenolic compounds, such as phenolic acids and tannins, can be a promising approach to tick control. The objective of this study was to evaluate the effects of Punica granatum red peel and Acacia saligna leaf extracts on Rhipicephalus (Boophilus) annulatus GST activity in order to reduce the resistance of cattle to acaricide. The results showed that P. granatum ethanol extract (70%) contained the highest total phenol content (350 ± 1.2μM GAE g-1), the highest condensed tannin content (270 ± 1.3μM CE g-1) and the highest hydrolysable tannin content (70 ± 5.0μM TAE g-1). Adult immersion test with a dosage of 100mg ml-1 of A. saligna ethanol extracts had a significant mortality of 50% and 75% after 24h and 96h, respectively (p < 0.01). A simple and reproducible procedure was established to purify the whole R. annulatus GST (wRaGST) while a full-length cDNA of GST was cloned from a cDNA library of the local Egyptian cattle tick R. (B.) annulatus (rRaGST). Aqueous extracts of P. granatum inhibited both wRaGST and rRaGST with values of IC50 = 0.114 and 0.07µgml-1, respectively, compared to A. saligna extracts (IC50 values = 2.08 and 1.35µgml, respectively). These inhibitory effects were attributed to the presence of a high tannin concentration (≥ 80%). HPLC analysis indicated the presence of gallic acid and catechin in both extracts, in addition to the rutin, which was only observed in A. saligna extracts. The addition of a tannin inhibitor, polyethylene glycol, suggested the existence of other phenolic compounds in combination with catechins responsible for inhibiting the activity of these extracts. Non-competitive behaviour of catechins may be helpful in preventing, or at least delaying, the development of chemical acaricide resistancein R. annulatus.

Differential responses of a pi-class glutathione S-transferase (CnGSTp) expression and antioxidant status between golden and brown noble scallops under pathogenic stress

Glutathione S-transferases (GSTs) play important roles in immunity by protecting organisms against the damage of reactive oxygen species (ROS). In this study, a pi-class GST cDNA sequence was first cloned from noble scallop Chlamys nobilis (named CnGSTp). The full length cDNA of CnGSTp was 922 bp, encoding a cytosolic protein of 202 amino acids residues, with predicted molecular masses of 23.1 kDa. Then an acute Vibrio Parahaemolyticus challenge experiment was conducted by using the Golden and Brown noble scallops with different total carotenoids content (TCC), and CnGSTp expression level, TCC and ROS level was separately determined. The results showed that ROS and CnGSTp expression levels were significantly up-regulate under Vibrio Parahaemolyticus challenge than the control group (P < 0.05). The Golden scallops showed significantly higher CnGSTp expression level and lower ROS level in hemocytes than the Brown ones (P < 0.05). Moreover, there is a significantly positive correlation between TCC and ROS in the Golden scallops. The present results revealed that CnGSTp plays important roles in immune response and carotenoids play assistant roles in antioxidant defense system under pathogenic stress in the noble scallop.

RNA Interference to Control Asian Corn Borer Using dsRNA from a Novel Glutathione-S-Transferase Gene of Ostrinia furnacalis (Lepidoptera: Crambidae).

Glutathione-S-transferases (GST) comprise a multifunctional protein superfamily, which plays important roles as detoxifiers and antioxidants in insects. The GST in Asian corn borer has not been previously characterized. In this study, we cloned, characterized, and expressed the complete GST genes from the midgut of Asian corn borer. Furthermore, we designed htL4440-OfGST vector to exploit this gene for RNA interference (RNAi) strategy to control this pest. A complete GST cDNA sequence in Asian corn borer was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends technology. The gene was 887bp in length and contained a 705bp open reading frame and 5′ UTR and 3′ UTR lengths of 89 and 93bp, respectively. The putative sequence encoded a putative 234 amino acid residue peptide and had a predicted molecular weight of ~26kDa. The GST protein of Asian corn borer is hydrophilic and may have a 30 amino acid signal peptide with a cleavage site between L30 and K31. A recombination vector pET28a-OfGST was constructed for purification and antibody preparation. Western blotting analysis showed that this protein reached the maximum expression level around 24 h in Asian corn borer larvae fed the plant toxin 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one. A second vector, htL4440-OfGST, was constructed to generate the dsRNA of the GST gene. A larval feeding bioassay showed that the expressed dsRNA significantly reduced the detoxification ability of Asian corn borer larvae and increased mortality rate up to 54%. Our data indicated that GST plays very important roles in detoxifying in Asian corn borer and can be used as an RNAi method to control this pest in the field.

