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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The V3 loop of the HIV (human immunodeficiency virus)-1 envelope glycoprotein gp120 likely plays a role in HIV-1 infectivity. Although the amino acid sequence of the V3 loop is hypervariable, it contains a conserved region, Gly-Pro-Gly-Arg, that shows similarity to the active-site Gly-Pro-Cys-Arg sequence of inter-alpha-trypsin and trypstatin proteinase inhibitors. The purpose of the present work was to identify proteinases recognizing substrates with basic amino acids in the P1 substrate site that are present in MOLT-4 cells, a human CD4-positive T helper lymphocyte cell line, and to characterize these enzymes in terms of substrate, pH and ionic-strength preferences, size and susceptibility to various inhibitors, including 24- and 36-amino-acid-long V3 loop peptides. Extraction of MOLT-4 cells at low ionic strength solubilized nearly all of the trypsin-like activity, which was separable into five peaks of activity by chromatography on Mono-Q: Peaks 1, 2a, 2b, 3 and 4. All showed a neutral pH optimum, and all except Peak 4 showed optimal activity at high ionic strength. Peak 1 preferred Tos-Gly-Pro-Arg, p-nitroanilide (-pNA) substrate; Peaks 2-4 preferred benzyloxycarbonyl-Val-Leu-Gly-Arg-pNA. Peak 1, a zinc-dependent enzyme with serine and histidine in the active site, exhibited an M(r) of 75,000 on Superose 12 and was poorly inhibited by V3 loop peptides. Peak 2 contained two overlapping peaks, called 2a and 2b, that exhibited properties of zinc-dependent metalloproteinases. Gel filtration of Peak 2 activities revealed a major peak of activity at 81 kDa and a shoulder centred at 240 kDa. Each was modestly inhibited by V3 loop peptides. Peak 3, a zinc-dependent proteinase, exhibited a molecular mass of 100 kDa by gel filtration and was particularly sensitive to inhibition by V3 loop peptides. Peak 4 exhibited a molecular mass of 1100 kDa by gel filtration and was not inhibited by V3 loop peptides. None of these enzymes could be classified as mast-cell tryptase, and material in MOLT-4 cells cross-reactive with anti-(human tryptase) antibodies was not detected. Whether any of the MOLT-4 proteinases described in this study play a role in HIV-1 infectivity remains to be examined.
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PMID:Separation and partial characterization of proteinases with substrate specificity for basic amino acids from human MOLT-4 T lymphocytes: identification of those inhibited by variable-loop-V3 peptides of HIV-1 (human immunodeficiency virus-1) envelope glycoprotein. 831 3

In enveloped viruses, post-translational proteolytic activation is a critical step for the fusion activity and thus for the infectivity of the virus. In addition to the membrane receptors for the viruses, proteolytic activation is indispensable for effective virus spread in the infected host and it is a prime determinant for pathogenicity. Here we described the host cellular processing proteases, tryptase Clara and tryptase TL2, which proteolytically activate the infectivity of influenza A and Sendai viruses in the respiratory tract and HIV-1 in human CD4+ T cells, respectively. A novel trypsin-like protease, designated tryptase Clara, was purified from rat lung. The enzyme is localized in Clara cells of the bronchial epithelium and is secreted into the airway lumen. The enzyme specifically recognizes the consensus cleavage motif Gln(Glu)-X-Arg of influenza A and Sendai viruses and proteolytically activates the envelope fusion glycoproteins of the progeny viruses extracellularly in the airway lumen. Human mucus protease inhibitor and pulmonary surfactant in airway fluid inhibited the proteolytic activation of these viruses and also suppressed multiple cycles of viral replication in vitro. These results suggest that an imbalance between the amount of tryptase Clara and that of endogenous inhibitors in airway fluid is a prime determinant for pneumopathogenicity of the viruses. Therefore supplementing an endogenous inhibitor at therapeutic doses may protect against virus infection. In HIV-1 infection, binding of the gp120 envelope glycoprotein to the CD4 receptor is not sufficient in itself to allow virus entry, and an additional component(s) in the membrane is required for cell infection as a cofactor. We isolated a serine protease named tryptase TL2, in the membrane of CD4+ lymphocytes, which specifically binds to the V3 loop of HIV-1 gp120 as a cofactor. After binding, tryptase TL2 proteolytically processed gp120 into two protein species of 70 and 50 kDa and the cleavage was suppressed by a neutralizing antibody against the V3 loop. The amino acids that constitute the cleavage sites in the V3 loop of almost all HIV isolates are variable, but they are restricted to those which are susceptible to chymotryptic and/or tryptic enzyme. The multi-substrate specificity of tryptase TL2, which has tryptic and chymotryptic specificities, may correspond tot he variability of the V3 loop. The selective cleavage of the V3 loop by tryptase TL2 may lead to a conformational change of gp120, resulting in the dissociation of gp120 from gp41, exposing the fusogenic domain of the transmembrane protein gp41 following virus-host cell fusion.
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PMID:Cellular proteases involved in the pathogenicity of enveloped animal viruses, human immunodeficiency virus, influenza virus A and Sendai virus. 886 54

