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)

Hepatitis C virus (HCV) causes chronic hepatitis, liver cirrhosis and hepatocellular carcinoma in addition to acute hepatitis. The HCV genome encodes two envelope glycoproteins, E1 and E2. To investigate the role of E1 and E2 in HCV infection, we used a recombinant vesicular stomatitis virus (VSV), VSVdeltaG*, harboring the green fluorescent protein gene instead of the VSV G envelope protein gene. It was complemented with the native form of E1 and E2, or E1 or E2 alone, to make HCV pseudotypes VSVdeltaG*(HCV), VSVdeltaG*(E1), and VSVdeltaG*(E2). Neither E1 nor E2 expression was detected on the cell surface, as reported. Unlike previous reports, infectious activities of VSVdeltaG*(HCV), VSVdeltaG*(E1) and VSVdeltaG*(E2) pseudotypes were detected under conditions where VSV was completely neutralized by anti-VSV. We could enhance the infectious titers 100-fold by sonication upon virus harvest. Bovine lactoferrin efficiently inhibited infection by VSVdeltaG*(HCV) as well as VSVdeltaG*(E2), as the interaction between E2 and lactoferrin has been thought to contribute to the inhibition of HCV infectivity. VSVdeltaG*(HCV) infected many adherent cell lines, including hepatic cell lines, but not most hematopoietic cell lines. Treatment of cells with trypsin, tunicamycin, or sulfated polysaccharides before infection reduced the infectivity of VSVdeltaG*(HCV) by about 90%, suggesting that a cell surface protein(s) with sugar chains plays an important role in HCV infection. The VSV pseudotypes developed here would be useful for analyzing the early stages of HCV infection.
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PMID:Efficient formation of vesicular stomatitis virus pseudotypes bearing the native forms of hepatitis C virus envelope proteins detected after sonication. 1571 60

Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The precursor activity of serum Gc protein was reduced in all influenza virus-infected patients. These patient sera contained alpha-N-acetylgalactosaminidase (Nagalase) that deglycosylates Gc protein. Deglycosylated Gc protein cannot be converted to MAF, thus it loses the MAF precursor activity, leading to immunosuppression. An influenza virus stock contained a large amount of Nagalase activity. A sucrose gradient centrifugation analysis of the virus stock showed that the profile of Nagalase activity corresponds to that of hemagglutinating activity. When these gradient fractions were treated with 0.01% trypsin for 30 min, the Nagalase activity of each fraction increased significantly, suggesting that the Nagalase activity resides on an outer envelope protein of the influenza virion and is enhanced by the proteolytic process. After disruption of influenza virions with sodium deoxycholate, fractionation of the envelope proteins with mannose-specific lectin affinity column along with electrophoretic analysis of the Nagalase peak fraction revealed that Nagalase is the intrinsic component of the hemagglutinin (HA). Cloned HA protein exhibited Nagalase activity only if treated with trypsin. Since both fusion capacity and Nagalase activity of HA protein are expressed by proteolytic cleavage, Nagalase activity appears to be an enzymatic basis for the fusion process. Thus, Nagalase plays dual roles in regulating both infectivity and immunosuppression.
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PMID:Pathogenic significance of alpha-N-acetylgalactosaminidase activity found in the hemagglutinin of influenza virus. 1584 73

Baculovirus occlusion-derived virions (ODVs) are released from occlusion bodies by the alkaline environment of the insect midgut. The ODV envelope protein P74 is required for oral infectivity. A soluble form of the Autographa californica multiple nucleopolyhedrovirus P74 protein, P74sol, was engineered as part of a chimeric protein with jellyfish green fluorescent protein (GFP). P74sol-GFP was overproduced by the baculovirus expression system and purified away from the wild-type P74. Brush border membrane vesicles (BBMVs) were prepared from the midguts of third-instar Helicoverpa zea larvae. When P74sol-GFP was incubated under alkaline conditions with BBMVs, a P74sol-GFP product with a smaller molecular mass was produced. Immunoblots indicated that the smaller product was generated by N-terminal cleavage of P74. This cleavage was prevented by soybean trypsin inhibitor. Analysis of the peptide sequences of P74 homologues identified a conserved trypsin cleavage site that could generate the observed P74sol-GFP BBMV-specific cleavage product.
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PMID:Evidence for proteolytic cleavage of the baculovirus occlusion-derived virion envelope protein P74. 1591 41

Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The precursor activity of serum Gc protein was lost or reduced in HIV-infected patients. These patient sera contained alpha-N-acetylgalactosaminidase (Nagalase), which deglycosylates serum Gc protein. Deglycosylated Gc protein cannot be converted to MAF and thus loses MAF precursor activity, leading to immunosuppression. Nagalase in the blood stream of HIV-infected patients was complexed with patient immunoglobulin G, suggesting that this enzyme is immunogenic, seemingly a viral gene product. In fact, Nagalase was inducible by treatment of cultures of HIV-infected patient peripheral blood mononuclear cells with a provirus-inducing agent. This enzyme was immunoprecipitable with polyclonal anti-HIV but not with anticellular constitutive enzyme or with antitumor Nagalase. The kinetic parameters (km value of 1.27 mM and pH optimum of 6.1), of the patient serum Nagalase were distinct from those of constitutive enzyme (km value of 4.83 mM and pH optimum of 4.3). This glycosidase should reside on an envelope protein capable of interacting with cellular membranous O-glycans. Although cloned gp160 exhibited no Nagalase activity, treatment of gp160 with trypsin expressed Nagalase activity, suggesting that proteolytic cleavage of gp160 to generate gp120 and gp41 is required for Nagalase activity. Cloned gp120 exhibited Nagalase activity while cloned gp41 showed no Nagalase activity. Since proteolytic cleavage of protein gp160 is required for expression of both fusion capacity and Nagalase activity, Nagalase seems to be an enzymatic basis for fusion in the infectious process. Therefore, Nagalase appears to play dual roles in viral infectivity and immunosuppression.
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PMID:Pathogenic significance of alpha-N-acetylgalactosaminidase activity found in the envelope glycoprotein gp160 of human immunodeficiency virus Type 1. 1654 13

The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV.
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PMID:Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein. 1672 70

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.
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PMID:Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells. 1700 19

Previous work has shown that the secretion of enveloped hepatitis B virus (HBV) DNA and the HBV middle envelope protein (MHBs) are sensitive to glucosidase inhibition. Here, it is shown that HBV DNA secretion remains depressed after the removal of the glucosidase inhibitor and long after glucosidase function returns to normal. For example, glyco-processing and the secretion of alpha-1 anti-trypsin returned to normal within 3 h of the removal of the glucosidase inhibitor. In contrast, the secretion of HBV did not return to normal for more than 7 days after the removal of the inhibitor. Consistent with the inhibition of HBV virion secretion, the levels of HBV L and HBV M proteins were also reduced by treatment with the glucosidase inhibitor and remained reduced for 7 days after compound withdrawal. The implications of the prolonged antiviral effect against HBV and the use of glucosidase inhibitors as antiviral agents are discussed.
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PMID:alpha-Glucosidase inhibitors have a prolonged antiviral effect against hepatitis B virus through the sustained inhibition of the large and middle envelope glycoproteins. 1717 30

