日本血吸虫中国大陆株26kD GST基因在大肠杆菌中的高效表达

用大肠杆菌高效表达日本血吸虫中国大陆株 2 6k D GST基因 (Sj2 6)并观察表达产物诱导的免疫保护效果 .将 Sj2 6基因亚克隆至 p ET2 8b(+)中构建重组表达质粒 Sj2 6/ p ET,转化大肠杆菌 BL 2 1 (DE3) .Sj2 6在诱导条件下及未诱导条件下均获得高效表达 .表达产物 (r Sj2 6GST)以包含体形式表达 ,分子量为 2 8k D左右 ,能用 6× His亲和层析柱纯化 ,纯化产量为 55～ 60 mg/ L大肠杆菌培养物 .免疫印迹及 ELSA实验证实 r Sj2 6GST具有良好抗原性 .用 r Sj2 6GST不加佐剂直接背部皮下免疫昆明系小鼠 ,获得了 1 9.6% (P<0 .0 5)的减虫率及 31 .9% (P<0 .0 5)的成熟虫体减虫率 ;且免疫组检获的虫体中未成熟虫体的比例为 32 % (66/ 2 0 6) ,明显高于对照组的 1 9.6% (56/2 85) (P<0 .0 1 ) ,说明 r Sj2 6GST免疫不仅可诱导抗日本血吸虫的部分攻击感染作用 ,而且能抑制部分虫体的发育 .     

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.     

1.3-A resolution structure of human glutathione S-transferase with S-hexyl glutathione bound reveals possible extended ligandin binding site

Cytosolic glutathione S-transferases (GSTs) play a critical role in xenobiotic binding and metabolism, as well as in modulation of oxidative stress. Here, the high-resolution X-ray crystal structures of homodimeric human GSTA1-1 in the apo form and in complex with S-hexyl glutathione (two data sets) are reported at 1.8, 1.5, and 1.3A respectively. At this level of resolution, distinct conformations of the alkyl chain of S-hexyl glutathione are observed, reflecting the nonspecific nature of the hydrophobic substrate binding site (H-site). Also, an extensive network of ordered water, including 75 discrete solvent molecules, traverses the open subunit-subunit interface and connects the glutathione binding sites in each subunit. In the highest-resolution structure, three glycerol moieties lie within this network and directly connect the amino termini of the glutathione molecules. A search for ligand binding sites with the docking program Molecular Operating Environment identified the ordered water network binding site, lined mainly with hydrophobic residues, suggesting an extended ligand binding surface for nonsubstrate ligands, the so-called ligandin site. Finally, detailed comparison of the structures reported here with previously published X-ray structures reveal a possible reaction coordinate for ligand-dependent conformational changes in the active site and the C-terminus.     

Role of mutation Y6F on the binding properties of Schistosoma japonicum glutathione S-transferase

The role of the hydroxyl group of tyrosine 6 in the binding of Schistosoma japonicum glutathione S-transferase has been investigated by isothermal titration calorimetry (ITC). A site-specific replacement of this residue with phenylalanine produces the Y6F mutant, which shows negative cooperativity for the binding of reduced glutathione (GSH). Calorimetric measurements indicated that the binding of GSH to Y6F dimer is enthalpically driven over the temperature range investigated. A concomitant net uptake of protons upon binding of GSH to Y6F mutant was detected carrying out calorimetric experiments in various buffer systems with different heats of ionization. The entropy change is favorable at temperatures below 26 °C for the first site, being entropically favorable at all temperatures studied for the second site. The enthalpy change of binding is strongly temperature-dependent, arising from a large negative ΔC°_p1=−3.45±0.62 kJ K⁻¹ mol⁻¹ for the first site, whereas a small ΔC°_p2=−0.33±0.05 kJ K⁻¹ mol⁻¹ for the second site was obtained. This large heat capacity change is indicative of conformational changes during the binding of substrate.     

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.     

