Immunoprophylaxis against filarial parasite,Brugia malayi: potential of excretory-secretory antigens in inducing immunity

The role of excretory-secretory antigens in inducing immunity in the host againstBrugia malayi microfilariae and infective larvae was studied byin vitro antibody dependent cell-mediated reaction as well asin vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised againstBrugia malayi microfilarial and infective larval excretory-secretory antigens(Bm Mf ESA andBm L₃ ESA) promoted the adherence of peritoneal exudate cells toBrugia malayi microfilariae and infective larvaein vitro and induced cytotoxicity to the parasites within 48 h. The antiBm Mf ESA serum was more effective than antiBm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further,Mastomys natalensis immunized againstBm Mf ESA and L₃ ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory-secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.     

Probing the mechanism of GSH activation in Schistosoma haematobium glutathione-S-transferase by site-directed mutagenesis and X-ray crystallography

During turnover, the catalytic tyrosine residue (Tyr10) of the sigma class Schistosoma haematobium wild-type glutathione-S-transferase is expected to switch alternately in and out of the reduced glutathione-binding site (G-site). The Tyrout10 conformer forms a pi-cation interaction with the guanidinium group of Arg21. As in other similar glutathione-S-transferases, the catalytic Tyr has a low pKa of 7.2. In order to investigate the catalytic role of Tyr10, and the structural and functional roles of Arg21, we carried out structural studies on two Arg21 mutants (R21L and R21Q) and a Tyr10 mutant, Y10F. Our crystallographic data for the two Arg21 mutants indicate that only the Tyrout10 conformation is populated, thereby excluding a role of Arg21 in the stabilisation of the out conformation. However, Arg21 was confirmed to be catalytically important and essential for the low pKa of Tyr10. Upon comparison with structural data generated for reduced glutathione-bound and inhibitor-bound wild-type enzymes, it was observed that the orientations of Tyr10 and Arg35 are concerted and that, upon ligand binding, minor rearrangements occur within conserved residues in the active site loop. These rearrangements are coupled to quaternary rigid-body movements at the dimer interface and alterations in the localisation and structural order of the C-terminal domain.     

Setaria cervi: kinetic studies of filarial glutathione synthetase by high performance liquid chromatography

The bovine filarial worm Setaria cervi was found to have abundance of glutathione synthetase (GS; EC 6.3.2.3) activity, the enzyme being involved in catalysing the final step of glutathione (GSH) biosynthesis. A RP-HPLC method involving precolumn derivatization with o-phthalaldehyde has been followed for the estimation of GS activity in crude filarial preparations. Subcellular fractionation of the enzyme was undertaken and it was confirmed to be a soluble protein residing mainly in cytosolic fraction. Attempts to determine the Km value for L-gamma-glutamyl-L-cysteine gave a distinctly nonlinear double-reciprocal plot in which data obtained at relatively high dipeptide concentrations (>1 mM) extrapolate to a Km value of about 400 microM whereas data obtained at lower concentrations (<0.1 mM) extrapolate to a value of about 33 microM. Km was determined to be around 950 and 410 microM for ATP and glycine, respectively. The effect of various amino acids was studied on enzyme activity at 1mM concentration. L-cystine caused a significant enzyme inhibition of 11%. Preincubation with N-ethylmaleimide also resulted in significant inhibition of GS activity.     

Structure of the extracellular glutathione S-transferase OvGST1 from the human pathogenic parasite Onchocerca volvulus

Onchocerciasis or river blindness, caused by the filarial worm Onchocerca volvulus, is the world’s second leading infectious cause of blindness. In order to chronically infect the host, O. volvulus has evolved molecular strategies that influence and direct immune responses away from the modes most damaging to it. The O. volvulus GST1 (OvGST1) is a unique glutathione S-transferase (GST) in that it is a glycoprotein and possesses a signal peptide that is cleaved off in the process of maturation. The mature protein starts with a 25-amino-acid extension not present in other GSTs. In all life stages of the filarial worm, it is located directly at the parasite-host interface. Here, the OvGST1 functions as a highly specific glutathione-dependent prostaglandin D synthase (PGDS). The enzyme therefore has the potential to participate in the modulation of immune responses by contributing to the production of parasite-derived prostanoids and restraining the host’s effector responses, making it a tempting target for chemotherapy and vaccine development. Here, we report the crystal structure of the OvGST1 bound to its cofactor glutathione at 2.0 Å resolution. The structure reveals an overall structural homology to the haematopoietic PGDS from vertebrates but, surprisingly, also a large conformational change in the prostaglandin binding pocket. The observed differences reveal a different vicinity of the prostaglandin H₂ binding pocket that demands another prostaglandin H₂ binding mode to that proposed for the vertebrate PGDS. Finally, a putative substrate binding mode for prostaglandin H₂ is postulated based on the observed structural insights.     

