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:3.2.1.96 (endoglycosidase H)
1,826 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exocytic organelles undergo profound reorganization during myoblast differentiation and fusion. Here, we analyzed whether glycoprotein processing and targeting changed during this process by using vesicular stomatitis virus (VSV) G protein and influenza virus hemagglutinin (HA) as models. After the induction of differentiation, the maturation and transport of the VSV G protein changed dramatically. Thus, only half of the G protein was processed and traveled through the Golgi, whereas the other half remained unprocessed. Experiments with the VSV tsO45 mutant indicated that the unprocessed form folded and trimerized normally and then exited the ER. It did not, however, travel through the Golgi since brefeldin A recalled it back to the ER. Influenza virus HA glycoprotein, on the contrary, acquired resistance to endoglycosidase H and insolubility in Triton X-100, indicating passage through the Golgi. Biochemical and morphological assays indicated that the HA appeared at the myotube surface. A major fraction of the Golgi-processed VSV G protein, however, did not appear at the myotube surface, but was found in intracellular vesicles that partially colocalized with the regulatable glucose transporter. Taken together, the results suggest that, during early myogenic differentiation, the VSV G protein was rerouted into developing, muscle-specific membrane compartments. Influenza virus HA, on the contrary, was targeted to the myotube surface.
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PMID:Differential targeting of vesicular stomatitis virus G protein and influenza virus hemagglutinin appears during myogenesis of L6 muscle cells. 949 Jul 23

Endo-alpha-D-mannosidase is an enzyme involved in N-linked oligosaccharide processing which through its capacity to cleave the internal linkage between the glucose-substituted mannose and the remainder of the polymannose carbohydrate unit can provide an alternate pathway for achieving deglucosylation and thereby make possible the continued formation of complex oligosaccharides during a glucosidase blockade. In view of the important role which has been attributed to glucose on nascent glycoproteins as a regulator of a number of biological events, we chose to further define the in vivo action of endomannosidase by focusing on the well characterized VSV envelope glycoprotein (G protein) which can be formed by the large array of cell lines susceptible to infection by this pathogen. Through an assessment of the extent to which the G protein was converted to an endo-beta-N-acetylglucosaminidase (endo H)-resistant form during a castanospermine imposed glucosidase blockade, we found that utilization of the endomannosidase-mediated deglucosylation route was clearly host cell specific, ranging from greater than 90% in HepG2 and PtK1 cells to complete absence in CHO, MDCK, and MDBK cells, with intermediate values in BHK, BW5147.3, LLC-PK1, BRL, and NRK cell lines. In some of the latter group the electrophoretic pattern after endo H treatment suggested that only one of the two N-linked oligosaccharides of the G protein was processed by endomannosidase. In the presence of the specific endomannosidase inhibitor, Glcalpha1-->3(1-deoxy)mannojirimycin, the conversion of the G protein into an endo H-resistant form was completely arrested. While the lack of G protein processing by CHO cells was consistent with the absence of in vitro measured endomannosidase activity in this cell line, the failure of MDBK and MDCK cells to convert the G protein into an endo H-resistant form was surprising since these cell lines have substantial levels of the enzyme. Similarly, we observed that influenza virus hemagglutinin was not processed in castanospermine-treated MDCK cells. Our findings suggest that studies which rely on glucosidase inhibition to explore the function of glucose in controlling such critical biological phenomena as intracellular movement or quality control should be carried out in cell lines in which the glycoprotein under study is not a substrate for endomannosidase action.
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PMID:Processing of viral envelope glycoprotein by the endomannosidase pathway: evaluation of host cell specificity. 962 Nov 13