Characterization of glutathione S‐transferase in the saturniid moth,Samia Cynthia pryeri (Lep.: Saturniidae)

An enzyme, which possesses glutathione S‐transferase (GST) activity, has been found in the midgut of the saturniid moth, Samia cynthia pryeri. The enzyme was initially purified into homogeneity by ammonium sulphate fractionation, affinity chromatography, and ion‐exchange chromatography. The resulting enzyme revealed a single band with a molecular mass of 23 kDa by sodium dodecyl sulfate polyacrylamide electrophoresis under reduced conditions. When tested with 1‐chloro‐2,4‐dinitrobenzene, a universal substrate of GST, the purified remnants had an optimum pH of 8.0 for enzymatic activity, and was fairly stable at pH 5–9 and at temperatures below 40°C. The enzyme was also responsive to 4‐hydroxynonenal, a cytotoxic lipid‐peroxidation product. The present GST was inhibited by organophosphorus and pyrethroid insecticides including fenitrothion, permethrin and deltamethrin.     

Influence of the H‐site residue 108 on human glutathione transferase P1‐1 ligand binding: Structure‐thermodynamic relationships and thermal stability

The effect of the Y108V mutation of human glutathione S‐transferase P1‐1 (hGST P1‐1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D‐structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H‐site) and glutathione binding site (G‐site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H‐site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (K_d ～ 0.5 μM) when compared with those of the parent compounds, K ～ 13 μM, K ～ 25 μM. The EA moiety of the conjugate binds in the H‐site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔC_p values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information.     

Molecular cloning and enzymatic expression of the 28-kDa glutathione S-transferase of Schistosoma japonicum: evidence for sequence variation but lack of consistent vaccine efficacy in the murine host

Glutathione S-transferases (GSTs) have long been regarded as attractive vaccine (and drug) targets in schistosomes due to their suspected role in detoxification processes. Indeed, the 28-kDa GST of Schistosoma mansoni (SmGST28) has proven efficacy as an antigen for protective immunity reducing worm burden, female fecundity and egg viability. In contrast, the vaccinating effects of the bacterial expressed homologue of Philippine S. japonicum (SjpGST28) have proved disappointing, possibly because this recombinant form was an incomplete sequence, lacking five N-terminal amino acids which may have affected its vaccination efficacy. Here we describe the cloning and functional enzymatic expression of a complete cDNA encoding SjpGST28. We report also on the immunogenicity and vaccine efficacy of this molecule as a purified recombinant protein and as a DNA plasmid vaccine in the murine model. We further describe the cloning of several complete cDNAs encoding the Chinese homologue of SjpGST28 and the identification of 3 SjcGST28 sequence variants which are probably encoded by distinct alleles.     

Structural and catalytic role of two conserved tyrosines in Delta-class glutathione S-transferase from Locusta migratoria

Glutathione S-transferases (GSTs) are an important family of detoxifying enzymes and play a key role in pesticide resistance in the insect. Tyrosine is essential for its detoxification function. In the present study, two conserved tyrosine residues are located at positions 108 and 116 in H-site of LmGSTD1. To elucidate how the two residues participate in the catalytic process and keeping structural stability, four mutants, Y108A, Y108E, Y116A, and Y116E, were generated. It was found that the four mutants affected the specific activity of LmGSTD1 in various degrees, depending on the types of substrate and reaction mechanism. Steady-state kinetics assay revealed that Y108E and Y116E had a significant influence on GSH-binding ability, which indicates the two tyrosine residues of H-site contribute to topology rearrangement of G-site. Both Y116A and Y116E exhibited lower CDNB-binding affinity, suggesting that Y116 takes part in hydrophobic substrate binding. The thermostability assay, intrinsic, and 8-anilino-1-naphthalenesulfonic acid (ANS) florescence results showed that the two tyrosine residues were involved in regulation of active-site conformation. Finally, homology modeling provided evidence that the two tyrosines in H-site participate in hydrophobic substrate binding. Furthermore, Y108 is closer to the S atom of S-hexylglutathione. In conclusion, the two tyrosines in LmGSTD1 are important residues in both the catalytic process and protein stability.     