Multiple Glutathione S-Transferase Genes in Heortia vitessoides (Lepidoptera: Crambidae): Identification and Expression Patterns.

To elucidate the role of glutathione S-transferases (GSTs) in Heortia vitessoides Moore (Lepidoptera: Crambidae), one of the most destructive defoliating pests in Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae) forests, 16 GST cDNAs were identified in the transcriptome of adult H. vitessoides. All cDNAs included a complete open reading frame and were designated HvGSTd1–HvGSTu2. A phylogenetic analysis showed that the 16 HvGSTs were classified into seven different cytosolic classes; three in delta, two in epsilon, three in omega, three in sigma, one in theta, two in zeta, and two in unclassified. The expression patterns of these HvGSTs in various larval and adult tissues, following exposure to half the lethal concentrations (LC50s) of chlorantraniliprole and beta-cypermethrin, were determined using real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of the 16 HvGSTs were found to differ among various larval and adult tissues. Furthermore, the RT-qPCR confirmed that the transcription levels of nine (HvGSTd1, HvGSTd3, HvGSTe2, HvGSTe3, HvGSTo3, HvGSTs1, HvGSTs3, HvGSTu1, and HvGSTu2) and six (HvGSTd1, HvGSTd3, HvGSTe2, HvGSTo2, HvGSTs1, and HvGSTu1) HvGST genes were significantly higher in the fourth-instar larvae following exposure to the insecticides chlorantraniliprole and beta-cypermethrin, respectively. These genes are potential candidates involved in the detoxification of these two insecticides. Further studies utilizing the RNA interference approach are required to enhance our understanding of the functions of these genes in this forest pest.

Identification and characterization of two distinct sigma-class glutathione-S-transferase from freshwater bivalve Cristaria plicata.

Glutathione-S-transferases (GSTs) are multifunctional phase II detoxification enzymes that catalyze the attachment of electrophilic substrates to glutathione, and play an important role in protecting organisms against the toxicity of reactive oxygen species. In this study, two distinct sigma-class GST (CpGSTσ1 and 2) cDNA sequences were cloned from freshwater bivalve Cristaria plicata. The full length cDNA of CpGSTσ1 and 2 was 826 bp and 1609 bp, which encoded 213 and 248 amino acid residues, respectively. Their transcripts were expressed in all detected tissues and the highest expression level was in hepatopancreas from C. plicata. The expression level of CpGSTσ1 and 2 in hepatopancreas and hemocytes showed a significantly increased trend after bacterial challenge. The recombinant CpGSTσ1 was successfully expressed as a soluble form in Escherichia coli DE3. The specific activity of recombinase toward CDNB was 46.965 ± 0.082 μmol/min/mg, and its optimum temperature and pH was 37 °C and 9.0, respectively. The recombinant of CpGSTσ1 could bear 6 M urea and 8% SDS, when the concentration of urea was 8 M and its activity was only below 20%. The results might provide a better perspective on the mechanisms of resistance to bacterial infection in molluscs.

Cloning and characterization of two glutathione S-transferases cDNAs in Foc4 and expression under exogenous oxidative stress