Hepatoprotective effects of Solanum nigrum Linn extract against CCl(4)-induced oxidative damage in rats

Solanum nigrum L. (SN) is an herbal plant that has been used as hepatoprotective and anti-inflammation agent in Chinese medicine. In this study, the protective effects of water extract of SN (SNE) against liver damage were evaluated in carbon tetrachloride (CCl4)-induced chronic hepatotoxicity in rats. Sprague-Dawley (SD) rats were orally fed with SNE (0.2, 0.5, and 1.0 g kg(-1) bw) along with administration of CCl4 (20% CCl4/corn oil; 0.5 mL kg(-1) bw) for 6 weeks. The results showed that the treatment of SNE significantly lowered the CCl4-induced serum levels of hepatic enzyme markers (GOT, GPT, ALP, and total bilirubin), superoxide and hydroxyl radical. The hepatic content of GSH, and activities and expressions of SOD, GST Al, and GST Mu that were reduced by CCl4 were brought back to control levels by the supplement of SNE. Liver histopathology showed that SNE reduced the incidence of liver lesions including hepatic cells cloudy swelling, lymphocytes infiltration, hepatic necrosis, and fibrous connective tissue proliferation induced by CCl4 in rats. Therefore, the results of this study suggest that SNE could protect liver against the CCl4-induced oxidative damage in rats, and this hepatoprotective effect might be contributed to its modulation on detoxification enzymes and its antioxidant and free radical scavenger effects.     

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.     

Characterization of human thioredoxin-like-1: potential involvement in the cellular response against glucose deprivation

The thioredoxin system, composed of thioredoxin (Trx) and thioredoxin reductase (TrxR), emerges as one of the most important thiol-based systems involved in the maintenance of the cellular redox balance. Thioredoxin-like-1 (TXL-1) is a highly conserved protein comprising an N-terminal Trx domain and a C-terminal domain of unknown function. Here we show that TXL-1 is a substrate for the cytosolic selenoprotein TrxR-1. In situ hybridization experiments demonstrates high expression of Txl-1 mRNA in various areas of central nervous system and also in some reproductive organs. Glucose deprivation, but not hydrogen peroxide treatment, reduced the levels of endogenous TXL-1 protein in HEK-293 cell line. Conversely, overexpression of TXL-1 protects against glucose deprivation-induced cytotoxicity. Taken together, the finding that Txl-1 mRNA is highly expressed in tissues which use glucose as a primary energy source and the modulation of TXL-1 levels upon glucose deprivation indicate that TXL-1 might be involved in the cellular response to sugar starvation stress.     

Structural Analysis of Cysteine S-Nitrosylation: A Modified Acid-Based Motif and the Emerging Role of Trans-Nitrosylation

S-Nitrosylation, the selective and reversible addition of nitric oxide (NO) moiety to cysteine (Cys) sulfur in proteins, regulates numerous cellular processes. In recent years, proteomic approaches that are capable of identifying nitrosylated Cys residues have been developed. However, the features underlying the specificity of Cys modification with NO remain poorly defined. Previous studies suggested that S-nitrosylated Cys may be flanked by an acid-base motif or hydrophobic areas and show high reactivity, low pK_a, and high sulfur atom exposure. In the current study, we prepared an extensive, manually curated data set of proteins with S-nitrosothiols, accounting for a variety of biochemical functions, organisms of origin, and physiological responses to NO. Analysis of this generic NO-Cys data set revealed that proximal acid-base motif, Cys pK_a, sulfur atom exposure, and Cys conservation or hydrophobicity in the vicinity of the modified Cys do not define the specificity of S-nitrosylation. Instead, this analysis revealed a revised acid-base motif, which is located more distantly to the Cys and has its charged groups exposed. We hypothesize that, rather than being strictly used for direct activation of Cys, the modified acid-base motif is engaged in protein-protein interactions thereby contributing to trans-nitrosylation as an important and widespread mechanism for reversible modification of Cys with NO moiety. For proteins lacking the revised motif, we discuss alternative mechanisms including a potential role of nitrosoglutathione as a trans-acting agent.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