Diuretic drug binding to human glutathione transferase P1‐1: potential role of Cys‐101 revealed in the double mutant C47S/Y108V

The diuretic drug ethacrynic acid (EA), both an inhibitor and substrate of pi class glutathione S‐transferase (GST P1‐1), has been tested in clinical trials as an adjuvant in chemotherapy. We recently studied the role of the active site residue Tyr‐108 in binding EA to the enzyme and found that the analysis was complicated by covalent binding of this drug to the highly reactive Cys‐47. Previous attempts to eliminate this binding by chemical modification yielded ambiguous results and therefore we decided here to produce a double mutant C47S/Y108V by site directed mutagenesis and further expression in Escherichia coli and the interaction of EA and its GSH conjugate (EASG) examined by calorimetric studies and X‐ray diffraction. Surprisingly, in the absence of Cys‐47, Cys‐101 (located at the dimer interface) becomes a target for modification by EA, albeit at a lower conjugation rate than Cys‐47. The Cys‐47 → Ser mutation in the double mutant enzyme induces a positive cooperativity between the two subunits when ligands with affinity to G‐site bind to enzyme. However, this mutation does not seem to affect the thermodynamic properties of ligand binding to the electrophilic binding site (H‐site) and the thermal or chemical stability of this double mutant does not significantly affect the unfolding mechanism in either the absence or presence of ligand. Crystal structures of apo and an EASG complex are essentially identical with a few exceptions in the H‐site and in the water network at the dimer interface. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of putative hydrophobic substrate binding site residues of a Delta class glutathione transferase from Anopheles dirus

To date, investigations of the hydrophobic substrate site of the insect Delta class glutathione transferase are limited in number. In the present study, putative hydrophobic site residues of AdGSTD4-4 have been proposed and characterized. These residues are Gln-112, Thr-174, Phe-212, Arg-214, Tyr-215 and Phe-216. It was found that Gln-112 does not contribute significantly to the catalytic properties of AdGSTD4-4. Arg-214, Tyr-215 and Phe-216 made contributions to catalytic properties and the rate-limiting step. Thr-174 and Phe-212 appeared to be important in enzymatic catalysis by stabilizing the active site β1-α1 loop on which the critical catalytic residue Ser-9 is located. The aromatic Phe-212 pi cloud appears to be important for interactions with its hydrophobic size representing an almost equally important factor. The data suggests that these residues are not directly involved in catalysis but exert their influence through secondary interactions. In addition, active site rearrangements occur to bring different residues into play even for conjugation through the same mechanisms. Therefore, due to the conformational rearrangements topologically equivalent residues observed in crystal structures may not perform equivalent roles in catalysis in different GST classes.     

Structure of a Drosophila sigma class glutathione S-transferase reveals a novel active site topography suited for lipid peroxidation products

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75A resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.     

Fasciola gigantica and Schistosoma mansoni: vaccine potential of recombinant glutathione S-transferase (rFgGST26) against infections in mice

Recombinant Fasciola gigantica glutathione S-transferase (rFgGST26) was expressed in Escherichia coli. This protein had 86% and 56% sequence identity with 26 kDa GST from Fasciolahepatica and Schistosoma mansoni, respectively. Polyclonal antibody raised in ICR mice against rFgGST26 recognized immunoblotted 26 kDa native GSTs from F. gigantica and S. mansoni. rFgGST26 was used as a vaccine in combination with Freund’s adjuvant to evaluate the induction of immune responses and protection against F. gigantica and S. mansoni infection in mice. Mice were immunized via subcutaneous (s.c.), intramuscular (i.m.) or intradermal (i.d.) routes. Strong protection (77-84%) against F. gigantica was observed in all routes. Immunization via s.c. route induced immune response with IgG1 isotype predominating, while i.m. and i.d. routes resulted in mixed IgG1/IgG2a immune responses. Passive intraperitoneal transfer of IgG1 predominating antisera from s.c. rFgGST26-immunized donors to naïve recipient mice resulted in 47% protection against F. gigantica infection. This suggests that the mechanism of resistance depends on the presence of specific antibody against rFgGST26. Immunization with rFgGST26 via i.m. and i.d. routes resulted in significant cross protection (55%) against S. mansoni infection in the i.d. route with mixed IgG1/IgG2a response with IgG1 isotype predominating. This indicated that rFgGST26 is a good vaccine candidate against F. gigantica in mice and could also provide cross protection against S. mansoni.     