In order to identify two putative glutathione S-transferase (GSTs) genes in Fusarium oxysporum f. sp. cubense race 4 (Foc4), cDNA sequences of the entire coding regions of the two genes were cloned from Foc4 using RT-PCR method. Subsequently, the two genes were named Fogst1 and Fogst2 respectively. The length of open reading frame of Fogst1 was 609 bp and encoded a protein including 202 amino acid residues, Fogst2 possessed an open reading frame with 693 bp which encoded a 230-amino acid protein. Phylogenetic analysis showed that Fogst1 belonged to sigma (σ) subtype members of the GSTs superfamily, and Fogst2 was a new member of an unknown subfamily in the GSTs superfamily. To verify the expression of Fogst1 and Fogst2, the recombinant prokaryotic expression vector pET28a-Fogst1 and pET28a-Fogst2 were constructed and transformed into Escherichia coli expression strain BL21(DE3). The soluble recombinant proteins Fogst1 and Fogst2 were obtained after being induced by IPTG. GSTs activity assays showed that both of the two recombinant proteins had specific activity with CDNB. For real time RT- PCR analysis, the mycelium samples of Foc4 were collected after treatment by H₂O₂ for 1, 5, 12, 24 hours. The results showed that the expression of Fogst1 and Fogst2 were significantly up-regulated in the first 5 hours, and then decreased and returned to normal level. These results suggested that Fogst1 and Fogst2 may be involved in the process of Foc4 resistance to exogenous oxidative stress.

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.

북방전복 (Haliotis discus hannai)에서 분리한 Glutathione S-transferase 유전자의 분자생물학적 고찰 및 발현분석

본 연구에서는 북방전복 (Haliotis discus hannai)의 대용량 염기서열 분석을 통해 GST유전자의 전장 cDNA를 동정하였다. 북방전복 GST 유전자의 총 길이는 1669 bp로 672 bp의 ORF는 총 223개의 아미노산을 코딩하고 있으며 등전점은 5.69, 분자량은 25.8 kDa으로 예측되었다. 북방전복 GST아미노산 서열은 둥근전복과 지중해 담치와 같은 패류의 GSTA와 가장 유사성이 높았으며 계통수 분석을 통해 GSTA와 하나의 그룹을 이루었다. 북방전복 GST에는 GSTA의 특징을 갖는 두 site (N-말단의 G-site, C-말단의 H-site)가 보존되어 있었고 효소활성과 구조 유지에 중요한 잔기가 종간에 매우 보존되어 있었다. 북방전복 GST 유전자의 mRNA는 관찰된 모든 조직에서 발현하고 있었으며, 특히 외투막, 아가미, 간췌장, 소화관에서 높은 발현이 확인되었다. 북방전복의 GST는 비브리오균을 인위감염 시킨 전복의 간췌장에서 감염 후 1시간 뒤 발현이 급격히 증가했다가 3시간까지 증가한 뒤 감소하였고, 혈구세포에서는 감염 3시간 경과 후 발현 정도가 최고로 증가했다가 감소하였다. 따라서 북방전복 GST는 alpha class GST의 특징을 가지며 병원체 감염에 따른 면역반응 조절에 관여할 것이라 생각되며 병원균 감염에 따른 바이오마커로 활용가능 할 것이라 예상된다. Glutathione S-transferases (GSTs) are a superfamily of detoxification enzymes that primarily catalyze the nucleophilic addition of reduced glutathione to both endogenous and exogenous electrophiles. In this study, we isolated and characterized a full-length of alpha class GST cDNA from the abalone (Haliotis discus hannai). The abalone GST cDNA encodes a 223-amino acid polypeptide with a calculated molecular mass of 25.8 kDa and isoelectric point of 5.69. Multiple alignments and phylogenetic analysis with the deduced abalone GST protein revealed that it belongs to the alpha class GSTs and showed strong homology with disk abalone (Haliotis discus discus) putative alpha class GST. Abalone GST mRNA was ubiquitously detected in all tested tissues. GST mRNA expression was comparatively high in the mantle, gill, liver, and digestive duct, however, lowest in the hemocytes. Expression level of abalone GST mRNA in the mantle, gill, liver, and digestive duct was 182.7-fold, 114.8-fold, 4675.8-fold, 406.1-fold higher than in the hemocytes, respectively. Expression level of abalone GST mRNA in the liver was peaked at 6 h post-infection with Vibrio parahemolyticus and decreased at 12 h post-infection. While the expression level of abalone GST mRNA in the hemocytes was drastically increased at 3 h post-infection with Vibrio parahemolyticus. These results suggest that abalone GST is conserved through evolution and may play roles similar to its mammalian counterparts.

