Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant major surface antigen (P30), which was produced as a glutathione S-transferase (EC 2.5.1.18) fusion protein of Toxoplasma gondii, was found to be able to activate macrophages to kill T. gondii in vitro. The macrophage activation was due to P30 in the fusion protein, not to glutathione S-transferase.
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PMID:Toxoplasmacidal activity of macrophages activated by recombinant major surface antigen (P30) of Toxoplasma gondii. 185

A gamma gt11 cDNA library was constructed from poly(U)-Sepharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-125I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degrees C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.
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PMID:cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica. 220 Oct 27

To investigate the failure of high-level production of hepatitis B viral (HBV) surface antigen (HBsAg), including three authentic forms, large (L), middle (M) and major/small (S) HBsAg, in Escherichia coli, we employed the high-expression vector pGEX containing the glutathione S-transferase-encoding gene (GST) to study HBsAg production. Different fragments of HBV DNA containing the entire pre-S1/pre-S2/S region (for L protein), or partial pre-S1, pre-S2, pre-S1/pre-S2 and pre-S2/S region (for M protein), were fused downstream from the GST gene, in order to obtain five plasmids which encode GST-HBsAg fusion proteins. SDS-PAGE analyses revealed that cells containing plasmids with a full-length S region (pGLS and pGMS) produced undetectable GST-HBsAg fusion proteins, in contrast to those cells harboring plasmids without the S region (pGS1, pGS2 and pGS1S2), which synthesized fusion proteins in 3-10% of the total cellular protein. Using an immunoblot method to screen HBsAg production in cells which harbored plasmids derived from exonuclease BAL 31-digested pGLS, we obtained eight positive clones. Nucleotide sequence analyses of plasmids from the positive clones revealed that termination, deletion or frameshift occurred at the regions encoding either the first or the third transmembrane domain of the major HBsAg. Correlation between the production level of GST-HBsAg fusion proteins and their constituent and arrangement of amino acids (aa) at the last 20 aa among 15 clones suggested that the fusion protein ended with a longer stretch of or a higher ratio of hydrophobic aa had a lower production in E. coli.
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PMID:Deletion or alteration of hydrophobic amino acids at the first and the third transmembrane domains of hepatitis B surface antigen enhances its production in Escherichia coli. 764 92

Hepatitis delta virus (HDV) encodes two proteins, the small hepatitis delta antigen (SHDAg) and large hepatitis delta antigen (LHDAg). Both proteins are identical except for the presence of additional 19 amino acids at the C terminus of LHDAg. While SHDAg is required for HDV RNA replication, LHDAg inhibits replication and is required together with hepatitis B surface antigen for the assembly of HDV. The C-terminal last 4 amino acids of LHDAg (Cys-Arg-Pro-Gln) is an isoprenylation motif. It has previously been shown that the mutation of the Cys inhibited the assembly of HDV. In order to discern whether this effect is due to change of amino acid residue or abolition of isoprenylation, we constructed several LHDAg mutants of the terminal three amino acid residues and tested their abilities to be packaged with HBsAg by cotransfection experiments. We also made GST-fusion proteins of these mutants and tested their abilities to be isoprenylated in rabbit reticulocyte lysate system. We found that some, but not all, of the substitutions of the amino acid residues other than the Cys also inhibited isoprenylation and that the status of isoprenylation of these mutant proteins correlated well with their abilities to be packaged with HBsAg into virions. This result indicates that isoprenylation, rather than the primary amino acid sequence, is required for LHDAg packaging. Furthermore, we found that the attachment of an isoprenylation motif to SHDAg did not enable it to be packaged with HBsAg and that the deletions of any 5 amino acids in the last 15 amino acids (amino acids 196 to 210) unique to the LHDAg abolished the packaging ability. In contrast, the deletion of 33 amino acids (amino acids 163 to 195) upstream of the last C-terminal 19 amino acids of LHDAg did not interfere with its packaging ability. Therefore, we conclude that the 15 amino acids upstream of the isoprenylation site of LHDAg are also essential for HDV assembly, and a large portion of the alleged C-terminal Pro/Gly-rich region (amino acids 146 to 195) is not required for the assembly process.
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PMID:Isoprenylation of large hepatitis delta antigen is necessary but not sufficient for hepatitis delta virus assembly. 811 40