Actin protein up-regulated upon infection and development of the filarial parasite, Wuchereria bancrofti (Spirurida: Onchocercidae), in the vector mosquito, Culex quinquefasciatus (Diptera: Culicidae)

Detection and identification of humoral proteins, which are up-regulated in Culex quinquefasciatus upon infection by Wuchereria bancrofti, is important in tracing out the biochemical consequences of the filarial parasite development in the vector mosquito. Analysis of the haemolymph of infected mosquitoes through SDS-PAGE and RP-HPLC showed up-regulation of five proteins of molecular weights 40, 66, 22, 14, and 7-kDa. Among these, only the 40-kDa was unknown and the others were comparable with those already reported as transferrin, attacin, lysozyme, and defensin, respectively. In the present study, the 40-kDa protein up-regulated upon infection was identified as actin through nano-LC-MS/MS analysis. Actin is known to be one of the cytoskeletal proteins up-regulated in the haemolymph, as part of the innate immune system, of Escherichia coli challenged Drosophila melanogaster larvae. For the first time, we have observed an increased level of actin in the haemolymph of W. bancrofti-infected Cx. quinquefasciatus. However, the exact mechanism of actin involvement in the immune system of this mosquito is yet to be studied.     

Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein

Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.     

gamma-Glutamyl transpeptidase GGT4 initiates vacuolar degradation of glutathione S-conjugates in Arabidopsis

The xenobiotic monochlorobimane is conjugated to glutathione in the cytosol of Arabidopsis thaliana, transported to the vacuole, and hydrolyzed to cysteine S-bimane [Grzam, A., Tennstedt, P., Clemens, S., Hell, R. and Meyer, A.J. (2006) Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase. FEBS Lett. 580, 6384-6390]. The work here identifies gamma-glutamyl transpeptidase 4 (At4g29210, GGT4) as the first step of vacuolar degradation of glutathione conjugates. Hydrolysis of glutathione S-bimane is blocked in ggt4 null mutants of A. thaliana. Accumulation of glutathione S-bimane in mutants and in wild-type plants treated with the high affinity GGT inhibitor acivicin shows that GGT4 is required to initiate the two step hydrolysis sequence. GGT4:green fluorescent protein fusions were used to demonstrate that GGT4 is localized in the lumen of the vacuole.     

Transgenic tobacco plants overexpressing glyoxalase enzymes resist an increase in methylglyoxal and maintain higher reduced glutathione levels under salinity stress

The mechanism behind enhanced salt tolerance conferred by the overexpression of glyoxalase pathway enzymes was studied in transgenic vis-à-vis wild-type (WT) plants. We have recently documented that salinity stress induces higher level accumulation of methylglyoxal (MG), a potent cytotoxin and primary substrate for glyoxalase pathway, in various plant species [Yadav, S.K., Singla-Pareek, S.L., Ray, M., Reddy, M.K. and Sopory, S.K. (2005) MG levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 337, 61-67]. The transgenic tobacco plants overexpressing glyoxalase pathway enzymes, resist an increase in the level of MG that increased to over 70% in WT plants under salinity stress. These plants showed enhanced basal activity of various glutathione related antioxidative enzymes that increased further upon salinity stress. These plants suffered minimal salinity stress induced oxidative damage measured in terms of the lipid peroxidation. The reduced glutathione (GSH) content was high in these transgenic plants and also maintained a higher reduced to oxidized glutathione (GSH:GSSG) ratio under salinity. Manipulation of glutathione ratio by exogenous application of GSSG retarded the growth of non-transgenic plants whereas transgenic plants sustained their growth. These results suggest that resisting an increase in MG together with maintaining higher reduced glutathione levels can be efficiently achieved by the overexpression of glyoxalase pathway enzymes towards developing salinity stress tolerant plants.     

Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase

Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione-bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the gamma-glutamylcysteine-bimane intermediate (gamma-EC-B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine-bimane without intermediate. Only co-infiltration of monochlorobimane with Cd2+ and Cu2+ increased gamma-EC-B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null-mutants. In the absence of elevated heavy metal concentrations glutathione-conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate-limiting.     

