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:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glycoprotein precursor of the highly cytopathic Zairian virus HIV1-NDK synthesized in CEM leukemic cells displayed a molecular mass of 140 kDa (gp140) as compared to the 160 kDa of gp160 of HIV1-LAV prototype strain. This precursor was cleaved to produce a smaller than prototype extra-cellular envelope glycoprotein (gp100) and a transmembrane component with a usual size (gp41). Immunoprecipitates from tunicamycin-treated infected cells demonstrated the presence of a non-glycosylated precursor of 100 kDa for HIV1-LAV prototype strain and 90 kDa for HIV1-NDK. Digestion of labeled precipitates with a mixture of endoglycosidase F and glycopeptidase F reduced the size of HIV1-LAV gp160 and gp120 to 100 and 60 kDa, respectively, while HIV1-NDK gp140 and gp100, after treatment with the same enzymes, displayed an apparent molecular mass of 90 kDa and 55 kDa, respectively. From these data we conclude that HIV1-LAV gp120 and HIV1-NDK gp100 differ both in their proteic moiety (60 kDa and 55 kDa, respectively) and in their carbohydrate moiety (60 kDa and 45 kDa, respectively). These differences could not be deduced from the available gene sequences of the two viruses. A chimeric virus containing the first 124 amino acid residues of the envelope glycoprotein coded by HIV1-LAV sequence and the rest by HIV1-NDK displayed normal size envelope glycoproteins, demonstrating the involvement of this N-terminal sequence in the alteration of the molecular mass characteristic of HIV1-NDK gp140 and gp100. Finally, characterization of the gag gene products from both strains demonstrated that HIV1-NDK p18 and p15 have a slower electrophoretic mobility as compared to its HIV1-LAV counterparts. Therefore, structural properties of HIV1-NDK env and gag products, reflected by their unusual electrophoretic mobilities, may be responsible for HIV1-NDK biological properties.
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PMID:Structural variability of env and gag gene products from a highly cytopathic strain of HIV-1. 164 54

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.
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PMID:gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing. 189 96

FeLV-FAIDS, an immunodeficiency-inducing isolate of feline leukemia virus, is composed of a pathogenic but replication-defective genome (molecular clone 61C) and a replication-competent but non-immunodeficiency-inducing variant genome (molecular clone 61E). The chimeric virus EECC, composed of the 5' gag-pol of 61E fused to the env-3' LTR of 61C, also induces immunodeficiency. The 61C (or EECC) gp80 can be distinguished from that of 61E on the basis of antigenic recognition, size, and rate of posttranslational processing. We found that the nascent precursor polypeptides of the two viruses were the same size; however, the 61E gp80 rapidly shifted to a smaller size and was subsequently cleaved to gp70, whereas EECC gp80 maintained its nascent size and was cleaved to gp70 only after a prolonged time. Endo-beta-N-acetyl glucosaminidase H and N-glycanase digestions of newly formed glycoproteins resulted in a similar banding pattern for both viruses, indicating that both contained the same number of oligosaccharide side chains and that all of these were high mannose sugars. The metabolic inhibitors of glycosylation, castanospermine or N-methyldeoxynojirimycin, prevented both the rapid trimming of 61E gp80 and its cleavage to gp70. Treatment with mannosidase inhibitors, however, did not affect 61E gp80 processing or size, suggesting that retention of glucose residues on EECC was responsible for these distinguishing properties of the glycoprotein. The pathological consequence of aberrant viral glycoprotein processing was evaluated in feline 3201 T lymphocytes, which are infectable by both 61E and EECC but are killed only by EECC. As in fibroblasts, the EECC glycoprotein produced in lymphocytes was larger, antigenically distinct, and processed more slowly than was the glycoprotein of 61E. Castanospermine treatment of 61E-infected 3201 T cells, however, not only abrogated the antigenic differences between the 61E and EECC glycoproteins but also resulted in a cytopathic effect. Our results suggest that (i) intracellular accumulation of EECC envelope glycoprotein may occur consequent to retention of glucose residues on carbohydrate side chains and (ii) a strong correlation exists between delayed glycoprotein processing and cytopathicity in FeLV-FAIDS-infected T lymphocytes.
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PMID:Characterization and significance of delayed processing of the feline leukemia virus FeLV-FAIDS envelope glycoprotein. 216 20

The carbohydrate moiety of the envelope glycoprotein gp51 of bovine leukemia virus, American strain, was studied. The virus was grown in ovine, bovine, porcine, bat and rat cells of various organ specificities. The gp51 was purified by immunoaffinity chromatography from virions of ten different virus-producing cells derived from various body organs of different species. Highly purified glycoproteins (single band in PAGE) were compared for their electrophoretic mobility, for the presence of epitopes by a battery of monoclonal antibodies, and for the glycosylation pattern by lectin blot analysis. Electrophoretic analysis of all tested glycoproteins deglycosylated by glycopeptidase F detected the same polypeptide backbone according to PAGE. The glycoproteins produced in rat cells migrated faster in PAGE, as detected in cells or in virions, than those produced in ovine cells. The pattern of their glycosylation was found to be dependent on the type of cells used for virus production. The differences in glycosylation were most pronounced when comparing the glycoprotein produced in ovine cells versus bat or rat cells. Changes in epitope expression were also detected. The differences in the patterns of glycosylation and in the accessibility of epitopes owing to the virus production in various kind of cells are discussed from virus infectivity and vaccine points of view.
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PMID:Envelope glycoprotein gp51 of bovine leukemia virus is differently glycosylated in cells of various species and organ origin. 768 44

Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.
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PMID:Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells. 768 70

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been shown to be extensively modified by N-linked glycosylation; however, the presence of O-linked carbohydrates on the glycoprotein has not been firmly established. We have found that enzymatic deglycosylation of the HIV-1 envelope glycoprotein with neuraminidase and O-glycosidase results in a decrease in the apparent molecular weight of the envelope glycoprotein. This result was observed in both vaccinia virus recombinant-derived envelope glycoproteins and glycoproteins derived from the IIIB, SG3, and HXB2, strains of HIV-1. The decrease in molecular weight was also observed when the envelope glycoprotein had been deglycosylated with N-glycanase F after treatment with neuraminidase and O-glycosidase, indicating that the decrease in apparent molecular weight was not attributable to the removal of N-linked carbohydrate. Treatment with neuraminidase, O-glycosidase, and N-glycanase F was found to be necessary to remove all radiolabel from [3H]glucosamine-labelled envelope glycoprotein, a result seen for both recombinant and HIV-1-derived envelope glycoprotein. [3H]glucosamine-labelled carbohydrates liberated by O-glycosidase treatment were separated by paper chromatography and were found to be of a size consistent with O-linked oligosaccharides. We, therefore, conclude that the HIV-1 envelope glycoprotein is modified by the addition of O-linked carbohydrates.
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PMID:Human immunodeficiency virus type 1 envelope glycoprotein is modified by O-linked oligosaccharides. 825 57

We have previously demonstrated that human immunodeficiency virus (HIV) envelope glycoproteins have specific carbohydrate-binding properties for mannosyl/N-acetylglucosaminyl residues presented at high density on a carrier in vitro. Here, we investigated whether HIV envelope glycoprotein gp120 was able to interact with surface membrane carbohydrates of CD4+ cells by means of such lectin-carbohydrate interactions. CD4-free tryptic glycopeptides, prepared from the membrane of CD4+ monocytic U937 cells and partially purified by ConA-agarose affinity chromatography, could be eluted by mannan but not by methyl-alpha-mannose or methyl-alpha-glucose, which strongly suggests that they displayed oligomannosidic structures. These glycopeptides bound in a mannosyl-specific manner to radiolabeled recombinant gp120. Deglycosylation with N-glycanase which, as expected, strongly diminished binding of the glycopeptides to concanavalin A also abolished their interaction with gp120. In addition, the glycopeptides inhibited HIV infection of both U937 and CD4+ lymphoid CEM cells when preincubated with the virus. These findings indicate that, independently of the binding to CD4, mannosyl structures on CD4+ cells may play a role through lectin-carbohydrate interactions in envelope glycoprotein binding to a putative coreceptor(s) of HIV.
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PMID:Inhibition of human immunodeficiency virus infection of CD4+ cells by CD4-free glycopeptides from monocytic U937 cells. 882 18

Using a CD4-binding assay to assess the conformation of the human immunodeficiency virus envelope glycoprotein (CHO+ Env), we studied the effect of treatment with various glycosidases on the stability of Env in denaturing environments and in biological media: cleavage from Env of either high-mannose-type glycans (HMT- Env) by endoglycosidase H or sialic acid residues (Sial- Env) by sialidase did not alter Env stability whereas its complete deglycosylation (CHO- Env) by N-glycanase had a large effect. The influence of glycan removal on Env sensitivity to proteases was also studied. Thrombin cleavage within V3 was affected by N-glycanase treatment; both HMT- Env and CHO- Env displayed an increased sensitivity to other endoproteases. Thus, partial deglycosylation increases Env sensitivity to proteases but only its total deglycosylation alters its stability.
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PMID:Effect of various glycosidase treatments on the resistance of the HIV-1 envelope to degradation. 910 16

We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines.
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PMID:Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules. 991 84

The E2 envelope glycoprotein of virulent Shimen strain and avirulent C-strain of Classical swine fever virus (CSFV) has 5 and 6 potential glycosylation sites, respectively, and the potential glycosylation site 986N is unique to C-strain. To study the differences in glycosylation between the virus pair, the E2 genes (removing signal sequence and transmembrane anchor regions) of the two strains fused with the melittin signal sequence were expressed in the Sf9 insect cells. The recombinant E2 proteins were secreted into the medium of Sf9 cells in dimer form with different molecular weight (MW). Deglycosylation of the recombinant E2 proteins by endo H and PNGase F showed that the deglycosalated Shimen-E2 and HCLV-E2 have the same MW, indicating that the different MW between Shimen-E2 and HCLV-E2 proteins came from different glycosylation. Site-directed mutagenesis in the potential glycosylation site at 986N demonstrated that the mutated Shimen-E2 protein had the same MW as the wild-type HCLV-E2 protein, while the mutated HCLV-E2 had the same MW as the wild-type Shimen-E2 protein. We suggest that the different MW between Shimen-E2 and HCLV-E2 is resulted from the different glycosylation on 986 N glycosylation site.
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PMID:[Differences in glycosylation of the E2 protein between virulent Shimen strain and a virulent C-strain of classical swine fever virus]. 1796 57


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