To characterize human herpesvirus 8 (HHV-8) gB, the open reading frame was PCR amplified from the HHV-8-infected cell line BCBL-1 and cloned into an expression vector. To facilitate detection of expressed HHV-8 gB, the cytoplasmic tail of the glycoprotein was tagged with the influenza hemagglutinin (HA) epitope. Expression of tagged HHV-8 gB (gB-HA), as well as the untagged form, was readily detected in CHO-K1 cells and several lymphoblastoid cell lines (LCLs). HHV-8 gB-HA was sensitive to endoglycosidase H treatment, and immunofluorescence revealed that HHV-8 gB-HA was detectable in the perinuclear region of CHO-K1 cells. These observations suggest that HHV-8 gB is not processed in the Golgi and localizes to the endoplasmic reticulum or nuclear membrane. Because both HHV-8 and EBV are gamma-herpesviruses, the ability of HHV-8 gB to interact with and functionally complement EBV gp110 was examined. HHV-8 gB-HA and EBV gp110 co-immunoprecipitated, indicating formation of hetero-oligomers. However, HHV-8 gB-HA and HHV-8 gB failed to restore the infectivity of gp110-negative EBV mutants. These findings indicate that although HHV-8 gB and EBV gp110 have similar patterns of intracellular localization and can interact, there is not sufficient functional homology to allow efficient complementation.
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PMID:Human herpesvirus-8 glycoprotein B interacts with Epstein-Barr virus (EBV) glycoprotein 110 but fails to complement the infectivity of EBV mutants. 983 4

Polyphosphoinositides regulate numerous steps in membrane transport. The levels of individual phosphatidylinositols are controlled by specific lipid kinases, whose activities and localization are in turn regulated by a variety of effectors. Here we have examined the effect of overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase activity, on biosynthetic and postendocytic traffic in polarized Madin-Darby canine kidney cells. Endogenous frequenin was identified in these cells by polymerase chain reaction, Western blotting, and indirect immunofluorescence. Adenoviral-mediated overexpression of frequenin had no effect on early Golgi transport of membrane proteins, as assessed by acquisition of resistance to endoglycosidase H. However, delivery of newly synthesized influenza hemagglutinin from the trans-Golgi network to the apical cell surface was severely inhibited in cells overexpressing frequenin, whereas basolateral delivery of the polymeric immunoglobulin receptor was unaffected. Overexpression of frequenin did not affect postendocytic trafficking steps including apical and basolateral recycling and basal-to-apical transcytosis. We conclude that frequenin, and by inference, phosphatidylinositol 4-kinase, plays an important and selective role in apical delivery in polarized cells.
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PMID:Overexpression of frequenin, a modulator of phosphatidylinositol 4-kinase, inhibits biosynthetic delivery of an apical protein in polarized madin-darby canine kidney cells. 1082 56

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15 degrees C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.
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PMID:Dissecting the role of the golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface. 1089 Sep

The occurrence of sulfate substituents on several positions of glycoprotein N-linked oligosaccharides prompted us to determine the subcellular localization and temporal relationships of the addition of these anionic groups employing as a model system the hemagglutinin (HA) produced by influenza virus-infected Madin-Darby canine kidney (MDCK) cells. It became apparent from a study of the HA glycoprotein in subcellular fractions resolved by Nycodenz gradient centrifugation following pulse-chase radiolabeling that sulfation of the complex N-linked oligosaccharides occurs only after they have been processed to an endo-beta-N-acetylglucosaminidase-resistant state and have reached the medial/trans Golgi and the trans Golgi network (TGN), with the former carrying out most of the sulfation activity. Hydrazine/nitrous acid/NaBH(4) treatment of the HA from the subcellular fractions indicated that C-3 of the galactose as well as C-6 of the N-acetylglucosamine residues of the N-acetyllactosamine chains became sulfated in these post ER fractions, as did the C-6 of the outer N-acetylglucosamine of the di-N-acetylchitobiose core. Consistent with the specificities of the stepwise assembly of the N-acetyllactosamine branches, we observed that the 3'-phosphoadenosine 5'-phosphosulfate (PAPS):GlcNAc-6-O-sulfotransferase migrated in the gradient to a medial/trans Golgi position while in contrast the PAPS:Gal-3-O-sulfotransferase was found in both Golgi and TGN locations. In accordance with the concept that beta-galactosylation must precede the sulfation catalyzed by the latter enzyme, we observed the presence of UDP-Gal:GlcNAc galactosyltransferase in both these sites in the MDCK cells. The presence of the Gal-3-O-sulfotransferase in the TGN is particularly important in the influenza virus-infected cells, as it makes possible the addition of terminal anionic groups after removal of the sialic acid residues by the viral neuraminidase.
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PMID:Sulfation of the N-linked oligosaccharides of influenza virus hemagglutinin: temporal relationships and localization of sulfotransferases. 1108 16

