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)

LIMPII (lysosomal integral membrane protein II) is one of a family of proteins structurally related to the cell surface glycoprotein CD36. We recently defined a single structural domain on CD36 that mediates binding to adhesive glycoprotein thrombospondin-1 (TSP1). The CD36-TSP1 interaction is known to play a role in platelet-tumor and platelet-monocyte adhesion, angiogenesis, and in monocyte uptake of apoptotic cells. To test whether LIMPII also binds TSP1, a LIMPII peptide corresponding to the TSP1 binding domain of CD36 was expressed as a recombinant glutathione S-transferase (GST) fusion protein. In solid phase binding assays, purified 125I-TSP1 bound to immobilized GST/LIMPII in a time-dependent and saturable manner. Inhibition by excess unlabeled TSP1 or EDTA demonstrated specificity. LIMPII.TSP1 complex formation was specifically blocked by soluble LIMPII fusion protein, by monospecific rabbit IgG directed against the LIMPII peptide and by CD36 fusion proteins containing the TSP1 binding domain. Transfection of Bowes melanoma cells with a chimeric LIMPII cDNA that targets expression to the plasma membrane conferred the ability to bind 125I-TSP1 and to adhere to TSP1-coated surfaces. This study defines a TSP1 binding site conserved between LIMPII and CD36 and suggests that cell surface LIMPII may function in some circumstances as an adhesion receptor for TSP1. Computer-assisted homology searches suggest that the TSP1 recognition motif identified from study of CD36 family members may be widely expressed in nature.
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PMID:Lysosomal integral membrane protein II binds thrombospondin-1. Structure-function homology with the cell adhesion molecule CD36 defines a conserved recognition motif. 947 26

The pseudorabies virus (PrV) gene homologous to herpes simplex virus type 1 (HSV-1) UL53, which encodes HSV-1 glycoprotein K (gK), has recently been sequenced (J. Baumeister, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 69:5560-5567, 1995). To identify the corresponding protein, a rabbit antiserum was raised against a 40-kDa glutathione S-transferase-gK fusion protein expressed in Escherichia coli. In Western blot analysis, this serum detected a 32-kDa polypeptide in PrV-infected cell lysates as well as a 36-kDa protein in purified virion preparations, demonstrating that PrV gK is a structural component of virions. After treatment of purified virions with endoglycosidase H, a 34-kDa protein was detected, while after incubation with N-glycosidase F, a 32-kDa protein was specifically recognized. This finding indicates that virion gK is modified by N-linked glycans of complex as well as high-mannose type. For functional analysis, the UL53 open reading frame was interrupted after codon 164 by insertion of a gG-lacZ expression cassette into the wild-type PrV genome (PrV-gKbeta) or by insertion of the bovine herpesvirus 1 gB gene into a PrV gB- genome (PrV-gK(gB)). Infectious mutant virus progeny was obtained only on complementing gK-expressing cells, suggesting that gK has an important function in the replication cycle. After infection of Vero cells with either gK mutant, only single infected cells or small foci of infected cells were visible. In addition, virus yield was reduced approximately 30-fold, and penetration kinetics showed a delay in entry which could be compensated for by phenotypic gK complementation. Interestingly, the plating efficiency of PrV-gKbeta was similar to that of wild-type PrV on complementing and noncomplementing cells, pointing to an essential function of gK in virus egress but not entry. Ultrastructurally, virus assembly and morphogenesis of PrV gK mutants in noncomplementing cells were similar to wild-type virus. However, late in infection, numerous nucleocapsids were found directly underneath the plasma membrane in stages typical for the entry process, a phenomenon not observed after wild-type virus infection and also not visible after infection of gK-complementing cells. Thus, we postulate that presence of gK is important to inhibit immediate reinfection.
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PMID:Pseudorabies virus glycoprotein gK is a virion structural component involved in virus release but is not required for entry. 949 48