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity and cell penetration. Recombinant forms of gB being used for the development of subunit vaccines are able to induce virus-neutralizing antibodies and protective efficacy in animal models. To gain structural information about the protein, we have determined the location of the disulfide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by expression in Chinese hamster ovary cells. The purified protein, which contains virtually the entire extracellular domain of herpes simplex virus type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptides were characterized by using mass spectrometry and amino acid sequence analysis. The conditions of cleavage (4 M urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three different HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule were found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys-571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to be similar in the corresponding gBs from other herpesviruses because the 10 cysteines listed above are always conserved in the corresponding protein sequences.
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PMID:Disulfide bonds of herpes simplex virus type 2 glycoprotein gB. 889 56

Tryptase Clara, a trypsin-like protease localized exclusively in and secreted from Clara cells to the bronchial epithelium of rat, proteolytically activates the infectivity of influenza A virus [H. Kido, Y. Yokogoshi, K. Sakai, M. Tashiro, Y. Kishino, A. Fukutomi, and N. Katunuma (1992) J. Biol. Chem. 267, 13573-13579]. We report here that human mucus protease inhibitor (MPI), a major inhibitor of granulocyte elastase in the lining fluids of the human respiratory tract, significantly inhibited proteolytic activation of the infectivity of influenza A and Sendai viruses by tryptase Clara in vitro and multi-cycles of mouse-adapted influenza A virus replication in rat lungs in vitro. Recombinant MPI and the C- but not the N-terminal domain of the MPI inhibited both the proteolytic activity of tryptase Clara and the activation of virus infection. The 50% inhibitory concentrations of recombinant MPI and the C-terminal domain for tryptase Clara with Sendai virus envelope glycoprotein as substrate were 7.4 and 61.6 nM, respectively. These results indicate that MPI is a defensive compound against virus infection. Since there is evidence suggesting that concentrations of MPI in respiratory fluids are insufficient for prevention of virus infection, administration of MPI in the airway may be useful for treatment of these virus infections.
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PMID:Human mucus protease inhibitor in airway fluids is a potential defensive compound against infection with influenza A and Sendai viruses. 908 5

Semliki Forest virus (SFV) has been shown previously to fuse efficiently with cholesterol- and sphingolipid-containing liposomal model membranes in a low-pH-dependent manner. Several steps can be distinguished in this process, including low-pH-induced irreversible binding of the virus to the liposomes, facilitated by target membrane cholesterol, and subsequent fusion of the viral membrane with the liposomal bilayer, specifically catalyzed by target membrane sphingolipid. Binding and fusion are mediated by the heterodimeric viral envelope glycoprotein E2/E1. At low pH the heterodimer dissociates, and the E1 monomers convert to a homotrimeric structure, the presumed fusion-active conformation of the viral spike. In this paper, we demonstrate that SFV-liposome fusion is specifically inhibited by Zn2+ ions. The inhibition is at the level of the fusion reaction itself, since virus-liposome binding was found to be unaffected. Zn2+ did not inhibit E2/E1 dissociation, but severely inhibited exposure of an acid-specific epitope on E1, E1 homotrimer formation, and acquisition of trypsin-resistance. It is concluded that virus--liposome binding solely requires low-pH-induced E2/E1 heterodimer dissociation, while fusion depends on further rearrangements in the E1 spike protein. As these rearrangements occur subsequent to the binding step, their precise course, including the formation of a fusion complex, may be influenced by interaction of E1 with target membrane lipids.
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PMID:Membrane fusion activity of Semliki Forest virus in a liposomal model system: specific inhibition by Zn2+ ions. 937 4