Replication of human influenza A/H3N2 and A/H1N1 viruses was studied in human CACO-2 cells, a continuous line of intestinal epithelial differentiated cells. Hemagglutinin (HA) was cleaved in these cells by an endogenous protease. Thus, infectious virus was produced that underwent multiple cycle replication and plaque formation in the absence of trypsin added to the media. Cleavage of de novo-synthesized HA occurred at a late stage of the exocytic pathway as indicated by pulse-chase labeling and by experiments employing endoglycosidase H and brefeldin A treatment. However, surface-labeling experiments employing biotinylation suggested that there is no cleavage at the plasma membrane. Unlike HA of serotypes H5 and H7 cleaved at multibasic cleavage sites by furin, the HAs with monobasic cleavage sites analyzed here were not cleaved in CACO-2 cells in the presence of aprotinin, a natural inhibitor of trypsinlike proteases. Growing CACO-2 cells were able to cleave HA of incoming virus, although influenza virus activating protease was not detected in culture medium. These observations indicate that the activating enzyme of CACO-2 cells is a trypsinlike protease functioning in the trans-Golgi network and presumably endosomes. In support of this concept immune staining with antibodies specific to human and bovine trypsin revealed the presence of a trypsinlike protease in CACO-2 cells. Unlike MDCK and CV-1 cells undergoing rapid apoptosis after influenza virus infection, CACO-2 cells showed no apoptosis but displayed cytopathic effects with necrotic signs significantly later after infection. It follows from these data that, depending on the cell type, influenza virus may kill cells either by apoptosis or by necrosis.
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PMID:Human influenza A viruses are proteolytically activated and do not induce apoptosis in CACO-2 cells. 1295 Oct 33

P42, encoded by a colinear transcript of Influenza C virus RNA segment 6 (M gene), is an integral membrane protein which is cleaved by signal peptidase to generate M1' and CM2 composed of N-terminal 259 amino acids and C-terminal 115 amino acids, respectively. Herein, the biochemical features of P42 were investigated. N-glycosylated form of P42, designated P44, forms disulphide-linked dimers and tetramers. P44 is transported to the Golgi apparatus, but not to the trans-Golgi, since P44 is completely sensitive to endoglycosidase H. P44 and P42 are unstable irrespective of N-glycosylation or oligomerization. 26S proteasome inhibitor, lactacystin prevented the degradation of P42 as well as M1', but not that of P44 efficiently, suggesting that P44 is degraded by another protease besides the 26S proteasome.
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PMID:Biochemical properties of the P42 protein encoded by RNA segment 6 of influenza C virus. 1474 95

Sialoglycopeptide (SGP) is referred as the glycopeptide in hen's egg yolk, which has an N-linked, complex-type, disialyl biantennary oligosaccharide with an alpha-(2-->6)-sialyl N-acetyllactosamine residue. The residue is known as a binding ligand of type-A human influenza virus hemagglutinin. We describe herein a simple synthesis of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination that makes use of the natural source ingredient, SGP, and the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M). Its inhibitiory activity for influenza virus hemagglutination is 40 times higher than that of SGP, and its competitive inhibition is determined to be over 300 times higher than that of fetuin. These results indicate that a sialoglycopolymer having a multivalent sialo-oligosaccharide could potentially be used for the prevention of influenza virus infection.
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PMID:Chemoenzymatic synthesis and application of a sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination. 1671 73

The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection.
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PMID:Thiazolides, a new class of anti-influenza molecules targeting viral hemagglutinin at the post-translational level. 1963 39


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