Characterization of glutathione S-transferases from Sus scrofa, Cydia pomonella and Triticum aestivum: Their responses to cantharidin

Glutathione S-transferases (GSTs) play a key role in detoxification of xenobiotics in organisms. However, their other functions, especially response to the natural toxin cantharidin produced by beetles in the Meloidae and Oedemeridae families, are less known. We obtained GST cDNAs from three sources: Cydia pomonella (CpGSTd1), Sus scrofa (SsGSTα1), and Triticum aestivum (TaGSTf3). The predicted molecular mass is 24.19, 25.28 and 24.49kDa, respectively. These proteins contain typical N-terminal and C-terminal domains. Recombinant GSTs were heterologously expressed in Escherichia coli as soluble fusion proteins. Their optimal activities are exhibited at pH 7.0–7.5 at 30°C. Activity of CpGSTd1 is strongly inhibited by cantharidin and cantharidic acid, but is only slightly suppressed by the demethylated analog of cantharidin and cantharidic acid. Enzymatic assays revealed that cantharidin has no effect on SsGSTα1 activity, while it significantly stimulates TaGSTf3 activity, with an EC50 value of 0.3852mM. Activities of these proteins are potently inhibited by the known GST competitive inhibitor: S-hexylglutathione (GTX). Our results suggest that these GSTs from different sources share similar structural and biochemical characteristics. Our results also suggest that CpGSTd1 might act as a binding protein with cantharidin and its analogs.

Transcriptional responses of mu-, pi- and omega-class glutathione S-transferase genes in the hepatopancreas of Cipangopaludina cahayensis exposed to microcystin-LR

The glutathione S-transferases (GSTs) play important roles in detoxification of microcystins (MCs), but the responses of GSTs to MC-LR have not been well characterized in freshwater gastropod, Cipangopaludina cahayensis. In the present study, we cloned full-length cDNAs of mu- and pi-class GSTs (GSTM and GSTP) and partial cDNA of omega-class GST (GSTO), and determined the transcriptional responses of the three GST genes to different concentrations of MC-LR (0, 1, 10 and 100 μg/L) in C. cahayensis. The full-length cDNAs of GSTM and GSTP of C. cahayensis were 813 and 820 bp, containing an open reading frame (ORF) of 648 bp (encoding 215 amino acids) and 624 bp (encoding 207 amino acids), respectively. The mRNA expression of GSTM and GSTO significantly decreased after exposure to 10 μg/L MC-LR, and the mRNA expression of GSTP significantly decreased after 100 μg/L MC-LR exposure. This might contribute to the detoxication of MCs in C. cahayensis, which is consistent with its sedentary life and filter-feeder status. The mRNA expression of the three GST isoforms in C. cahayensis could be used as biomarkers for water contamination.

Identification and expression pattern of a glutathione S‐transferase in Echinochloa crus‐galli

SummaryPlant glutathione S‐transferase (GST) forms a major part of the herbicide detoxification enzyme network in plants. A GST cDNA was isolated from Echinochloa crus‐galli and characterised. The gene, designated EcGST1 (E. crus‐galli GeneBank no: JX518596), has a 684 bp open reading frame predicted to encode a 25 kD protein. Sequence alignment showed that EcGST1 is a GST homologue. Its expression in response to quinclorac treatment was monitored in seedlings (leaves and roots) and adult plants (leaves, roots, stems and seeds) of quinclorac‐resistant (R) and susceptible (S) biotypes of E. crus‐galli. EcGST1 expression was 1.5–3 times greater in the R plants than in the S plants. However, after exposure to quinclorac, the difference in the expression levels of EcGST1 in R plants, compared with S plants, increased to a ratio of 6–10. Enhanced EcGST1 levels should enable greater quinclorac detoxification following quinclorac stimulation in R plants. GST‐based metabolism may be partially responsible for resistance to quinclorac in E. crus‐galli. The results suggest a new resistance mechanism for this R biotype in Chinese rice fields.