The envelope glycoprotein G of vesicular stomatitis virus induces membrane fusion at acidic pH. A highly conserved amino terminal region spanning residues 123 to 137 has previously been identified as an internal fusion domain. Here we have substituted specific amino acids within a carboxy terminal region, conserved in five vesiculoviruses encompassing residues 395 to 418, and studied the effect of these mutations on membrane fusion at acid pH and pH-dependent conformational change. Substitution of conserved Gly 395, Gly 404, Gly 406, Asp 409, and Asp 411 with Glu, Ala, Ala, Asn, and Asn, respectively, decreased the cell-cell fusion efficiency, as well as reduced the pH threshold of membrane fusion. Mutation of Gly 404 and Asp 409 to Lys and Ala, respectively, abolished the fusion activity. Mutant Gly 404 Lys also showed markedly altered resistance to trypsin digestion at acidic pH. These results suggest that the region between amino acids 395 to 418 is important for the fusogenic activity of the G protein. The possible role of this domain in conformational changes involved in fusion activity of VSV G is also discussed.
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PMID:Mutations in a carboxy-terminal region of vesicular stomatitis virus glycoprotein G that affect membrane fusion activity. 950 Oct 39

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.
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PMID:Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes. 1048

Human cytomegalovirus (HCMV) is known to down-regulate the expression of human leukocyte antigen (HLA) class I, the process of which involves a subset of virus genes. Infection of human foreskin fibroblast (HFF) cells with UV-inactivated HCMV (UV-HCMV), however, resulted in an increase in HLA class I presentation on the cell surface in the absence of HCMV gene expression. Heparin, which inhibits the interaction of virus particles with cell surface heparan sulfate proteoglycans (HSPGs), blocked the effect of UV-HCMV on HLA class I expression. Pretreatment of cells with heparinase I decreased in a dose-dependent manner the effect of UV-HCMV on HLA class I expression enhancement. Sodium chlorate, which is known to inhibit the sulfation of HSPGs, gave a similar result. Pretreatment of UV-HCMV with trypsin or monoclonal antibody reactive with the envelope glycoprotein gB reduced the increase in HLA class I expression on the HFF cell surface by UV-HCMV. RT-PCR analysis demonstrated that the increase in HLA class I presentation on the HFF cell surface was due to an increase in HLA class I transcription. Thus, binding of HCMV particles to cell surface HSPGs appears to be required for the stimulation of HLA class I expression. It is also possible that virus entry, in addition to binding to HSPGs, may be involved in the stimulation of HLA class I expression, since the UV-HCMV entered the cells and all treatments to block virus binding to HSPGs would necessarily prevent virus entry.
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PMID:Human cytomegalovirus binding to heparan sulfate proteoglycans on the cell surface and/or entry stimulates the expression of human leukocyte antigen class I. 1156 34

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gin-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsin-type serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-alpha and -beta. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.
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PMID:A novel influenza A virus activating enzyme from porcine lung: purification and characterization. 1267 14

The endothelial cell line ECV304, derived from human umbilical cord and identified to be susceptible to dengue virus type 2 (DEN-2) infection, was used to study the molecular mechanism of DEN-2 binding to endothelial cells. DEN-2 was found by virus overlay protein-binding assays (VOPBAs) to bind to three ECV304 cell membrane proteins with molecular masses of 29, 34 and 43 kDa. Only a single protein of 29 kDa was observed when VOPBAs were carried out using preparations of trypsin-treated ECV304 cells. Pre-incubation of live ECV304 cells in culture or cell membrane proteins in modified VOPBAs with the recombinant DEN-2 envelope glycoprotein (rEgp) inhibited DEN-2 infection and blocked virus binding to the three proteins identified. These results indicate that DEN-2 rEgp could bind to three proteins on the surface of ECV304 cells. This virus-cell interaction may be associated with the receptor complex specific for DEN-2 infection of endothelial cells.
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PMID:Dengue virus type 2 infects human endothelial cells through binding of the viral envelope glycoprotein to cell surface polypeptides. 1457 14


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