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Sample records for 60-kda glycoprotein gp60

  1. Development and Application of a gp60-Based Typing Assay for Cryptosporidium viatorum

    PubMed Central

    Elwin, K.; Winiecka-Krusnell, J.; Chalmers, R. M.; Xiao, L.; Lebbad, M.

    2015-01-01

    The apicomplexan intestinal parasites of the genus Cryptosporidium take a major toll on human and animal health and are frequent causes of waterborne outbreaks. Several species and genotypes can infect humans, including Cryptosporidium viatorum, which, to date, has only been found in humans. Molecular characterization of Cryptosporidium spp., critical to epidemiological analyses, is commonly based on gp60 gene analysis, which appears to require bespoke species- or group-specific PCR primers due to extensive genetic diversity across the genus. In this study, we amplified, sequenced, and characterized the gp60 gene of C. viatorum for the first time. Moreover, we developed and validated a gp60 typing assay for this species and applied it to 27 isolates originating from Asia, Africa, and Central America. A single subtype family, XVa, was identified containing multiple alleles. PMID:25832304

  2. Cryptosporidium parvum GP60 subtypes in dairy cattle from Buenos Aires, Argentina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryptosporidium parvum from 73 dairy calves less than two months old from Buenos Aires province (Argentina) were molecularly characterized using sequence analysis of the GP60 gene. Seventy five sequences were obtained, and seven different subtypes were identified, all belonging to the IIa subtype f...

  3. Endothelial Cell-Surface Gp60 Activates Vesicle Formation and Trafficking via Gi-Coupled Src Kinase Signaling Pathway

    PubMed Central

    Minshall, Richard D.; Tiruppathi, Chinnaswamy; Vogel, Stephen M.; Niles, Walter D.; Gilchrist, Annette; Hamm, Heidi E.; Malik, Asrar B.

    2000-01-01

    We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein Gi, and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial 125I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11–amino acid Gαi carboxyl-terminal peptide inhibited endothelial 125I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of Gαi with the caveolin-1, whereas dn-Src inhibited Gαi binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60

  4. Highly sensitive non-isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species, genotypes and subgenotypes infective to humans, and its implications.

    PubMed

    Pangasa, Aradhana; Jex, Aaron R; Nolan, Matthew J; Campbell, Bronwyn E; Haydon, Shane R; Stevens, Melita A; Gasser, Robin B

    2010-05-01

    The high-resolution analysis of genetic variation has major implications for the identification of parasites and micro-organisms to species and subspecies as well as for population genetic and epidemiological studies. In this study, we critically assessed the effectiveness of a PCR-based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium, a genus of parasitic protists of major human and animal health importance globally. This gene displays substantial intraspecific variability in sequence, particularly in a TCA (perfect and imperfect) microsatellite region, is present as a single copy in the nuclear genome and is used widely as a marker in molecular epidemiological studies of Cryptosporidium hominis and C. parvum, the two predominant species that infect humans. The results of this study demonstrated an exquisite capacity of REF to detect nucleotide variability in the gp60 gene within each of the two species. The differentiation of genotypes/subgenotypes based on REF analysis was supported by targeted sequencing, allowing the detection of levels of variation as low as a single-nucleotide transversion for amplicons of approximately 1 kb in size. The high-throughput potential and relatively low-cost of REF make it a particularly useful tool for large-scale genetic analyses of C. hominis and C. parvum. REF could also be utilized for comparative surveys of genetic variability across large nuclear genomic regions. Such analyses of Cryptosporidium in clinical and environmental samples by REF have important implications for identifying sources of infection, modes of transmission and/or possible infectivity to humans, thus assisting in the surveillance and control of cryptosporidiosis. Given its excellent mutation detection capacity, REF should find broad applicability to various single-copy genes as well as a wide range of other protozoan and metazoan parasites.

  5. Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis.

    PubMed

    Alba-Fierro, Carlos A; Pérez-Torres, Armando; Toriello, Conchita; Pulido-Camarillo, Evelyn; López-Romero, Everardo; Romo-Lozano, Yolanda; Gutiérrez-Sánchez, Gerardo; Ruiz-Baca, Estela

    2016-01-01

    Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response. PMID:27051673

  6. Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis.

    PubMed

    Alba-Fierro, Carlos A; Pérez-Torres, Armando; Toriello, Conchita; Pulido-Camarillo, Evelyn; López-Romero, Everardo; Romo-Lozano, Yolanda; Gutiérrez-Sánchez, Gerardo; Ruiz-Baca, Estela

    2016-01-01

    Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response.

  7. Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis

    PubMed Central

    Alba-Fierro, Carlos A.; Pérez-Torres, Armando; Toriello, Conchita; Pulido-Camarillo, Evelyn; Romo-Lozano, Yolanda; Gutiérrez-Sánchez, Gerardo

    2016-01-01

    Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response. PMID:27051673

  8. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-05-15

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  9. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2006-10-31

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  10. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-08-28

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  11. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-07-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  12. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2005-08-09

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  13. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-16

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  14. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2010-11-02

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  15. Glycoprotein synthesis

    DOEpatents

    Schultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-02-27

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  16. Glycoprotein synthesis

    DOEpatents

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

    2009-07-14

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  17. Glycoprotein synthesis

    DOEpatents

    Shultz, Peter G.; Wang, Lei; Zhang, Zhiwen

    2007-04-03

    Methods for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid into a protein and attaching one or more saccharide moieties to the unnatural amino acid. Another method involves incorporating an unnatural amino acid that includes a saccharide moiety into a protein. Proteins made by both methods can be further modified with additional sugars.

  18. Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus–ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor

    PubMed Central

    Mocan, Lucian; Matea, Cristian; Tabaran, Flaviu A; Mosteanu, Ofelia; Pop, Teodora; Mocan, Teodora; Iancu, Cornel

    2015-01-01

    We present a method of enhanced laser thermal ablation of HepG2 cells based on a simple gold nanoparticle (GNP) carrier system such as serum albumin (Alb), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. HepG2 or hepatocytes were treated with Alb-GNPs at various concentrations and various incubation times, and further irradiated using a 2 W, 808 nm laser. Darkfield microscopy and immunochemical staining was used to demonstrate the selective internalization of Alb-GNPs inside the HepG2 cells via Gp60 receptors targeting. The postirradiation apoptotic rate of HepG2 cells treated with Alb-GNPs ranged from 25.8% (for 5 μg/mL) to 48.2% (for 50 μg/mL) at 60 seconds, while at 30 minutes the necrotic rate increased from 35.7% (5 μg/mL) to 52.3% (50 μg/mL), P-value <0.001. Significantly lower necrotic rates were obtained when human hepatocytes were treated with Alb-GNPs in a similar manner. We also showed by means of immunocytochemistry that photothermal treatment of Alb-conjugated GNPs in liver cancer initiates Golgi apparatus–endoplasmic reticulum dysfunction with consequent caspase-3 apoptotic pathway activation and cellular apoptosis. The presented results may become a new method of treating cancer cells by selective therapeutic vectors using nanolocalized thermal ablation by laser heating. PMID:26346915

  19. Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus-ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor.

    PubMed

    Mocan, Lucian; Matea, Cristian; Tabaran, Flaviu A; Mosteanu, Ofelia; Pop, Teodora; Mocan, Teodora; Iancu, Cornel

    2015-01-01

    We present a method of enhanced laser thermal ablation of HepG2 cells based on a simple gold nanoparticle (GNP) carrier system such as serum albumin (Alb), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. HepG2 or hepatocytes were treated with Alb-GNPs at various concentrations and various incubation times, and further irradiated using a 2 W, 808 nm laser. Darkfield microscopy and immunochemical staining was used to demonstrate the selective internalization of Alb-GNPs inside the HepG2 cells via Gp60 receptors targeting. The postirradiation apoptotic rate of HepG2 cells treated with Alb-GNPs ranged from 25.8% (for 5 μg/mL) to 48.2% (for 50 μg/mL) at 60 seconds, while at 30 minutes the necrotic rate increased from 35.7% (5 μg/mL) to 52.3% (50 μg/mL), P-value <0.001. Significantly lower necrotic rates were obtained when human hepatocytes were treated with Alb-GNPs in a similar manner. We also showed by means of immunocytochemistry that photothermal treatment of Alb-conjugated GNPs in liver cancer initiates Golgi apparatus-endoplasmic reticulum dysfunction with consequent caspase-3 apoptotic pathway activation and cellular apoptosis. The presented results may become a new method of treating cancer cells by selective therapeutic vectors using nanolocalized thermal ablation by laser heating.

  20. Glycoproteins: Occurrence and Significance

    NASA Astrophysics Data System (ADS)

    Wittmann, Valentin

    Protein glycosylation is regarded as the most complex form of post-translational modification leading to a heterogeneous expression of glycoproteins as mixtures of glycoforms. This chapter describes the structure and occurrence of glycoproteins with respect to their glycan chains. Discussed are different carbohydrate-peptide linkages including GPI anchors, common structures of N- and O-glycans, and the structure of glycosaminoglycans contained in proteoglycans. Also covered are the bacterial cell wall polymer peptidoglycan and the glycopeptide antibiotics of the vancomycin group. Properties and functions of the glycans contained in glycoproteins are dealt with in the next chapter of this book.

  1. Analysis of Glycoproteins for Biomarker Discovery

    PubMed Central

    He, Jintang; Liu, Yashu; Wu, Jing; Lubman, David M.

    2012-01-01

    Summary Glycoproteins play an important role in cell signaling and cell-cell interaction. The alterations of glycoproteins are often relevant to progression of diseases and these changed glycoproteins can be important biomarkers. The lectin-based glycoproteomic technology has extensively been used for high-throughput screening of potential glycoprotein biomarkers. Here we describe a multi-lectin affinity chromatography and label-free quantitative glycoproteomic approach for discovery of glycoprotein biomarkers relevant to differentiation of glioblastoma stem cells. PMID:23625399

  2. Lubrication by glycoprotein brushes.

    NASA Astrophysics Data System (ADS)

    Zappone, Bruno; Ruths, Marina; Greene, George W.; Israelachvili, Jacob

    2006-03-01

    Grafted polyelectrolyte brushes show excellent lubricating properties under water and have been proposed as a model to study boundary lubrication in biological system. Lubricin, a glycoprotein of the synovial fluid, is considered the major boundary lubricant of articular joints. Using the Surface Force Apparatus, we have measured normal and friction forces between model surfaces (negatively charged mica, positively charged poly-lysine and aminothiol, hydrophobic alkanethiol) bearing adsorbed layers of lubricin. Lubricin layers acts like a versatile anti-adhesive, adsorbing on all the surfaces considered and creating a repulsion similar to the force between end-grafted polymer brushes. Analogies with polymer brushes also appear from bridging experiment, where proteins molecules are end-adsorbed on two opposing surfaces at the same time. Lubricin `brushes' show good lubricating ability at low applied pressures (P<0.5MPa), especially on negatively charged surfaces like mica. At higher load, the adsorbed layers wears and fails lubricating the surfaces, while still protecting the underlying substrate from wearing. Lubricin might thus be a first example of biological polyelectrolytes providing `brush-like' lubrication and wear-protection.

  3. Folding of synthetic homogeneous glycoproteins in the presence of a glycoprotein folding sensor enzyme.

    PubMed

    Dedola, Simone; Izumi, Masayuki; Makimura, Yutaka; Seko, Akira; Kanamori, Akiko; Sakono, Masafumi; Ito, Yukishige; Kajihara, Yasuhiro

    2014-03-10

    UDP-glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re-glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re-glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.

  4. Salivary Mucin 19 Glycoproteins

    PubMed Central

    Culp, David J.; Robinson, Bently; Cash, Melanie N.; Bhattacharyya, Indraneel; Stewart, Carol; Cuadra-Saenz, Giancarlo

    2015-01-01

    Saliva functions in innate immunity of the oral cavity, protecting against demineralization of teeth (i.e. dental caries), a highly prevalent infectious disease associated with Streptococcus mutans, a pathogen also linked to endocarditis and atheromatous plaques. Gel-forming mucins are a major constituent of saliva. Because Muc19 is the dominant salivary gel-forming mucin in mice, we studied Muc19−/− mice for changes in innate immune functions of saliva in interactions with S. mutans. When challenged with S. mutans and a cariogenic diet, total smooth and sulcal surface lesions are more than 2- and 1.6-fold higher in Muc19−/− mice compared with wild type, whereas the severity of lesions are up to 6- and 10-fold higher, respectively. Furthermore, the oral microbiota of Muc19−/− mice display higher levels of indigenous streptococci. Results emphasize the importance of a single salivary constituent in the innate immune functions of saliva. In vitro studies of S. mutans and Muc19 interactions (i.e. adherence, aggregation, and biofilm formation) demonstrate Muc19 poorly aggregates S. mutans. Nonetheless, aggregation is enhanced upon adding Muc19 to saliva from Muc19−/− mice, indicating Muc19 assists in bacterial clearance through formation of heterotypic complexes with salivary constituents that bind S. mutans, thus representing a novel innate immune function for salivary gel-forming mucins. In humans, expression of salivary MUC19 is unclear. We find MUC19 transcripts in salivary glands of seven subjects and demonstrate MUC19 glycoproteins in glandular mucous cells and saliva. Similarities and differences between mice and humans in the expression and functions of salivary gel-forming mucins are discussed. PMID:25512380

  5. Phosphorylation of the multidrug resistance associated glycoprotein

    SciTech Connect

    Mellado, W.; Horwitz, S.B.

    1987-11-03

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistant phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl/sub 2/ was enhanced a minimum of 2-fold by 10 ..mu..M cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by (..gamma..-/sup 32/P)ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance.

  6. Structure and Function of RSV Surface Glycoproteins

    PubMed Central

    McLellan, Jason S.; Ray, William C.; Peeples, Mark E.

    2014-01-01

    The two major glycoproteins on the surface of the RSV virion, the attachment glycoprotein (G) and the fusion (F) glycoprotein, control the initial phases of infection. G targets the ciliated cells of the airways, and F causes the virion membrane to fuse with a target cell membrane. The F protein is the major target for antiviral drug development, and both G and F glycoproteins are the antigens targeted by neutralizing antibodies induced by infection. In this chapter we review the structure and function of the RSV surface glycoproteins, including recent X-ray crystallographic data of the F glycoprotein in its pre- and postfusion conformations, and discuss how this information informs antigen selection and vaccine development. PMID:24362685

  7. Effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

    SciTech Connect

    Schwarz, P.M.; Elbein, A.D.

    1985-11-25

    Influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. To determine what structural features of the oligosaccharide were required for fucosylation influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, (5,6-TH)fucose and (1- UC)mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the ( UC)mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the ( UC)mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the (TH)fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the ( UC)mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.

  8. A sweet code for glycoprotein folding.

    PubMed

    Caramelo, Julio J; Parodi, Armando J

    2015-11-14

    Glycoprotein synthesis is initiated in the endoplasmic reticulum (ER) lumen upon transfer of a glycan (Glc3Man9GlcNAc2) from a lipid derivative to Asn residues (N-glycosylation). N-Glycan-dependent quality control of glycoprotein folding in the ER prevents exit to Golgi of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones (calnexin and calreticulin) that recognize monoglucosylated polymannose protein-linked glycans, lectin-associated oxidoreductase acting on monoglucosylated glycoproteins (ERp57), a glucosyltransferase that creates monoglucosylated epitopes in protein-linked glycans (UGGT) and a glucosidase (GII) that removes the glucose units added by UGGT. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded glycoproteins or in not completely assembled multimeric glycoprotein complexes. Glycoproteins that fail to properly fold are eventually driven to proteasomal degradation in the cytosol following the ER-associated degradation pathway, in which the extent of N-glycan demannosylation by ER mannosidases play a relevant role in the identification of irreparably misfolded glycoproteins.

  9. Functional roles of membrane glycoprotein CD36.

    PubMed

    Daviet, L; McGregor, J L

    1996-01-01

    Cell-cell and cell-extracellular matrix interactions are mediated by a number of membrane glycoproteins. On the basis of structural homologies, several families of cell adhesion molecules (integrins, selectins, immunoglobulins, cadherins, leucine-rich glycoproteins) have been established. Since 1991, a new family of CD36-like proteins has been identified. CD36 is a cell surface glycoprotein that interacts with a large variety of ligands. CD36 has been implicated in thrombosis, vascular biology, lipid metabolism and atherogenesis. In this review, we aim to summarize our present knowledge on this important, multifunctional glycoprotein. PMID:21043590

  10. Clinical applications of bacterial glycoproteins.

    PubMed

    Fulton, Kelly M; Smith, Jeffrey C; Twine, Susan M

    2016-01-01

    There is an ongoing race between bacterial evolution and medical advances. Pathogens have the advantages of short generation times and horizontal gene transfer that enable rapid adaptation to new host environments and therapeutics that currently outpaces clinical research. Antibiotic resistance, the growing impact of nosocomial infections, cancer-causing bacteria, the risk of zoonosis, and the possibility of biowarfare all emphasize the increasingly urgent need for medical research focussed on bacterial pathogens. Bacterial glycoproteins are promising targets for alternative therapeutic intervention since they are often surface exposed, involved in host-pathogen interactions, required for virulence, and contain distinctive glycan structures. The potential exists to exploit these unique structures to improve clinical prevention, diagnosis, and treatment strategies. Translation of the potential in this field to actual clinical impact is an exciting prospect for fighting infectious diseases. PMID:26971465

  11. [Biological role of heterogeneous glycoprotein structures].

    PubMed

    Jakab, Lajos

    2016-07-01

    Carbohydrate molecules connected mostly with covalent junctions to protein chains are called glycoproteins. These carbohydrate molecules are attached to the protein core in different qualities and order. When the protein core is connected with acidic components such as uronic acid or SO4 radicals, they are called proteoglycans. The currently used name "glycosaminoglycan" in this case is not entirely correct. In the living world polymannane structures occur, too. Glycoproteins do not only exceptionally hold acidic groups but they have neuraminic acid derivatives. Tissue, cellular and matrix structures, and mostly all serum "proteins" are mainly glycoproteins. In the everyday clinical practice glycoproteins are mentioned as proteins. Nevertheless, the inadequate use of the concept may cause errors in the attitudes, too. This paper aims to correct this notion, because the term of "glycobiology" has already been expanded to be an independent scientific field. The practical clinical consequences of recent knowledge in this field are also summarized including novel findings on glycoprotein structures and functions. The importance of the quantity of carbohydrates, and their structural arrangements are also presented. In short, significance of glycoprotein-carbohydrate structures, as well as their physiological and pathological roles are reviewed in order to introduce the field of "glycobiology". Orosomucoid and immunoglobulins are discussed separately. Orv. Hetil., 2016, 157(30), 1185-1192.

  12. Chemical synthesis of intentionally misfolded homogeneous glycoprotein: a unique approach for the study of glycoprotein quality control.

    PubMed

    Izumi, Masayuki; Makimura, Yutaka; Dedola, Simone; Seko, Akira; Kanamori, Akiko; Sakono, Masafumi; Ito, Yukishige; Kajihara, Yasuhiro

    2012-05-01

    Biosynthesis of glycoproteins in the endoplasmic reticulum employs a quality control system, which discriminates and excludes misfolded malfunctional glycoproteins from a correctly folded one. As chemical tools to study the glycoprotein quality control system, we systematically synthesized misfolded homogeneous glycoproteins bearing a high-mannose type oligosaccharide via oxidative misfolding of a chemically synthesized homogeneous glycopeptide. The endoplasmic reticulum folding sensor enzyme, UDP-glucose:glycoprotein glucosyltransferase (UGGT), recognizes a specific folding intermediate, which exhibits a molten globule-like hydrophobic nature.

  13. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway.

    PubMed

    Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Horimoto, Satoshi; Sugimoto, Takehiro; Ishikawa, Tokiro; Takeda, Shunichi; Yamamoto, Takashi; Suzuki, Tadashi; Kamiya, Yukiko; Kato, Koichi; Mori, Kazutoshi

    2015-11-23

    Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ-ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ-ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER.

  14. Forcible destruction of severely misfolded mammalian glycoproteins by the non-glycoprotein ERAD pathway

    PubMed Central

    Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Horimoto, Satoshi; Sugimoto, Takehiro; Ishikawa, Tokiro; Takeda, Shunichi; Yamamoto, Takashi; Suzuki, Tadashi; Kamiya, Yukiko

    2015-01-01

    Glycoproteins and non-glycoproteins possessing unfolded/misfolded parts in their luminal regions are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L with distinct mechanisms. Two-step mannose trimming from Man9GlcNAc2 is crucial in the ERAD-L of glycoproteins. We recently showed that this process is initiated by EDEM2 and completed by EDEM3/EDEM1. Here, we constructed chicken and human cells simultaneously deficient in EDEM1/2/3 and analyzed the fates of four ERAD-L substrates containing three potential N-glycosylation sites. We found that native but unstable or somewhat unfolded glycoproteins, such as ATF6α, ATF6α(C), CD3-δ–ΔTM, and EMC1, were stabilized in EDEM1/2/3 triple knockout cells. In marked contrast, degradation of severely misfolded glycoproteins, such as null Hong Kong (NHK) and deletion or insertion mutants of ATF6α(C), CD3-δ–ΔTM, and EMC1, was delayed only at early chase periods, but they were eventually degraded as in wild-type cells. Thus, higher eukaryotes are able to extract severely misfolded glycoproteins from glycoprotein ERAD and target them to the non-glycoprotein ERAD pathway to maintain the homeostasis of the ER. PMID:26572623

  15. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

    PubMed

    Song, Ehwang; Mechref, Yehia

    2015-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers.

  16. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment

    PubMed Central

    Song, Ehwang; Mechref, Yehia

    2016-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer’s and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers. PMID:26330015

  17. Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

    PubMed

    Song, Ehwang; Mechref, Yehia

    2015-01-01

    Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers. PMID:26330015

  18. Traceless labeling of glycoproteins and its application to the study of glycoprotein-protein interactions.

    PubMed

    Yang, Yung-Lin; Lee, Yen-Pin; Yang, Yen-Ling; Lin, Po-Chiao

    2014-02-21

    A new chemical method for the traceless labeling of glycoproteins with synthetic boronic acid (BA)-tosyl probes was successfully developed. The BA moiety acts as an affinity head to direct the formation of a cyclic boronate diester with the diol groups of glycans. Following this step, the electrophilic tosyl group is displaced by an SN2 reaction with a nucleophilic residue of the boronated glycoprotein, and finally, a reporter group is tagged onto the glycoprotein via an ether linkage. In the presence of polyols, a competition reaction recovers the native glycan of the tagged glycoprotein, conserving its biological significance. The BA-tosyl probes were used successfully for the specific labeling of glycosylated fetuins in a mixed protein pool and from crude Escherichia coli (E. coli) lysate. Further, a BA-tosyl-functionalized glass slide was used to fabricate glycoprotein microarrays with highly conserved glycans. By interacting with various lectins (carbohydrate-binding proteins), such as Concanavalin A (Con A) and wheat germ agglutinin (WGA), the types of carbohydrates and specific linkages of glycoproteins (α or β) could be systematically monitored. It is believed that the newly developed method will greatly accelerate the understanding of glycoproteins.