Isolation and Characterization of a Theta Glutathione S-transferase Gene from Panax ginseng Meyer

Plants have versatile detoxification systems to encounter the phytotoxicity of the wide range of natural and synthetic compounds present in the environment. Glutathione S-transferase (GST) is an enzyme that detoxifies natural and exogenous toxic compounds by conjugation with glutathione (GSH). Recently, several roles of GST giving stress tolerance in plants have demonstrated, but little is known about the role of ginseng GSTs. Therefore, this work aimed to provide further information on the GST gene present in Panax ginseng genome as well as its expression and function. A GST cDNA (PgGST) was isolated from P. ginseng cDNA library, and it showed the amino acid sequence similarity with theta type of GSTs. PgGST in ginseng plant was induced by exposure to metals, plant hormone, heavy metals, and high light irradiance. To improve the resistance against environmental stresses, full-length cDNA of PgGST was introduced into Nicotiana tabacum. Overexpression of PgGST led to twofold increase in GST-specific activity compared to the non-transgenic plants, and the GST overexpressed plant showed resistance against herbicide phosphinothricin. The results suggested that the PgGST isolated from ginseng might have a role in the protection mechanism against toxic materials such as heavy metals and herbicides.

Transgenic alfalfa plants co-expressing glutathione S-transferase (GST) and human CYP2E1 show enhanced resistance to mixed contaminates of heavy metals and organic pollutants

Transgenic alfalfa plants simultaneously expressing human CYP2E1 and glutathione S-transferase (GST) were generated from hypocotyl segments by the use of an Agrobacterium transformation system for the phytoremediation of the mixed contaminated soil with heavy metals and organic pollutants. The transgenic alfalfa plants were screened by a combination of kanamycin resistance, PCR, GST and CYP2E1 activity and Western blot analysis. The capabilities of mixed contaminants (heavy metals-organic compounds) resistance of pKHCG transgenic alfalfa plants became markedly increased compared with the transgenic alfalfa plants expressing single gene (GST or CYP2E1) and the non-transgenic control plants. The pKHCG alfalfa plants exhibited strong resistance towards the mixtures of cadmium (Cd) and trichloroethylene (TCE) that were metabolized by the introduced GST and CYP2E1 in combination. Our results show that the pKHCG transgenic alfalfa plants have good potential for phytoremediation because they have cross-tolerance towards the complex contaminants of heavy metals and organic pollutants. Therefore, these transgenic alfalfa plants co-expressing GST and human P450 CDNAs may have a great potential for phytoremediation of mixed environmental contaminants.

A delta-class glutathione transferase from the Chinese mitten crab Eriocheir sinensis: cDNA cloning, characterization and mRNA expression

Glutathione-S-transferases (GSTs), a major superfamily of antioxidative enzymes, play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). The delta-class GST cDNA was cloned and sequenced by rapid amplification of cDNA ends (RACE) from the Chinese mitten crab, Eriocheir sinensis. The 938 bp E. sinensis GST gene, encoding a polypeptide of 216 amino acids, showed significant similarity to homologous genes in insects. The deduced amino acid sequence of the crab GST contains conserved features of this gene family including the G-site (1–82 aa, tripeptide glutathione binding site) in the N-terminal region and an H-site (88–213 aa, substrate binding site) in the C-terminus. Additionally, a kinase C phosphorylation site (ITI) and one putative N-linked glycosylation site (DNIT) for N-linked carbohydrate chains were also identified. Quantitative real-time RT-PCR (qRT-PCR) was employed to investigate the distribution of GST mRNA in different tissues and its temporal expression in haemocytes of crabs challenged with Aeromonas hydrophila. Different levels of GST mRNA expression were detected in haemocytes, hepatopancreas and muscle, while it was not detected in the gills, intestines and stomach. GST transcription was significantly induced in haemocytes at 6 h post-bacterial challenge ( P < 0.05) and dropped to basal levels at 12 h, presumably down-regulated by increased bacteremia by that time. These findings suggest that GST could play a critical role in immunity, and this positive feedback mechanism can allow for efficient activation of the early innate immune response to infection.