The evolution of catalytic efficiency and substrate promiscuity in human theta class 1-1 glutathione transferase

Theta class glutathione transferases (GST) from various species exhibit markedly different catalytic activities in conjugating the tripeptide glutathione (GSH) to a variety of electrophilic substrates. For example, the human theta 1-1 enzyme (hGSTT1-1) is 440-fold less efficient than the rat theta 2-2 enzyme (rGSTT2-2) with the fluorogenic substrate 7-amino-4-chloromethyl coumarin (CMAC). Large libraries of hGSTT1-1 constructed by error-prone PCR, DNA shuffling, or saturation mutagenesis were screened for improved catalytic activity towards CMAC in a quantitative fashion using flow cytometry. An iterative directed evolution approach employing random mutagenesis in conjunction with homologous recombination gave rise to enzymes exhibiting up to a 20,000-fold increase in k(cat)/K(M) compared to hGSTT1-1. All highly active clones encoded one or more mutations at residues 32, 176, or 234. Combinatorial saturation mutagenesis was used to evaluate the full complement of natural amino acids at these positions, and resulted in the isolation of enzymes with catalytic rates comparable to those exhibited by the fastest mutants obtained via directed evolution. The substrate selectivities of enzymes resulting from random mutagenesis, DNA shuffling, and combinatorial saturation mutagenesis were evaluated using a series of distinct electrophiles. The results revealed that promiscuous substrate activities arose in a stochastic manner, as they did not correlate with catalytic efficiency towards the CMAC selection substrate. In contrast, chimeric enzymes previously constructed by homology-independent recombination of hGSTT-1 and rGSTT2-2 exhibited very different substrate promiscuity profiles, and showed a more defined relationship between evolved and promiscuous activities.     

Molecular cloning and characterization of a sigma‐class glutathione S‐Transferase from the freshwater mussel Hyriopsis cumingii

A full‐length cDNA of a sigma‐like glutathione S‐transferase (GST) was identified from Hyriopsis cumingii (HcGSTS). The deduced amino acid sequence of HcGSTS was found to comprise 203 amino acid residues and to contain the distinct highly conserved glutathione binding site of N‐terminal and the relatively diverse substrate binding site of C‐terminal. Alignment analysis and phylogenetic relationship suggested that the HcGSTS is a sigma‐class GST. The mRNA of HcGSTS was constitutively expressed in all tested tissues, the strongest expression being in the hepatopancreas. The mRNA expression of HcGSTS was significantly up‐regulated (P < 0.05) in all assessed tissues after stimulation of the mussels with peptidoglycan (PGN) and LPS, the only exception being when the gills were challenged with PGN. The expression of HcGSTS mRNA in kidney and foot was also significantly up‐regulated (P < 0.05) by microcystin‐LR. Recombinant HcGSTS exhibited high activity towards the substrate 1‐chloro‐2,4‐dinitrobenzene. The optimal pH was 8.0 and temperature 35 °C.     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

The combined use of photoaffinity labeling and surface plasmon resonance‐based technology identifies multiple salicylic acid‐binding proteins

Salicylic acid (SA) is a small phenolic molecule that not only is the active ingredient in the multi‐functional drug aspirin, but also serves as a plant hormone that affects diverse processes during growth, development, responses to abiotic stresses and disease resistance. Although a number of SA‐binding proteins (SABPs) have been identified, the underlying mechanisms of action of SA remain largely unknown. Efforts to identify additional SA targets, and thereby elucidate the complex SA signaling network in plants, have been hindered by the lack of effective approaches. Here, we report two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance‐based technology to identify and evaluate candidate SABPs from Arabidopsis. Using these approaches, multiple proteins, including the E2 subunit of α‐ketoglutarate dehydrogenase and the glutathione S‐transferases GSTF2, GSTF8, GSTF10 and GSTF11, were identified as SABPs. Their association with SA was further substantiated by the ability of SA to inhibit their enzymatic activity. The photoaffinity labeling and surface plasmon resonance‐based approaches appear to be more sensitive than the traditional approach for identifying plant SABPs using size‐exclusion chromatography with radiolabeled SA, as these proteins exhibited little to no SA‐binding activity in such an assay. The development of these approaches therefore complements conventional techniques and helps dissect the SA signaling network in plants, and may also help elucidate the mechanisms through which SA acts as a multi‐functional drug in mammalian systems.