  19. Characterization of disease-associated N-linked glycoproteins.

    PubMed

    Tian, Yuan; Zhang, Hui

    2013-02-01

    N-linked glycoproteins play important roles in biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death. Specific N-linked glycoprotein changes are associated with disease progression and identification of these N-linked glycoproteins has potential for use in disease diagnosis, prognosis, and prediction of treatments. In this review, we summarize common strategies for N-linked glycoprotein characterization and applications of these strategies to identification of glycoprotein changes associated with disease states. We also review the N-linked glycoproteins altered in diseases such as breast cancer, lung cancer, and prostate cancer. Although assays for these glycoproteins have potential clinical utility, research is needed to translate these glycoproteins to clinical biomarkers.

  20. Characterization of disease-associated N-linked glycoproteins

    PubMed Central

    Tian, Yuan; Zhang, Hui

    2013-01-01

    N-linked glycoproteins play important roles in biological processes, including cell-to-cell recognition, growth, differentiation, and programmed cell death. Specific N-linked glycoprotein changes are associated with disease progression and identification of these N-linked glycoproteins has potential for use in disease diagnosis, prognosis, and prediction of treatments. In this review, we summarize common strategies for N-linked glycoprotein characterization and applications of these strategies to identification of glycoprotein changes associated with disease states. We also review the N-linked glycoproteins altered in diseases such as breast cancer, lung cancer, and prostate cancer. Although assays for these glycoproteins have potential clinical utility, research is needed to translate these glycoproteins to clinical biomarkers. PMID:23255236

  1. Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins.

    PubMed

    Lajko, Michelle; Haddad, Alexander F; Robinson, Carolyn A; Connolly, Sarah A

    2015-09-01

    Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus-cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein-protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

  2. Sweet new world: glycoproteins in bacterial pathogens.

    PubMed

    Schmidt, M Alexander; Riley, Lee W; Benz, Inga

    2003-12-01

    In eukaryotes, the combinatorial potential of carbohydrates is used for the modulation of protein function. However, despite the wealth of cell wall and surface-associated carbohydrates and glycoconjugates, the accepted dogma has been that prokaryotes are not able to glycosylate proteins. This has now changed and protein glycosylation in prokaryotes is an accepted fact. Intriguingly, in Gram-negative bacteria most glycoproteins are associated with virulence factors of medically significant pathogens. Also, important steps in pathogenesis have been linked to the glycan substitution of surface proteins, indicating that the glycosylation of bacterial proteins might serve specific functions in infection and pathogenesis and interfere with inflammatory immune responses. Therefore, the carbohydrate modifications and glycosylation pathways of bacterial proteins will become new targets for therapeutic and prophylactic measures. Here we discuss recent findings on the structure, genetics and function of glycoproteins of medically important bacteria and potential applications of bacterial glycosylation systems for the generation of novel glycoconjugates.

  3. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function.

    PubMed

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewis(a) substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

  4. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function

    PubMed Central

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review. PMID:25324850

  5. Cell wall O-glycoproteins and N-glycoproteins: aspects of biosynthesis and function.

    PubMed

    Nguema-Ona, Eric; Vicré-Gibouin, Maïté; Gotté, Maxime; Plancot, Barbara; Lerouge, Patrice; Bardor, Muriel; Driouich, Azeddine

    2014-01-01

    Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER) and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensins (EXTs), the O-glycan chains of arabinogalactan proteins (AGPs) are highly heterogeneous consisting mostly of (i) a short oligo-arabinoside chain of three to four residues, and (ii) a larger β-1,3-linked galactan backbone with β-1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose, or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core β,2-xylose, core α1,3-fucose residues, and Lewis(a) substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins, only EXTs and AGPs are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review.

  6. Unusual molecular architecture of the machupo virus attachment glycoprotein.

    PubMed

    Bowden, Thomas A; Crispin, Max; Graham, Stephen C; Harvey, David J; Grimes, Jonathan M; Jones, E Yvonne; Stuart, David I

    2009-08-01

    New World arenaviruses, which cause severe hemorrhagic fever, rely upon their envelope glycoproteins for attachment and fusion into their host cell. Here we present the crystal structure of the Machupo virus GP1 attachment glycoprotein, which is responsible for high-affinity binding at the cell surface to the transferrin receptor. This first structure of an arenavirus glycoprotein shows that GP1 consists of a novel alpha/beta fold. This provides a blueprint of the New World arenavirus attachment glycoproteins and reveals a new architecture of viral attachment, using a protein fold of unknown origins.

  7. Native functionality and therapeutic targeting of arenaviral glycoproteins.

    PubMed

    Crispin, Max; Zeltina, Antra; Zitzmann, Nicole; Bowden, Thomas A

    2016-06-01

    Surface glycoproteins direct cellular targeting, attachment, and membrane fusion of arenaviruses and are the primary target for neutralizing antibodies. Despite significant conservation of the glycoprotein architecture across the arenavirus family, there is considerable variation in the molecular recognition mechanisms used during host cell entry. We review recent progress in dissecting these infection events and describe how arenaviral glycoproteins can be targeted by small-molecule antivirals, the natural immune response, and immunoglobulin-based therapeutics. Arenaviral glycoprotein-mediated assembly and infection pathways present numerous opportunities and challenges for therapeutic intervention. PMID:27104809

  8. Swainsonine: an inhibitor of glycoprotein processing.

    PubMed Central

    Elbein, A D; Solf, R; Dorling, P R; Vosbeck, K

    1981-01-01

    Swainsonine, an indolizidine alkaloid, inhibits the processing of asparagine-linked glycoproteins in both cell-free extracts and animal cells in culture. Thus, in a liver particulate enzyme preparation, swainsonine at 0.1-1.0 microM inhibited the mannosidase that releases [3H]mannose from a high mannose glycopeptide but only slightly inhibited the release of glucose from a glucose-labeled glycopeptide. MDCK and Chinese hamster ovary cells in culture incorporate [2-3H]mannose and [6-3H]glucosamine into both high mannose and complex types of oligosaccharides. When these cells were incubated with swainsonine and then labeled with mannose or glucosamine, there was a dramatic decrease in the amount of label in the complex type of glycopeptide and a substantial increase in the radioactivity in the high mannose type. This change was monitored by the increase in radioactivity that became susceptible to digestion by endoglucosaminidase H with increasing concentrations of swainosine. The endoglucosaminidase H-released oligosaccharide(s) from swainsonine-treated cells was larger and more homogeneous than that from controls and eluted from Bio-Gel P-4 at the position of Man9GlcNAc. Several tissue culture cell lines were grown in the presence of swainsonine to determine its effect on cell surface glycoproteins. Cells grown in the alkaloid showed an increased capacity to bind Escherichia coli B886, a bacterium that binds to high mannose glycoproteins. These cells also showed an increasing binding of [3H]concanavalin A. PMID:6801650

  9. Swainsonine: an inhibitor of glycoprotein processing.

    PubMed

    Elbein, A D; Solf, R; Dorling, P R; Vosbeck, K

    1981-12-01

    Swainsonine, an indolizidine alkaloid, inhibits the processing of asparagine-linked glycoproteins in both cell-free extracts and animal cells in culture. Thus, in a liver particulate enzyme preparation, swainsonine at 0.1-1.0 microM inhibited the mannosidase that releases [3H]mannose from a high mannose glycopeptide but only slightly inhibited the release of glucose from a glucose-labeled glycopeptide. MDCK and Chinese hamster ovary cells in culture incorporate [2-3H]mannose and [6-3H]glucosamine into both high mannose and complex types of oligosaccharides. When these cells were incubated with swainsonine and then labeled with mannose or glucosamine, there was a dramatic decrease in the amount of label in the complex type of glycopeptide and a substantial increase in the radioactivity in the high mannose type. This change was monitored by the increase in radioactivity that became susceptible to digestion by endoglucosaminidase H with increasing concentrations of swainosine. The endoglucosaminidase H-released oligosaccharide(s) from swainsonine-treated cells was larger and more homogeneous than that from controls and eluted from Bio-Gel P-4 at the position of Man9GlcNAc. Several tissue culture cell lines were grown in the presence of swainsonine to determine its effect on cell surface glycoproteins. Cells grown in the alkaloid showed an increased capacity to bind Escherichia coli B886, a bacterium that binds to high mannose glycoproteins. These cells also showed an increasing binding of [3H]concanavalin A. PMID:6801650

  10. Secondary cell-wall-specific glycoprotein(s) from French bean hypocotyls.

    PubMed Central

    Wojtaszek, P; Bolwell, G P

    1995-01-01

    Specific labeling of secondary cell walls of tracheary elements and of xylary and phloem fibers has been observed when wheat germ agglutinin (WGA) and anti-WGA antibodies were used during ultrastructural studies of French bean (Phaseolus vulgaris L.) hypocotyls. In this report we demonstrate that at least part of this labeling is due to the presence of secondary cell-wall-specific glycoproteins. Three major novel glycoproteins with relative molecular weights of 55,000, 86,000, and 90,000, purified by means of WGA-Sepharose affinity chromatography, have been characterized. Their amino acid composition indicates that they are not the members of known classes of structural cell-wall proteins, since they contain no hydroxyproline, a lower level of glycine than seen in glycine-rich proteins, and very little proline. N-terminal sequences of all three proteins show no significant homology with other proteins. Antibodies were raised against electrophoretically pure 90-kD glycoprotein. These were used to localize this protein in secondary cell walls of xylem tracheary elements and in xylary and phloem fibers, i.e. in the same compartments where labeling with WGA has been observed. To our knowledge this is one of the first biochemical and ultrastructural demonstrations of secondary cell-wall-specific glycoproteins. PMID:7630932

  11. [Lactoferrin - a glycoprotein of great therapeutic potentials].

    PubMed

    Lauterbach, Ryszard; Kamińska, Ewa; Michalski, Piotr; Lauterbach, Jan Paweł

    2016-01-01

    Lactoferrin is an iron-binding glycoprotein, which is present in most biological fluids with particularly high levels in colostrum and in mammalian milk. Bovine lactoferrin is more than 70% homologous with human lactoferrin. Most of the clinical trials have used bovine lactoferrin for supplementation. This review summarizes the recent advances in explaining the mechanisms, which are responsible for the multifunctional roles of lactoferrin, and presents its potential prophylactic and therapeutic applications. On the ground of the results of preliminary clinical observations, authors suggest beneficial effect of lactoferrin supplementation on the prevalence of necrotizing enterocolitis in infants with birth weight below 1250 grams. PMID:27442696

  12. The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

    ERIC Educational Resources Information Center

    Estelrich, J.; Pouplana, R.

    1988-01-01

    Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

  13. Enhanced detection of glycoproteins in polyacrylamide gels.

    PubMed

    Muñoz, G; Marshall, S; Cabrera, M; Horvat, A

    1988-05-01

    A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel. PMID:3394948

  14. Enhanced detection of glycoproteins in polyacrylamide gels.

    PubMed

    Muñoz, G; Marshall, S; Cabrera, M; Horvat, A

    1988-05-01

    A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.

  15. Glycosylation modulates arenavirus glycoprotein expression and function

    SciTech Connect

    Bonhomme, Cyrille J. Capul, Althea A. Lauron, Elvin J. Bederka, Lydia H. Knopp, Kristeene A. Buchmeier, Michael J.

    2011-01-20

    The glycoprotein of lymphocytic choriomeningitis virus (LCMV) contains nine potential N-linked glycosylation sites. We investigated the function of these N-glycosylations by using alanine-scanning mutagenesis. All the available sites were occupied on GP1 and two of three on GP2. N-linked glycan mutations at positions 87 and 97 on GP1 resulted in reduction of expression and absence of cleavage and were necessary for downstream functions, as confirmed by the loss of GP-mediated fusion activity with T87A and S97A mutants. In contrast, T234A and E379N/A381T mutants impaired GP-mediated cell fusion without altered expression or processing. Infectivity via virus-like particles required glycans and a cleaved glycoprotein. Glycosylation at the first site within GP2, not normally utilized by LCMV, exhibited increased VLP infectivity. We also confirmed the role of the N-linked glycan at position 173 in the masking of the neutralizing epitope GP-1D. Taken together, our results indicated a strong relationship between fusion and infectivity.

  16. Podoplanin - a small glycoprotein with many faces

    PubMed Central

    Ugorski, Maciej; Dziegiel, Piotr; Suchanski, Jaroslaw

    2016-01-01

    Podoplanin is a small membrane glycoprotein with a large number of O-glycoside chains and therefore it belongs to mucin-type proteins. It can be found on the surface of many types of normal cells originating from various germ layers. It is present primarily on the endothelium of lymphatic vessels, type I pneumocytes and glomerular podocytes. Increased levels of podoplanin or its neo-expression have been found in numerous types of human carcinomas, but it is especially common in squamous cell carcinomas, such as cervical, larynx, oral cavity, skin and lung cancer. This small sialomucin is also seen on the surface of cancer-associated fibroblasts (CAFs) in lung adenocarcinomas, as well as in breast and pancreatic tumors. In most cancers, a high level of podoplanin expression, both in cancer cells, as well as in CAFs, is correlated with an increased incidence of metastasis to lymph nodes and shorter survival time of patients. Little is known about the biological role of podoplanin, however research carried out on mice with a knock-out gene of this glycoprotein shows that the presence of podoplanin determines normal development of lungs, the lymphatic system and heart. Podoplanin on cancer cells and CAFs seems to play an important role in the development and progression of various cancers. However, its role in these processes is both unclear and controversial. In this review, the role of podoplanin in both physiological processes and carcinogenesis is discussed. PMID:27186410

  17. Podoplanin - a small glycoprotein with many faces.

    PubMed

    Ugorski, Maciej; Dziegiel, Piotr; Suchanski, Jaroslaw

    2016-01-01

    Podoplanin is a small membrane glycoprotein with a large number of O-glycoside chains and therefore it belongs to mucin-type proteins. It can be found on the surface of many types of normal cells originating from various germ layers. It is present primarily on the endothelium of lymphatic vessels, type I pneumocytes and glomerular podocytes. Increased levels of podoplanin or its neo-expression have been found in numerous types of human carcinomas, but it is especially common in squamous cell carcinomas, such as cervical, larynx, oral cavity, skin and lung cancer. This small sialomucin is also seen on the surface of cancer-associated fibroblasts (CAFs) in lung adenocarcinomas, as well as in breast and pancreatic tumors. In most cancers, a high level of podoplanin expression, both in cancer cells, as well as in CAFs, is correlated with an increased incidence of metastasis to lymph nodes and shorter survival time of patients. Little is known about the biological role of podoplanin, however research carried out on mice with a knock-out gene of this glycoprotein shows that the presence of podoplanin determines normal development of lungs, the lymphatic system and heart. Podoplanin on cancer cells and CAFs seems to play an important role in the development and progression of various cancers. However, its role in these processes is both unclear and controversial. In this review, the role of podoplanin in both physiological processes and carcinogenesis is discussed. PMID:27186410

  18. Evidence for glycoprotein transport into complex plastids.

    PubMed

    Peschke, Madeleine; Moog, Daniel; Klingl, Andreas; Maier, Uwe G; Hempel, Franziska

    2013-06-25

    Diatoms are microalgae that possess so-called "complex plastids," which evolved by secondary endosymbiosis and are surrounded by four membranes. Thus, in contrast to primary plastids, which are surrounded by only two membranes, nucleus-encoded proteins of complex plastids face additional barriers, i.e., during evolution, mechanisms had to evolve to transport preproteins across all four membranes. This study reveals that there exist glycoproteins not only in primary but also in complex plastids, making transport issues even more complicated, as most translocation machineries are not believed to be able to transport bulky proteins. We show that plastidal reporter proteins with artificial N-glycosylation sites are indeed glycosylated during transport into the complex plastid of the diatom Phaeodactylum tricornutum. Additionally, we identified five endogenous glycoproteins, which are transported into different compartments of the complex plastid. These proteins get N-glycosylated during transport across the outermost plastid membrane and thereafter are transported across the second, third, and fourth plastid membranes in the case of stromal proteins. The results of this study provide insights into the evolutionary pressure on translocation mechanisms and pose unique questions on the operating mode of well-known transport machineries like the translocons of the outer/inner chloroplast membranes (Toc/Tic).

  19. Ammonia transport in the kidney by Rhesus glycoproteins

    PubMed Central

    Verlander, Jill W.

    2014-01-01

    Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport. PMID:24647713

  20. Ammonia transport in the kidney by Rhesus glycoproteins.

    PubMed

    Weiner, I David; Verlander, Jill W

    2014-05-15

    Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4(+) with a new model in which specific and regulated transport of both NH3 and NH4(+) across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport.

  1. Role of envelope glycoproteins in intracellular virus maturation

    SciTech Connect

    Matsuoka, Y.

    1988-01-01

    The possible role viral glycoproteins in intracellular maturation was studied by using two different viruses, avian infectious bronchitis virus (IBV), a coronavirus, and Punta Toro virus (PTV), a bunyavirus. Using the antibiotic tunicamycin, which inhibits glycosylation of N-linked glycoproteins, it was shown that coronavirus particles are formed in the absence of glycosylation. Analysis of the protein composition of these particles indicated that they contain an unglycosylated form of the membrane-associated E1 glycoprotein but lack the E2 spike glycoprotein. A cDNA clone derived from the PTV M RNA genome segment, which encodes the G1 and G2 glycoproteins, was cloned into vaccinia virus. Studies by indirect immunofluorescence microscopy revealed that the glycoproteins synthesized from this recombinant were found to accumulate intracellularly at the Golgi complex, where virus budding usually takes place. Surface immunoprecipitation and {sup 125}I-protein A binding assays also demonstrated that a majority of the glycoproteins are retained intracellularly and are not transported to the cellular surface. The sequences which encode the G1 and G2 glycoproteins were independently cloned into vaccinia virus as well.

  2. Decoration of proteins with sugar chains: recent advances in glycoprotein synthesis.

    PubMed

    Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro

    2014-10-01

    Chemical or chemoenzymatic synthesis is an emerging approach to produce homogeneous glycoproteins, which are hard to obtain by conventional biotechnology methods. Recent advances in the synthetic methodologies for the decoration of protein molecules with oligosaccharides provide several remarkable syntheses of homogeneous glycoproteins. This short review highlights several of the latest syntheses of glycoproteins including therapeutically important glycoproteins, a highly glycosylated protein, and unnatural glycoproteins in order to illustrate the power of the modern glycoprotein synthesis. Structurally defined glycoproteins are a novel material for understanding the molecular basis of glycoprotein functions and for the development of the next generation of biopharmaceuticals.

  3. Mass spectrometry-based proteomics of fungal wall glycoproteins.

    PubMed

    Yin, Qing Yuan; de Groot, Piet W J; de Koster, Chris G; Klis, Frans M

    2008-01-01

    The manifold functions of fungal wall glycoproteins include maintenance of cell wall integrity, homotypic and heterotypic adhesion, biofilm formation, acquisition of iron and sterols, protein degradation and coping with oxidative stress. Transcriptome studies indicate that the expression levels of most cell wall glycoproteins can vary widely and are tightly controlled. However, owing to their complex and variable glycosylation, fungal wall glycoproteins are difficult to analyze using traditional proteomics approaches. Recent advances in mass spectrometry-based proteomics have enabled rapid and sensitive identification and quantitation of fungal wall glycoproteins; this will be particularly useful for studying the dynamics of the subproteome of fungal wall glycoproteins, and for the development of novel vaccines and diagnostic tools.

  4. Aberrant expression of a chemokinetic glycoprotein in psoriatic skin.

    PubMed

    Rajaraman, S; Schmalsteig, F C; Brysk, M M; Hendrick, S J; Solomon, A R

    1987-05-01

    Clinically involved and uninvolved skin samples of 6 psoriatic patients, 4 samples each of normal skin specimens, basal cell carcinoma and keratoacanthoma were studied by an indirect immunofluorescence technique. The monospecific antibody used in this study was directed against a 30 kD glycoprotein, normally expressed by the terminally differentiated corneocytes. Functional characterization of this glycoprotein was evaluated by neutrophil cell movement assays. The involved and uninvolved skin of psoriatic patients expressed the 30 kD glycoprotein not only in the stratum corneum but in all the viable epidermal layers as well. Functional studies revealed this glycoprotein to be a potent chemokinetic molecule. These results suggest that the 30 kD glycoprotein is an intrinsic chemokinetic molecule of the terminally differentiated corneocytes, and its precocious and aberrant expression in psoriatic epidermis is potentially responsible for some of the pathophysiologic aspects of psoriasis. PMID:3302266

  5. Solubilization of glycoproteins of envelope viruses by detergents

    SciTech Connect

    Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.; Isaeva, E.S.; Zhdanov, V.M.

    1986-11-20

    The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-..beta..-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis. Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines.

  6. The turnover rate of rabbit urinary Tamm–Horsfall glycoprotein

    PubMed Central

    Grant, Anne M. S.; Neuberger, Albert

    1973-01-01

    1. The turnover rate of urinary Tamm–Horsfall glycoprotein in rabbits was determined by two different methods. The first involved measurement of the pool size of the glycoprotein in rabbit kidney and the daily urinary excretion rate by a radioimmunoassay from which the turnover rate was calculated. 2. The second method made use of the incorporation in vivo of Na214CO3 and sodium [14C]acetate. After a single intramuscular injection of one of these compounds, urine collections were made every 24h and the glycoprotein was isolated and its specific radioactivity was determined. 3. Incorporation of the label into urinary HCO3−, urea and plasma fibrinogen was also examined. The specific radio-activities of the O-acetyl, sialic acid, aspartic acid and glutamic acid residues isolated from the Tamm–Horsfall glycoprotein were compared and their half-lives were compared with that of the intact glycoprotein. The two methods gave results in quite close agreement and indicated a half-life for the glycoprotein of approx. 9h. 4. An attempt was made to localize the glycoprotein within the kidney and within the cell. It is present throughout the kidney, but was not detected in the brush-border fraction isolated from the proximal tubules. From differential cell-centrifugation studies, the glycoprotein seemed to be predominantly present in the soluble fraction (100000g supernatant). This suggests that it is either largely a soluble cytoplasmic component or is very loosely bound to a membrane, being readily released under the gentlest homogenization procedure. 5. The half-life of Tamm–Horsfall glycoprotein in human kidney was found by the radioimmunoassay method to be approx. 16h. The similarity between the composition of Tamm–Horsfall glycoprotein and human erythropoietin is discussed. PMID:4780692

  7. Array-based analysis of secreted glycoproteins for rapid selection of a single cell producing a glycoprotein with desired glycosylation.