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.
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PMID:Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5. 813 57

In order to assess associations between the genetic polymorphism of L-myc and glutathione S-transferase M1 (GST M1) and the risk of hepatocellular carcinoma (HCC), a total of 46 surgically treated HCC patients who were seropositive in hepatitis B surface antigen (HBsAg) and 88 HBs-Ag positive controls were recruited for this study. L-myc and GST M1 genetic polymorphism was examined using a polymerase chain reaction-based restriction fragment length polymorphism assay on DNA extracted from liver and peripheral blood samples. There was no significant difference in GST M1 genotypes between HCC patients and matched controls. A gene dosage trend of association with HCC risk was observed for L-myc genotype. The dose-response relationship remained statistically significant in the multiple logistic regression analysis.
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PMID:L-myc, GST M1 genetic polymorphism and hepatocellular carcinoma risk among chronic hepatitis B carriers. 863 54

This study was carried out to elucidate the effect of glutathione S-transferase (GST) Ml and Tl polymorphisms on the aflatoxin-related hepatocarcinogenesis among chronic carriers of hepatitis B surface antigen (HBsAg). A total of 32 newly diagnosed hepatocellular carcinoma (HCC) cases and 73 age-matched controls selected from a cohort of 4,841 chronic HBsAg carriers who had been followed for 5 years were studied. The level of aflatoxin B1 (AFB1)-albumin adducts in their serum samples collected at the recruitment was examined by competitive enzyme-linked immunosorbance assay, and genotypes of GST M1 and T1 were determined by PCR. There was a dose-response relationship between serum level of AFB1-albumin adducts and risk of HCC. The biological gradients between serum AFB1-albumin adducts level and HCC risk were observed among chronic HBsAg carriers who had null genotypes of GST M1 and/or T1 but not among those who had non-null genotypes. The multivariate-adjusted odds ratios of developing HCC for those who had low and high serum levels of AFB1-albumin adducts compared with those who had a undetectable adduct level as the referent (odds ratio = 1.0) were 4.1 and 12.4, respectively, for HBsAg carriers with null GST M1 genotype (P < .01, on the basis of the significance test for trend); 0.7 and 1.4 for those with non-null GST Ml genotype (P = .98); 1.8 and 10.2 for those with null GST T1 genotype (P < .05); and 1.3 and 0.8 for those with non-null GST T1 genotype (P = .93). The interaction between serum AFB1-albumin adduct level and polymorphisms of GST M1 and T1 was at marginal statistical significance levels (.05 < P < .10).
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PMID:Chronic hepatitis B carriers with null genotypes of glutathione S-transferase M1 and T1 polymorphisms who are exposed to aflatoxin are at increased risk of hepatocellular carcinoma. 865 16

