Schistosoma Japonicum Glutathione S-transferase Research Articles

Glutathione S-transferase influences the fecundity of Schistosoma japonicum

The aim of this study was to investigate the effect of Schistosoma japonicum glutathione S-transferase (SjGST) on the developmental stages of the parasite. We found that the mRNA levels of GST were higher in schistosomula obtained from the host and the eggs than that in other developmental stages. SjGST was mainly distributed in the egg shells, teguments of the worms, and part of the parenchyma of the worms. GST knockdown with RNA interference in S. japonicum worms resulted in a silencing rate higher than 80%. The egg reduction rate (18%) and abnormal egg ratio (28%) were significantly higher (P < 0.05) in the GST-silenced group than in the negative control group. These results indicate that SjGST plays an important role in the fecundity of S. japonicum, specifically in egg formation.

Schistosoma mansoni cercarial elastase (SmCE): differences in immunogenic properties of native and recombinant forms.

The Schistosoma mansoni cercarial elastase (SmCE) has previously been shown to be poorly immunogenic in mice. However, a minority of mice were able to produce antibodies against SmCE after multiple immunizations with crude preparations containing the enzyme. These mice were partially protected against challenge infections of S. mansoni. In the present study, we show that in contrast to the poor immunogenicity of the enzymatically active native form of SmCE derived from a crude preparation (cercarial transformation fluid), immunization of CBA/Ca mice with two enzymatically inactive forms, namely purified native SmCE or a recombinant SmCE fused to recombinant Schistosoma japonicum glutathione S-transferase (rSmCE-SjGST), after adsorption onto aluminum hydroxide adjuvant, induced specific anti-SmCE immunoglobulin G (IgG) in all mice within 2 weeks of the second immunization. The IgG antibody response to rSmCE-SjGST was mainly of the IgG1 subclass. These results suggest that inactive forms of the antigen could be used to obtain the optimum immunogenic effects as a vaccine candidate against schistosomiasis. Mice immunized with the rSmCE-SjGST on alum had smaller mean worm burdens and lower tissue egg counts when compared with adjuvant alone- and recombinant SjGST-injected controls. The native SmCE was antigenically cross-reactive with homologous enzymes of Schistosoma haematobium and Schistosoma margrebowiei.

Fluorometric titration assay of affinity of tight-binding nonfluorescent inhibitor of glutathione S-transferase.

To determine inhibition constant (K(i)) of tight-binding inhibitor, the putative method estimated an apparent K(i) from the response of initial rates to total concentrations of the inhibitor considering its depletion during binding for conversion into the true K(i), but was impractical with glutathione S-transferase of sophisticated kinetics. A fluorometric titration assay of dissociation constant (K(d)) was thus proposed. Schistosoma japonicum glutathione S-transferase (SjGST) action on a nonfluorescent divalent pro-inhibitor and glutathione yielded a divalent product in active site to act as a tight-binding inhibitor, whose binding quenched fluorescence of SjGST at 340 nm under the excitation at 280 nm. K(d) was estimated from the response of fluorescence of SjGST at 340 nm to total concentrations of the divalent product considering its depletion during binding. By fluorometric titration assay, K(d) of two tested nonfluorescent divalent products varied from subnanomolar to nanomolar, but both were resistant to change of SjGST levels and consistent with their apparent K(i) estimated via the putative method. Hence, fluorometric titration assay of K(d) of nonfluorescent tight-binding inhibitors/ligands was effective to GST and may be universally applicable to common enzymes/proteins; affinities of tight-binding inhibitors of GST can be approximated by their apparent K(i) estimated via the putative method.

