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)

We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoprotein precursor gp160 (rgp160) behaves as a mannosyl/N-acetylglucosaminyl (GlcNAc) binding protein. If such a carbohydrate-binding property were of biological relevance it should be shared by other related primate immunodeficiency viruses such as HIV-2. The present study confirms this hypothesis and extends these findings by showing that HIV-2 recombinant gp140 (rgp140) specifically interacts with three affinity matrices substituted by synthetic or natural carbohydrate structures: D-mannose-divinylsulphone-agarose, para-aminophenyl-beta-D-GlcNAc-agarose and the natural glycoprotein, bovine fetuin, also coupled to agarose. Binding of rpg140 to the matrices was inhibited by alpha-D-Man17-BSA (where BSA is bovine serum albumin), beta-D-GlcNAc47-BSA and fetuin, and by glycopeptides derived from pronase-treated porcine thyroglobulin. Glycopeptides obtained after endoglycosidase H treatment of thyroglobulin had a limited inhibitory effect, whereas beta-D-Gal17-BSA and beta-D-glucan had no effect. These results indicate that, like HIV-1 envelope glycoprotein, HIV-2 rgp140 interacts with high-mannose and with the mannosyl core of complex-type N-linked glycans, as well as with the N-acetylglucosaminyl core of oligosaccharidic structures.
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PMID:Mannosyl/N-acetyl-beta-D-glucosaminyl binding properties of the envelope glycoprotein of human immunodeficiency virus type 2. 128 Oct 21

Two monoclonal antibodies designated BAT085 and G3-136 were raised by immunizing BALB/c mice with gp120 purified from human immunodeficiency virus type 1 (HIV-1) IIIB-infected H9 cell extracts. Among three HIV-1 laboratory isolates (IIIB, MN, and RF), BAT085 neutralized only IIIB infection of CEM-SS cells, whereas G3-136 neutralized both IIIB and RF. These antibodies also neutralized a few primary HIV-1 isolates in the infection of activated human peripheral blood mononuclear cells. In indirect immunofluorescence assays, BAT085 bound to H9 cells infected with IIIB or MN, while G3-136 bound to H9 cells infected with IIIB or RF, but not MN. Using sequence-overlapping synthetic peptides of HIV-1 IIIB gp120, the binding site of BAT085 and G3-136 was mapped to a peptidic segment in the V2 region (amino acid residues 169 to 183). The binding of these antibodies to immobilized gp120 was not inhibited by the antibodies directed to the principal neutralization determinant in the V3 region or to the CD4-binding domain of gp120. In a competition enzyme-linked immunosorbent assay, soluble CD4 inhibited G3-136 but not BAT085 from binding to gp120. Deglycosylation of gp120 by endo-beta-N-acetylglucosaminidase H or reduction of gp120 by dithiothreitol diminished its reactivity with G3-136 but not with BAT085. These results indicate that the V2 region of gp120 contains multiple neutralization determinants recognized by antibodies in both a conformation-dependent and -independent manner.
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PMID:Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120. 137 May 58

Sulfation is a posttranslational modification of proteins which occurs on either the tyrosine residues or the carbohydrate moieties of some glycoproteins. In the case of secretory proteins, sulfation has been hypothesized to act as a signal for export from the cell. We have shown that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor (gp160) as well as the surface (gp120) and transmembrane (gp41) subunits can be specifically labelled with 35SO42-. Sulfated HIV-1 envelope glycoproteins were identified in H9 cells infected with the IIIB isolate of HIV-1 and in the cell lysates and culture media of cells infected with vaccinia virus recombinants expressing a full-length or truncated, secreted form of the HIV-1 gp160 gene. N-glycosidase F digestion of 35SO4(2-)-labelled envelope proteins removed virtually all radiolabel from gp160, gp120, and gp41, indicating that sulfate was linked to the carbohydrate chains of the glycoprotein. The 35SO42-label was at least partially resistant to endoglycosidase H digestion, indicating that some sulfate was linked to complex carbohydrates. Brefeldin A, a compound that inhibits the endoplasmic reticulum to Golgi transport of glycoproteins, was found to inhibit the sulfation of the envelope glycoproteins. Envelope glycoproteins synthesized in cells treated with chlorate failed to incorporate 35SO42-. However, HIV glycoproteins were still secreted from cells in the presence of chlorate, indicating that sulfation is not a requirement for secretion of envelope glycoproteins. Sulfation of HIV-2 and simian immunodeficiency virus envelope glycoproteins has also been demonstrated by using vaccinia virus-based expression systems. Sulfation is a major determinant of negative charge and could play a role in biological functions and antigenic properties of HIV glycoproteins.
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PMID:Sulfation of the human immunodeficiency virus envelope glycoprotein. 143