Two epsilon glutathione S-transferase cDNAs from the common cutworm, Spodoptera litura: Characterization and developmental and induced expression by insecticides

Two Spodoptera litura glutathione S-transferase cDNAs ( Slgste2 and Slgste3) which were cloned from a midgut cDNA, encoded two structurally distinct proteins with a predicted molecular mass of 25 and 24 kDa, respectively. Slgste2 and Slgste3 were single-copy genes in the S. litura genome and there was no intron within the genes. The transcripts and proteins of Slgste2 and Slgste3 were predominately present in the midgut of the 5th and 6th instar larvae. The apparent Vmax of the purified SlGSTE2 and SlGSTE3 recombinant proteins towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. Slgste2 expression was up-regulated by 1-naphthyl methylcarbamate (carbaryl), 1,1,1-trichloro-2,2-bis-(p-chlorophenyl) ethane (DDT), deltamethrin, tebufenozide (RH5992) and Bacillus thuringiensis (Bt), but not affected by malathion, while Slgste3 expression was slightly up-regulated by carbaryl, Bt and DDT, but not affected by RH5992, malathion and deltamethrin. The results suggest that Slgste2 and Slgste3 may play roles in detoxifying various insecticides in S. litura.

Glutathione S-transferase as a biomarker in the Antarctic bivalve Laternula elliptica after exposure to the polychlorinated biphenyl mixture Aroclor 1254

Glutathione S-transferases (GSTs) are a family of multifunctional enzymes involved in cellular detoxification that catalyze the attachment of electrophilic substrates to glutathione. Two classes of GSTs related to the rho and sigma classes of enzymes in Antarctic bivalves have been cloned from Laternula elliptica. The full-length cDNA of rho class GST (leGSTr) is 1530 bp in length and contains an open reading frame (ORF) of 672 bp encoding 223 amino acid residues. The deduced amino acid sequences of this gene have 41% and 40% identity to rho class GSTs from Ctenopharyngodon idella and Pleuronectes platessa, respectively. The sigma class GST (leGSTs) cDNA, however, is 1127 bp in length and contains an ORF of 696 bp encoding 231 amino acid residues. The deduced amino acid sequences share only 22% identity with sigma class GST from Xenopus laevis. The transcriptional expression of leGSTr, leGSTs, and leGSTp cloned in our previous study were examined using real-time polymerase chain reaction in response to exposure to a polychlorinated biphenyl (PCB) mixture. The expressions of these three GST transcripts were rapidly upregulated, although they showed different expression levels and patterns within each isoform. Moreover, leGSTs was the most upregulated in the gill and digestive gland in response to PCB exposure. The recombinant GSTs were highly expressed in transformed Escherichia coli, and their kinetic properties were studied with various substrates. As a result, the three classes of GSTs were found to have diverse biological functions and were responsible for different enzymatic features.

Molecular identification of glutathione S-transferase gene and cDNAs of two isotypes from northern quahog (Mercenaria mercenaria)

Glutathione S-transferases (GSTs) are universally xenobiotic detoxifying enzymes which have been shown to play a unique detoxifying role in northern quahogs. GST consists of two distinct domains: N-terminal domain which contributes residues to form G-site (GSH-binding site) and C-terminal domain which provides residues to form a hydrophobic H-site (second substrate-binding site). In this study, glutathione S-transferases (GSTs) gene and cDNAs of two isotypes from the northern quahog (Mercenaria mercenaria) were cloned and characterized for the first time. Two full-length GST cDNAs were obtained and named as GST Pi-1 and Pi-2, respectively. Both cDNAs have an open reading frame of 624 bp, which encodes a 207-amino acid protein. Multiple sequences alignment analysis shows that the deduced amino acid sequences of GST Pi-1 and Pi-2 have high homology with other Pi class GSTs. The evolutionary relationship assessment indicates that the two deduced amino acid sequences are closely related to Pi class GSTs. The GST Pi-1 gene is an intronless gene so that it has the same sequence as the transcript. The digested peptide fragments of the purified northern quahog GSTs were analyzed by tandem mass spectrometry and the results confirm the existence of the translation products of the GST Pi-1 and Pi-2 transcripts. The predicted three-dimensional structures of GST Pi-1 and Pi-2 showed that both proteins have two domains, N-terminal domain and C-terminal domain. The conserved phenylalanine-48 serving as the ball in ball and socket style interface in all reported Pi class GSTs was found in both proteins. These results suggest that GST Pi-1 and Pi-2 from the northern quahog belong to the Pi class GSTs which may be involved in polychlorinated biphenyl (PCB) dechlorination.