    PubMed

    Park, Sunyoung; Kim, Wanjung; Kim, Yongtae; Son, Young Dok; Lee, Sang-Chul; Kim, Eunkyung; Kim, Sung Ho; Kim, Jung Hoe; Kim, Hak-Sung

    2010-07-01

    The therapeutic efficacy and in vivo biological function of a glycoprotein is significantly affected by its glycosylation profile. For the development of glycoproteins with therapeutic applications, selection of cell lines producing a glycoprotein with adequate glycoform is crucial. Here, we demonstrate an array-based analysis of secreted glycoproteins for rapid and efficient selection of a single cell producing a glycoprotein with desirable glycosylation. Our approach relies on microengraving and interrogation of glycoproteins produced by individual cells in a microwell array in terms of glycosylation profile as well as the produced amount. On the basis of statistical analysis of the interrogation, single cells which are predicted to produce a desired glycoprotein are selected, retrieved, and expanded. We applied the approach to human recombinant erythropoietin (rhEPO)-producing CHO cells and verified the selection of a single CHO cell that produces rhEPO with a high sialylation degree. Human erythropoietin (hEPO) bearing highly sialylated oligosaccharide was shown to display a much longer plasma half-life, resulting in high therapeutic efficacy. This method may find widespread use in the clonal selection for the production of other glycoproteins with specific glycosylation as well as analysis of the heterogeneity in cell populations in a high-throughput manner.

  8. EBV glycoproteins: where are we now?

    PubMed Central

    Hutt-Fletcher, Lindsey M

    2015-01-01

    Glycoproteins are critical to virus entry, to spread within and between hosts and can modify the behavior of cells. Many viruses carry only a few, most found in the virion envelope. EBV makes more than 12, providing flexibility in how it colonizes its human host. Some are dedicated to getting the virus through the cell membrane and on toward the nucleus of the cell, some help guide the virus back out and on to the next cell in the same or a new host. Yet others undermine host defenses helping the virus persist for a lifetime, maintaining a presence that is mostly tolerated and serves to perpetuate EBV as one of the most common infections of man. PMID:26843889

  9. The effect of ginger extract on glycoproteins of Raji cells.

    PubMed

    Zamani, Zahra; Nassir-Ud-Din; Kohan, Haleemeh Kabini; Kadivar, Mehdi; Kalyee, Zahra; Rad, Behzad Laame; Iravani, Ayda; Rahimi, Nourooz Ali; Wahabi, Farideh; Sadeghi, Sedigheh; Pourfallah, Fatemeh; Arjmand, Mohammad

    2014-01-15

    Protein glycosylation is associated with the development and progression of specific diseases, including cancers. The ginger rhizome is known to have anti-cancer and anti-fungal properties. This investigation was carried out to study the effect of ginger on glycoproteins of Raji cells. A 10% yield of ginger extract was mixed with 0.01% DMSO and added to 6 x 10(4) Raji cells at different concentrations for 24, 48 and 72 h at 37 degrees C. Their half maximal inhibitory concentration (IC50) was determined and analyzed statistically using Graphpad prism software. Cell extracts were prepared and their glycoproteins purified using lectin-affinity chromatography (Q proteome total glycoprotein and O glycoprotein kits) and SDS PAGE was carried out. IC50 of ginger extract on Raji cells was 20 microg mL(-1) at 72 h with < 0.01 significance. Silver staining of purified glycoprotiens in Raji cells indicated the presence of O-glycans and N-glycans. N-linked mannose and N-linked sialic acids were detected with the total glycoprotein kit. O-linked galactose and O-linked sialic acids were identified with the O-glycoprotein. Ginger reduced the expression of O-linked sialic acid and also N-linked mannose on Raji cells but had no effect on other glycoproteins. Sialic acid is now well known as a cancer marker and investigations are on to use it as a drug-target in cancerous tissues.

  10. Lectin binding and surface glycoprotein pattern of human macrophage populations.

    PubMed

    Kreipe, H; Radzun, H J; Schumacher, U; Parwaresch, M R

    1986-01-01

    In the present study unstimulated and stimulated human blood monocytes, untreated and phorbol ester treated U-937 cells, as well as human peritoneal and alveolar macrophages were studied with respect to their surface membrane properties. Binding of different lectins and electrophoretic patterns of tritium labeled surface glycoproteins were compared. The analysis of surface glycoproteins could be interpreted as evidence for a common origin of the analysed cell populations. Furthermore, banding patterns of glycoproteins might be useful to define certain activation states within monocyte/macrophage differentiation. In contrast, lectin binding pattern did not clearly discriminate macrophage subpopulations. PMID:3102412

  11. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Beta-2-glycoprotein I immunological test system....5430 Beta-2-glycoprotein I immunological test system. (a) Identification. A beta-2-glycoprotein I... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of...

  12. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Beta-2-glycoprotein III immunological test system....5440 Beta-2-glycoprotein III immunological test system. (a) Identification. A beta-2-glycoprotein III... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of...

  13. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Beta-2-glycoprotein I immunological test system....5430 Beta-2-glycoprotein I immunological test system. (a) Identification. A beta-2-glycoprotein I... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of...

  14. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Beta-2-glycoprotein III immunological test system....5440 Beta-2-glycoprotein III immunological test system. (a) Identification. A beta-2-glycoprotein III... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of...

  15. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Beta-2-glycoprotein I immunological test system....5430 Beta-2-glycoprotein I immunological test system. (a) Identification. A beta-2-glycoprotein I... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of...

  16. Detection of glycoproteins in the Acanthamoeba plasma membrane

    SciTech Connect

    Paatero, G.I.L. ); Gahmberg, C.G. )

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.

  17. Regenerated bacterial cellulose microfluidic column for glycoproteins separation.

    PubMed

    Chen, Chuntao; Zhu, Chunlin; Huang, Yang; Nie, Ying; Yang, Jiazhi; Shen, Ruiqi; Sun, Dongping

    2016-02-10

    To analysis and separate glycoproteins, a simple strategy to prepare regenerated bacterial cellulose (RBC) column with concanavalin A (Con A) lectin immobilized in microfluidic system was applied. RBC was filled into microchannel to fabricate RBC microcolumn after bacterial cellulose dissolved in NaOH-sulfourea water solution. Lectin Con A was covalently connected onto RBC matrix surface via Schiff-base formation. Lysozyme (non-glycoprotein) and transferrin (glycoprotein) were successfully separated based on their different affinities toward the immobilized Con A. Overall, the RBC microfluidic system presents great potential application in affinity chromatography of glycoproteins analysis, and this research represents a significant step to prepare bacterial cellulose (BC) as column packing material in microfluidic system. What is more, troublesome operations for lectin affinity chromatography were simplified by integrating the microfluidic chip onto a HPLC (High Performance Liquid Chromatography) system.

  18. Using Single Lectins to Enrich Glycoproteins in Conditioned Media.

    PubMed

    Sethi, Manveen K; Fanayan, Susan

    2015-08-03

    Lectins are sugar-binding proteins that can recognize and bind to carbohydrates conjugated to proteins and lipids. Coupled with mass spectrometry technologies, lectin affinity chromatography is becoming a popular approach for identification and quantification of glycoproteins in complex samples such as blood, tumor tissues, and cell lines. Given the commercial availability of a large number of lectins that recognize diverse sugar structures, it is now possible to isolate and study glycoproteins for biological and medical research. This unit provides a general guide to single-lectin-based enrichment of glycoproteins from serum-free conditioned media. Due to the unique carbohydrate specificity of most lectins and the complexity of the samples, optimization steps may be required to evaluate different elution buffers and methods as well as binding conditions, for each lectin, for optimal recovery of bound glycoproteins.

  19. Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

    PubMed

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. PMID:24889823

  20. Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

    PubMed

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies.

  1. Oxygen radicals stimulate guinea pig gallbladder glycoprotein secretion in vitro

    SciTech Connect

    Hale, W.B.; Turner, B.; LaMont, J.T. )

    1987-11-01

    In several animal models of cholelithiasis, and in humans with gallstones, hypersecretion of gallbladder mucin is observed. This study was undertaken to determine the effect of oxygen radicals on guinea pig gallbladder glycoprotein secretion in organ culture. Mucosal explants were incubated with ({sup 3}H)glucosamine hydrochloride to label glycoproteins, then exposed to oxygen radicals generated by chelated ferric iron and ascorbic acid. Marked stimulation of glycoprotein release was observed after a 30-min exposure to the oxygen radical-generating system, and the effect was inhibited by mannitol. The stimulatory effect of hydroxyl radical was not accompanied by leakage of intracellular lactate dehydrogenase. Parallel experiments with human granulocytes activated with f-Met-Leu-Phe and coincubated with gallbladder explants revealed similar results. These results indicate that oxygen radicals, especially the hydroxyl radical (OH), are capable of stimulating rapid release of mucous-type glycoproteins from gallbladder epithelium.

  2. KDN-containing glycoprotein from loach skin mucus.

    PubMed

    Nakagawa, H; Hama, Y; Sumi, T; Li, S C; Li, Y T

    2001-01-01

    It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc. PMID:14533798

  3. Concanavalin A binding glycoprotein in human stratum corneum

    SciTech Connect

    Brysk, M.M.; Miller, J.

    1984-03-01

    A mannose-containing 40K glycoprotein has been identified in the stratum corneum of normal human epidermis. It is apparently membrane-bound and in the intact epidermis it is inaccessible to either concanavalin A or to trypsin. After it is detergent-solubilized, it can be labeled with concanavalin A or destroyed with trypsin. There is little or none of this glycoprotein in the viable cells of the epidermis.

  4. KDN-containing glycoprotein from loach skin mucus.

    PubMed

    Nakagawa, H; Hama, Y; Sumi, T; Li, S C; Li, Y T

    2001-01-01

    It has been widely recognized that the mucus coat of fish plays a variety of important physical, chemical, and physiological functions. One of the major constituents of the mucus coat is mucus glycoprotein. We found that sialic acids in the skin mucus of the loach, Misgurnus anguillicaudatus, consisted predominantly of KDN. Subsequently, we isolated KDN-containing glycoprotein from loach skin mucus and characterized its chemical nature and structure. Loach mucus glycoprotein was purified from the Tris-HCl buffer extract of loach skin mucus by DEAE-cellulose chromatography, Nuclease P1 treatment, and Sepharose CL-6B gel filtration. The purified mucus glycoprotein was found to contain 38.5 KDN, 0.5% NeuAc, 25.0% GalNAc, 3.5% Gal, 0.5% GlcNAc and 28% amino acids. Exhaustive Actinase digestion of the glycoprotein yielded a glycopeptide with a higher sugar content and higher Thr and Ser contents. The molecular size of this glycopeptide was approximately 1/12 of the intact glycoprotein. These results suggest that approximately 11 highly glycosylated polypeptide units are linked in tandem through nonglycosylated peptides to form the glycoporotein molecule. The oligosaccharide alditols liberated from the loach mucus glycoprotein by alkaline borohydride treatment were separated by Sephadex G-25 gel filtration and HPLC. The purified sugar chains were analyzed b --> 6GalNAc-ol, KDNalpha2 --> 3(GalNAcbeta1 --> 14)GalNAc-ol, KDNalpha2 --> 6(GalNAcalpha1 --> 3)GalNAc-ol, KDNalpha2 --> 6(Gal3alpha1--> 3)GalNAc-ol, and NeuAcalpha2 --> 6Gal NAc-ol. It is estimated that one loach mucus glycoprotein molecule contains more than 500 KDN-containing sugar chains that are linked to Thr and Ser residues of the protein core through GalNAc.

  5. Localization of the papain cleavage site of H-2 glycoproteins.

    PubMed Central

    Ewenstein, B M; Freed, J H; Mole, L E; Nathenson, S G

    1976-01-01

    The antigenic products of the murine H-2K and H-2D genes are glycoproteins of about 45,000 molecular weight which are tightly integrated within the cell surface membrane. A glycoprotein fragment (FAg, antigenic fragment) of 37,000 daltons carrying the carbohydrate, antigenic sites, and the associated putative beta2-microglobulin of 12,000 daltons can be generated by papain cleavage either of the native molecules in the cell membrane or of immune precipitates made from the antigen solubilized by nonionic detergent. Partial NH2-terminal sequence analyses of the native H-2 glycoprotein and of the papain-cleaved glycoprotein fragment establish that the fragment is, in fact, the NH2-terminal portion of the native molecule. Thus, the cleavage by papain proteolysis is near the COOH-terminus, and removal of the COOH-terminal portion (Fm, membrane fragment) converts the glycoprotein to a water-soluble form. This observation suggests that the NH2-terminus of the native glycoprotein extends out of the hydrophobic bilayer of the cell membrane, and that the COOH-terminus contains the membrane binding region and is buried within the bilayer. PMID:1062805

  6. Homology modelling of human P-glycoprotein.

    PubMed

    Domicevica, Laura; Biggin, Philip C

    2015-10-01

    P-glycoprotein (P-gp) is an ATP-binding cassette transporter that exports a huge range of compounds out of cells and is thus one of the key proteins in conferring multi-drug resistance in cancer. Understanding how it achieves such a broad specificity and the series of conformational changes that allow export to occur form major, on-going, research objectives around the world. Much of our knowledge to date has been derived from mutagenesis and assay data. However, in recent years, there has also been great progress in structural biology and although the structure of human P-gp has not yet been solved, there are now a handful of related structures on which homology models can be built to aid in the interpretation of the vast amount of experimental data that currently exists. Many models for P-gp have been built with this aim, but the situation is complicated by the apparent flexibility of the system and by the fact that although many potential templates exist, there is large variation in the conformational state in which they have been crystallized. In this review, we summarize how homology modelling has been used in the past, how models are typically selected and finally illustrate how MD simulations can be used as a means to give more confidence about models that have been generated via this approach.

  7. Antifreeze glycoprotein agents: structural requirements for activity.

    PubMed

    Carvajal-Rondanelli, Patricio A; Marshall, Sergio H; Guzman, Fanny

    2011-11-01

    Antifreeze glycoproteins (AFGPs) are considered to be the most efficient means to reduce ice damage to cell tissues since they are able to inhibit growth and crystallization of ice. The key element of antifreeze proteins is to act in a non-colligative manner which allows them to function at concentrations 300-500 times lowers than other dissolved solutes. During the past decade, AFGPs have demonstrated tremendous potential for many pharmaceutical and food applications. Presently, the only route to obtain AFGPs involves the time consuming and expensive process of isolation and purification from deep-sea polar fishes. Unfortunately, it is not amenable to mass production and commercial applications. The lack of understanding of the mechanism through which the AFGPs inhibit ice growth has also hampered the realization of industrial and biotechnological applications. Here we report the structural motifs that are essential for antifreeze activity of AFGPs, and propose a unified mechanism based on both recent studies of short alanine peptides and structure activity relationship of synthesized AFGPs.

  8. N-glycoprotein analysis discovers new up-regulated glycoproteins in colorectal cancer tissue.

    PubMed

    Nicastri, Annalisa; Gaspari, Marco; Sacco, Rosario; Elia, Laura; Gabriele, Caterina; Romano, Roberto; Rizzuto, Antonia; Cuda, Giovanni

    2014-11-01

    Colorectal cancer is one of the leading causes of death due to cancer worldwide. Therefore, the identification of high-specificity and -sensitivity biomarkers for the early detection of colorectal cancer is urgently needed. Post-translational modifications, such as glycosylation, are known to play an important role in cancer progression. In the present work, we used a quantitative proteomic technique based on (18)O stable isotope labeling to identify differentially expressed N-linked glycoproteins in colorectal cancer tissue samples compared with healthy colorectal tissue from 19 patients undergoing colorectal cancer surgery. We identified 54 up-regulated glycoproteins in colorectal cancer samples, therefore potentially involved in the biological processes of tumorigenesis. In particular, nine of these (PLOD2, DPEP1, SE1L1, CD82, PAR1, PLOD3, S12A2, LAMP3, OLFM4) were found to be up-regulated in the great majority of the cohort, and, interestingly, the association with colorectal cancer of four (PLOD2, S12A2, PLOD3, CD82) has not been hitherto described.

  9. Reglucosylation by UDP-glucose:glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation

    PubMed Central

    Tannous, Abla; Patel, Nishant; Tamura, Taku; Hebert, Daniel N.

    2015-01-01

    UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1) is a central quality control gatekeeper in the mammalian endoplasmic reticulum (ER). The reglucosylation of glycoproteins supports their rebinding to the carbohydrate-binding ER molecular chaperones calnexin and calreticulin. A cell-based reglucosylation assay was used to investigate the role of UGT1 in ER protein surveillance or the quality control process. UGT1 was found to modify wild-type proteins or proteins that are expected to eventually traffic out of the ER through the secretory pathway. Trapping of reglucosylated wild-type substrates in their monoglucosylated state delayed their secretion. Whereas terminally misfolded substrates or off-pathway proteins were most efficiently reglucosylated by UGT1, the trapping of these mutant substrates in their reglucosylated or monoglucosylated state did not delay their degradation by the ER-associated degradation pathway. This indicated that monoglucosylated mutant proteins were actively extracted from the calnexin/calreticulin binding-reglucosylation cycle for degradation. Therefore trapping proteins in their monoglucosylated state was sufficient to delay their exit to the Golgi but had no effect on their rate of degradation, suggesting that the degradation selection process progressed in a dominant manner that was independent of reglucosylation and the glucose-containing A-branch on the substrate glycans. PMID:25428988

  10. Isolation and characterization of lung connective-tissue glycoproteins.

    PubMed Central

    Lafuma, C; Moczar, M; Robert, L

    1982-01-01

    1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function. PMID:7115303

  11. Role of zona pellucida glycoproteins during fertilization in humans.

    PubMed

    Gupta, Satish Kumar

    2015-04-01

    In the last decade, scientific investigations pertaining to the role of zona pellucida (ZP) glycoproteins during fertilization in humans have led to new insights. This has been achieved using purified native/recombinant human zona proteins and transgenic mice expressing human ZP glycoproteins. The proposed model in mice of ZP glycoprotein-3 (ZP3) acting as primary sperm receptor and ZP glycoprotein-2 (ZP2) as secondary sperm receptor has been modified for sperm-egg binding in humans. ZP glycoprotein-1 (ZP1), ZP3, and ZP glycoprotein-4 (ZP4) have been shown to bind to the capacitated human sperm. ZP2 binds to the acrosome-reacted human spermatozoa. Further, the eggs obtained from transgenic mice expressing human ZP2 alone or in conjunction with other human instead of mouse zona proteins showed binding of human sperm, suggesting that ZP2 might also play a role in sperm-egg binding. This function has been mapped to a domain corresponding to amino acid residues 51-144 of ZP2. In contrast to mice, where ZP3 is the primary agonist for inducing the acrosome reaction, in humans, the acrosome reaction can be mediated by ZP1, ZP3, and ZP4. The effect of mutations in the genes encoding zona proteins on the ZP morphology and infertility has not been established. Further, the role of autoantibodies against ZP in women with 'unexplained infertility' leading to poor outcome of in vitro fertilization is currently controversial and needs further investigations. Understanding the role of ZP glycoproteins during human fertilization facilitates the development of new contraceptives and strategies to overcome the problem of infertility.

  12. Glycoproteins from the cell wall of Phaseolus coccineus.

    PubMed

    O'Neill, M A; Selvendran, R R

    1980-04-01

    1. The use of a modified sodium chlorite/acetic acid delignification procedure for the solubilization of a hydroxyproline-rich glycoprotein fraction from the depectinated cell walls of Phaseolus coccineus is described. 2. The crude glycoprotein was associated with some pectic material; hydroxyproline and serine were the most abundant amino acids, and arabinose, galactose and galacturonic acid the predominant monosaccharides. 3. The bulk of the hydroxyproline is O-glycosidically substituted with tetra- and tri-arabinofuranosides. From methylation analysis the linkages in these arabinosides could be inferred. 4. Ion-exchange chromatography of the crude glycoprotein gave one major and two minor hydroxyproline-rich fractions, with similar amino acid but different monosaccharide composition. 5. In the major fraction, serine appears to be O-glycosidically substituted with a single galactopyranoside residue that can be removed by the action of alpha-galactosidase but not beta-galactosidase. Removal of arabinofuranoside residues by partial acid hydrolysis greatly enhanced the action of alpha-galactosidase. 6. Methylation followed by carboxy reduction with LiAl2H4 has shown the presence of (1 leads to 4)-linked galacturonic acid in the crude glycoprotein fraction but not in the major fraction from the ion-exchange column. Hence the bulk of the pectic material is not associated with the major glycoprotein component. It is suggested that the glycoprotein is held in the wall by phenolic cross-links. 7. Similarities with the glycopeptide moiety of potato lectin provides further evidence for a class of hydroxyproline-rich glycoproteins with common features.

  13. P-glycoprotein activity and biological response

    SciTech Connect

    Vaalburg, W. . E-mail: w.vaalburg@pet.umcg.nl; Hendrikse, N.H.; Elsinga, P.H.; Bart, J.; Waarde, A. van

    2005-09-01

    P-glycoprotein (P-gp) is a transmembrane drug efflux pump encoded by the MDR-1 gene in humans. Most likely P-gp protects organs against endogenous and exogenous toxins by extruding toxic compounds such as chemotherapeutics and other drugs. Many drugs are substrates for P-gp. Since P-gp is also expressed in the blood-brain barrier, P-gp substrates reach lower concentrations in the brain than in P-gp-negative tissues. Failure of response to chemotherapy of malignancies can be due to intrinsic or acquired drug resistance. Many tumors are multidrug resistant (MDR); resistant to several structurally unrelated chemotherapeutic agents. Several mechanisms are involved in MDR of which P-gp is studied most extensively. P-gp extrudes drugs out of tumor cells resulting in decreased intracellular drug concentrations, leading to the MDR phenotype. Furthermore, the MDR-1 gene exhibits several single nucleotide polymorphisms, some of which result in different transport capabilities. P-gp functionality and the effect of P-gp modulation on the pharmacokinetics of novel and established drugs can be studied in vivo by positron emission tomography (PET) using carbon-11 and fluorine-18-labeled P-gp substrates and modulators. PET may demonstrate the consequences of genetic differences on tissue pharmacokinetics. Inhibitors such as calcium-channel blockers (verapamil), cyclosporin A, ONT-093, and XR9576 can modulate the P-gp functionality. With PET the effect of P-gp modulation on the bioavailability of drugs can be investigated in humans in vivo. PET also allows the measurement of the efficacy of newly developed P-gp modulators.

  14. Physical Properties of the Glycoprotein Mucin

    NASA Astrophysics Data System (ADS)

    Matthews, Garrett; Davis, William; Superfine, Richard; Boucher, Richard

    2003-03-01

    Epithelial cell surfaces are covered by a protective gel known as mucus. The physiological function of this gel depends on its rheological properties, and these properties are largely derived from the secreted glycoprotein mucin. The genetic disease Cystic Fibrosis (CF) is characterized by the adhesion of thick, viscous mucus on these tissues. In the lungs, this results in the interruption of mucus transport thus compromising the first line of defense against pathogens in these tissues. In order to restore the flow of tracheobronchial mucus out of the body, knowledge of the molecular and physical properties of mucin and mucin solutions would be greatly beneficial. The present model for these molecules is that of a long linear strand consisting of highly glycosylated regions linked by cystein-rich globular regions. It is thought that the globular regions may interact either through intermolecular disulfide bonds or through hydrophobic interactions. It has also been speculated that the glycosylated regions may have lectin-like interactions. In the present work, single mucin molecules were imaged at high resolution using atomic force microscopy (AFM). Phase mode imaging was used to map the interactions between functionalized AFM tips and the molecular topography. Additionally, using force-distance curves with the AFM, the adhesion between mucin bound tips and cell surface glycocalyx and glycocalyx-like model surfaces, was measured. And, finally, the viscoelastic properties of mucin solutions were measured using the recently developed technique, single particle tracking microrheology. A model is being developed that will incorporate the properties of mucins beginning at the single molecule and ending with the bulk viscoelastic properties.