An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gp120/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gp120 detected at the end of a 20-h chase was in the culture medium. Therefore gp120 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gp160. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gp160, as well as the apparent instability of the gp120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gp120 and gp160 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gp160 occurs in the absence of CD4, and that the stable surface expression of envelope gp120/41 complex may require additional factors not present in transfected cells.
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PMID:Stable expression of the human immunodeficiency virus type 1 envelope glycoprotein in transfected L cells. 149 50

Apparently conflicting results have been reported regarding the role of env glycoprotein glycans in human immunodeficiency virus type 1 (HIV-1) infectivity and cytopathogenicity. Whereas we have shown that enzymic removal of carbohydrates from mature envelope glycoproteins has only limited effect on the ability of HIV-1 to bind to CD4 and to infect target cells, sugar analogues that interfere with the glycosylation process of the nascent molecule markedly reduce virus infectivity. Here we have investigated the effect of a glucosidase inhibitor, 1-deoxynojirimycin (dNM), on the bioactivity and immunoreactivity of precursor gp160 produced by recombinant vaccinia virus-infected BHK-21 cells (rgp160). dNM (4 mM) did not affect the amount of rgp160 recovered nor its secretion from the cells. As described by other authors the effect of dNM was incomplete, resulting in the production of rgp160, the glycosylation of which was heterogeneous with respect to apparent Mr distribution and to sensitivity to endoglycosidase H and endoglycosidase F, all the species being susceptible to N-glycanase. A major reduction of the binding to CD4+ cells was noted with rgp 160 produced by dNM-treated cells using a quantitative indirect immunofluorescence assay and labelling with polyclonal human anti-HIV IgG. Similarly, dNM treatment altered the accessibility to murine monoclonal antibody 110-4 of the exposed V3 loop of HIV-1 gp120 by at least 10-fold, as determined by either ELISA capture assay or immunoaffinity purification. Such bioactivity and conformation modifications, which result from the abnormal folding of the nascent glycoprotein due to aberrant glycosylation, may account for the impaired HIV-1 infectivity elicited by dNM.
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PMID:Effect of a glucosidase inhibitor on the bioactivity and immunoreactivity of human immunodeficiency virus type 1 envelope glycoprotein. 167 78

The processing and transport of the envelope glycoprotein complex of feline immunodeficiency virus (FIV) in the persistently infected Crandell feline kidney (CRFK) cell line were investigated. Pulse-chase analyses revealed that the glycoprotein is synthesized as a precursor with an Mr of 145,000 (gp145) and is quickly trimmed to a molecule with an Mr of 130,000 (gp130). Treatment of gp130 with endoglycosidase H (endo H) resulted in a protein with an Mr of 75,000, indicating that nearly half the weight of the gp130 precursor consists of endo H-sensitive glycans during biosynthesis. Chase periods of up to 8 h revealed intermediates during the further processing of this glycoprotein precursor. Initially, two minor protein species with apparent Mrs of 100,000 and 90,000 were detected along with gp130. At later chase times these two species appeared to migrate as a single dominant species with an Mr of 95,000 (gp95). Concomitant with the appearance of gp95 was another protein with an Mr of approximately 40,000 (gp40). Chase periods of up to 8 h revealed that approximately half of the precursor was processed into the gp95-gp40 complex within 4 h. gp95 was efficiently transported from the cell into the culture medium by 1 to 2 h after labeling, whereas gp40 was not observed to be released from infected CRFK cells. Analysis of the processing in the presence of monensin, castanospermine, and swainsonine also suggests the existence of these intermediates in the processing of this lentivirus glycoprotein. As with human immunodeficiency virus, virus produced in the presence of glucosidase inhibitors and reduced infectivity for T-lymphocyte cultures.
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PMID:Processing of the glycoprotein of feline immunodeficiency virus: effect of inhibitors of glycosylation. 184 41

A detailed kinetic and quantitative analysis of the early and late biosynthetic events undergone by the human immunodeficiency virus type 1 envelope protein expressed by a recombinant vaccinia virus was performed. Early folding events that occurred in the endoplasmic reticulum included disulfide bond formation (t1/2 approximately 10 min), folding of envelope protein into a form competent to bind CD4 (t1/2 approximately 15 min), and specific and transient association and dissociation with GRP78-BiP (t1/2 approximately 25 min). After initial folding, envelope protein monomers formed noncovalently associated dimers with high efficiency (t1/2 approximately 30 min). Studies with brefeldin A, a compound that inhibits endoplasmic reticulum-to-Golgi transport, suggested that assembly occurred in the endoplasmic reticulum while cleavage of gp160 into gp120/gp41 subunits occurred in a post-endoplasmic reticulum compartment. Transport to the Golgi was monitored by modification of N-linked sugars to forms partially resistant to endoglycosidase H. The kinetics of endoglycosidase H resistance were nearly identical to the kinetics of gp160 cleavage (t1/2 approximately 80 min). Cleavage efficiency was strongly cell type dependent, ranging from 13 to 70%. By contrast, approximately 50% of the gp120 generated by the cleavage event was shed (t1/2 approximately 120 min) regardless of the cell type used. The results are discussed in terms of the overall biosynthetic pathway of the envelope protein and provide a framework with which to assess the effects of mutations on structure and function.
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PMID:Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein. 190 May 40

