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)

CD83 is a 45-kDa glycoprotein and member of the immunoglobulin (Ig) superfamily. It is the best known marker for mature dendritic cells. Although the precise function of CD83 is not known, its selective expression and upregulation together with the costimulators CD80 and CD86 suggests an important role of CD83 in the induction of immune responses. To perform functional studies and to elucidate its mode of action it is vital to obtain recombinant expressed and highly purified CD83 molecules. Therefore, the external Ig domain of human CD83 (hCD83ext) was expressed as a GST fusion protein (GST-hCD83ext) and the soluble protein was purified under native conditions. The fusion protein was purified using GSTrap columns followed by anion-exchange chromatography. GST-hCD83ext was then cleaved using thrombin and soluble hCD83ext was further purified using GSTrap columns and finally by a preparative gel filtration as a polishing step and used for further characterization. The purified GST-hCD83 fusion protein was also used to generate monoclonal anti-CD83 antibodies in a rat system. Two different monoclonal antibodies were generated. Using these antibodies, CD83 was specifically recognized in FACS and Western blot analyses. Furthermore, we showed that native CD83 is glycosylated and that this glycosylation influences the binding of the antibodies in Western blot analyses. Finally, the purified hCD83ext protein was analyzed by one-dimensional NMR and these analyses strongly indicate that hCD83ext is folded and could therefore be used for further structural and functional studies.
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PMID:Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies. 1192 61

Trimucytin is a powerful platelet aggregation inducer isolated from the venom of Taiwan habu snake (Trimeresurus mucrosquamatus). In this study, we found that the snake venom protein, crovidisin, which prevents collagen-platelet interaction through its high-affinity binding to collagen, inhibited competitively trimucytin-induced aggregation of washed human platelets with a pA(2) value of 6.65. The ability of trimucytin in triggering platelet aggregation was suppressed by a monoclonal antibody (A2-IIE10) raised against the alpha2 subunit of alpha2beta1 integrin (glycoprotein Ia/IIa), indicating that platelet alpha2beta1 integrin plays a central role in trimucytin's platelet reactivity. Many studies have localized the major reactive site of alpha2beta1 integrin to the I-domain of alpha2 subunit. However, Escherichia coli-produced recombinant alpha2 I-domain (GST-alpha2 fusion protein) blocking collagen-induced platelet aggregation failed to inhibit aggregation of platelets in response to trimucytin. Based on these findings, it is concluded that the platelet reactivity of trimucytin is alpha2beta1 integrin-dependent, while the I-domain present in the alpha2 subunit is not involved. This novel snake venom protein would be useful for mapping the functional domain of alpha2beta1 integrin.
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PMID:Trimucytin, a collagen-like snake venom protein, activates platelets independent of I-domain within alpha2 subunit of alpha2beta1 integrin. 1195 6

The gamma2 human herpesvirus-8 (HHV-8) or Kaposi's sarcoma associated herpesvirus (KSHV) ORFs 22 and 47 are counterparts to glycoproteins gH and gL, respectively, that are conserved among the members of herpesviruses. To define HHV-8 gH and gL, rabbit polyclonal antibodies were raised against GST-gH and GST-gL fusion proteins. Anti-gL and anti-gH antibodies reacted with the surface of virus carrying BCBL-1 cells. Both antibodies immunoprecipitated the HHV-8 envelope associated 120 kDa and 41-42 kDa proteins. In transfected COS-1 cells, gH was expressed as an endo-H sensitive 110 kDa glycoprotein, which was absent on the surface of the cells. However, after co-transfection with gL, gH was detected as an endo-H resistant 120 kDa glycoprotein, and was expressed on the surface of the cells. Non-covalent complex formation between gH and gL was detected in the transfected COS-1 cells. Anti-gH and anti-gL antibodies neutralized HHV-8 infectivity in the absence of complement, individually and more efficiently together. However, virus binding to the target cells was not inhibited. These studies suggest that HHV-8 gL is required for gH processing and expression on the cell surface membranes, and gH/gL complex plays an important role in the post-binding step of HHV-8 infection.
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PMID:Characterization of gamma2-human herpesvirus-8 glycoproteins gH and gL. 1211 12

To investigate the immune response to anti-rabies vaccination in the principal recipient (the domestic dog), four truncated fragments of the rabies virus glycoprotein were expressed as glutathione S-transferase fusion proteins. Immune sera from vaccinated rabbits and dogs were then used to probe for reactivity with these expressed proteins. In two rabbits and four dogs tested, the dominant antibody response to non-conformational antigenic sites appeared to be directed to a region of the glycoprotein between amino acids 222 and 332. The N-terminal fragment of the glycoprotein was also significantly antigenic. Further studies to assess whether the antibody response to the internal domain could neutralize the rabies Challenge Virus Standard (CVS) strain, using antibody depletion, suggested that this fraction did contribute to the ability of post-vaccination sera to neutralize and therefore protect against infection.
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PMID:Canine vaccine recipients recognize an immunodominant region of the rabies virus glycoprotein. 1238 1

The binding of von Willebrand factor (VWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3- kinase (PI 3-kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3-kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3-kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)-fusion protein coprecipitated the p85 subunit of PI 3-kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3-kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3- kinase.
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PMID:Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3-kinase. 1239 36