  15. Linkage of a membrane skeleton to integral membrane glycoproteins in human platelets. Identification of one of the glycoproteins as glycoprotein Ib.

    PubMed Central

    Fox, J E

    1985-01-01

    Experiments were performed to determine whether platelets contain a membrane skeleton. Platelets were labeled by a sodium periodate/sodium [3H]borohydride method and lysed with Triton X-100. Much of the filamentous actin could be sedimented at low g forces (15,600 g, 4 min), but some of the actin filaments required high-speed centrifugation for their sedimentation (100,000 g, 3 h). The latter filaments differed from those in the low-speed pellet in that they could not be depolymerized by Ca2+ and could not be sedimented at low g forces even from Triton X-100 lysates of platelets that had been activated with thrombin. Actin-binding protein sedimented with both types of filaments, but 3H-labeled membrane glycoproteins were recovered mainly with the high-speed filaments. The primary 3H-labeled glycoprotein recovered with this "membrane skeleton" was glycoprotein (GP) Ib. Approximately 70% of the platelet GP Ib was present in this skeleton. Several other minor glycoproteins, including greater than 50% of the GP Ia and small amounts of three unidentified glycoproteins of Mr greater than 200,000, were also recovered with the membrane skeleton. The Triton X-100 insolubility of GP Ib, GP Ia, a minor membrane glycoprotein of 250,000 Mr, and actin-binding protein resulted from their association with actin filaments as they were rendered Triton X-100-soluble when actin filaments were depolymerized with deoxyribonuclease I and co-isolated with actin filaments on sucrose gradients. When isolated platelet plasma membranes were extracted with Triton X-100, actin, actin-binding protein, and GP Ib were recovered as the Triton X-100 residue. These studies show that unstimulated platelets contain a membrane skeleton composed of actin filaments and actin-binding protein that is distinct from the rest of the cytoskeleton and is attached to GP Ib, GP Ia, and a minor glycoprotein of 250,000 Mr on the plasma membrane. Images PMID:2932470

  16. Glycoprotein labeling with click chemistry (GLCC) and carbohydrate detection.

    PubMed

    Wu, Zhengliang L; Huang, Xinyi; Burton, Andrew J; Swift, Karl A D

    2015-08-14

    Molecular labeling and detection techniques are essential to research in life science. Here, a method for glycoprotein labeling/carbohydrate detection through glycan replacement, termed glycoprotein labeling with click chemistry (GLCC), is described. In this method, a glycoprotein is first treated with specific glycosidases to remove certain sugar residues, a procedure that creates acceptor sites for a specific glycosyltransferase. A 'clickable' monosaccharide is then installed onto these sites by the glycosyltransferase. This modified glycoprotein is then conjugated to a reporter molecule using a click chemistry reaction. For glycoproteins that already contain vacant glycosylation sites, deglycosylation is not needed before the labeling step. As a demonstration, labeling on fetal bovine fetuin, mouse immunoglobulin IgG and bacterial expressed human TNFα and TNFβ are shown. Compared to traditional ways of protein labeling, labeling at glycosylation sites with GLCC is considerably more specific and less likely to have adverse effects, and, when utilized as a method for carbohydrate detection, this method is also highly specific and sensitive.

  17. Intracellular processing of the Newcastle disease virus fusion glycoprotein

    SciTech Connect

    Morrison, T.; Ward, L.J.; Semerjian, A.

    1985-03-01

    The fusion glycoprotein (Fo) of Newcastle disease virus is cleaved at an intracellular site into F1 and F2. This result was confirmed by comparing the transit time of the fusion protein to the cell surface with the time course of cleavage of Fo. The time required for cleavage of half of the pulse-labeled Fo protein is ca. 40 min faster than the half time of the transit of the fusion protein to the cell surface. To determine the cell compartment in which cleavage occurs, use was made of inhibitors which block glycoprotein migration at specific points and posttranslational modifications known to occur in specific cell membranes. Cleavage of Fo is inhibited by carbonyl cyanide m-chlorophenylhydrazone; thus, cleavage does not occur in the rough endoplasmic reticulum. Monensin blocks the incorporation of Newcastle disease virus glycoproteins into virions and blocks the cleavage of the fusion glycoprotein. However, Fo cannot be radioactively labeled with (/sup 3/H) fucose, whereas F1 is readily labeled. These results argue that cleavage occurs in the trans Golgi membranes or in a cell compartment occupied by glycoproteins quite soon after their transit through the trans Golgi membranes. The implications of the results presented for the transit times of the fusion protein between subcellular organelles are discussed.

  18. The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins.

    PubMed

    Schwarz, P M; Elbein, A D

    1985-11-25

    The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure. PMID:3932356

  19. P-Glycoprotein Transport of Neurotoxic Pesticides.

    PubMed

    Lacher, Sarah E; Skagen, Kasse; Veit, Joachim; Dalton, Rachel; Woodahl, Erica L

    2015-10-01

    P-glycoprotein (P-gp) has been associated with a number of neurodegenerative diseases, including Parkinson's disease, although the mechanisms remain unclear. Altered transport of neurotoxic pesticides has been proposed in Parkinson's disease, but it is unknown whether these pesticides are P-gp substrates. We used three in vitro transport models, stimulation of ATPase activity, xenobiotic-induced cytotoxicity, and inhibition of rhodamine-123 efflux, to evaluate P-gp transport of diazinon, dieldrin, endosulfan, ivermectin, maneb, 1-methyl-4-phenyl-4-phenylpyridinium ion (MPP(+)), and rotenone. Diazinon and rotenone stimulated ATPase activity in P-gp-expressing membranes, with Vmax values of 22.4 ± 2.1 and 16.8 ± 1.0 nmol inorganic phosphate/min per mg protein, respectively, and Km values of 9.72 ± 3.91 and 1.62 ± 0.51 µM, respectively, compared with the P-gp substrate verapamil, with a Vmax of 20.8 ± 0.7 nmol inorganic phosphate/min per mg protein and Km of 0.871 ± 0.172 μM. None of the other pesticides stimulated ATPase activity. We observed an increased resistance to MPP(+) and rotenone in LLC-MDR1-WT cells compared with LLC-vector cells, with 15.4- and 2.2-fold increases in EC50 values, respectively. The resistance was reversed in the presence of the P-gp inhibitor verapamil. None of the other pesticides displayed differential cytotoxicity. Ivermectin was the only pesticide to inhibit P-gp transport of rhodamine-123, with an IC50 of 0.249 ± 0.048 μM. Our data demonstrate that dieldrin, endosulfan, and maneb are not P-gp substrates or inhibitors. We identified diazinon, MPP(+), and rotenone as P-gp substrates, although further investigation is needed to understand the role of P-gp transport in their disposition in vivo and associations with Parkinson's disease.

  20. Retroviral env glycoprotein trafficking and incorporation into virions.

    PubMed

    Murakami, Tsutomu

    2012-01-01

    Together with the Gag protein, the Env glycoprotein is a major retroviral structural protein and is essential for forming infectious virus particles. Env is synthesized, processed, and transported to certain microdomains at the plasma membrane and takes advantage of the same host machinery for its trafficking as that used by cellular glycoproteins. Incorporation of Env into progeny virions is probably mediated by the interaction between Env and Gag, in some cases with the additional involvement of certain host factors. Although several general models have been proposed to explain the incorporation of retroviral Env glycoproteins into virions, the actual mechanism for this process is still unclear, partly because structural data on the Env protein cytoplasmic tail is lacking. This paper presents the current understanding of the synthesis, trafficking, and virion incorporation of retroviral Env proteins.

  1. Processing of virus-specific glycoproteins of varicella zoster virus

    SciTech Connect

    Namazue, J.; Campo-Vera, H.; Kitamura, K.; Okuno, T.; Yamanishi, K.

    1985-05-01

    Monoclonal antibodies to varicella zoster virus (VZV) glycoproteins were used to study the processing of three glycoproteins with molecular weights of 83K-94K (gp 2), 64K (gp 3), and 55K (gp 5). Immunoprecipitation experiments performed with VZV-infected cells, pulse labeled with (/sup 3/H)glucosamine in the presence of tunicamycin, suggest that O-linked oligosaccharide is present on the glycoprotein of gp 2. Use of the enzyme endo-beta-N-acetylglucosaminidase H revealed that the fully processed form of gp 3 had high-mannose type and that of gp 5 had only complex type of N-linked oligosaccharides. Experiments with monensin suggest that the precursor form (116K) of gp 3 is cleaved during the processing from Golgi apparatus to cell surface membrane. The extension of O-linked oligosaccharide chain and the complex type of N-linked oligosaccharide chains also occurs during this processing.

  2. Square-wave voltammetry assays for glycoproteins on nanoporous gold

    PubMed Central

    Pandey, Binod; Bhattarai, Jay K.; Pornsuriyasak, Papapida; Fujikawa, Kohki; Catania, Rosa; Demchenko, Alexei V.; Stine, Keith J.

    2014-01-01

    Electrochemical enzyme-linked lectinsorbent assays (ELLA) were developed using nanoporous gold (NPG) as a solid support for protein immobilization and as an electrode for the electrochemical determination of the product of the reaction between alkaline phosphatase (ALP) and p-aminophenyl phosphate (p-APP), which is p-aminophenol (p-AP). Glycoproteins or concanavalin A (Con A) and ALP conjugates were covalently immobilized onto lipoic acid self-assembled monolayers on NPG. The binding of Con A – ALP (or soybean agglutinin – ALP) conjugate to glycoproteins covalently immobilized on NPG and subsequent incubation with p-APP substrate was found to result in square-wave voltammograms whose peak difference current varied with the identity of the glycoprotein. NPG presenting covalently bound glycoproteins was used as the basis for a competitive electrochemical assay for glycoproteins in solution (transferrin and IgG). A kinetic ELLA based on steric hindrance of the enzyme-substrate reaction and hence reduced enzymatic reaction rate after glycoprotein binding is demonstrated using immobilized Con A–ALP conjugates. Using the immobilized Con A-ALP conjugate, the binding affinity of immunoglobulin G (IgG) was found to be 105 nM, and that for transferrin was found to be 650 nM. Minimal interference was observed in the presence of 5 mg mL−1 BSA as a model serum protein in both the kinetic and competitive ELLA. Inhibition studies were performed with methyl D-mannoside for the binding of TSF and IgG to Con A-ALP; IC50 values were found to be 90 μM and 286 μM, respectively. Surface coverages of proteins were estimated using solution depletion and the BCA protein concentration assay. PMID:24611035

  3. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of beta-2-glycoprotein III aids in the diagnosis of an inherited deficiency of this serum protein and a variety of...

  4. Magnetic enzyme reactors for isolation and study of heterogeneous glycoproteins

    NASA Astrophysics Data System (ADS)

    Korecká, Lucie; Ježová, Jana; Bílková, Zuzana; Beneš, Milan; Horák, Daniel; Hradcová, Olga; Slováková, Marcela; Viovy, Jean-Louis

    2005-05-01

    The newly developed magnetic micro- and nanoparticles with defined hydrophobicity and porosity were used for the preparation of magnetic enzyme reactors. Magnetic particles with immobilized proteolytic enzymes trypsin, chymotrypsin and papain and with enzyme neuraminidase were used to study the structure of heterogeneous glycoproteins. Factors such as the type of carrier, immobilization procedure, operational and storage stability, and experimental conditions were optimized.

  5. Cancer Biomarker Discovery: Lectin-Based Strategies Targeting Glycoproteins

    PubMed Central

    Clark, David; Mao, Li

    2012-01-01

    Biomarker discovery can identify molecular markers in various cancers that can be used for detection, screening, diagnosis, and monitoring of disease progression. Lectin-affinity is a technique that can be used for the enrichment of glycoproteins from a complex sample, facilitating the discovery of novel cancer biomarkers associated with a disease state. PMID:22710864

  6. Glycoproteins identified from heart failure and treatment models.

    PubMed

    Yang, Shuang; Chen, Lijun; Sun, Shisheng; Shah, Punit; Yang, Weiming; Zhang, Bai; Zhang, Zhen; Chan, Daniel W; Kass, David A; van Eyk, Jennifer E; Zhang, Hui

    2015-01-01

    Conduction abnormalities can lead to dyssynchronous contraction, which significantly worsens morbidity and mortality of heart failure. Cardiac resynchronization therapy (CRT) can reverse ventricular remodeling and improve cardiac function. Although the underlying molecular changes are unknown, the use of a canine model of dyssynchronous heart failure (DHF) and CRT has shown that there are global changes across the cardiac proteome. This study determines changes in serum glycoprotein concentration from DHF and CRT compared to normal. We hypothesize that CRT invokes protective or advantageous pathways that can be reflected in the circulating proteome. Two prong discovery approaches were carried out on pooled normal, DHF, and CRT samples composed of individual canine serum to determine the overall protein concentration and the N-linked glycosites of circulating glycoproteins. The level of the glycoproteins was altered in DHF and CRT compared to control sera, with 63 glycopeptides substantially increased in DHF and/or CRT. Among the 32 elevated glycosite-containing peptides in DHF, 13 glycopeptides were reverted to normal level after CRT therapy. We further verify the changes of glycopeptides using label-free LC-MS from individual canine serum. Circulating glycoproteins such as alpha-fetoprotein, alpha-2-macroglobulin, galectin-3-binding protein, and collectin-10 show association to failing heart and CRT treatment model.

  7. QUANTITATIVE MASS SPECTROMETRIC ANALYSIS OF GLYCOPROTEINS COMBINED WITH ENRICHMENT METHODS

    PubMed Central

    Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

    2015-01-01

    Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:148–165, 2015. PMID:24889823

  8. Characterization and mapping of a nonessential pseudorabies virus glycoprotein

    SciTech Connect

    Wathen, M.W.; Wathen, L.M.K.

    1986-04-01

    Antigenic variants of pseudorabies virus (PRV) containing mutations in a viral glycoprotein with a molecular weight of 82,000 (gIII) were isolated by selecting for resistance to a complement-dependent neutralizing monoclonal antibody (MCA82-2) directed against gIII. These mutants were completely resistant to neutralization with MCA82-2 in the presence of complement. Two mutants selected for further studies either did not express gIII or expressed an improperly processed form of the glycoproteins. The mutations were also associated with an altered plaque morphology (syncytium formation). The gIII gene was mapped by the marker rescue of a gIII/sup -/ mutant with cloned restriction enzyme fragments to the long unique region of the PRV genome between 0.376 and 0.383 map units. This corresponds to the map location of a glycoprotein described by Robbins et al. Since gIII is nonessential for viral replication in cell culture and has several other characteristics in common with the herpes simplex virus glycoprotein gC, gIII may represent the PRV equivalent to herpes simplex virus gC.

  9. Glycoprotein secretion in a tracheal organ culture system

    SciTech Connect

    Warunek, D.J.

    1985-01-01

    Glycoprotein secretion in the rat trachea was studied in vitro, utilizing a modified, matrix embed/perfusion chamber. Baseline parameters of the culture environment were determined by enzymatic and biochemical procedures. The effect of pilocarpine on the release of labelled glycoproteins from the tracheal epithelium was assessed. After a single stimulation with the drug, there was a significant increase in the release of /sup 14/C-glucosamine and /sup 3/H-fucose-labelled glycoprotein. The response was dose-dependent. Similar results were obtained after a second exposure to pilocarpine. However, no dose response was observed. Morphological analyses of the tracheal epithelial secretory cells by Alcian Blue/Periodic Acid Schiff staining showed a significant decrease in the total number of Alcian Blue staining cells and an increase in the mixed cell population after a single exposure to pilocarpine. Second stimulation with the drug showed that the trachea was able to respond again, this time with a further decrease in the number of Alcian Blue staining cells and a decrease in the PAS staining cells as well. Carbohydrate analyses after the first simulation with pilocarpine showed increased levels of N-acetyl neuraminic acid and the neutral carbohydrates, fucose and galactose, in the precipitated glycoproteins.

  10. Glycoprotein expression by adenomatous polyps of the colon

    NASA Astrophysics Data System (ADS)

    Roney, Celeste A.; Xie, Jianwu; Xu, Biying; Jabour, Paul; Griffiths, Gary; Summers, Ronald M.

    2008-03-01

    Colon cancer is the second leading cause of cancer related deaths in the United States. Specificity in diagnostic imaging for detecting colorectal adenomas, which have a propensity towards malignancy, is desired. Adenomatous polyp specimens of the colon were obtained from the mouse model of colorectal cancer called adenomatous polyposis coli-multiple intestinal neoplasia (APC Min). Histological evaluation, by the legume protein Ulex europaeus agglutinin I (UEA-1), determined expression of the glycoprotein α-L-fucose. FITC-labelled UEA-1 confirmed overexpression of the glycoprotein by the polyps on fluorescence microscopy in 17/17 cases, of which 13/17 included paraffin-fixed mouse polyp specimens. In addition, FITC-UEA-1 ex vivo multispectral optical imaging of 4/17 colonic specimens displayed over-expression of the glycoprotein by the polyps, as compared to non-neoplastic mucosa. Here, we report the surface expression of α-L-fucosyl terminal residues by neoplastic mucosal cells of APC specimens of the mouse. Glycoprotein expression was validated by the carbohydrate binding protein UEA-1. Future applications of this method are the development of agents used to diagnose cancers by biomedical imaging modalities, including computed tomographic colonography (CTC). UEA-1 targeting to colonic adenomas may provide a new avenue for the diagnosis of colorectal carcinoma by CT imaging.

  11. Changes of glycoprotein patterns in sera of humans under stress.

    PubMed

    Barisic, K; Lauc, G; Dumic, J; Pavlovic, M; Flogel, M

    1996-02-01

    Stress exhibits adverse effects on many vital processes in which glycoproteins play a significant role(e.g. cell-cell/matrix interactions, immune response, neoplastic growth, implantation, prenatal development), yet only scarce attention has been directed towards studying stress induced changes in glycoprotein patterns. Using SDS-electrophoresis, blotting and digoxigenin-labelled lectins (Sambucus nigra agglutinin, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Maackia amurensis agglutinin and peanut (Arachis hypogaea) agglutinin),sera were analysed from 30 individuals chosen randomly from a severely stressed population of 309 male volunteers with no specific medical symptoms. Significant changes were found in glycoprotein pattern and content, compared with healthy controls of matching age and sex. Occasionally minor non-specific deviations from the reference values for several analytes (haemoglobin, glucose, bilirubin and alanine aminotransferase) were detected in the tested group, but glycoprotein GP4S (Mr = 45 000), detected by Datura stramonium agglutinin and Sambucus nigra agglutinin, appeared in 96.7% of samples of the stressed population. The same population also revealed an approximately 500-fold increase of GP37 in comparison with the control sera. These results suggest that stress, as a non-specific syndrome, induces specific biochemical changes, which could be of diagnostic relevance as risk makers before any more serious symptoms of stress-related consequences have developed.

  12. Blepharmone: a conjugation-inducing glycoprotein in the ciliate blepharisma.

    PubMed

    Miyake, A; Beyer, J

    1974-08-16

    Gamone 1 of Blepharisma intermedium was isolated, identified as a slightly basic glycoprotein (mizoleclular weight, 2 x 10(5)), and designated as blepharmnone. At the concentration of 6 x 10(-8) milligram per milliliter, it specifically transforms matinig type 2 cells, so that they can conjugate in about 2 hours.

  13. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    SciTech Connect

    Morrison, A.I. ); Keeble, S.; Watt, F.M. )

    1988-08-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of ({sup 14}C)galactose- or ({sup 14}C)mannose- and ({sup 14}C)glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.

  14. Isolation and characterization of glycoproteins from canine tracheal pouch secretions.

    PubMed

    Liao, T H; Blumenfeld, O O; Park, S S

    1979-04-25

    Canine tracheal pouch secretions were solubilized with 1% sodium dodecyl sulfate and visualized by sodium dodecyl sulfate-agarose-acrylamide gel electrophoresis. Intact mucus, and water-soluble and insoluble fractions of mucus were shown to be composed of high molecular weight glycoproteins (Mr greater than or equal to 3 . 10(6)) and three major classes of proteins of lower molecular weight (Mr approximately 4 . 10(5), 2 . 10(5), and 6 . 10(4)). When the mucus secretions were further treated with a reducing agent, the glycoproteins were dissociated into subunits which appeared on the gel as three discrete bands. Separation of the high molecular weight glycoproteins from the other proteins was achieved by gel filtration on Biogel A-15m in the presence of 1% dodecyl sulfate following reduction and alkylation of mucus. These glycoproteins were further resolved, using DEAE cellulose chromatography in the presence of 6 M urea, into two protein fractions. Both fractions contained approximately 87% carbohydrate, high amounts of serine and threonine but differed significantly in contents of N-acetyl glucosamine and sialic acid; their mobility on gel electrophoresis was also different. Significant contents of cysteine were noted in both fractions. Results of this study indicate that the canine tracheal pouch preparations provide normal tracheal secretions which bear similarity in structure to the tracheobronchial secretions obtained from human patients. PMID:454656

  15. EXPRESSION OF THE MAIZE MOSAIC VIRUS GLYCOPROTEIN IN INSECT CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize mosaic virus (genus Nucleorhabdovirus, family Rhabdoviridae) is transmitted in a persistent-propagative manner by Peregrinus maidis, the corn planthopper. Like other rhabdoviruses, the MMV genome encodes a surface glycoprotein that is likely involved in virus attachment and entry into host ce...

  16. A new Ebola virus nonstructural glycoprotein expressed through RNA editing.

    PubMed

    Mehedi, Masfique; Falzarano, Darryl; Seebach, Jochen; Hu, Xiaojie; Carpenter, Michael S; Schnittler, Hans-Joachim; Feldmann, Heinz

    2011-06-01

    Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP₁,₂) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP₁,₂, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

  17. Development of glycoprotein capture-based label-free method for the high-throughput screening of differential glycoproteins in hepatocellular carcinoma.

    PubMed

    Chen, Rui; Tan, Yexiong; Wang, Min; Wang, Fangjun; Yao, Zhenzhen; Dong, Liwei; Ye, Mingliang; Wang, Hongyang; Zou, Hanfa

    2011-07-01

    A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.

  18. Glycoprotein L Disruption Reveals Two Functional Forms of the Murine Gammaherpesvirus 68 Glycoprotein H▿

    PubMed Central

    Gillet, Laurent; May, Janet S.; Colaco, Susanna; Stevenson, Philip G.

    2007-01-01

    The herpesvirus glycoprotein H (gH) and gL associate to form a heterodimer that plays a central role in virus-driven membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. In order to define the role that gL plays in gamma-2 herpesvirus infections, we disrupted its coding sequence in murine gammaherpesvirus-68 (MHV-68). MHV-68 lacking gL folded gH into a conformation antigenically distinct from the form that normally predominates on infected cells. gL-deficient virions bound less well than the wild type to epithelial cells and fibroblasts. However, they still incorporated gH and remained infectious. The cell-to-cell spread of gL-deficient viruses was remarkably normal, as was infection, dissemination, and latency establishment in vivo. Viral membrane fusion was therefore gL independent. The major function of gL appeared to be allowing gH to participate in cell binding prior to membrane fusion. This function was most important for the entry of MHV-68 virions into fibroblasts and epithelial cells. PMID:17050601

  19. [Acid and basic glycoproteins of human saliva. 2. Investigation of glycoprotein of parotid saliva].