Glutathione S-Transferases Interact with AMP-Activated Protein Kinase: Evidence for S-Glutathionylation and Activation In Vitro

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

Regioselective Covalent Immobilization of Catalytically Active Glutathione S-Transferase on Glass Slides

The high selectivity of protein farnesyltransferase was used to regioselectively append farnesyl analogues bearing bioorthogonal alkyne and azide functional groups to recombinant Schistosoma japonicum glutathione S-transferase (GSTase) and the active modified protein was covalently attached to glass surfaces. The cysteine residue in a C-terminal CVIA sequence appended to N-terminally His(6)-tagged glutathione S-transferase (His(6)-GSTase-CVIA) was post-translationally modified by incubation of purified protein or cell-free homogenates from E. coli M15/pQE-His(6)-GSTase-CVIA with yeast protein farnesyltransferase (PFTase) and analogues of farnesyl diphosphate (FPP) containing ω-azide and alkyne moieties. The modified proteins were added to wells on silicone-matted glass slides whose surfaces were modified with PEG units containing complementary ω-alkyne and azide moieties and covalently attached to the surface by a Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition. The wells were washed and assayed for GSTase activity by monitoring the increase in A(340) upon addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GT). GSTase activity was substantially higher in the wells spotted with alkyne (His(6)-GSTase-CVIA-PE) or azide (His(6)-GSTase-CVIA-AZ) modified glutathione-S-transferase than in control wells spotted with farnesyl-modified enzyme (His(6)-GSTase-CVIA-F).

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Protein Acyltransferase Function of Purified Calreticulin: The Exclusive Role of P-Domain in Mediating Protein Acylation Utilizing Acyloxycoumarins and Acetyl CoA as the Acyl Group Donors

The distinct biochemical function of endoplasmic reticulum (ER) protein Calreticulin (CR) catalyzing the transfer of acyl group from acyloxycoumarin to a receptor protein was termed calreticulin transacylase (CRTAase). The present study, unlike the previous reports of others utilizing CR-deficient cells alone, dealt with the recombinant CR domains of Heamonchus contortus (rhCRTAase) in order to examine their CRTAase activity. P-domain of rhCR unlike N- and C-domains was found to be endowed with CRTAase function. We have also observed for the first time acetyl CoA, as a substrate for rhCRTAase/P-domain mediated acetylation of recombinant Schistosoma japonicum glutathione S-transferase (rGST). rhCRTAase/P-domain were also found to undergo autoacylation by acyloxycoumarins. Also, the isolated autoacylated rhCRTAase/P-domain in non-denatured form alone exhibited the ability to transfer acyl group to rGST indicating the stable intermediate nature of acylated CR. P-domain catalyzed acetylation of rGST by 7,8-Diacetoxy-4-methylcoumarin or acetyl CoA resulted in the modification of several lysine residues in common was evidenced by LC-MS/MS analysis. The putative site of the binding of acyloxycoumarins with CR was predicted by computational blind docking studies. The results showed the involvement of two lysine residues Lys-173 and Lys-174 present in P-domain for binding acyloxycoumarins and acetyl CoA thus highlighting that the active site for the CRTAase activity would reside in the P-domain of CR. Certain ER proteins are known to undergo acetylation under the physiological conditions involving acetyl CoA. These results demonstrating CRTAase mediated protein acetylation by acetyl CoA may hint at CR as the possible protein acetyltransferase of the ER lumen.

Protein acyltransferase function of purified calreticulin. Part 1: characterization of propionylation of protein utilizing propoxycoumarin as the propionyl group donor

We have earlier reported that an endoplasmic reticulum luminal protein calreticulin (CR) mediated the acetylation of certain receptor proteins such as glutathione S-transferase (GST) by polyphenolic acetates, leading to irreversible inhibition. This function of calreticulin was termed calreticulin transacetylase. In this communication, we have demonstrated for the first time the ability of the purified recombinant calreticulin of a parasitic nematode Haemonchus contortus to transfer propionyl group from 7,8-Dipropoxy-4-methylcoumarin (DPMC) to recombinant Schistosoma japonicum glutathione S-transferase (rGST). Calreticulin transacetylase exhibited hyperbolic kinetics and yielded K(m) (140 microM) and V(max) (105 units) when the concentration of DPMC was varied keeping the concentration of rGST constant. rGST thus propionylated was found to positively interact with anti-acetyl lysine antibody. Also, the nanoscale LC-MS/MS analysis identified the propionylation sites on three lysine residues: Lys-11, -180 and -181 of rGST. These results highlight the transacylase function of calreticulin (CRTAase).