In recent years, there have been a number of efforts to identify genes that are expressed in mature ovarian follicles in response to an ovulatory dose of LH or its homologue hCG. This review keys on 20 ovulation-specific genes that we have identified by the molecular procedure known as differential display. The objective is to use this sampling of genes to illustrate the diversity in the temporal and spatial patterns of expression of genes in the ovary following the stimulus of this gonadal target tissue by a single glycoprotein hormone. The specific genes that are surveyed include 5-aminolevulinate synthase; early growth response protein-1; gamma-glutamylcysteine synthetase; cyclooxygenase-2; epiregulin; pituitary adenylate cyclase-activating polypeptide; tumor necrosis factor-stimulated gene-6; regulator of G-protein signaling protein-2; adrenodoxin; steroidogenic acute regulatory protein; 3alpha-hydroxysteroid dehydrogenase; CD63, a disintegrin and metalloproteinase with thrombospondin motifs; tissue inhibitor of metalloproteinase-1; carbonyl reductase, a G-protein-coupled receptor; pancreatitis-associated protein-III; glutathione S-transferase; and metallothionein-1. The ovulatory expression of these different genes is predominantly within the granulosa layer of mature follicles. However, there were also instances of expression in the thecal and stromal tissue of the ovary, as well as in vascular endothelial cells and in luteal tissue. The overwhelming impression is that the molecular events of ovulation are far more complex, and therefore more highly ordered, than originally imagined.
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PMID:Temporal and spatial patterns of ovarian gene transcription following an ovulatory dose of gonadotropin in the rat. 1244 39

A 1.1 bp fragment of E2 gene of Chinese classical swine fever virus(CSFV) Shimen strain, a standard virulent strain, was amplified by RT-PCR from total RNA of cell cultures infected by CSFV, and cloned into pGEM T vector. The nucleotide sequence of this fragment was sequenced by Sanger's method and the amino acid sequence was deduced. Compared with the corresponding region of Alfort, Brescia and C strain of CSFV, the nucleotide sequence homology is 84.7%, 92.6% and 95.2% respectively, and the amino acid sequence 89.4%, 92.6% and 94.6%, respectively, we subcloned 1.1 bp of E2 gene cDNA into baculovirus transfer vector and successfully constructed two recombinant baculoviruses expressing GST-E2 and GST-GFP-E2 fusion protein respectively by homologous recombination in sf-9 cell. Furthermore, we also constructed recombinant eukaryotic expression vector pcE2 containing E2 gene in frame and transfected COS-7 cell by lipofectamine, the indirect immunofluorescence assay (IFA) showed that the expressed E2 protein can be recognized by E2 specific monoclonal antibody the pcE2 DNA was directly injected into BALB/c mice intramuscular(i.m.) and the CSFV E2-specific antibodies was measured by enzyme-linked immunosorbent assay(ELISA) the ELISA results indicated the E2-specific antibodies was induced in inoculated mice and virus neutralization assays also indicate single inoculations of plasmids expressing CSFV E2 glycoprotein raised neutralizing antibody in BALB/c mice. these results will be beneficial to investigate the possibility of DNA vaccine against CSFV.
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PMID:[Molecular cloning and expression of E2 gene of the Chinese classical swine fever virus(shimen strain) and preliminary studies of its DNA vaccine]. 1254 87

Rabies virus glycoprotein (G) is a trimeric type I transmembrane glycoprotein that mediates both receptor recognition and low pH-induced membrane fusion. Electron microscopy has indicated that the ectodomain of protein G is made of a globular head and a stem. In order to characterize the putative stem region at the molecular level, we designed two peptides, P(S) and P(L), which were produced as GST fusion proteins in bacteria. Peptide P(S) extends from amino acid (aa) 374 to aa 428 whereas peptide P(L) extends from aa 368 down to the end of the ectodomain of G (aa 439). Their secondary and quaternary structures have been studied with spectroscopic and biophysical methods. We show that these isolated peptides are monomeric and poorly structured in aqueous solution. However, circular dichroism (CD) in presence of 2,2,2-trifluoroethanol and NMR data indicate that this region may adopt a alpha-helical conformation in the complete glycoprotein.
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PMID:Spectroscopic characterization of two peptides derived from the stem of rabies virus glycoprotein. 1278 63

We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibalpha (GpIbalpha) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)-induced platelet activation. To begin, we examined filamin binding to GpIbalpha in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbalpha's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbalpha's filamin A-binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbalpha cytoplasmic domain fusion protein. One peptide (residues 557-575; designated "A4 peptide") inhibited filamin A binding to the GST-GpIbalpha cytoplasmic domain fusion protein and competed with GpIbalpha for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbalpha and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbalpha regulates proaggregatory tyrosine kinase signaling.
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PMID:Filamin A binding to the cytoplasmic tail of glycoprotein Ibalpha regulates von Willebrand factor-induced platelet activation. 1279 64

Tva is the receptor for subgroup A Rous sarcoma virus, and it contains a single LDL-A module which is the site of virus interaction. In this study, we expressed the entire extracellular region of Tva (referred to as Ecto-Tva) as a GST fusion protein and characterized its refolding properties. We demonstrated that the correct folding of the Ecto-Tva protein, like that of the Tva LDL-A module, is calcium dependent. We used the IAsys system to measure the kinetics of binding between the surface (SU) subunit of the viral glycoprotein and Tva in real time. We found that the Ecto-Tva protein and the Tva LDL-A module displayed similar affinities for SU, providing direct evidence that the LDL-A module of Tva is the only viral interaction domain of the receptor. Furthermore, misfolded Tva proteins displayed lower binding affinities to SU, largely due to a decrease in their association rates, suggesting that a high association rate between SU and Tva is crucial for efficient virus-host interaction. Furthermore, we found that calcium did not influence the overall binding affinity between Tva and SU. These results indicate that, although calcium is important in facilitating correct folding of the LDL-A module of Tva, it is not essential for ligand binding. Thus, these results may have broad implications for the mechanism of protein folding and ligand recognition of the LDL receptor and other members of the LDL receptor superfamily.
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PMID:Kinetic analysis of binding interaction between the subgroup A Rous sarcoma virus glycoprotein SU and its cognate receptor Tva: calcium is not required for ligand binding. 1280 52


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