    PubMed

    Mirković, S

    1991-01-01

    We applied the standard diagnostic electrophoretic method on lyophilized human parotid saliva under the appropriate conditions (pH = 8.6 voltage 90 V and time of 30 seconds). The variation of the essential electrophoretic parameters (volume, time, voltage and pH) gave the best protein separation results in the natural range (pH = 7). Also in all cases, except pH = 11, the catodic side was richer in fractions than the anodic one; this was the qualitative characteristic of the protein component of the parotid saliva. Consequently, the protein content of the parotid saliva was rich in basic elements with the typical electrophoregram and densitogram for human serum and mixed saliva. The Pol-E agarose film method is appropriate for investigation and detection of the protein content in human, especially parotid saliva. It also enables differentiation of samples of mixed and parotid saliva on the basis of appropriate densitograms which are the consequence of different protein and especially glycoprotein components of the content.

  20. Platelet receptor expression and shedding: glycoprotein Ib-IX-V and glycoprotein VI.

    PubMed

    Gardiner, Elizabeth E; Andrews, Robert K

    2014-04-01

    Quantity, quality, and lifespan are 3 important factors in the physiology, pathology, and transfusion of human blood platelets. The aim of this review is to discuss the proteolytic regulation of key platelet-specific receptors, glycoprotein(GP)Ib and GPVI, involved in the function of platelets in hemostasis and thrombosis, and nonimmune or immune thrombocytopenia. The scope of the review encompasses the basic science of platelet receptor shedding, practical aspects related to laboratory analysis of platelet receptor expression/shedding, and clinical implications of using the proteolytic fragments as platelet-specific biomarkers in vivo in terms of platelet function and clearance. These topics can be relevant to platelet transfusion regarding both changes in platelet receptor expression occurring ex vivo during platelet storage and/or clinical use of platelets for transfusion. In this regard, quantitative analysis of platelet receptor profiles on blood samples from individuals could ultimately enable stratification of bleeding risk, discrimination between causes of thrombocytopenia due to impaired production vs enhanced clearance, and monitoring of response to treatment prior to change in platelet count.

  1. Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine.

    PubMed

    Wang, Yang; Zhou, Xuan; Yu, Qing; Duan, Yuanmeng; Huang, Binbin; Hong, Guoying; Zhou, Ayi; Jin, Litai

    2014-06-01

    A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time-consuming steps needed for poststains. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to that of Pro-Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis was performed to confirm the specificity of the newly developed method. PMID:24668852

  2. Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine.

    PubMed

    Wang, Yang; Zhou, Xuan; Yu, Qing; Duan, Yuanmeng; Huang, Binbin; Hong, Guoying; Zhou, Ayi; Jin, Litai

    2014-06-01

    A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time-consuming steps needed for poststains. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to that of Pro-Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis was performed to confirm the specificity of the newly developed method.

  3. [Prokaryotic expression and immunogenicity analysis of glycoprotein from infectious hematopoietic necrosis virus].

    PubMed

    Xu, Li-ming; Liu, Hong-bai; Yin, Jia-sheng; Lu, Tong-yan

    2013-09-01

    In order to detect Infectious hematopoietic necrosis virus with immunological methods, the surface glycoprotein of a recent IHNV-Sn isolated from farmed rainbow trout ( Oncorhynchus mykiss ) in China was amplified and cloned into pET27b(+) vector (designated as pET27b-G ). The expression of recombinant plasmid pET27b-G in E. coli BL21(DE3) was induced and determined by SDS-PAGE analysis. The predicted molecular weight of glycoprotein protein was approximately 55 kD and was confirmed in this study. The inclusion body of glycoprotein was treated with urea at different urea concentrations, and dialyzed into PBS buffer. Purified glycoprotein with high concentration was obtained after dialyzed in the PBS buffer. Antisera against glycoprotein were produced from immunized rabbits. The prepared antisera could react specifically with both the recombinant glycoprotein and natural glycoprotein of the IHNV-Sn isolated in the test of indirect ELISA, and the titer against the recombinant glycoprotein was 1:20,000. IFA showed that the antisera can recognize the glycoprotein located on the surface of IHNV-Sn and IHNV reference strain. These results indicated that the expressed glycoprotein was immunogenical and antigenical and could be functional as the natural IHNV glycoprotein. These results established a foundation for further study on vaccine and rapid diagnosis of IHNV.

  4. Frostbite Protection in Mice Expressing an Antifreeze Glycoprotein

    PubMed Central

    Heisig, Martin; Mattessich, Sarah; Rembisz, Alison; Acar, Ali; Shapiro, Martin; Booth, Carmen J.; Neelakanta, Girish; Fikrig, Erol

    2015-01-01

    Ectotherms in northern latitudes are seasonally exposed to cold temperatures. To improve survival under cold stress, they use diverse mechanisms to increase temperature resistance and prevent tissue damage. The accumulation of anti-freeze proteins that improve cold hardiness occurs in diverse species including plants, arthropods, fish, and amphibians. We previously identified an Ixodes scapularis anti-freeze glycoprotein, named IAFGP, and demonstrated its cold protective function in the natural tick host and in a transgenic Drosophila model. Here we show, in a transgenic mouse model expressing an anti-freeze glycoprotein, that IAFGP protects mammalian cells and mice from cold shock and frostbite respectively. Transgenic skin samples showed reduced cell death upon cold storage ex vivo and transgenic mice demonstrated increased resistance to frostbite injury in vivo. IAFGP actively protects mammalian tissue from freezing, suggesting its application for the prevention of frostbite, and other diseases associated with cold exposure. PMID:25714402

  5. Marine Natural Products with P-Glycoprotein Inhibitor Properties

    PubMed Central

    Lopez, Dioxelis; Martinez-Luis, Sergio

    2014-01-01

    P-glycoprotein (P-gp) is a protein belonging to the ATP-binding cassette (ABC) transporters superfamily that has clinical relevance due to its role in drug metabolism and multi-drug resistance (MDR) in several human pathogens and diseases. P-gp is a major cause of drug resistance in cancer, parasitic diseases, epilepsy and other disorders. This review article aims to summarize the research findings on the marine natural products with P-glycoprotein inhibitor properties. Natural compounds that modulate P-gp offer great possibilities for semi-synthetic modification to create new drugs and are valuable research tools to understand the function of complex ABC transporters. PMID:24451193

  6. Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions

    PubMed Central

    Ujike, Makoto; Taguchi, Fumihiro

    2015-01-01

    The envelopes of coronaviruses (CoVs) contain primarily three proteins; the two major glycoproteins spike (S) and membrane (M), and envelope (E), a non-glycosylated protein. Unlike other enveloped viruses, CoVs bud and assemble at the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC). For efficient virion assembly, these proteins must be targeted to the budding site and to interact with each other or the ribonucleoprotein. Thus, the efficient incorporation of viral envelope proteins into CoV virions depends on protein trafficking and protein–protein interactions near the ERGIC. The goal of this review is to summarize recent findings on the mechanism of incorporation of the M and S glycoproteins into the CoV virion, focusing on protein trafficking and protein–protein interactions. PMID:25855243

  7. [Purification and analysis of Cimicifuga foetida glycoprotein ( CF- I )].

    PubMed

    Sun, Yu-jun; Chen, Yan; Song, Zhi-yin; Zhou, Dong-wen

    2007-02-01

    A kind of glycoprotein ( CF- I ) was extracted from Cimicifuga foetida and purified by DEAE-Cellulose ( DEAE-52) and Sepharose CL-4B. It was identified to be homogeneous glycoprotein complex by electrophoresis and fast protein liquid chromatography (FPLC). alpha-glucosidic bond was detrermined by IR. Typical absorption of polysaccharide was shown in its IR spectrum. It had no typical absorption of nucleic acid or pigment by UV scanning. Glucose, galactose, mannose and arabinose were identified in CF- I with the molar ratio of 11. 94: 2. 18: 1. 38: 1 by GC. Its average MW was estimated to be 5. 8 x 10' by gel filtration. The content of total saccharide, protein and acid polysaccharide was 78% , 14. 4% and 23% respectively. It might be composed of 1-->4 and 1-->6 linked glucopyranose by periodate oxidation and Smith degration.

  8. Rheologic studies on middle ear effusions and their mucus glycoproteins.

    PubMed

    FitzGerald, J E; Green, G G; Birchall, J P; Pearson, J P

    1989-04-01

    The properties of pooled thick and thin middle ear effusions, from children with otitis media with effusion, were studied by viscometry. Mucus glycoproteins were responsible for effusion viscosity. Their percentage by weight in thick and thin effusions was 25% and 8.2%, respectively. N-acetylcysteine and 0.2 mol/L of mercaptoethanol caused a 39% viscosity drop in a 5-mg/mL glycoprotein solution, whereas S-carboxymethylcysteine had no effect. Treatment of thick effusions with 0.2 mol/L of mercaptoethanol initially caused a viscosity decrease followed by a gradual increase. Higher reducing agent concentrations (0.5 mol/L) caused a more rapid decrease followed by a rapid increase, presumably by causing nonspecific aggregation of reduced protein molecules. These results suggest that the concentration of and the time that a mucolytic is in the middle ear would be of prime importance in achieving the desired decrease in viscosity.

  9. Ice growth in supercooled solutions of antifreeze glycoproteins

    NASA Technical Reports Server (NTRS)

    Harrison, K.; Hallett, J.; Burcham, T. S.; Feeney, R. E.; Kerr, W. L.

    1987-01-01

    The effects of different degrees of supercooling on the habit and rates of growth of ice crystals from solutions of antifreeze glycoproteins are reported. To isolate the influence of different solutions and supercooling alone, a system was devised that nucleated crystals in the middle of a uniformly supercooled sample. Alternatively, single crystals of selected orientation were inserted into free liquid surface. A crystallization rate up to five times greater than that in pure water was found. A mechanism explaining these results is suggested.

  10. Structural Insights into the Human Metapneumovirus Glycoprotein Ectodomain

    PubMed Central

    Leyrat, Cedric; Paesen, Guido C.; Charleston, James; Renner, Max

    2014-01-01

    Human metapneumovirus is a major cause of respiratory tract infections worldwide. Previous reports have shown that the viral attachment glycoprotein (G) modulates innate and adaptive immune responses, leading to incomplete immunity and promoting reinfection. Using bioinformatics analyses, static light scattering, and small-angle X-ray scattering, we show that the extracellular region of G behaves as a heavily glycosylated, intrinsically disordered polymer. We discuss potential implications of these findings for the modulation of immune responses by G. PMID:25031352

  11. Alpha-2-HS-glycoprotein phenotype frequencies in Cook Islanders.

    PubMed

    Abe, S; Kurisaki, E; Mizusawa, I; Hiraiwa, K

    1991-01-01

    The polymorphism of the alpha 2-HS-glycoprotein (A2HS) was analysed in Rarotonga and Mangaia, the Cook Islands. The A2HS*2 frequency was found to be the highest value among all populations studied up to now. There was a significant difference in A2HS*2 gene frequencies between the two populations, Rarotonga (0.62) and Mangaia (0.76). PMID:2050386

  12. Mucus glycoprotein secretion by tracheal explants: effects of pollutants

    SciTech Connect

    Last, J.A.; Kaizu, T.

    1980-04-01

    Tracheal slices incubated with radioactive precursors in tissue culture medium secrete labeled mucus glycoproteins into the culture medium. We have used an in vivtro approach, a combined method utilizing exposure to pneumotoxins in vivo coupled with quantitation of mucus secretion rates in vitro, to study the effects of inhaled pollutants on mucus biosynthesis by rat airways. In addition, we have purified the mucus glycoproteins secreted by rat tracheal explants in order to determine putative structural changes that might by the basis for the observed augmented secretion rates after exposure of rats to H2SO4 aerosols in combination with high ambient levels of ozone. After digestion with papain, mucus glycoproteins secreted by tracheal explants may be separated into five fractions by ion-exchange chromatography, with recovery in high yield, on columns of DEAE-cellulose. Each of these five fractions, one neutral and four acidic, migrates as a single unique spot upon cellulose acetate electrophoresis at pH values of 8.6 and 1.2. The neutral fraction, which is labeled with (3H) glucosamine, does not contain radioactivity when Na2 35SO4 is used as the precursor. Acidic fractions I to IV are all labeled with either 3H-glucosamine or Na2 35SO4 as precursor. Acidic fraction II contains sialic acid as the terminal sugar on its oligosaccharide side chains, based upon its chromatographic behavior on columns of wheat-germ agglutinin-Agarose. Treatment of this fraction with neuraminidase shifts its elution position in the gradient to a lower salt concentration, coincident with acidic fraction I. After removal of terminal sialic acid residues with either neuraminidase or low pH treatment, the resultant terminal sugar on the oligosaccharide side chains is fucose. These results are identical with those observed with mucus glycoproteins secreted by cultured human tracheal explants and purified by these same techniques.

  13. Requirements within the Ebola Viral Glycoprotein for Tetherin Antagonism

    PubMed Central

    Vande Burgt, Nathan H.; Kaletsky, Rachel L.; Bates, Paul

    2015-01-01

    Tetherin is an interferon-induced, intrinsic cellular response factor that blocks release of numerous viruses, including Ebola virus, from infected cells. As with many viruses targeted by host factors, Ebola virus employs a tetherin antagonist, the viral glycoprotein (EboGP), to counteract restriction and promote virus release. Unlike other tetherin antagonists such as HIV-1 Vpu or KSHV K5, the features within EboGP needed to overcome tetherin are not well characterized. Here, we describe sequences within the EboGP ectodomain and membrane spanning domain (msd) as necessary to relieve tetherin restriction of viral particle budding. Fusing the EboGP msd to a normally secreted form of the glycoprotein effectively promotes Ebola virus particle release. Cellular protein or lipid anchors could not substitute for the EboGP msd. The requirement for the EboGP msd was not specific for filovirus budding, as similar results were seen with HIV particles. Furthermore trafficking of chimeric proteins to budding sites did not correlate with an ability to counter tetherin. Additionally, we find that a glycoprotein construct, which mimics the cathepsin-activated species by proteolytic removal of the EboGP glycan cap and mucin domains, is unable to counteract tetherin. Combining these results suggests an important role for the EboGP glycan cap and msd in tetherin antagonism. PMID:26516900

  14. Boron dependent membrane glycoproteins in symbiosome development and nodule organogenesis

    PubMed Central

    Redondo-Nieto, Miguel; Reguera, María; Bonilla, Ildefonso

    2008-01-01

    During the last two decades, we have analyzed the roles of boron (B) in the development of the legume-rhizobia symbiosis and nodule organogenesis. As in other plant tissues, B is needed for the maintenance of nodule cell wall structure. Moreover, several symbiotic events including rhizobial infection, nodule cell invasion and symbiosome development that involve membrane related functions (i.e., vesicle targeting, secretion, or cell surface interactions) are affected by B deficiency. Using anti-rhamnogalacturonan II (anti-RGII) antiserum and immunological techniques, we recently described membrane glycoproteins (RGII-glycoproteins) developmentally regulated in Pisum sativum nodules, which are not detected by the antibody in B-deficient nodules. RGII-glycoproteins appeared related with development processes involving extensive membrane synthesis, like symbiosome maturation or cell growth, both of them negatively affected by B deficiency. Here, we suggest that, besides maintaining cell wall structure, B is both stabilizing components of the membrane glycocalyx and promoting interactions between cell surfaces glycoconjugates that are important during the establishment of the symbiosis and during nodule development. Moreover, we hypothesize that B is playing a similar role during plant or animal embryogenesis and development. PMID:19841651

  15. Identification of glycoproteins from mouse skin tumors and plasma

    PubMed Central

    Tian, Yuan; Kelly-Spratt, Karen S.; Kemp, Christopher J.; Zhang, Hui

    2010-01-01

    Plasma has been the focus of testing different proteomic technologies for the identification of biomarkers due to its ready accessibility. However, it is not clear if direct proteomic analysis of plasma can be used to discover new marker proteins from tumor that are associated with tumor progression. Here, we reported that such proteins can be detected in plasma in a chemical induced skin cancer mouse model. We analyzed glycoproteins from both benign papillomas and malignant carcinomas from mice using our recently developed platform, solid-phase extraction of glycopeptides (SPEG) and mass spectrometry, and identified 463 unique N-linked glycosites from 318 unique glycoproteins. These include most known extracellular proteins that have been reported to play roles in skin cancer development such as thrombospondin, cathepsins, epidermal growth factor receptor, cell adhesion molecules, cadherins, integrins, tuberin, fibulin, TGFβ receptor, etc. We further investigated whether these tumor proteins could be detected in plasma from tumor bearing mice using isotope labeling and 2D-LC-MALDI-MS/MS. Two tumor glycoproteins, Tenascin-C and Arylsulfatase B, were identified and quantified successfully in plasma from tumor bearing mice. This result indicates that analysis of tumor associated proteins in tumors and plasma by method using glycopeptide capture, isotopic labeling, and mass spectrometry can be used as a discovery tool to identify candidate tumor proteins that may be detected in plasma. PMID:21072318

  16. Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum.

    PubMed

    Trombetta, E S; Helenius, A

    2000-03-20

    Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway. PMID:10725325

  17. Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens.

    PubMed

    Sheppard, Neil C; Brinckmann, Sarah A; Gartlan, Kate H; Puthia, Manoj; Svanborg, Catharina; Krashias, George; Eisenbarth, Stephanie C; Flavell, Richard A; Sattentau, Quentin J; Wegmann, Frank

    2014-10-01

    Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens. PMID:24844701

  18. Hantavirus Gc glycoprotein: evidence for a class II fusion protein.

    PubMed

    Tischler, Nicole D; Gonzalez, Angel; Perez-Acle, Tomas; Rosemblatt, Mario; Valenzuela, Pablo D T

    2005-11-01

    Hantavirus cell entry is promoted by its envelope glycoproteins, Gn and Gc, through cell attachment and by fusion between viral and endosomal membranes at low pH. However, the role of Gn and Gc in receptor binding and cell fusion has not yet been defined. In this work, a sequence presenting characteristics similar to those of class II fusion peptides (FPs) of alphavirus E1 and flavivirus E proteins is identified within the hantavirus Gc glycoprotein. A three-dimensional comparative molecular model based on crystallographic data of tick-borne encephalitis virus E protein is proposed for the Andes virus (ANDV) Gc ectodomain, which supports a feasible class II fusion-protein fold. In vitro experimental evidence is provided for the binding activity of the ANDV FP candidate to artificial membranes, as demonstrated by fluorescence anisotropy assays. Taken together, these results support the hypothesis that the Gc glycoprotein of hantaviruses and of other members of the family Bunyaviridae directs the viral fusion activity and that it may be classified as a class II viral fusion protein.

  19. A double responsive smart upconversion fluorescence sensing material for glycoprotein.

    PubMed

    Guo, Ting; Deng, Qiliang; Fang, Guozhen; Yun, Yaguang; Hu, Yongjin; Wang, Shuo

    2016-11-15

    A novel strategy was developed to prepare double responsive smart upconversion fluorescence material for highly specific enrichment and sensing of glycoprotein. The novel double responsive smart sensing material was synthesized by choosing Horse radish peroxidase (HRP) as modal protein, the grapheme oxide (GO) as support material, upconversion nanoparticles (UCNPs) as fluorescence signal reporter, N-isopropyl acrylamide (NIPAAM) and 4-vinylphenylboronic acid (VPBA) as functional monomers. The structure and component of smart sensing material was investigated by transmission electron microscopy (TEM), Scanning electron microscopy (SEM), X-ray photoelectron spectroscopic (XPS) and Fourier transform infrared (FTIR), respectively. These results illustrated the smart sensing material was prepared successfully. The recognition characterizations of smart sensing material were evaluated, and results showed that the fluorescence intensity of smart sensing material was reduced gradually, as the concentration of protein increased, and the smart sensing material showed selective recognition for HRP among other proteins. Furthermore, the recognition ability of the smart sensing material for glycoprotein was regulated by controlling the pH value and temperature. Therefore, this strategy opens up new way to construct smart material for detection of glycoprotein. PMID:27236725

  20. Identification and expression of a human cytomegalovirus early glycoprotein.

    PubMed Central

    Chang, C P; Vesole, D H; Nelson, J; Oldstone, M B; Stinski, M F

    1989-01-01

    A human cytomegalovirus early gene which possesses three temporally regulated promoters is located in the large unique component of the viral genome between 0.054 and 0.064 map units (C.-P. Chang, C.L. Malone, and M.F. Stinski, J. Virol. 63:281-290, 1989). This gene contains a major open reading frame (ORF) located 233 bases downstream of the cap site of an early unspliced RNA. The major ORF predicts a polypeptide of 17 kilodaltons (kDa) which contains a glycoproteinlike signal and anchor domains as well as potential N-glycosylation sites. Antisera were prepared against synthetic peptides derived from amino acid sequences within the major ORF. The antisera detected a viral glycoprotein of 48 kDa in infected cells and recognized the in vitro-translated 17-kDa protein early-gene product. The viral glycoprotein, designated gp48, was modified by N-linked glycans and possibly O-linked glycans. The synthesis of gp48 occurred in the absence of viral DNA replication but accumulated to the highest levels at late times after infection. Since gp48 was found in the virion, it is considered an early structural glycoprotein. Images PMID:2545908

  1. Glycoproteins That Exhibit Extensive Size Polymorphisms in Dictyostelium Discoideum

    PubMed Central

    Smith, E.; Gooley, A. A.; Hudson, G. C.; Williams, K. L.

    1989-01-01

    Electrophoretic variants which arise from amino acid substitutions, leading to charge differences between proteins are ubiquitous and have been used extensively for genetic analysis. Less well documented are polymorphisms in the size of proteins. Here we report that a group of glycoproteins, which share a common carbohydrate epitope, vary in size in different isolates of the cellular slime mould, Dictyostelium discoideum. One of these proteins, PsA, a developmentally regulated prespore-specific surface glycoprotein, has previously been shown to exist in three size forms due to allelic variation at the pspA locus on linkage group I. In this report, a second glycoprotein, PsB, which is also prespore specific but found inside prespore cells, is studied. PsB maps to linkage group II and exhibits at least four different sizes in the isolates examined. We propose that the size polymorphisms are the product of allelic variation at the pspB locus, due to differences in the number of repeat units. PMID:2731733

  2. A double responsive smart upconversion fluorescence sensing material for glycoprotein.

    PubMed

    Guo, Ting; Deng, Qiliang; Fang, Guozhen; Yun, Yaguang; Hu, Yongjin; Wang, Shuo

    2016-11-15

    A novel strategy was developed to prepare double responsive smart upconversion fluorescence material for highly specific enrichment and sensing of glycoprotein. The novel double responsive smart sensing material was synthesized by choosing Horse radish peroxidase (HRP) as modal protein, the grapheme oxide (GO) as support material, upconversion nanoparticles (UCNPs) as fluorescence signal reporter, N-isopropyl acrylamide (NIPAAM) and 4-vinylphenylboronic acid (VPBA) as functional monomers. The structure and component of smart sensing material was investigated by transmission electron microscopy (TEM), Scanning electron microscopy (SEM), X-ray photoelectron spectroscopic (XPS) and Fourier transform infrared (FTIR), respectively. These results illustrated the smart sensing material was prepared successfully. The recognition characterizations of smart sensing material were evaluated, and results showed that the fluorescence intensity of smart sensing material was reduced gradually, as the concentration of protein increased, and the smart sensing material showed selective recognition for HRP among other proteins. Furthermore, the recognition ability of the smart sensing material for glycoprotein was regulated by controlling the pH value and temperature. Therefore, this strategy opens up new way to construct smart material for detection of glycoprotein.