Nine glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, and gL) have been identified in bovine herpesvirus 1 (BHV-1). gM has been identified in many other alpha-, beta-, and gammaherpesviruses, in which it appears to play a role in membrane penetration and cell-to-cell fusion. We sought to express BHV-1 open reading frame U(L)10, which encodes gM, and specifically identify the glycoprotein. We corrected a frameshift error in the published sequence and used the corrected sequence to design coterminal peptides from the C terminus. These were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fusion protein containing the 63 C-terminal amino acids from the corrected gM sequence engendered antibodies that immunoprecipitated a 30-kDa protein from in vitro translation reactions programmed with the U(L)10 gene. Proteins immunoprecipitated by this antibody from virus-infected cells ran at 36 and 43 kDa in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 43 and 48 kDa in nonreducing SDS-PAGE. Only the larger of the pair was present in virions. A 7-kDa protein was released from gM by reducing agents. The 7-kDa protein was not recognized in Western blots probed with the anti-gM antibody but reacted specifically with antibodies prepared against BHV-1 U(L)49.5, previously reported to be a 9-kDa protein associated with an unidentified 39-kDa protein (X. Liang, B. Chow, C. Raggo, and L. A. Babiuk, J. Virol. 70:1448-1454, 1996). This is the first report of a small protein covalently bound to any herpesvirus gM. Similar patterns of hydrophobic domains and cysteines in all known gM and U(L)49.5 homologs suggest that these two proteins may be linked by disulfide bonds in all herpesviruses.
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PMID:Bovine herpesvirus 1 glycoprotein M forms a disulfide-linked heterodimer with the U(L)49.5 protein. 952 25

To cast light on the mechanisms of drug-resistance, experimental brain tumors were immunohistochemically evaluated for expression of glutathione S-transferase (GST)-alpha, mu, pi, p-glycoprotein and apoptosis-related factors, such as bcl-2 and p53, as well as by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) method. Rat brain tumors induced by means of prenatal exposure to ethylnitrosourea (ENU) were treated with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and/or vincristine. Tumors more than 2 mm in size were considered to be drug resistant. The expression of GST-mu was strongly positive in ACNU-treated brain tumors, while p-glycoprotein was overexpressed in vincristine-treated brain tumors. Neither p53 nor bcl-2 expression directly correlated with apoptosis identified by TUNEL method, but tumors lacking apoptotic cells always demonstrated the expression of either GST-mu or p-glycoprotein. These results indicate that tumors resistant to chemotherapy might not be susceptible to induction of apoptosis, and therefore that mechanisms of drug resistance are related to programmed cell death in brain tumors.
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PMID:Drug resistance and apoptosis in ENU-induced rat brain tumors treated with anti-cancer drugs. 952 10

The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1- cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1-, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.
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PMID:The Cys-rich region of hepatitis A virus cellular receptor 1 is required for binding of hepatitis A virus and protective monoclonal antibody 190/4. 955 57

An antibody made against the herpes simplex virus 1 US5 gene predicted to encode glycoprotein J was found to react strongly with two proteins, one with an apparent Mr of 23,000 and mapping in the S component and one with a herpes simplex virus protein with an apparent Mr of 43,000. The antibody also reacted with herpes simplex virus type 2 proteins forming several bands with apparent Mrs ranging from 43,000 to 50,000. Mapping studies based on intertypic recombinants, analyses of deletion mutants, and ultimately, reaction of the antibody with a chimeric protein expressed by in-frame fusion of the glutathione S-transferase gene to an open reading frame antisense to the gene encoding glycoprotein B led to the definitive identification of the new open reading frame, designated UL27.5. Sequence analyses indicate the conservation of a short amino acid sequence common to US5 and UL27.5. The coding sequence of the herpes simplex virus UL27.5 open reading frame is strongly homologous to the sequence encoding the carboxyl terminus of the herpes simplex virus 2 UL27.5 sequence. However, both open reading frames could encode proteins predicted to be significantly larger than the mature UL27.5 proteins accumulating in the infected cells, indicating that these are either processed posttranslationally or synthesized from alternate, nonmethionine-initiating codons. The UL27.5 gene expression is blocked by phosphonoacetate, indicating that it is a gamma2 gene. The product accumulated predominantly in the cytoplasm. UL27.5 is the third open reading frame found to map totally antisense to another gene and suggests that additional genes mapping antisense to known genes may exist.
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PMID:UL27.5 is a novel gamma2 gene antisense to the herpes simplex virus 1 gene encoding glycoprotein B. 962 Oct 69