Our results demonstrate that the formation of intracellular complexes between the envelope glycoprotein precursor gp160 of human immunodeficiency virus type 1 and CD4 is a major event, leading to the disappearance of CD4 at the cell surface of infected U937 cells. Using both productively and defectively infected clones of U937 cells, we assessed the effect of CD4-gp160 intracellular association on the maturation of both proteins. Pulse-chase labeling followed by sequential immunoprecipitation was used to analyze the processing of both free and associated CD4 and gp160, and the results showed that the trimming, proteolytic cleavage, and degradation of gp160 were completely abrogated after intracellular binding to CD4. Similarly, the maturation process which normally transforms 80% of CD4 to a partially endoglycosidase H-resistant species was also impaired subsequent to the formation of these complexes. A comparison of gp160 maturation either in free form or as a CD4 complex revealed that neither inefficient transport nor degradation of gp160 can account for the observed blockage of CD4 maturation. Moreover, this impairment was independent of gp120 and gp41, since a defective clone of human immunodeficiency virus type 1-infected cells, unable to cleave gp160, showed binding of CD4 and inhibition of CD4 transport and maturation with the same efficiency as occurred in productively infected cells. Expression of gp160 is thus necessary and sufficient to cause CD4 receptor down-modulation for both productively and defectively infected cells.
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PMID:Inhibition of gp160 and CD4 maturation in U937 cells after both defective and productive infections by human immunodeficiency virus type 1. 194 41

Feline immunodeficiency virus (FIV) structural proteins were identified using sera obtained from experimentally inoculated cats. Proteins analysed by both radioimmunoprecipitation and Western blotting were specific for FIV infection and failed to cross-react with either antisera to feline leukaemia virus of feline syncytium-forming virus. Western blot analysis of purified virus revealed immunoreactive proteins with apparent Mr of 65K, 50K, 40K, 32K, 24K, 15K and 10K. The major core structural proteins of the virus were isolated by reverse phase HPLC and the aminoterminal sequences of p10 and p24 were determined. Monoclonal antibodies specific for p24 suggested the presence of a precursor protein that could be detected in 35[S]methionine/cysteine-labelled, virus-infected cell extracts. This putative precursor protein possessed an apparent Mr of 50K (Pr50gag). Further analysis revealed the presence of two additional proteins of 130K and 40K. Experiments utilizing tunicamycin, endoglycosidase H and glycopeptidase F revealed that p130 and p40 exhibited properties characteristic of glycoproteins. Our studies also indicated that FIV is immunologically related to other lentiviruses.
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PMID:Biochemical and immunological characterization of the major structural proteins of feline immunodeficiency virus. 215 3

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) plays a major role in the down-regulation of its receptor, CD4. Using a transient-expression system, we investigated the interaction of the HIV-1 envelope glycoprotein with CD4 during their movement through the intracellular membrane traffic. In singly transfected cells, the envelope glyprotein gp160 was synthesized, glycosylated, and localized predominantly in the endoplasmic reticulum. Only a minor fraction of gp160 was proteolytically cleaved, producing gp120 and gp41, and gp120 was secreted into the medium. On the other hand, the CD4 molecule, when expressed alone, was properly glycosylated and transported efficiently to the cell surface. However, when gp160 and CD4 were coexpressed in the same cell, the cell surface delivery of CD4 was greatly reduced. In coexpressing cells, CD4 formed a specific intracellular complex with gp160 as both proteins could be immunoprecipitated by antibodies against either the gp160 or CD4 (OKT4) but not by OKT4A, a blocking antibody against CD4. The specific gp160-CD4 complex was localized predominantly in the endoplasmic reticulum, and the CD4 in the complex did not acquire endoglycosidase H resistance. The present studies demonstrated that a specific intracellular interaction between gp160 and CD4 was responsible for the cell surface down-regulation of CD4 in cells expressing both the envelope glycoprotein of HIV-1 and its receptor, CD4.
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PMID:Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane. 224 95


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