  3. Requirements within the Ebola Viral Glycoprotein for Tetherin Antagonism.

    PubMed

    Vande Burgt, Nathan H; Kaletsky, Rachel L; Bates, Paul

    2015-10-01

    Tetherin is an interferon-induced, intrinsic cellular response factor that blocks release of numerous viruses, including Ebola virus, from infected cells. As with many viruses targeted by host factors, Ebola virus employs a tetherin antagonist, the viral glycoprotein (EboGP), to counteract restriction and promote virus release. Unlike other tetherin antagonists such as HIV-1 Vpu or KSHV K5, the features within EboGP needed to overcome tetherin are not well characterized. Here, we describe sequences within the EboGP ectodomain and membrane spanning domain (msd) as necessary to relieve tetherin restriction of viral particle budding. Fusing the EboGP msd to a normally secreted form of the glycoprotein effectively promotes Ebola virus particle release. Cellular protein or lipid anchors could not substitute for the EboGP msd. The requirement for the EboGP msd was not specific for filovirus budding, as similar results were seen with HIV particles. Furthermore trafficking of chimeric proteins to budding sites did not correlate with an ability to counter tetherin. Additionally, we find that a glycoprotein construct, which mimics the cathepsin-activated species by proteolytic removal of the EboGP glycan cap and mucin domains, is unable to counteract tetherin. Combining these results suggests an important role for the EboGP glycan cap and msd in tetherin antagonism. PMID:26516900

  4. Identification of a glycoprotein produced by enterotoxigenic Escherichia coli.

    PubMed

    Lindenthal, C; Elsinghorst, E A

    1999-08-01

    Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for Tib

  5. Identification of a Glycoprotein Produced by Enterotoxigenic Escherichia coli

    PubMed Central

    Lindenthal, Christoph; Elsinghorst, Eric A.

    1999-01-01

    Enterotoxigenic Escherichia coli (ETEC) strain H10407 is capable of invading epithelial cell lines derived from the human ileocecum and colon in vitro. Two separate chromosomally encoded invasion loci (tia and tib) have been cloned from this strain. These loci direct nonadherent and noninvasive laboratory strains of E. coli to adhere to and invade cultured human intestinal epithelial cells. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane protein that is directly correlated with the adherence and invasion phenotypes. TibA is synthesized as a 100-kDa precursor (preTibA) that must be modified for biological activity. Outer membranes of recombinant E. coli expressing TibA or preTibA were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose. The presence of glycoproteins was detected by oxidization of carbohydrates with periodate and labeling with hydrazide-conjugated digoxigenin. Only TibA could be detected as a glycoprotein. Complementation experiments with tib deletion mutants of ETEC strain H10407 demonstrate that the TibA glycoprotein is expressed in H10407, that the entire tib locus is required for TibA synthesis, and that TibA is the only glycoprotein produced by H10407. Protease treatment of intact H10407 cells removes the carbohydrates on TibA, suggesting that they are surface exposed. TibA shows homology with AIDA-I from diffuse-adhering E. coli and with pertactin precursor from Bordetella pertussis. Both pertactin and AIDA-I are members of the autotransporter family of outer membrane proteins and are afimbrial adhesins that play an important role in the virulence of these organisms. Analysis of the predicted TibA amino acid sequence indicates that TibA is also an autotransporter. Analysis of the tib locus DNA sequence revealed an open reading frame with similarity to RfaQ, a glycosyltransferase. The product of this tib locus open reading frame is proposed to be responsible for Tib

  6. Characterization of a pseudorabies virus glycoprotein gene with homology to herpes simplex virus type 1 and type 2 glycoprotein C.

    PubMed Central

    Robbins, A K; Watson, R J; Whealy, M E; Hays, W W; Enquist, L W

    1986-01-01

    A pseudorabies virus (Becker strain) glycoprotein gene was located in the UL region at map position 0.40. The gene was identified by using open reading frame Escherichia coli plasmid expression vectors and specific antibody reagents. A 1.55-kilobase unspliced transcript from the gene was detected in pseudorabies virus-infected tissue culture cells. The DNA sequence revealed a single open reading frame of 1,437 base pairs encoding 479 amino acids. The predicted primary translation product has a molecular weight of 50,860 and contains features of a typical herpesvirus glycoprotein. An E. coli expression plasmid was constructed that contained essentially all of the open reading frame for this gene. Antibodies raised in rabbits against the protein expressed in bacteria by this plasmid immunoprecipitated pseudorabies virus-specific glycoproteins of 92,000 and 74,000 daltons from infected cell extracts. It is likely that these two forms represent different glycosylation states of the protein. Images PMID:3009851

  7. Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors

    SciTech Connect

    Koener, J.F.; Leong, J.A.C. )

    1990-01-01

    A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots.

  8. Isolation and characterization of calcium binding glycoproteins of cardiac sarcolemmal vesicles

    SciTech Connect

    Michalak, M.; Fliegel, L.; Wlasichuk, K. )

    1990-04-05

    Two major Ca2(+)-binding glycoproteins Mr 120,000 and 100,000 were isolated from 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonic acid -solubilized bovine heart sarcolemma membrane. Peroxidase-conjugated concanavalin A and wheat germ agglutinin lectins bind strongly to the isolated 120- and 100-kDa glycoproteins. Treatment with endoglycosidase F resulted in conversion of the 120-kDa glycoprotein to a form migrating at about 97 kDa. Treatment of the 100-kDa band with endoglycosidase F produced form of about 80 kDa. Endoglycosidase H digestion removes only 5% of the mass of both glycoproteins. the carbohydrate structure of both glycoproteins, is therefore, predicted to be at least 75% complex structure and 25% high mannose or hybrid structure. The 120- and 100-kDa glycoproteins are the major Ca2(+)-binding proteins in the sarcolemma membranes. Intact and endoglycosidase-treated glycoproteins bind 45Ca2+ as analyzed by a 45Ca2+ overlay technique. Using polyclonal antibodies, the 120- and 100-kDa glycoproteins were identified in muscle plasma membranes (ventricles, atria, and uterus smooth muscle). They were, however, not present in non-muscle tissues such as pancreas, liver, and kidney. The 120- and 100-kDa glycoproteins appear to be homologous molecules as judged by their similar V8 protease peptide maps, cross-reactivity with polyclonal antibody, and other physicochemical properties.

  9. Recent advances in glycoprotein production for structural biology: toward tailored design of glycoforms.

    PubMed

    Kamiya, Yukiko; Satoh, Tadashi; Kato, Koichi

    2014-06-01

    Because of the complexity, heterogeneity, and flexibility of the glycans, the structural analysis of glycoproteins has been eschewed until recently, with a few prominent exceptions. This aversion may have branded structural biologists as glycophobics. However, recent technological advancements in glycoprotein expression systems, employing genetically engineered production vehicles derived from mammalian, insect, yeast, and even bacterial cells, have yielded encouraging breakthroughs. The major advance is the active control of glycoform expression of target glycoproteins based on the genetic manipulation of glycan biogenetic pathways, which was previously overlooked, abolished, or considered unmanageable. Moreover, synthetic and/or chemoenzymatic approaches now enable the preparation of glycoproteins with uniform glycoforms designed in a tailored fashion.

  10. Mannostatin A, a new glycoprotein-processing inhibitor

    SciTech Connect

    Tropea, J.E.; Kaushal, G.P.; Pastuszak, I.; Mitchell, M.; Elbein, A.D. ); Aoyagi, Takaaki ); Molyneux, R.J. )

    1990-10-01

    Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal {alpha}-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward {alpha}- and {beta}-glucosidase and galactosidase as well as {beta}-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal {alpha}-mannosidases, with estimated IC{sub 50}'s of 70 nM, 450 nM, and 160 nM, respectively. The type of inhibition was competitive in nature. This compound also proved to be an effective competitive inhibitor of the glycoprotein-processing enzyme mannosidase II (IC{sub 50} of about 10-15 nM with p-nitrophenyl {alpha}-D-mannopyranoside as substrate, and about 90 nM with ({sup 3}H)mannose-labeled GlcNAc-Man{sub 5}GlcNAc as substrate). However, it was virtually inactive toward mannosidase I. The N-acetylated derivative of mannostatin A had no inhibitory activity. In cell culture studies, mannostatin A also proved to be a potent inhibitor of glycoprotein processing. Thus, in influenza virus infected Madin Darby canine kidney (MDCK) cells, mannostatin A blocked the normal formation of complex types of oligosaccharides on the viral glycoproteins and caused the accumulation of hybrid types of oligosaccharides. This observation is in keeping with other data which indicate that the site of action of mannostatin A is mannosidase II. Thus, mannostatin A represents the first nonalkaloidal processing inhibitor and adds to the growing list of chemical structures that can have important biological activity.

  11. (Hydroxyproline-rich glycoproteins of the plant cell wall)

    SciTech Connect

    Varner, J.E.

    1990-01-01

    We are studying the chemistry and architecture of plant cells walls, the extracellular matrices that taken together shape the plant and provide mechanical support for the plant. Cell walls are dynamic structures that regulate, or are the site of, many physiological processes, in addition to being the cells' first line of defense against invading pathogens. In the past year we have examined the role of the cell wall enzyme ascorbic acid oxidase as related to the structure of the wall and its possible interactions with hydroxyproline-rich glycoproteins of the wall.

  12. HIV Entry and Envelope Glycoprotein-mediated Fusion*

    PubMed Central

    Blumenthal, Robert; Durell, Stewart; Viard, Mathias

    2012-01-01

    HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process. PMID:23043104

  13. Ebolavirus Glycoprotein Directs Fusion through NPC1+ Endolysosomes

    PubMed Central

    Simmons, James A.; D'Souza, Ryan S.; Ruas, Margarida; Galione, Antony; Casanova, James E.

    2015-01-01

    Ebolavirus, a deadly hemorrhagic fever virus, was thought to enter cells through endolysosomes harboring its glycoprotein receptor, Niemann-Pick C1. However, an alternate model was recently proposed in which ebolavirus enters through a later NPC1-negative endosome that contains two-pore Ca2+ channel 2 (TPC2), a newly identified ebolavirus entry factor. Here, using live cell imaging, we obtained evidence that in contrast to the new model, ebolavirus enters cells through endolysosomes that contain both NPC1 and TPC2. PMID:26468524

  14. Glycoprotein import: a common feature of complex plastids?

    PubMed

    Peschke, Madeleine; Hempel, Franziska

    2013-10-01

    Complex plastids evolved by secondary endosymbiosis and are, in contrast to primary plastids, surrounded by 3 or 4 envelope membranes. Recently, we provided evidence that in diatoms proteins exist that get N-glycosylated during transport across the outermost membrane of the complex plastid. This gives rise to unique questions on the transport mechanisms of these bulky proteins, which get transported across up to 3 further membranes into the plastid stroma. Here we discuss our results in an evolutionary context and speculate about the existence of plastidal glycoproteins in other organisms with complex plastids.

  15. Glycoprotein screening in colorectal cancer based on differentially expressed Tn antigen.

    PubMed

    Wei, Hongyun; Cheng, Zongyong; Ouyang, Chunhui; Zhang, Yu; Hu, Yanyan; Chen, Shuijiao; Wang, Chunlian; Lu, Fanggen; Zhang, Jie; Wang, Yongjun; Liu, Xiaowei

    2016-09-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide, and the identification of new biomarkers for CRC is valuable for its diagnosis and treatment. We aimed to screen differentially expressed glycoproteins (especially O-glycoproteins) and to identify diagnostic or therapeutic candidates for colorectal cancer (CRC) based on different Tn antigen expression levels. Fresh cancer tissues and adjacent healthy tissues were obtained from CRC patients and classified into three groups based on their Tn antigen expression: CRC with negative Tn expression (CRC Tn‑), CRC with positive Tn expression (CRC Tn+) and normal control without Tn expression (NC). Protein extractions were separated and identified by iTRAQ technology. Glycoproteins and O-glycoproteins were selected using UniProt and DAVID. Deep bioinformatic analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KO), was used to annotate this O-glycoprotein interaction network. Subsequently, two O‑glycoproteins were verified by western blotting and immunohistochemistry in either LS174T cells or CRC tissues. We found that 330 differentially expressed proteins were identified by iTRAQ between CRC Tn‑ and NC tissues, 317 between CRC Tn+ and NC tissues, and 316 between CRC Tn‑ and Tn+ tissues. Of the 316 proteins, 55 glycoproteins and 19 O‑glycoproteins were identified and analyzed via deep informatics. Namely, different Tn antigen expression levels in CRC led to differential protein expression patterns, especially for glycoproteins and O‑glycoproteins. Decorin and SORBS1, two representative functional O-glycoproteins, were significantly downregulated in the CRC Tn+ tissues compared with the level in the CRC Tn‑ or NC tissues. Based on this deep bioinformatic analysis, Decorin and SORBS1 are hypothesized to be involved in the TGF‑β and PPAR‑γ signaling pathways, respectively. PMID:27432485

  16. Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

    PubMed Central

    Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.

    2014-01-01

    ABSTRACT The rodent arenavirus glycoprotein complex encodes a stable signal peptide (SSP) that is an essential structural component of mature virions. The SSP, GP1, and GP2 subunits of the trimeric glycoprotein complex noncovalently interact to stud the surface of virions and initiate arenavirus infectivity. Nascent glycoprotein production undergoes two proteolytic cleavage events: first within the endoplasmic reticulum (ER) to cleave SSP from the remaining precursor GP1/2 (glycoprotein complex [GPC]) glycoprotein and second within the Golgi stacks by the cellular SKI-1/S1P for GP1/2 processing to yield GP1 and GP2 subunits. Cleaved SSP is not degraded but retained as an essential glycoprotein subunit. Here, we defined functions of the 58-amino-acid lymphocytic choriomeningitis virus (LCMV) SSP in regard to glycoprotein complex processing and maturation. Using molecular biology techniques, confocal microscopy, and flow cytometry, we detected SSP at the plasma membrane of transfected cells. Further, we identified a sorting signal (FLLL) near the carboxyl terminus of SSP that is required for glycoprotein maturation and trafficking. In the absence of SSP, the glycoprotein accumulated within the ER and was unable to undergo processing by SKI-1/S1P. Mutation of this highly conserved FLLL motif showed impaired glycoprotein processing and secretory pathway trafficking, as well as defective surface expression and pH-dependent membrane fusion. Immunoprecipitation of SSP confirmed an interaction between the signal peptide and the GP2 subunit; however, mutations within this FLLL motif disrupted the association of the GP1 subunit with the remaining glycoprotein complex. PMID:25352624

  17. Disulfide Bonds in Hepatitis C Virus Glycoprotein E1 Control the Assembly and Entry Functions of E2 Glycoprotein

    PubMed Central

    Wahid, Ahmed; Helle, François; Descamps, Véronique; Duverlie, Gilles; Penin, François

    2013-01-01

    Class II membrane fusion proteins have been described in viruses in which the envelope proteins are derived from a precursor polyprotein containing two transmembrane glycoproteins arranged in tandem. Although the second protein, which carries the membrane fusion function, is in general well characterized, the companion protein, which is a protein chaperone for the folding of the fusion protein, is less well characterized for some viruses, like hepatitis C virus (HCV). To investigate the role of the class II companion glycoprotein E1 of HCV, we chose to target conserved cysteine residues in the protein, and we systematically mutated them in a full-length infectious HCV clone by reverse genetics. All the mutants were infectious, albeit with lower titers than the wild-type virus. The reduced infectivity was in part due to a decrease in viral assembly, as revealed by measurement of intracellular infectivity and by quantification of core protein released from cells transfected with mutant genomes. Analyses of mutated proteins did not show any major defect in folding. However, the mutations reduced virus stability, and they could also affect the density of infectious viral particles. Mutant viruses also showed a defect in cell-to-cell transmission. Finally, our data indicate that HCV glycoprotein E1 can also affect the fusion protein E2 by modulating its recognition by the cellular coreceptor CD81. Therefore, in the context of HCV, our data identify an additional function of a class II companion protein as a molecule that can control the binding capacity of the fusion protein. PMID:23175356

  18. The relationship between glycan structures and expression levels of an endoplasmic reticulum-resident glycoprotein, UDP-glucose: Glycoprotein glucosyltransferase 1.

    PubMed

    Daikoku, Shusaku; Seko, Akira; Son, Sang-Hyun; Suzuki, Katsuhiko; Ito, Yukishige; Kanie, Osamu

    2015-06-19

    In this article, we report a relationship between glycan structures and expression levels of a recombinant ER-resident glycoprotein, uridine 5'-diphosphate-glucose: glycoprotein glucosyltransferase (UGGT1). The function of glycan structures attached to a glycoprotein is actively studied; however, the glycan structures of recombinant, and not endogenous, glycoproteins have not been examined. In this study, we indicate a relationship between the glycan structure and the level of protein expression. Expression levels were controlled utilizing a series of vectors (pFN21K, pFN22K, pFN23K, and pFN24K HaloTag CMV Flexi Vectors). Qualitative and semi-quantitative confirmation of glycan structures was achieved with tandem mass spectrometry. The results of this study indicate that glycan structures are similar to endogenous glycans at low expression levels.

  19. Chemosensitization potential of P-glycoprotein inhibitors in malaria parasites.

    PubMed

    Alcantara, Laura M; Kim, Junwon; Moraes, Carolina B; Franco, Caio H; Franzoi, Kathrin D; Lee, Sukjun; Freitas-Junior, Lucio H; Ayong, Lawrence S

    2013-06-01

    Members of the ATP-binding cassette (ABC)-type transporter superfamily have been implicated in multidrug resistance in malaria, and various mechanistic models have been postulated to explain their interaction with diverse antimalarial drugs. To gain insight into the pharmacological benefits of inhibiting ABC-type transporters in malaria chemotherapy, we investigated the in vitro chemosensitization potential of various P-glycoprotein inhibitors. A fluorescent chloroquine derivative was synthesized and used to assess the efflux dynamics of chloroquine in MDR and wild type Plasmodium falciparum parasites. This novel BODIPY-based probe accumulated in the digestive vacuole (DV) of CQ-sensitive parasites but less so in MDR cells. Pre-exposure of the MDR parasites to non-cytocidal concentrations of unlabeled chloroquine resulted in a diffused cytoplasmic retention of the probe whereas a similar treatment with the CQR-reversing agent, chlorpheniramine, resulted in DV accumulation. A diffused cytoplasmic distribution of the probe was also obtained following treatment with the P-gp specific inhibitors zosuquidar and tariquidar, whereas treatments with the tyrosine kinase inhibitors gefitinib or imatinib produced a partial accumulation within the DV. Isobologram analyses of the interactions between these inhibitors and the antimalarial drugs chloroquine, mefloquine, and artemisinin revealed distinct patterns of drug synergism, additivity and antagonism. Taken together, the data indicate that competitive tyrosine kinase and noncompetitive P-glycoprotein ATPase-specific inhibitors represent two new classes of chemosensitizing agents in malaria parasites, but caution against the indiscriminate use of these agents in antimalarial drug combinations.

  20. Isolation of oligomannose-type glycans from bean glycoproteins.

    PubMed

    Lu, Y; Ye, J; Wold, F

    1993-02-15

    We have isolated individual oligosaccharyl-asparagine derivatives from the total soluble glycoproteins from kidney beens (Phaseolus vulgaris) and from lima beans (Phaseolus limensis). The protein/glycoprotein mixture was digested exhaustively by pronase, and the glycan-containing fractions were separated from free amino acids and peptides by gel filtration. The oligosaccharyl-asparagine derivatives were finally fractionated on Dowex 50 (C. C. Huang, H.E. Meyer, and R. Montgomery, Carbohydr. Res. 13, 127-137, 1970), and the individual fractions were characterized by mass spectrometry, NMR, and ion exchange chromatography. With the procedures described, only oligomannose derivatives were obtained from the beans. In the case of kidney beans, six different derivatives were observed and characterized, Man9GlcNAc2Asn, two positional isomers of Man8GlcNAc2Asn, two positional isomers of Man7GlcNAc2Asn, and Man6GlcNAc2Asn. Under identical conditions the lima beans yielded primarily the Man9GlcNAc2Asn derivative along with a small amount of the two Man8GlcNAc2Asn derivatives. The oligomannose structures can be isolated in reasonable quantities (2-20 mg) from about 200 g of dry beans. PMID:8465965

  1. Characterization of immunomodulatory activities of honey glycoproteins and glycopeptides.

    PubMed

    Mesaik, M Ahmed; Dastagir, Nida; Uddin, Nazim; Rehman, Khalid; Azim, M Kamran

    2015-01-14

    Recent evidence suggests an important role for natural honey in modulating immune response. To identify active components responsible, this study investigated the immunomodulatory properties of glycoproteins and glycopeptides fractionated from Ziziphus honey. Honey proteins/peptides were fractionated by size exclusion chromatography into five peaks with molecular masses in the range of 2-450 kDa. The fractionated proteins exhibited potent, concentration-dependent inhibition of reactive oxygen species production in zymosan-activated human neutrophils (IC50 = 6-14 ng/mL) and murine macrophages (IC50 = 2-9 ng/mL). Honey proteins significantly suppressed the nitric oxide production by LPS-activated murine macrophages (IC50 = 96-450 ng/mL). Moreover, honey proteins inhibited the phagocytosis latex bead macrophages. The production of pro-inflammatory cytokines IL-1β and TNF-α by human monocytic cell line in the presence of honey proteins was analyzed. Honey proteins did not affect the production of IL-1β; however, TNF-α production was significantly suppressed. These findings indicated that honey glycoproteins and glycopeptides significantly interfere with molecules of the innate immune system.