The objective of this work is to examine the possible modulation of carcinogen metabolism (activation by cytochrome P450s and detoxification by conjugation via glutathione S-transferases [GST]) in relation to hepatitis B virus (HBV)-associated liver injury. In HBV transgenic mouse lineage 107.5, the hepatitis B surface antigen (HBsAg) is expressed at noncytopathic concentrations but after injection of an HBsAg-specific, major histocompatibility complex (MHC) class I restricted cytotoxic T-lymphocyte (CTL) clone, the mice develop a severe acute necroinflammatory liver disease that reaches maximum severity within 3 days and gradually subsides during the next 2 to 3 weeks. In this model, using immunohistochemical analysis, we observed an increase of P450s (CYP1A and 2A5), both involved in aflatoxin B1, metabolism, but minor changes or no changes for others (2B, 2C, 2E, 3A). There was a fivefold decrease in the total liver P450 microsomal content 3 days' post-CTL injection with the result that the relative proportion of CYP2A5 and 1A compared with other P450s is increased. Individual microsomal P450 enzyme contents estimated by Western blotting; Northern blot analysis of liver CYP messenger RNA (mRNA) levels as well as in vitro metabolism of specific substrates for different P450 isoenzymes were consistent with the immunohistochemical data. Immunohistochemical staining with antibodies to cytosolic pi class GST was increased 1 and 3 days postinjection followed by a progressive decrease at later time points (the same phenomenon was observed to a lesser extent for GST alpha). The activity of hepatic cytosols toward substrates specific for different subclasses of GST (mu, pi, alpha) showed that while GST mu was not changed in the CTL-injected HBV transgenic mice, GST pi and, to a lesser extent, alpha were increased as compared with controls. These results suggest that liver cell injury induced by a process of acute fulminant-like hepatitis can lead to the induction of some carcinogen metabolizing enzymes notably, Cyp 1A, 2A5 and GST pi in the mouse.
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PMID:Differential induction of carcinogen metabolizing enzymes in a transgenic mouse model of fulminant hepatitis. 878 38

Recently we identified human liver endonexin II (EII) as a specific hepatitis B surface antigen (HBsAg) binding protein. To investigate whether EII is also able to interact with the HBsAg envelope of the hepatitis delta virus (H delta V), immunoprecipitation experiments were performed. H delta V particles could be co-precipitated by polyclonal rabbit anti-EII, but not by rabbit anti-glutathiontransferase (GST pi) antibodies from an H delta V-enriched fraction containing EII or GST pi. These findings suggest that H delta V particles were co-precipitated by anti-EII as a consequence of the binding between HBsAg present in the H delta V envelope and EII. Furthermore, binding of H delta V particles to human hepatocytes could be inhibited by incubation of the liver cells with rabbit anti-EII IgG or the H delta V particles with anti-idiotypic (anti-HBsAg) antibodies, developed spontaneously in rabbits immunized with EII. These findings support the assumption that small HBsAg present in the H delta V envelope is important for the attachment to the hepatocytes and that EII plays an important role in this process.
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PMID:Hepatitis delta virus attaches to human hepatocytes via human liver endonexin II, a specific HBsAg binding protein. 879 May 57

Hepatitis C virus (HCV) produces chronic persistent liver infection in 1-2% of the U.S. population and is the leading cause of end stage liver disease in patients presenting for liver transplantation at our center. Efforts to cure persistent HCV infection are frequently unsuccessful, so the development of a HCV vaccine is a high priority. HCV envelope proteins are hypervariable so production of a recombinant surface antigen vaccine such as is available for hepatitis B is not likely to confer widespread, high level protective immunity. As the most highly conserved structural protein in the HCV genome, the core protein is one reasonable target for vaccine production. Presented here are data on the manufacture of recombinant core protein containing partial carboxy terminus deletions in an effort to increase the efficiency of core expression. The maltose binding protein (MBP) and glutathione S-transferase (GST) protein prokaryotic expression systems were used to study two different constructs, expressing the first 140 and 163 amino acids of the core region. Deletion of the 23 amino acids (aa) from aa141-163 led to a marked increase in the efficiency of protein production from < 1 to 3-4 mg/liter for both systems studied. Protein purification was accomplished using affinity chromatography (MBP) or inclusion body isolation (GST) as determined by SDS-PAGE gels and immunotransblot with HCV core protein-specific monoclonal antibody. Finally, the immune response to recombinant protein was assessed in BALB/c mice using a MBP HCV core fusion protein and an ELISA developed using GST HCV core protein as a target. In all mice of this strain, serum anti-HCV core antibody titer increased to 10(-4), two logs above background, following immunization in conjunction with Freund's complete adjuvant. These results represent an encouraging first step toward production of a core protein vaccine. Recombinant core protein is a useful tool to study the immune response to core protein and may be useful to further study the epidemiology and biology of the HCV virus.
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PMID:High efficiency prokaryotic expression and purification of a portion of the hepatitis C core protein and analysis of the immune response to recombinant protein in BALB/c mice. 882 96


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