Growth hormone (GH) transgenic amago salmon (Oncorhynchus masou) were generated with a construct containing the sockeye salmon GH1 gene fused to the metallothionein-B (MT-B) promoter from the same species. This transgene directed significant growth enhancement with transgenic fish reaching approximately four to five times greater weight than control salmon in F(2) and F(3) generations. This drastic growth enhancement by GH transgene is well known in fish species compared with mammals, however, such fish can show morphological abnormalities and physiological disorders like other GH transgenic animals. GH is known to have many acute effects, but currently there are no data describing the chronic effects of over-expression of GH on various hepatic genes in GH transgenic fish. Hepatic gene expression is anticipated to play very important roles in many physiological functions and growth performance of transgenic and control salmon. To examine these effects, we performed subtractive hybridization (using cDNA generated from liver RNA) in both directions to identify genes both increased and decreased in transgenic salmon relative to controls (576 clones were isolated and sequenced in total). Heme oxygenase, vitelline envelope protein, Acyl-coA binding protein, NADH dehydrogenase, mannose binding lectin-associated serine protease, hemopexin-like protein, leucyte-derived chemotaxin2 (LECT2), and many other genes were obtained in higher clone frequencies suggesting enhanced expression. In contrast, complement C3-1, lectin, rabin, alcohol dehydrogenase, Tc1-like transposase, Delta6-desaturase, and pentraxin genes were obtained in lower frequencies. Microarray analysis was also performed to obtain quantitative expression data for these subtracted cDNA clones. Analysis of fish across seasons was also conducted using both F(2) and F(3) salmon. Results of the microarray data essentially corresponded with those of the subtraction data when both F(2) and F(3) fish were completely immature, but the expression pattern was changed when fish approached maturation. Genes showing enhanced expression in GH transgenic fish in F(2) and F(3) by array analysis were vitelline envelope protein, hemopexin-like protein, heme-oxygenase, inter alpha-trypsin inhibitor, LECT2, GTP cyclohydrolase I feedback regulatory protein (GFRP), and bikunin. Reduced expression genes were lectin, Delta6-desaturase, apolipoprotein, and pentraxin. In particular, lectin was found to be highly suppressed in all F(2) and immature F(3) salmon. Further, serum lysozyme activity, one of innate immunity, was significantly (p<0.05) decreased in both F(2) and F(3) GH transgenic fish. These results indicate that the GH transgene fish had altered hepatic gene expression relating to iron-metabolism, innate immunity, reproduction, and growth.
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PMID:Changes in hepatic gene expression related to innate immunity, growth and iron metabolism in GH-transgenic amago salmon (Oncorhynchus masou) by cDNA subtraction and microarray analysis, and serum lysozyme activity. 1722 41

The bio-nanocapsules (BNCs) composed of the recombinant envelope L-protein of hepatitis B virus constitute efficient delivery vectors specifically targeting human hepatocytes. Here, we have tried to enhance the stability of the BNCs because the L-proteins in the BNCs were aggregated due to random disulfide bridging when stored for a long period at 4 degrees C. The envelope protein contains fourteen cysteine residues in the S domain. Aggregation of the envelope proteins might be avoided if unessential cysteine residues are replaced or removed because the irreversible alkylation of the free sulfhydryl group protects against the aggregation and enhances the efficiency of encapsulation. In this study, the possibility of reducing the number of cysteine residues in the S domain to enhance the stability of the BNCs was assessed. The replacement of each cysteine residue by site-directed mutation showed that nine of fourteen cysteine residues were not essential to obtaining BNCs secreted into the culture media. Furthermore, upon evaluating the combination of these mutations, it was found that eight residues of replacement were acceptable. The mutant BNCs with replaced eight cysteine residues were not only more resistant against trypsin, but also more effective in transducing genes into human hepatoma-derived HepG2 cells than the original type BNC. Thus, we demonstrated that the minimized number of cysteine residues in the S domain could enhance the stability of the BNCs.
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PMID:Characterization of bio-nanocapsule as a transfer vector targeting human hepatocyte carcinoma by disulfide linkage modification. 1730 5

The dengue virus envelope protein plays an essential role in viral entry by mediating fusion between the viral and host membranes. The crystal structure of the envelope protein shows a pocket (located at a "hinge" between Domains I and II) that can be occupied by ligand n-octyl-beta-D-glucoside (betaOG). Compounds blocking the betaOG pocket are thought to interfere with conformational changes in the envelope protein that are essential for fusion. Two fusion assays were developed to examine the anti-fusion activities of compounds. The first assay measures the cellular internalization of propidium iodide upon membrane fusion. The second assay measures the protease activity of trypsin upon fusion between dengue virions and trypsin-containing liposomes. We performed an in silico virtual screening for small molecules that can potentially bind to the betaOG pocket and tested these candidate molecules in the two fusion assays. We identified one compound that inhibits dengue fusion in both assays with an IC(50) of 6.8 microM and reduces viral titers with an EC(50) of 9.8 microM. Time-of-addition experiments showed that the compound was only active when present during viral infection but not when added 1h later, in agreement with a mechanism of action through fusion inhibition.
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PMID:A small molecule fusion inhibitor of dengue virus. 1980 Mar 68


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