  2. Stationary phases for the enrichment of glycoproteins and glycopeptides.

    PubMed

    Huang, Bao-Yu; Yang, Chun-Kai; Liu, Ching-Piao; Liu, Chuen-Ying

    2014-08-01

    The analysis of protein glycosylation is important for biomedical and biopharmaceutical research. Recent advances in LC-MS analysis have enabled the identification of glycosylation sites, the characterisation of glycan structures and the identification and quantification of glycoproteins and glycopeptides. However, this type of analysis remains challenging due to the low abundance of glycopeptides in complex protein digests, the microheterogeneity at glycosylation sites, ion suppression effects and the competition for ionisation by co-eluting peptides. Specific sample preparation is necessary for comprehensive and site-specific glycosylation analyses using MS. Therefore, researchers continue to pursue new columns to broaden their applications. The current manuscript covers recent literature published from 2008 to 2013. The stationary phases containing various chemical bonding methods or ligands immobilisation strategies on solid supports that selectively enrich N-linked or sialylated N-glycopeptides are categorised with either physical or chemical modes of binding. These categories include lectin affinity, hydrophilic interactions, boronate affinity, titanium dioxide affinity, hydrazide chemistry and other separation techniques. This review should aid in better understanding the syntheses and physicochemical properties of each type of stationary phases for enriching glycoproteins and glycopeptides. PMID:24729282

  3. Interaction of native and asialo rat sublingual glycoproteins with lectins.

    PubMed

    Wu, A M; Herp, A; Song, S C; Wu, J H; Chang, K S

    1995-01-01

    The binding properties of the rat sublingual glycoprotein (RSL) and its asialo product with lectins were characterized by quantitative precipitin(QPA) and precipitin inhibition(QPIA) assays. Among twenty lectins tested for QPA, native RSL reacted well only with Artocarpus integrifolia (jacalin), but weakly or not at all with the other lectins. However, its asialo product (asialo-RSL) reacted strongly with many Gal and GalNAc specific lectins-it bound best to three of the GalNAc alpha 1-->Ser/Thr (Tn) and/or Gal beta 1-->4GlcNAc (II) active lectins [jacalin, Wistaria floribunda and Ricinus communis agglutinins] and completely precipitated each of these three lectins. Asialo-RSL also reacted well with Abrus precatorius, Glycine max, Bauhinia purpurea alba, and Maclura pomifera agglutinins, and abrin-a, but not with Arachis hypogeae and Dolichos biflorus agglutinins. The interaction between asialo-RSL and lectins were inhibited by either Gal beta 1-->4GlcNAc, p-NO2-phenyl alpha-GalNAc or both. The mapping of the precipitation and inhibition profiles leads to the conclusion that the asialo rat sublingual glycoprotein provides important ligands for II (Gal beta 1-->4GlcNAc beta 1-->) and Tn (GalNAc alpha 1-->Ser/Thr) active lectins.

  4. The Lyssavirus glycoprotein: A key to cross-immunity.

    PubMed

    Buthelezi, Sindisiwe G; Dirr, Heini W; Chakauya, Ereck; Chikwamba, Rachel; Martens, Lennart; Tsekoa, Tsepo L; Stoychev, Stoyan H; Vandermarliere, Elien

    2016-11-01

    Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. PMID:27614701

  5. An altered platelet granule glycoprotein in patients with essential thrombocythemia.

    PubMed Central

    Booth, W J; Berndt, M C; Castaldi, P A

    1984-01-01

    The protein profiles of washed platelets from nine patients with essential thrombocythemia were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In four patients, an additional protein band (reduced Mr of 170,000) was clearly identified in both unstimulated platelet preparations and thrombin-released supernatant fractions. This band was also evident, though to a lesser extent, in three more patients, but it could not be located in the two remaining patients nor in any of ten controls. Subsequent characterization of the 170,000 reduced protein in one patient indicated that (a) it was glycosylated, as judged by periodic acid-Schiff staining, and (b) that native protein was a disulfide-linked multimer (possibly trimeric), which (c) partially bound to the activated platelet plasma membrane in the presence of calcium, and (d) was immune precipitated by anti-glycoprotein G antisera. The combined evidence is consistent with the 170,000 reduced protein being a modified form of the normal subunit of the platelet alpha-granule constituent, glycoprotein G (also termed thrombospondin and thrombin-sensitive protein). Images PMID:6365970

  6. The variable surface glycoproteins of Trypanosoma equiperdum are phosphorylated.

    PubMed Central

    Baltz, T; Giroud, C; Baltz, D; Duvillier, G; Degand, P; Demaille, J; Pautrizel, R

    1982-01-01

    The phosphoproteins from three Trypanosoma equiperdum variants were studied by labelling the parasites in vivo with 32P. Phosphoprotein analysis reveals the presence of a 58 000 mol. wt. phosphoprotein ( pp58 ) which is absent when live trypanosomes are pre-treated with proteinase K under conditions where only the surface coat containing the variable surface glycoprotein (VSG) is removed. Immunological and fingerprint analysis on labelled pp58 , purified from these variants by affinity chromatography on Concanavalin A-Sepharose, clearly identify this component as the VSG. Furthermore, the VSGs seem to be phosphorylated to the extent of 1 mol phosphate per mol glycoprotein. The phosphorylated region is located in the extreme C-terminal region representing approximately 10% of the total molecule. The phosphorylated residue is not an aliphatic or aromatic ester of serine, threonine, or tyrosine, nor an acyl phosphate involving an aspartyl or glutamyl residue, nor phosphohistidine. The evidence that VSGs are phosphorylated could have considerable implications for the transfer and function of these structures. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6821334

  7. Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens.

    PubMed

    Wegmann, Frank; Gartlan, Kate H; Harandi, Ali M; Brinckmann, Sarah A; Coccia, Margherita; Hillson, William R; Kok, Wai Ling; Cole, Suzanne; Ho, Ling-Pei; Lambe, Teresa; Puthia, Manoj; Svanborg, Catharina; Scherer, Erin M; Krashias, George; Williams, Adam; Blattman, Joseph N; Greenberg, Philip D; Flavell, Richard A; Moghaddam, Amin E; Sheppard, Neil C; Sattentau, Quentin J

    2012-09-01

    Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use. PMID:22922673

  8. [Immune efficacy of rabies virus glycoprotein expressed by baculovirus vector].

    PubMed

    Chen, Qi; Zhang, Shou-Feng; Liu, Ye; Fu, Yun-Hong; Sun, Cheng-Long; Yang, Yang; Gong, Ting; Song, Fei-Fei; Hu, Rong-Liang

    2012-09-01

    To construct a recombinant baculovirus expressing glycoprotein (GP) of RV SRV9 strain and test the immunological efficacy in mice, open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector Bacmid to construct the recombinant shuttle plasmid Bacmid-G and transfection was performed into S f9 cells with the recombinant shuttle plasmid. CPE appeared in cell cultures was identified by electronmicroscopy. Western-blot, IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products. Our results showed a recombinant baculovirus expressing GP protein of rabies virus SRV9 was obtained. The expression products possessed a favorable immunogenicity and fall immunized mice could develop 100% protective level of anti-rabies neutralizing antibody. In conclusion, The SRV9 glycoprotein expressed by the recombinant baculovirus in this study had good immunogenicity and could induce anti-rabies neutralizing antibody, which laid the foundation of further development of rabies subunit vaccine.

  9. Glycoprotein Gene Sequence Variation in Rhesus Monkey Rhadinovirus

    PubMed Central

    Shin, Young C.; Jones, Leandro R.; Manrique, Julieta; Lauer, William; Carville, Angela; Mansfield, Keith G.; Desrosiers, Ronald C.

    2010-01-01

    Gene sequences for seven glycoproteins from 20 independent isolates of rhesus monkey rhadinovirus (RRV) and of the corresponding seven glycoprotein genes from nine strains of the Kaposi’s sarcoma-associated herpesvirus (KSHV) were obtained and analyzed. Phylogenetic analysis revealed two discrete groupings of RRV gH sequences, two discrete groupings of RRV gL sequences and two discrete groupings of RRV gB sequences. We called these phylogenetic groupings gHa, gHb, gLa, gLb, gBa and gBb. gHa was always paired with gLa and gHb was always paired with gLb for any individual RRV isolate. Since gH and gL are known to be interacting partners, these results suggest the need of matching sequence types for function of these cooperating proteins. gB phylogenetic grouping was not associated with gH/gL phylogenetic grouping. Our results demonstrate two distinct, distantly-related phylogenetic groupings of gH and gL of RRV despite a remarkable degree of sequence conservation within each individual phylogenetic group. PMID:20172576

  10. Weak anion exchange chromatographic profiling of glycoprotein isoforms on a polymer monolithic capillary.

    PubMed

    Liu, Jing; Ren, Lianbing; Liu, Yunchun; Li, Hengye; Liu, Zhen

    2012-03-01

    High resolution separation of intact glycoproteins, which is essential for many aspects such as finger-print profiling, represents a great challenge because one glycoprotein can exhibit many isoforms with close physicochemical properties. Monolithic columns are important separation media for the separation of intact proteins due to its significant advantages such as easy preparation, high column efficiency and high permeability. However, there are few reports on high resolution profiling of intact glycoproteins. Herein, we presented a polymeric weak anion exchange (WAX) monolithic capillary for high resolution separation of glycoprotein isoforms. A base monolith was first prepared through ring-opening polymerization between tris(2,3-epoxypropyl)isocyanurate and tri(2-aminoethyl), and then modified through reacting with ammonia aqueous solution to convert the unreacted epoxide moieties into primary amino groups. The prepared monolithic capillary was characterized in terms of morphology, pore size, hydrophilicity and reproducibility. The obtained WAX monolithic capillary exhibited desired through-pores and mesopore size, stable skeleton and hydrophilic nature. The performance of the capillary was evaluated using several typical glycoproteins such as α(1)-acid glycoprotein (AGP) as mode analytes. Effects of the experimental parameters on the glycoform resolution were investigated. Under the optimized separation conditions, the tested glycoproteins were all resolved into distinct glycoforms. A comparative investigation with capillary zone electrophoresis (CZE) revealed that this WAX column provided better selectivity as more isoforms were observed, although the resolution of some glycoprotein isoforms decreased.

  11. Systemic alteration of cell-surface and secreted glycoprotein expression in malignant breast cancer cell lines.

    PubMed

    Timpe, Leslie C; Yen, Roger; Haste, Nicole V; Litsakos-Cheung, Christina; Yen, Ten-Yang; Macher, Bruce A

    2013-11-01

    Breast cancer cell lines express fewer transmembrane and secreted glycoproteins than nonmalignant ones. The objective of these experiments was to characterize the changes in the expression of several hundred glycoproteins quantitatively. Secreted and cell-surface glycoproteins were isolated using a glycoprotein capture protocol and then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared with those expressed by a nonmalignant set. The average number of spectral counts (proportional to relative protein abundance) and the total number of glycopeptides in the malignant samples were reduced to about two-thirds of the level in the nonmalignant samples. Most glycoproteins were expressed at a different level in the malignant samples, with nearly as many increasing as decreasing. The glycoproteins with reduced expression accounted for a larger change in spectral counts, and hence for the net loss of spectral counts in the malignant lines. Similar results were found when the glycoproteins were studied via identified glycosylation sites only, or through identified sites together with non-glycopeptides. The overall reduction is largely due to the loss of integrins, laminins and other proteins that form or interact with the basement membrane.

  12. Glycoprotein Biochemistry--Some Clinical Aspects of Interest to Biochemistry Students.

    ERIC Educational Resources Information Center

    Smith, Christopher A.; And Others

    1991-01-01

    Authors describe some clinical features of glycoprotein biochemistry, including recognition, selected blood glycoproteins, glycated proteins, histochemistry, and cancer. The material presented has largely been taught to medical laboratory students; however, it can be used to teach premedical students and pure biochemistry students. Includes two…

  13. Histochemical and structural analysis of mucous glycoprotein secreted by the gill of Mytilus edulis

    SciTech Connect

    Ahn, Hae-Young.

    1988-01-01

    Studies were carried out to characterized various mucous cells in the gill filament, to ascertain structural characteristics of the secreted mucous glycoproteins, and to determine the ability of the gill epithelium to incorporate ({sup 14}C)glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins. Using histochemical staining techniques, mucous cells containing neutral and acidic mucins were found in the lateral region, whereas mucous cells containing primarily neutral or sulfated mucins were found in the postlateral region. Serotonin, but not dopamine, stimulated the mucous secretion. In tissues pretreated with ({sup 14}C)glucosamine, the secreted glycoproteins contain incorporated radiolabel. Analysis by column chromatography using Bio-Gel P-2 and P-6 shows that the secretion contains two glycoprotein populations. Glycoprotein II has a molecular weight of 2.3 {times} 10{sup 4} daltons. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of glycoprotein I, about 70% of the radiolabel was removed from the protein. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contains N-acetylglucosamine (GluNAc), N-acetylgalactosamine (GalNAc), and galactose, fucose and mannose. Amino acid analysis shows that the glycoproteins are rich in serine, threonine and proline.

  14. Targeted entry via somatostatin receptors using a novel modified retrovirus glycoprotein that delivers genes at levels comparable to those of wild-type viral glycoproteins.

    PubMed

    Li, Fang; Ryu, Byoung Y; Krueger, Robin L; Heldt, Scott A; Albritton, Lorraine M

    2012-01-01

    Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.

  15. Loop-acting diuretics do not bind to Tamm-Horsfall urinary glycoprotein.

    PubMed

    Brunisholz, M C; Lynn, K L; Hunt, J S

    1987-09-01

    1. Binding between the radiolabelled loop-acting diuretics ([14C]frusemide, [14C]ethacrynic acid and [3H]bumetanide) and human Tamm-Horsfall glycoprotein or human serum albumin in vitro was evaluated by equilibrium dialysis. 2. The diuretic action and binding to urinary Tamm-Horsfall glycoprotein of the radiolabelled diuretics in vivo, after intravenous administration, were examined in rabbits. 3. In vitro, all three radiolabelled diuretics bound strongly to human serum albumin, but not to Tamm-Horsfall glycoprotein. 4. Radiolabelled frusemide and bumetanide, but not ethacrynic acid, caused a diuresis in rabbits, but no binding between the drugs and Tamm-Horsfall glycoprotein was seen in vivo. 5. Binding to Tamm-Horsfall glycoprotein does not appear to be an important mechanism in the action of loop diuretics.

  16. Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

    SciTech Connect

    Rayter, Z. ); McIlhinney, R. ); Gusterson, B. )

    1989-08-01

    Con A acceptor glycoproteins were analyzed by 2D-PAGE and {sup 125}I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21, was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.

  17. Separation of the bovine colostrum M-1 glycoprotein into two components

    PubMed Central

    Bezkorovainy, Anatoly; Grohlich, Dietmar

    1969-01-01

    1. Two glycoproteins were isolated from the M-1 acid glycoprotein fraction of bovine colostrum. 2. The lighter glycoprotein had a molecular weight of 7200, contained about 28·4% of carbohydrate, and had an absorption maximum at 275nm. The heavier glycoprotein had a molecular weight of 12000, contained 39·0% of carbohydrate, and had no absorption maxima in the 240–300nm. range of the spectrum. 3. The carbohydrate moiety of both glycoproteins was removable from the polypeptide moiety under the conditions of the β-elimination reaction. 4. Periodate oxidation experiments showed that sialic acid was linked to galactose in both proteins. ImagesFig. 3.Fig. 4. PMID:4311442

  18. Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants.

    PubMed

    Goh, John S Y; Chan, Kah Fai; Song, Zhiwei

    2015-01-01

    The degree of sialylation of therapeutic glycoproteins affects its circulatory half-life and efficacy because incompletely sialylated glycoproteins are cleared from circulation by asialoglycoprotein receptors present in the liver cells. Mammalian expression systems, often employed in the production of these glycoprotein drugs, produce heterogeneously sialylated products. Here, we describe how to produce highly sialylated glycoproteins using a Chinese hamster ovary (CHO) cell glycosylation mutant called CHO-gmt4 with human erythropoietin (EPO) as a model glycoprotein. The protocol describes how to isolate and characterize the CHO glycosylation mutants and how to assess the sialylation of the recombinant protein using isoelectric focusing (IEF). It further describes how to inactivate the dihydrofolate reductase (DHFR) gene in these cells using zinc finger nuclease (ZFN) technology to enable gene amplification and the generation of stable cell lines producing highly sialylated EPO.

  19. Demethoxycurcumin modulates human P-glycoprotein function via uncompetitive inhibition of ATPase hydrolysis activity.

    PubMed

    Teng, Yu-Ning; Hsieh, Yow-Wen; Hung, Chin-Chuan; Lin, Hui-Yi

    2015-01-28

    Curcuminoids are major components of Curcuma longa L., which is widely used as spice in food. This study aimed at identifying whether curcumin, demethoxycurcumin, and bisdemethoxycurcumin could modulate efflux function of human P-glycoprotein and be used as chemosensitizers in cancer treatments. Without altering P-glycoprotein expression levels and conformation, the purified curcuminoids significantly inhibited P-glycoprotein efflux function. In rhodamine 123 efflux and calcein-AM accumulation assays, demethoxycurcumin demonstrated the highest inhibition potency (inhibitory IC50 = 1.56 ± 0.13 μM) among the purified curcuminoids, as well as in the fold of reversal assays. Demethoxycurcumin inhibited P-glycoprotein-mediated ATP hydrolysis under concentrations of <1 μM and efficiently inhibited 200 μM verapamil-stimulated ATPase activity, indicating a high affinity of demethoxycurcumin for P-glycoprotein. These results suggested that demethoxycurcumin may be a potential additive natural product in combination with chemotherapeutic agents in drug-resistant cancers.

  20. Identification of glycoproteins containing specific glycans using a lectin-chemical method.

    PubMed

    Li, Yan; Shah, Punit; De Marzo, Angelo M; Van Eyk, Jennifer E; Li, Qianqian; Chan, Daniel W; Zhang, Hui

    2015-01-01

    Glycosylation is one of the most common protein modifications. Each glycoprotein can be glycosylated at multiple glycosites, and each glycosites can be modified by different glycans. Due to this heterogeneity of glycosylation, it has proven difficult to study the structure-function relationship of specific glycans and their affected glycoproteins. Here, we report a novel method for rapid and quantitative identification of glycoproteins containing specific glycans. Lectin affinity isolations are followed by chemical immobilization of the captured glycopeptides, allowing the identification of glycoproteins containing specific glycans by subsequent mass spectrometry. The application of the method should be useful to facilitate our understanding of how changes in glycan associate with diseases, and to discover novel glycoproteins with certain glycans that could serve as biomarkers or therapeutic targets.

  1. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

  2. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection. PMID:25271333

  3. Effect of reduced temperature on glycoprotein (Ig, HLA) processing and transport in lymphoid cells.

    PubMed

    Brand, M; Jansen, E; Ploegh, H L

    1985-07-01

    Secretion of Igs and surface expression of HLA antigens was examined in lymphoid cells as a function of temp. Upon reducing the temp from 37 to 20 degrees C a progressive decrease in the secretion of Ig and surface expression of HLA antigens was noted. When the status of the oligosaccharides present on these glycoproteins was examined, conversion of high-mannose [endo-beta-N-acetylglucosaminidase-(Endo H) sensitive] to complex-type (Endo H resistant) oligosaccharides diminished with decreasing temp. At no time was an accumulation of Endo H resistant glycoproteins seen intracellularly. These results show that the phenomenon observed for synthesis and intracellular transport of viral glycoproteins in epithelial cells at reduced temp, namely intracellular accumulation of viral glycoproteins carrying complex sugar moieties, does not necessarily apply to glycoprotein transport in lymphoid cells. A difference in subcellular organization of epithelial and lymphoid cells may be responsible for this discrepancy.

  4. Lectin-based analysis of fucosylated glycoproteins of human skim milk during 47 days of lactation.

    PubMed

    Lis-Kuberka, Jolanta; Kątnik-Prastowska, Iwona; Berghausen-Mazur, Marta; Orczyk-Pawiłowicz, Magdalena

    2015-12-01

    Glycoproteins of human milk are multifunctional molecules, and their fucosylated variants are potentially active molecules in immunological events ensuring breastfed infants optimal development and protection against infection diseases. The expression of fucosylated glycotopes may correspond to milk maturation stages. The relative amounts of fucosylated glycotopes of human skim milk glycoproteins over the course of lactation from the 2(nd) day to the 47(th) day were analyzed in colostrums, transitional and mature milk samples of 43 healthy mothers by lectin-blotting using α1-2-, α1-6-, and α1-3-fucose specific biotinylated Ulex europaeus (UEA), Lens culinaris (LCA), and Lotus tetragonolobus (LTA) lectins, respectively. The reactivities of UEA and LCA with the milk glycoproteins showed the highest expression of α1-2- and α1-6-fucosylated glycotopes on colostrum glycoproteins. The level of UEA-reactive glycoproteins from the beginning of lactation to the 14(th) day was high and relatively stable in contrast to LCA-reactive glycoproteins, the level of which significantly decreased from 2-3 to 7-8 days then remained almost unchanged until the 12(th)-14(th) days. Next, during the progression of lactation the reactivities with both lectins declined significantly. Eighty percent of α1-2- and/or α1-6-fucosylated glycoproteins showed a high negative correlation with milk maturation. In contrast, most of the analyzed milk glycoproteins were not recognized or weakly recognized by LTA and remained at a low unchanged level over lactation. Only a 30-kDa milk glycoprotein was evidently LTA-reactive, showing a negative correlation with milk maturation. The gradual decline of high expression of α1-2- and α1-6-, but not α1-3-, fucoses on human milk glycoproteins of healthy mothers over lactation was associated with milk maturation.

  5. Lectin-based analysis of fucosylated glycoproteins of human skim milk during 47 days of lactation.

    PubMed

    Lis-Kuberka, Jolanta; Kątnik-Prastowska, Iwona; Berghausen-Mazur, Marta; Orczyk-Pawiłowicz, Magdalena

    2015-12-01

    Glycoproteins of human milk are multifunctional molecules, and their fucosylated variants are potentially active molecules in immunological events ensuring breastfed infants optimal development and protection against infection diseases. The expression of fucosylated glycotopes may correspond to milk maturation stages. The relative amounts of fucosylated glycotopes of human skim milk glycoproteins over the course of lactation from the 2(nd) day to the 47(th) day were analyzed in colostrums, transitional and mature milk samples of 43 healthy mothers by lectin-blotting using α1-2-, α1-6-, and α1-3-fucose specific biotinylated Ulex europaeus (UEA), Lens culinaris (LCA), and Lotus tetragonolobus (LTA) lectins, respectively. The reactivities of UEA and LCA with the milk glycoproteins showed the highest expression of α1-2- and α1-6-fucosylated glycotopes on colostrum glycoproteins. The level of UEA-reactive glycoproteins from the beginning of lactation to the 14(th) day was high and relatively stable in contrast to LCA-reactive glycoproteins, the level of which significantly decreased from 2-3 to 7-8 days then remained almost unchanged until the 12(th)-14(th) days. Next, during the progression of lactation the reactivities with both lectins declined significantly. Eighty percent of α1-2- and/or α1-6-fucosylated glycoproteins showed a high negative correlation with milk maturation. In contrast, most of the analyzed milk glycoproteins were not recognized or weakly recognized by LTA and remained at a low unchanged level over lactation. Only a 30-kDa milk glycoprotein was evidently LTA-reactive, showing a negative correlation with milk maturation. The gradual decline of high expression of α1-2- and α1-6-, but not α1-3-, fucoses on human milk glycoproteins of healthy mothers over lactation was associated with milk maturation. PMID:26318738

  6. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

  7. Rapid intramolecular turnover of N-linked glycans in plasma membrane glycoproteins. Extension of intramolecular turnover to the core sugars in plasma membrane glycoproteins of hepatoma.