Anomalous Immunogenic Properties of Serine Proteases

It has previously been shown that a approximately 27 kDa serine protease of Schistosoma mansoni larvae, the cercarial elastase (CE), was a poor immunogen in as much as it failed to induce an antibody response. The CE has a critical role in enabling schistosome larvae to penetrate the skin of their definitive hosts, so the apparently poor immunogenicity of this enzyme is clearly of interest. To understand its lack of immunogenicity better and in particular to determine whether it is related to its proteolytic activity, we have measured antibody responses of mice to three different serine proteases. Groups of mice were immunized with porcine pancreatic trypsin (TRY), chymotrypsin (CHY) or elastase (ELA) and the resulting antibody response compared with antibody responses to two non-protease antigens, chicken egg albumin (OVA) and Schistosoma japonicum glutathione S-transferase (GST), all being administered with alum as an adjuvant. Of 12 mice that were injected five times at 14 day intervals with TRY, only one produced antibody reactive with this enzyme in ELISA. Immunizations with CHY or ELA induced somewhat better antibody responses than TRY, but the responses to the first and second injections of these two proteases nevertheless seemed comparatively lower than the responses to GST. Induction of antibody responses by OVA and GST was not affected when TRY was injected concomitantly. Thus, the antibody response to one of the serine proteases used in this study, mammalian trypsin, was anomalous.

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

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

Kinetic study on the irreversible thermal denaturation of Schistosoma japonicum glutathione s-transferase

The thermal unfolding pathway of the Schistosoma japonicum glutathione S-transferase (Sj26GST) was previously interpreted by applying equilibrium thermodynamics and a reversible two-state model (Kaplan et al., (1997) Protein Science, 6, 399–406), though weak support for this interpretation was provided. In our study, thermal denaturation of Sj26GST has been re-examined by differential scanning calorimetry in the pH range of 6.5–8.5 and in the presence of the substrate and S-hexylglutathione. Calorimetric traces were found to be irreversible and highly scan-rate dependent. Thermogram shapes, as well as their scan-rate dependence, can be globally explained by assuming that thermal denaturation takes place according to one irreversible step described by a first-order kinetic constant that changes with temperature, as given by an Arrhenius equation. On the basis of this model, values for the rate constant as a function of temperature and the activation energy have been determined. Data also indicate that binding of GSH or S-hexylglutathione just exert a very little stabilising effect on the dimeric structure of the molecule.

Long stretches of sequential and identical serine or alanine codons are compatible with an efficient full-length protein expression in Escherichia coli

The Schistosoma japonicum glutathione S-transferase (GST) recombinant cDNAs, carrying blocks of sequential and identical triplets, consisting of 15-30-45 GCT (Ala) codons or 15–30 and also up to 75 AGC (Ser) codons, are expressed efficiently in an Escherichia coli system in the form of full-length protein chains, as detected by Coomassie-stained SDS–polyacrylamide gels, and soluble fusion proteins are purified by GSH-affinity chromatography. High expression levels and high yields of purified recombinant proteins are achieved. The efficient protein expression is independent of the molecular context and position of the polySer/polyAla string inserted into the GST carrier (near the part of the gene encoding the N- or the C-terminus). These findings suggest that E. coli is a powerful biological system to express foreign genes carrying long stretches coding for Ser- or Ala-rich domains, which are not uncommon in eukaryotic proteins. Moreover, data reported here show that the negative effect of sequential serine codons on protein expression in bacteria, previously reported in the literature, is not a general phenomenon.

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29–763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST–SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST–3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST–3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.