Two endoplasmic reticulum PDI peroxidases increase the efficiency of the use of peroxide during disulfide bond formation

Disulfide bond formation in the endoplasmic reticulum by the sulfhydryl oxidase Ero1 family is thought to be accompanied by the concomitant formation of hydrogen peroxide. Since secretory cells can make substantial amounts of proteins that contain disulfide bonds, the production of this reactive oxygen species could have potentially lethal consequences. Here, we show that two human proteins, GPx7 and GPx8, labeled as secreted glutathione peroxidases, are actually endoplasmic reticulum-resident protein disulfide isomerase peroxidases. In vitro, the addition of GPx7 or GPx8 to a folding protein along with protein disulfide isomerase and peroxide enables the efficient oxidative refolding of a reduced denatured protein. Furthermore, both GPx7 and GPx8 interact with Ero1α in vivo, and GPx7 significantly increases oxygen consumption by Ero1α in vitro. Hence, GPx7 and GPx8 may represent a novel route for the productive use of peroxide produced by Ero1α during disulfide bond formation.     

Plasmodium vivax: molecular cloning, expression and characterization of glutathione S-transferase

Malaria parasite glutathione S-transferases (GSTs) are postulated to be essential for parasite survival by protecting the parasite against oxidative stress and buffering the detoxification of heme-binding compounds; therefore, GSTs are considered potential targets for drug development. In this study, we identified a Plasmodium vivax gene encoding GST (PvGST) and characterized the biochemical properties of the recombinant enzyme. The PvGST contained 618 bp that encoded 205 amino acids and shared a significant degree of sequence identity with GSTs from other Plasmodium species. The recombinant homodimeric enzyme had an approximate molecular mass of 50kDa and exhibited GSH-conjugating and GSH-peroxidase activities towards various model substrates. The optimal pH for recombinant PvGST (rPvGST) activity was pH 8.0, and the enzyme was moderately unstable at 37 degrees C. The K(m) values of rPvGST with respect to GSH and CDNB were 0.17+/-0.09 and 2.1+/-0.4mM, respectively. The significant sequence homology and similar biochemical properties of PvGST and Plasmodium falciparum GST (PfGST) indicate that they may have similar molecular structures. This information may be useful for the design of specific inhibitors for plasmodial GSTs as potential antimalarial drugs.     

The anti-cancer drug chlorambucil as a substrate for the human polymorphic enzyme glutathione transferase P1-1: kinetic properties and crystallographic characterisation of allelic variants

The commonly used anti-cancer drug chlorambucil is the primary treatment for patients with chronic lymphocytic leukaemia. Chlorambucil has been shown to be detoxified by human glutathione transferase Pi (GST P1-1), an enzyme that is often found over-expressed in cancer tissues. The allelic variants of GST P1-1 are associated with differing susceptibilities to leukaemia and differ markedly in their efficiency in catalysing glutathione (GSH) conjugation reactions. Here, we perform detailed kinetic studies of the allelic variants with the aid of three representative co-substrates. We show that the differing catalytic properties of the variants are highly substrate-dependent. We show also that all variants exhibit the same temperature stability in the range 10 °C to 45 °C. We have determined the crystal structures of GST P1-1 in complex with chlorambucil and its GSH conjugate for two of these allelic variants that have different residues at positions 104 and 113. Chlorambucil is found to bind in a non-productive mode to the substrate-binding site (H-site) in the absence of GSH. This result suggests that under certain stress conditions where GSH levels are low, GST P1-1 can inactivate the drug by sequestering it from the surrounding medium. However, in the presence of GSH, chlorambucil binds in the H-site in a productive mode and undergoes a conjugation reaction with GSH present in the crystal. The crystal structure of the GSH-chlorambucil complex bound to the *C variant is identical with the *A variant ruling out the hypothesis that primary structure differences between the variants cause structural changes at the active site. Finally, we show that chlorambucil is a very poor inhibitor of the enzyme in contrast to ethacrynic acid, which binds to the enzyme in a similar fashion but can act as both substrate and inhibitor.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

Role of Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in cardiac fibroblasts: protection against reactive oxygen and nitrogen species-induced cell injury

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.