Twenty-two monoclonal antibodies (MAbs) were generated to the gammaherpesvirus equine herpesvirus-2 (EHV-2). Using Western blot analysis, eight MAbs recognized an Escherichia coli glutathione S-transferase (GST)-glycoprotein B (gB) fusion protein and, using overlapping GST-gB fusion proteins, a neutralization epitope was mapped to amino acids 29-74. One of the gB-specific MAbs was used to characterize the glycosylation and kinetics of synthesis of EHV-2 gB. EHV-2 gB is synthesized as a 97 kDa polypeptide that is co-translationally modified to a 130 kDa high-mannose precursor that forms a 260 kDa dimer shortly after synthesis. Each 130 kDa precursor is endoproteolytically cleaved to disulphide-linked subunits of 75 and 58 kDa prior to further processing to complex oligosaccharide-containing subunits of 89 and 65/62 kDa. The 89 and 65/62 kDa subunits of EHV-2 gB contain 39 and 17 kDa of N-linked oligosaccharides, respectively, and do not contain any O-linked oligosaccharides. Western blot analysis of purified EHV-2 virions established that gB exists as a 320 kDa dimer in the virion envelope.
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PMID:Characterization of glycoprotein B of the gammaherpesvirus equine herpesvirus-2. 968 Jan 23

Varicella-zoster virus (VZV) is an alphaherpesvirus that is the causative agent of chickenpox and herpes zoster. VZV open reading frame 5 (ORF5) encodes glycoprotein K (gK), which is conserved among alphaherpesviruses. While VZV gK has not been characterized, and its role in viral replication is unknown, homologs of VZV gK in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) have been well studied. To identify the VZV ORF5 gene product, we raised a polyclonal antibody against a fusion protein of ORF5 codons 25 to 122 with glutathione S-transferase and used it to study the protein in infected cells. A 40,000-molecular-weight protein was detected in cell-free virus by Western blotting. In immunogold electron microscopic studies, VZV gK was in enveloped virions and was evenly distributed in the cytoplasm in infected cells. To determine the function of VZV gK in virus growth, a series of gK deletion mutants were constructed with VZV cosmid DNA derived from the Oka strain. Full and partial deletions in gK prevented viral replication when the gK mutant cosmids were transfected into melanoma cells. Insertion of the HSV-1 (KOS) gK gene into the endogenous VZV gK site did not compensate for the deletion of VZV gK. The replacement of VZV gK at a nonnative AvrII site in the VZV genome restored the phenotypic characteristics of intact recombinant Oka (rOka) virus. Moreover, gK complementing cells transfected with a full gK deletion mutant exhibited viral plaques indistinguishable from those of rOka. Our results are consistent with the studies of gK proteins of HSV-1 and PRV showing that gK is indispensable for viral replication.
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PMID:Characterization of Varicella-Zoster virus glycoprotein K (open reading frame 5) and its role in virus growth. 1019 16

Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on the E. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase from Schistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed in E. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.
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PMID:Indirect ELISAs based on recombinant and affinity-purified glycoprotein E of Aujeszky's disease virus to differentiate between vaccinated and infected animals. 1021 39

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin alpha(IIb)beta(3). We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (triangle upA1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor gamma-chain through interaction with Syk SH2 domains. GP Ib physically associates with the gamma-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cgamma2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated gamma-chain, phosphorylation of PLCgamma2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR gamma-chain and members of the Src family kinases, leading to phosphorylation of the gamma-chain, recruitment, and activation of Syk. Phosphorylation of PLCgamma2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.
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PMID:Glycoprotein Ib-V-IX, a receptor for von Willebrand factor, couples physically and functionally to the Fc receptor gamma-chain, Fyn, and Lyn to activate human platelets. 1047 89


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