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)

Since the Marburg (MBG) and Ebola (EBO) viruses have sequence homology and cause similar diseases, we hypothesized that they associate with target cells by similar mechanisms. Pseudotype viruses prepared with a luciferase-containing human immunodeficiency virus type 1 backbone and packaged by the MBG virus or the Zaire subtype EBO virus glycoproteins (GP) mediated infection of a comparable wide range of mammalian cell types, and both were inhibited by ammonium chloride. In contrast, they exhibited differential sensitivities to treatment of target cells with tunicamycin, endoglycosidase H, or protease (pronase). Therefore, while they exhibit certain functional similarities, the MBG and EBO virus GP interact with target cells by distinct processes.
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PMID:Distinct mechanisms of entry by envelope glycoproteins of Marburg and Ebola (Zaire) viruses. 1077 38

Stocks of simian immunodeficiency virus (SIV) from the supernatants of infected cell cultures were used to examine the sensitivity of envelope glycoprotein gp120 to enzymatic deglycosylation and the effects of enzyme treatment on infectivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis revealed little or no change in the mobility of virion-associated gp120 after digestion with high concentrations of N-glycosidase F, endoglycosidase F, endoglycosidase H, and endo-beta-galactosidase. Soluble gp120, which was not pelletable after the enzymatic reaction, was sensitive to digestion by the same enzymes within the same reaction mix and was only slightly less sensitive than gp120 that had been completely denatured by boiling in the presence of SDS and beta-mercaptoethanol. Digestion by three of the seven glycosidases tested significantly changed the infectivity titer compared to that of mock-treated virus. Digestion by endo-beta-galactosidase increased infectivity titers by about 2.5-fold, and neuraminidase from Newcastle disease virus typically increased infectivity titers by 8-fold. Most or all of the increase in infectivity titer resulting from treatment with neuraminidase could be accounted for by effects on the virus, not the cells; SIV produced in the presence of the sialic acid analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid also exhibited increased infectivity, and the effects could not be duplicated by neuraminidase treatment of cells. Digestion with mannosidase reduced infectivity by fivefold. Our results indicate that carbohydrates on native oligomeric gp120 as it exists on the surface of virus particles are largely occluded and are refractory to digestion by glycosidases. Furthermore, the sialic acid residues at the ends of carbohydrate side chains significantly reduce the inherent infectivity of SIV.
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PMID:Resistance of native, oligomeric envelope on simian immunodeficiency virus to digestion by glycosidases. 1107 15

The virucidal protein cyanovirin-N (CV-N) mediates its highly potent anti-human immunodeficiency virus activity, at least in part, through interactions with the viral envelope glycoprotein gp120. Here we dissect in further detail the mechanism of CV-N's glycosylation-dependent binding to gp120. Isothermal titration calorimetry (ITC) binding studies of CV-N with endoglycosidase H-treated gp120 showed that binding was completely abrogated by removal of high-mannose oligosaccharides from the glycoprotein. Additional ITC and circular dichroism spectral studies with CV-N and other glycoproteins as well showed that CV-N discriminately bound only glycoproteins that contain high-mannose oligosaccharides. Binding experiments with RNase B indicated that the single high-mannose oligosaccharide on that enzyme mediated all of its binding with CV-N (K(d) = 0.602 microM). A finer level of oligosaccharide selectivity of CV-N was revealed in affinity chromatography-liquid chromatography-mass spectrometry experiments, which showed that CV-N preferentially bound only oligomannose-8 (Man-8) and oligomannose-9 isoforms of RNase B. Finally, we biophysically characterized the interaction of CV-N with a purified, single oligosaccharide, Man-8. The binding affinity of Man-8 for CV-N is unusually strong (K(d) = 0.488 microM), several hundredfold greater than observed for oligosaccharides and their protein lectins (K(d) = 1 microM--1 mM), further establishing a critical role of high-mannose oligosaccharides in CV-N binding to glycoproteins.
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PMID:Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins. 1130 61

2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N-->A substitution produced significant decreases in 2G12 binding affinity to gp120(JR-CSF). Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120(JR-CSF). Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Man(alpha)1-->2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or N-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue N-butyl-deoxynojirimycin similarly implicated Man(alpha)1-->2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Man(alpha)1-->2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation.
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PMID:The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha1-->2 mannose residues on the outer face of gp120. 1207 29

A cell-free translation/glycosylation system derived from lepidopteran (Sf21) cells, which are widely used to express high yields of foreign active proteins that have post-translational modifications, was constructed. The insect cell extract was prepared using a Mini-Bomb cell disruption chamber by nitrogen pressure treatment, which stably retains translational and post-translational components. The gp120 mRNA was transcribed from the human immunodeficiency virus type-1 envelope glycoprotein gp120 gene with T7 RNA polymerase. When the gp120 mRNA was translated in the insect cell-free system, gp120 having a molecular mass of 100 kDa was detected by Western blot analysis. Synthesized gp120 and gp120 expressed in the intracellular fraction of recombinant-baculovirus-infected Sf21 cells had the same molecular mass, and they both had reduced mobility compared with gp120 secreted by recombinant baculovirus-infected Sf21 cells. In contrast, the 56-kDa gp120 protein, which corresponds to the polypeptide backbone of gp120, was synthesized in wheat germ and rabbit reticulocyte systems. The molecular mass of synthesized gp120 decreased from 100 kDa to 61 kDa after endoglycosidase H treatment, indicating that synthesized gp120 had been glycosylated with N-linked oligosaccharides. Furthermore, glycosylated gp120 was bound to human CD4 molecules expressed on the surface of quail cells. These results revealed that the insect cell-free system can synthesize gp120 that is folded in the proper conformation to provide a CD4-binding domain.
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PMID:A novel cell-free translation/glycosylation system prepared from insect cells. 1623


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