Design of potent inhibitors for Schistosoma japonica glutathione S-transferase

We implemented both structure-based drug design and the concept of polyvalency to discover a series of potent and unsymmetrical Schistosoma japonicum glutathione S-transferase ( SjGST) inhibitors 10– 12. This strategy achieved not only an excellent enhancement (10- to 490-fold) in the inhibitory potency, compared to the monofunctional analogues 1– 5, but was also an effective modification by selecting a hydrophobic moiety with a flexible linker. The designed compounds with a low micromolar hit demonstrate special values in refining the new generation of SjGST inhibitors. The stoichiometry of the binding is one inhibitor molecule per SjGST monomer via isothermal titration calorimetric measurement.

Crystal Structure of Non-Fused Glutathione S-Transferase from Schistosoma 709 japonicum in Complex with Glutathione

Schistosoma japonicum glutathione S-transferase (SjGST) is a common fusion tag in recombinant protein production, and its 3-dimensional structure has been studied in the context of drug design. We have determined the crystal structure of non-fused SjGST complexed with glutathione, and compare it to complexes between glutathione and SjGST fusion proteins.

Let E. coli modify your proteins

Until recently, all organisms from viruses to humans have used the same 20 amino acids to form proteins. In 2001, Peter Schultz's group reported the modification of the translation machinery of Escherichia coli to allow incorporation of additional novel amino acids into proteins [1xExpanding the genetic code of Escherichia coli. Wang, L. et al. Science. 2001; 292: 498–500Crossref | PubMedSee all References][1]. Now, this same laboratory has described the application of this technique to incorporate specifically a photocrosslinking amino acid, p-benzoyl-l-phenylalanine (pBpa), into proteins [2xAddition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Chin, J.W. et al. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11020–11024Crossref | PubMed | Scopus (278)See all References][2].First, the authors needed to select for an orthogonal aminoacyl-tRNA synthetase/tRNA pair that could incorporate pBpa specifically in response to the amber codon TAG. A strategy was devised to mutate the Methanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS). It was already known that this synthetase does not aminoacylate any endogenous E. coli tRNAs with tyrosine but can aminoacylate a mutant tyrosine amber suppressor. A library expressing mutated TyrRS was subjected to positive selection for chloramphenicol resistance based on the suppression of an amber stop codon in the gene encoding CAT. These cells were then challenged to grow in the presence of pBpa and chloramphenicol. At this point, these cells contained synthetases that could incorporate either a natural or unnatural (pBpa) amino acid. These synthetases were then transferred into cells containing the toxic barnase gene with amber mutations. Growth of these cells in the absence of pBpa provided selection against synthetases that could use natural amino acids. Five rounds of this positive and negative selection were performed. The sequences of six of these ‘selected’ synthetases were determined and used to model how pBpa complexes with the mutated TyrRS. Based on calculations related to the resistance to chloramphenicol in the presence or absence of pBpa, the authors conclude that they have achieved substantial in vivo specificity for the incorporation of pBpa in response to an amber codon using these mutated TyrRS.To prove the usefulness of this technology, the authors tested a modified Schistosoma japonicum glutathione S-transferase (SjGST). This protein is known to be a dimer of two identical subunits. By placing amber codons at specific sites in this gene, and using the modified TyrRS system, specific amino acids within SjGST were substituted with pBpa. One of these residues (Phe52) was known to interact between the SjGST subunits and the other (Tyr198) was known not to react. Upon irradiation, the purified SjGST (Phe-52-Bpa) was converted to a covalently linked homodimer as detected by SDS–PAGE, whereas the other purified SjGST (Tyr198-Bpa) remained as a monomer. This demonstrates the utility of this technique to incorporate this photocrosslinking agent specifically into proteins and crosslink them together. The goal of this technology is to increase the ability to identify specific amino acids involved in protein–protein interactions.

Microcalorimetric Study of the Effect of Hexa-histidine Tag and Denaturant on the Interaction Mechanism between Protein and Metal-Chelating Gel

A recombinant protein, Schistosoma japonicum glutathione-S-transferase (SjGST), was fused with a C-terminal hexa-histidine tag to obtain SjGST/His. Both proteins were used to probe the interaction mechanisms with the metal ions immobilized on chromatography gels. Isothermal titration calorimetry was used to directly measure the adsorption enthalpies (ΔHads) of both proteins with Ni-NTA and TALON (Co2+) commercial affinity resins, under the conditions of with and without the presence of a denaturant. The result reveals that SjGST/His had a lower ΔHads value with Ni-NTA than did SjGST, mainly attributed to the formation of more coordination bonds with or a stronger binding with Ni-NTA. Furthermore, the difference between the ΔHads values of SjGST/His onto TALON under the nature and denaturing conditions were insignificant, implying that the binding topography of the hexa-histidine tail with immobilized Co2+ was not significantly changed with the presence of a denaturant. In addition, this study shows that the proposed binding models and the directly measured adsorption heat can be combined to elucidate the difference in the interaction mechanisms of SjGST/His adsorption onto those two adsorbents from a thermodynamic perspective.

Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells

DNA sequence coding for a portion of DNA binding protein (amino acids 3–58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

52] Expression and analysis of properties of septin CDCrel-1 in exocytosis

This chapter describes the expression and analysis of properties of septin CDCrel- 1 in exocytosis. For in vitro studies of CDCrel-1, a fusion of CDCrel-1 is created with Schistosoma japonicum glutathione S-transferase (GST) in the vector pGEX-KG. Rat CDCreI-1 cDNA is amplified by polymerase chain reaction. Like other guanosine triphosphate (GTP)ases, septin proteins contain several motifs needed for GTP binding and hydrolysis. The ability of CDCrel-1 to bind and hydrolyze GTP, and the effect of the S58N mutation on properties is examined, using an assay for the septin Nedd5. Because syntaxin is a SNARE protein required for secretion, the hypothesis that CDCreI-1 interaction with syntaxin may regulate this process is tested. The insulin secreting pancreatic β cell line HIT-T15 contains the same isoforms of the SNARE proteins—namely, SNAP-25, VAMP2, and syntaxin-1A that are found in the neuron and needed for neurotransmitter release. Moreover, the use of this cell line permits to monitor the effect of transfection of wild type and GTP mutant CDCrel-1 on secretion.

Production and characterization of glutathione-S-transferase fused with a poly-histidine tag.

Schistosoma japonicum glutathione-S-transferase (SjGST) was genetically engineered with a poly-histidine tag at the C-terminus and highly expressed in Escherichia coli. Both SjGST and the tagged protein, SjGST/His, were purified with glutathione Sepharose 4B gels and subsequently studied for their activities, antibody-binding abilities, and metal affinities. The production level of active SjGST/His was higher than that of SjGST. Both proteins had similar specific catalytic activities and binding abilities with anti-SjGST antibody, while the antibody against poly-histidine recognized only SjGST/His. Proteolytic degradation was occasionally observed in aged dialyzed SjGST/His preparation. Under a native condition, the Co(2+)-chelated TANOL gel (Co-TANOL) had a better binding specificity to the tagged protein than did the Ni(2+)-chelated nitriloacetic acid (Ni-NTA) agarose gel. However, the binding capacity of the Ni-NTA gel for SjGST/His was 2-fold higher than that of the Co-TANOL one. To increase the native binding specificity of the Ni-NTA gel, 20 mM imidazole had to be added to the washing solution. In a denatured state, both gels could only capture SjGST/His, and the binding capacity of the Ni-NTA gel was nearly 2-fold higher than that of the Co-TANOL gel. The binding association constants of both gels with SjGST/His did not differ greatly under either condition. The study demonstrated that the C-terminal addition of the poly-histidine tag to SjGST increased the metal affinity of the enzyme to the Co-TANOL gel under both native and denaturing conditions and to the Ni-NTA gel under denaturing conditions, whereas the enzymatic activity and antibody-binding ability were not affected.