    PubMed

    Tauber, R; Park, C S; Becker, A; Geyer, R; Reutter, W

    1989-12-01

    Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl Acad. Sci. USA 80, 4026-4029]. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. Intramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with Mr of 85,000 (hgp85), 105,000 (hgp105), 115,000 (hgp115), 125,000 (hgp125), 175,000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin-A--Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgp105, hgp115 and hgp125 revealed the presence of N-linked oligosaccharides containing L-fucose, D-galactose, D-mannose and N-acetyl-D-glucosamine, but only of trace amounts of N-acetyl-D-galactosamine; hgp175 additionally contained significant amounts of N-acetyl-D-galactosamine, indicating the presence of both N- and O-linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N-linked oligosaccharides of hgp105, hgp115, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosaccharides of the high-mannose type. Half-lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse-chase experiments in vivo, using L-[3H]fucose as a marker of terminal sugars, D-[3H]mannose as marker of a core sugar and L-[3H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half-lives ranging over 41-90 h with a mean of 66 h. Compared to the

  8. Engineering yeast for producing human glycoproteins: where are we now?

    PubMed

    Laukens, Bram; De Visscher, Charlotte; Callewaert, Nico

    2015-01-01

    Yeast has advanced as an alternative for mammalian cell culture for the production of recombinant therapeutic glycoproteins. Engineered yeast strains not only allow to mimic the human N-glycosylation pathway but also specific types of human O-glycosylation. This is of great value for therapeutic protein production and indispensable to determine the structure-function relationships of glycans on recombinant proteins. However, as the technology matures, some limitations have come up that may hamper biomedical applications and must be considered to exploit the full potential of the unprecedented glycan homogeneity obtained on relevant biopharmaceuticals. In this special report, we focus on the recent developments in N- and O-glycosylation engineering in yeasts of industrial importance, to produce recombinant therapeutics with customized glycans.

  9. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    PubMed Central

    Liu, Bo

    2013-01-01

    Abstract Breastfeeding protects the neonate against pathogen infection. Major mechanisms of protection include human milk glycoconjugates functioning as soluble receptor mimetics that inhibit pathogen binding to the mucosal cell surface, prebiotic stimulation of gut colonization by favorable microbiota, immunomodulation, and as a substrate for bacterial fermentation products in the gut. Human milk proteins are predominantly glycosylated, and some biological functions of these human milk glycoproteins (HMGPs) have been reported. HMGPs range in size from 14 kDa to 2,000 kDa and include mucins, secretory immunoglobulin A, bile salt-stimulated lipase, lactoferrin, butyrophilin, lactadherin, leptin, and adiponectin. This review summarizes known biological roles of HMGPs that may contribute to the ability of human milk to protect neonates from disease. PMID:23697737

  10. Stereoselective Modulation of P-Glycoprotein by Chiral Small Molecules.

    PubMed

    Carocci, Alessia; Catalano, Alessia; Turi, Francesco; Lovece, Angelo; Cavalluzzi, Maria M; Bruno, Claudio; Colabufo, Nicola A; Contino, Marialessandra; Perrone, Maria G; Franchini, Carlo; Lentini, Giovanni

    2016-01-01

    Inhibition of drug efflux pumps such as P-glycoprotein (P-gp) is an approach toward combating multidrug resistance, which is a significant hurdle in current cancer treatments. To address this, N-substituted aryloxymethyl pyrrolidines were designed and synthesized in their homochiral forms in order to investigate the stereochemical requirements for the binding site of P-gp. Our study provides evidence that the chiral property of molecules could be a strategy for improving the capacity for interacting with P-gp, as the most active compounds of the series stereoselectively modulated this efflux pump. The naphthalene-1-yl analogue (R)-2-[(2,3-dichlorophenoxy)methyl]-1-(naphthalen-1-ylmethyl)pyrrolidine) [(R)-7 a] emerged foremost for its potency and stereoselectivity toward P-gp, with the S enantiomer being nearly inactive. The modulation of P-gp by (R)-7 a involved consumption of ATP, thus demonstrating that the compound behaves as a P-gp substrate.

  11. Small-angle scattering study of Aspergillus awamori glycoprotein glucoamylase

    NASA Astrophysics Data System (ADS)

    Schmidt, A. E.; Shvetsov, A. V.; Kuklin, A. I.; Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V.

    2016-01-01

    Glucoamylase from fungus Aspergillus awamori is glycoside hydrolase that catalyzes the hydrolysis of α-1,4- and α-1,6-glucosidic bonds in glucose polymers and oligomers. This glycoprotein consists of a catalytic domain and a starch-binding domain connected by an O-glycosylated polypeptide chain. The conformation of the linker, the relative arrangement of the domains, and the structure of the full-length enzyme are unknown. The structure of the recombinant glucoamylase GA1 was studied by molecular modelling and small-angle neutron scattering (SANS) methods. The experimental SANS data provide evidence that glucoamylase exists as a monomer in solution and contains a glycoside component, which makes a substantial contribution to the scattering. The model of full-length glucoamylase, which was calculated without taking into account the effect of glycosylation, is consistent with the experimental data and has a radius of gyration of 33.4 ± 0.6 Å.

  12. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    SciTech Connect

    Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

    2014-08-26

    Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

  13. Crystal Structure of the Human Cytomegalovirus Glycoprotein B

    PubMed Central

    Burke, Heidi G.; Heldwein, Ekaterina E.

    2015-01-01

    Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies. PMID:26484870

  14. MALDI linear TOF mass spectrometry of PEGylated (glyco)proteins.

    PubMed

    Seyfried, Birgit K; Siekmann, Jürgen; Belgacem, Omar; Wenzel, Ryan J; Turecek, Peter L; Allmaier, Günter

    2010-06-01

    PEGylation of proteins is a fast growing field in biotechnology and pharmaceutical sciences owing to its ability to prolong the serum half-life time of recombinant proteins. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) has been shown to be a powerful tool in the analysis of several PEGylated small proteins. Here we present data obtained with a standard secondary electron multiplier (SEM) and a high mass (HM) detector combined with a MALDI linear TOF MS system for the detection of PEGylated (glyco)proteins in the range of 60-600 kDa. Examples of MALDI TOF MS of small (interferon alpha2a), middle (human serum albumin (HSA)) and high molecular mass proteins (coagulation factor VIII and von Willebrand factor (vWF), both heavily glycosylated proteins) are presented. The particular challenge for the analysis was the heterogeneity of the (glyco)proteins in the high molecular weight range in combination with additional PEGylation, which even introduced more heterogeneity and was more challenging for interpretation. Nevertheless, the performance of MALDI linear TOF MS with both detector systems in terms molecular weight and heterogeneity determination depending on the m/z range was superior to the other methods. Although the SEM was able to obtain information about protein PEGylation in the mass range up to 100 kDa (e.g. PEGylated HSA), the HM system was crucial for detection of HM ions (e.g. PEGylated recombinant vWF), which was impossible with the standard SEM.

  15. Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays.

    PubMed

    Caragata, Michael; Shah, Alok K; Schulz, Benjamin L; Hill, Michelle M; Punyadeera, Chamindie

    2016-03-15

    Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the sample collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.

  16. Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.

    PubMed

    Toth, Ann M; Geisler, Christoph; Aumiller, Jared J; Jarvis, Donald L

    2011-12-01

    In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product.

  17. Preparation of Concanavalin A-Chelating Magnetic Nanoparticles for Selective Enrichment of Glycoproteins.

    PubMed

    Dong, Liping; Feng, Shun; Li, Shanshan; Song, Peipei; Wang, Jide

    2015-07-01

    In this work, a soft and nondestructive approach was developed to prepare concanavalin A-chelating magnetic nanoparticles (Con A-MNPs) for selective enrichment of glycoproteins. Ethylenediamine tetraacetic acid-modified-MNPs (EDTA-MNPs) were prepared by a one-pot chemical coprecipitation method first, and then, Cu(II) cations were used as bridge groups to immobilize Con A on EDTA-MNPs. The as-prepared absorbents with a mean diameter of 15 nm showed a strong magnetic response to an externally applied magnetic field. The results of thermogravimetric analysis showed the content of immobilized Con A was up to 28 wt %. For glycoprotein ovalbumin, the maximum capacity and equilibrium constant were 72.41 mg/g and 0.6035 L/mg, respectively. The as-prepared nanocomposites exhibited a remarkable selectivity for glycoproteins and can enrich glycoproteins specifically from a mixture of glycoprotein and nonglycoprotein even at a molar ratio of 1:600. It was also successfully applied for the enrichment of glycoproteins from real egg white samples. We expect that our finding will serve as a helpful template for others to design new adsorbents for enriching glycoproteins.

  18. Integrated glycoprotein immobilization method for glycopeptide and glycan analysis of cardiac hypertrophy.

    PubMed

    Yang, Shuang; Mishra, Sumita; Chen, Lijun; Zhou, Jian-Ying; Chan, Daniel W; Chatterjee, Subroto; Zhang, Hui

    2015-10-01

    Post-translational modifications of proteins can have a major role in disease initiation and progression. Incredible efforts have recently been made to study the regulation of glycoproteins for disease prognosis and diagnosis. It is essential to elucidate glycans and intact glycoproteins to understand the role of glycosylation in diseases. Sialylated N-glycans play crucial roles in physiological and pathological processes; however, it is laborious to study sialylated glycoproteins due to the labile nature of sialic acid residues. In this study, an integrated platform is developed for the analysis of intact glycoproteins and glycans using a chemoenzymatic approach for immobilization and derivatization of sialic acids. N-Glycans, deglycosylated proteins, and intact glycoproteins from heart tissues of wild type (WT) and transverse aortic constriction (TAC) mouse models were analyzed. We identified 291 unique glycopeptides from 195 glycoproteins; the comparative studies between WT and TAC mice indicate the overexpression of extracellular proteins for heart matrix remodeling and the down-regulation of proteins associated with energy metabolism in cardiac hypertrophy. The integrated platform is a powerful tool for the analysis of glycans and glycoproteins in the discovery of potential cardiac hypertrophy biomarkers.

  19. Preparation of biointeractive glycoprotein-conjugated hydrogels through metabolic oligosacchalide engineering.

    PubMed

    Iwasaki, Yasuhiko; Matsunaga, Aki; Fujii, Shuetsu

    2014-09-17

    In the current study, synthetic hydrogels containing metabolically engineered glycoproteins of mammalian cells were prepared for the first time and selectin-mediated cell adhesion on the hydrogel was demonstrated. A culture of HL-60 cells was supplemented with an appropriate volume of aqueous solution of N-methacryloyl mannosamine (ManMA) to give a final concentration of 5 mM. The cells were then incubated for 3 days to deliver methacryloyl groups to the glycoproteins of the cells. A transparent hydrogel was formed via redox radical polymerization of methacryloyl functionalized glycoproteins with 2-methacryloyloxyethyl phosphorylcholine and a cross-linker. Conjugation of the glycoproteins into the hydrogel was determined using Coomassie brilliant blue (CBB) and periodic acid-Schiff (PAS) staining. The surface density of P-selectin glycoprotein ligand-1 (PSGL-1) on the hydrogels was also detected using gold-colloid-labeled immunoassay. Finally, selectin-mediated cell adhesion on hydrogels containing glycoproteins was demonstrated. Selectin-mediated cell adhesion is considered an essential step in the progression of various diseases; therefore, hydrogels having glycoproteins could be useful in therapeutic and diagnostic applications.

  20. Sialic acid glycoproteins inhibit in vitro and in vivo replication of rotaviruses.

    PubMed Central

    Yolken, R H; Willoughby, R; Wee, S B; Miskuff, R; Vonderfecht, S

    1987-01-01

    We investigated the interactions of rotaviruses with glycoproteins and cells that support rotaviral replication. We found that a wide range of naturally occurring glycoproteins, including ovalbumins and ovomucoids from chicken and turkey eggs, and mucin derived from bovine submaxillary glands, inhibit the replication of rotaviruses in MA-104 cells. Our studies further indicated that the glycoproteins bind directly to rotaviruses and that virus-glycoprotein binding is dependent largely upon interactions with sialic acid oligosaccharides. We found that accessible sialic acid oligosaccharides are required for efficient rotavirus infection of MA-104 cells, thus demonstrating that sialic acid oligosaccharides play an important role in the interactions of rotaviruses with both glycoproteins and cells that support rotaviral replication. Bovine submaxillary mucin and chicken ovoinhibitor can also prevent the shedding of rotavirus antigen and the development of rotavirus gastroenteritis in a mouse model of rotavirus infection. Our findings document that a range of glycoproteins inhibit the in vivo and in vitro replication of rotaviruses and suggest that the alteration in the quantity or chemical composition of intestinal glycoproteins is a potential means for the modulation of enteric infections. Images PMID:3025257

  1. Immunogenicity in mice of human metapneumovirus with a truncated SH glycoprotein.

    PubMed

    Tedcastle, A B; Fenwick, F; Robinson, M J; Toms, G L

    2014-04-01

    The SH glycoprotein of human metapneumovirus (HMPV) is twice the size of that of human respiratory syncytial virus and possesses a large, hydrophilic luminal domain. The glycoprotein is located on the surface of the virion and of virus infected cells and, if immunogenic, might be expected to play a role in anti-viral immunity. Initial attempts to study anti-SH antibody immunogenicity were thwarted by the instability of the SH gene on passage both in human bronchial epithelial cells and in mice. Repeated passage of virus isolates in human bronchial epithelial cells in culture resulted in the appearance and eventual predominance of HMPV mutants lacking all or most of the luminal domain of SH coincidental with the loss of productive infection in mouse lungs. Where infection was established in mice with an early cell culture passage, the virus recovered from mouse lung differed markedly from the inoculum, carrying 19 coding mutations in the SH luminal domain. Immunization of mice with a mutant virus variant expressing only 14 amino acids of the luminal domain of SH induced a cross-reactive antibody response to both the F glycoprotein and the SH glycoprotein but a largely sub-group specific response to the G glycoprotein. Similar patterns of response were achieved by immunization with individual HMPV glycoproteins expressed from recombinant vaccinia viruses. Recombinant truncated SH glycoprotein induced sub-group cross-reactive antibodies capable of neutralizing wild-type virus. Recombinant F glycoprotein also induced cross-reactive neutralizing antibodies whilst recombinant G glycoprotein induced largely strain-specific, non-neutralizing antibodies.

  2. Bioactivity of proteins isolated from Lactobacillus plantarum L67 treated with Zanthoxylum piperitum DC glycoprotein.

    PubMed

    Song, S; Oh, S; Lim, K-T

    2015-06-01

    Lactobacilli in the human gastrointestinal tract have beneficial effects on the health of their host. To enhance these effects, the bioactivity of lactobacilli can be fortified through exogenous dietary or pharmacological agents, such as glycoproteins. To elucidate the inductive effect of Zanthoxylum piperitum DC (ZPDC) glycoprotein on Lactobacillus plantarum L67, we evaluated the radical-scavenging activity, anti-oxidative enzymes (SOD, GPx and CAT), growth rate, ATPase activity and β-galactosidase activity of this strain. When Lact. plantarum L67 was treated with ZPDC glycoprotein at different concentrations, the intensities of a few SDS-PAGE bands were slightly changed. The amount of a 23 kDa protein was increased upon treatment with increasing concentrations of ZPDC glycoprotein. The results of this study indicate that the radical-scavenging activity for O2(-) and OH¯, but not for the DPPH radical, increased in a concentration-dependent manner after treatment with ZPDC glycoprotein. The activation of anti-oxidative enzymes (SOD, GPx and CAT), growth rate and β-galactosidase activity also increased in a concentration-dependent manner in response to ZPDC glycoprotein treatment, whereas ATPase activity was decreased. In summary, ZPDC glycoprotein stimulated an increase in the bioactivity of Lact. plantarum L67. Significance and impact of the study: This study demonstrated that Lactobacillus plantarum L67 possesses anti-oxidative activity. This strain of lactic bacteria has been known to have various probiotic uses, such as yogurt starters and dietary additional supplements. We found, through this experiment, that the protein has a strong anti-oxidative character, and the activity can be enhanced by treatment with Zanthoxylum piperitum DC (ZPDC) glycoprotein. This study may be application of Lact. plantarum L67 treated by ZPDC glycoprotein in yogurt fermentation. It could be one of the avenues of minimizing yogurt postacidification during storage. In addition

  3. Characterization of I/F1 glycoprotein as a receptor for Mycoplasma pneumoniae.

    PubMed Central

    Hengge, U R; Kirschfink, M; König, A L; Nicklas, W; Roelcke, D

    1992-01-01

    Serologic evidence of anti-I and anti-Fl cold agglutinins occurring in mycoplasma infections led to the isolation of I/Fl glycoprotein from human erythrocyte membranes. Mycoplasma pneumoniae bound to purified I/Fl glycoprotein in a dose-dependent fashion depending on sialylated carbohydrate determinants. This was shown by the decreased binding of mycoplasmas to either sialidase-treated I/Fl glycoprotein (dot blot analysis) or sialidase-treated erythrocytes (hemagglutination test). Structural properties of the receptor for optimal binding could be explored by hemagglutination inhibition assays. Glycophorins were excluded as receptors. These results indicate that Fl (and I) antigens are receptors for M. pneumoniae. Images PMID:1370278

  4. Proposed pathway for biosynthesis of the S-layer glycoprotein of Bacillus alvei.

    PubMed Central

    Hartmann, E; Messner, P; Allmeier, G; König, H

    1993-01-01

    The outer surface of the murein sacculus of the eubacterium Bacillus alvei is covered by a surface layer (S-layer) glycoprotein. The glycan chain of this S-layer glycoprotein consists of trisaccharide repeating units with ManNAc, Gal, and Glc as constituents. From cell extracts of B. alvei, nucleotide-activated derivatives of ManNAc, Gal, Glc, and GlcNAc were isolated. Furthermore, GDP- and dolichyl-activated oligosaccharides were obtained. On the basis of the isolated putative glycoprotein precursors, a pathway for the biosynthesis of the oligosaccharide chain is proposed. PMID:8331079

  5. Selective binding of human cumulus cell-secreted glycoproteins to human spermatozoa during capacitation in vitro

    SciTech Connect

    Tesarik, J.; Kopecny, V.; Dvorak, M.

    1984-06-01

    The results of this study demonstrate that glycoproteins manufactured by human cumulus cells can be detected bound to human spermatozoa incubated in capacitational medium containing the labeled cumulus-cell secretions. Cumulus-cell-secreted glycoproteins were labeled with a mixture of /sup 3/H-methionine and /sup 3/H-tryptophan or with 3H-fucose, and the binding of the labeled compounds to spermatozoa was evaluated by autoradiography. The binding was highly selective, involving only approximately 1% of the samples of spermatozoa used. The results suggest that the binding of cumulus-cell-secreted glycoproteins to spermatozoa may represent a final and highly selective step in human sperm capacitation.

  6. A comprehensive study of interactions between lectins and glycoproteins for the development of effective theranostic nanoagents.

    PubMed

    Shipunova, V O; Nikitin, M P; Zelepukin, I V; Nikitin, P I; Deyev, S M; Petrov, R V

    2015-01-01

    A comprehensive study of the interactions between lectins and glycoproteins possessing different glycosylation profiles in the composition of nanoparticles was carried out in order to find specifically interacting protein pairs for the creation of novel classes of multifunctional nanoagets that based on protein-assisted selfassembly. We obtained information about specific interactions of certain lectins with selected glycoproteins as well as about the ability of certain monosaccharides to competitively inhibit binding of glycoproteins with lectins. These protein-mediated interactions may be involved in the formulation of self-assembled nanoparticles for therapy and diagnostics of various diseases. PMID:26518557

  7. Chemistry-enabled methods for the visualization of cell-surface glycoproteins in Metazoans.

    PubMed

    Chuh, Kelly N; Pratt, Matthew R

    2015-10-01

    The majority of cell-surface and secreted proteins are glycosylated, which can directly affect their macromolecular interactions, stability, and localization. Investigating these effects is critical to developing a complete understanding of the role of glycoproteins in fundamental biology and human disease. The development of selective and unique chemical reactions have revolutionized the visualization, identification, and characterization of glycoproteins. Here, we review the chemical methods that have been created to enable the visualization of the major types of cell-surface glycoproteins in living systems, from mammalian cells to whole animals.

  8. Enhancement of non-Candida antibody responses by Candida albicans cell wall glycoprotein.

    PubMed

    Domer, J E; Elkins, K L; Ennist, D L; Stashak, P W; Garner, R E; Baker, P J

    1987-11-01

    Two cell wall glycoprotein extracts from Candida albicans (glycoprotein [GP] and peptidoglucomannan [PGM]) were tested for their influence on antibody responses to type III pneumococcal polysaccharide and sheep erythrocytes. GP was isolated from lipid-extracted cell walls with ethylenediamine, whereas PGM was extracted with dilute sodium hydroxide. Both glycoproteins increased the number of antibody-producing plaque-forming cells in the spleens of mice immunized with type III polysaccharide or sheep erythrocytes, although PGM appeared to be about 10 times more effective. PGM could be administered up to 3 days prior to immunization with sheep erythrocytes to elicit enhancement; it did not have to be administered by the same route as the immunogen to cause significant enhancement. Enhancement did not appear to be the result of a direct mitogenic effect of GP and PGM on lymphocytes, nor did these glycoproteins appear to stimulate the production of B-cell growth factors or interleukin 2.

  9. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification....

  10. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification....

  11. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification....

  12. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification....

  13. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... the alpha-2-glycoproteins (a group of plasma proteins found in the alpha-2 group when subjected to... some cancers and genetically inherited deficiencies of these plasma proteins. (b) Classification....

  14. [Molecular Mechanism of Glycoprotein-induced Cell-Cell Fusion of Herpesviruses].

    PubMed

    Feng, Daishen; Jia, Renyong

    2016-01-01

    Herpesviridae is a large family comprising linear, double-stranded DNA viruses. Herpesviridae contains three subfamilies: α-, β- and γ-herpesviruses. The glycoproteins gB, gH and gL of each subfamily form the "core fusion function" in cell-cell fusion. Other herpesviruses also need additional glycoproteins to promote fusion, such as gD of the Herpes simplex virus, gp42 of the Epstein-Barr virus, and gO or UL128-131 of the Human cytomegalovirus. In contrast, glycoproteins gM or gM/gN of herpesvirus inhibit fusion. We describe the molecular mechanisms of glycoprotein-induced fusion and entry of herpesviruses. It will be helpful to further study the pathogenic mechanism of herpesvirus.

  15. Localization of P-glycoprotein at the nuclear envelope of rat brain cells

    SciTech Connect

    Babakhanian, Karlo; Bendayan, Moise; Bendayan, Reina . E-mail: r.bendayan@utoronto.ca

    2007-09-21

    P-Glycoprotein is a plasma membrane drug efflux protein implicated in extrusion of cytotoxic compounds out of a cell. There is now evidence that suggests expression of this transporter at several subcellular sites, including the nucleus, mitochondria, and Golgi apparatus. This study investigated the localization and expression of P-glycoprotein at the nuclear membrane of rat brain microvessel endothelial (RBE4) and microglial (MLS-9) cell lines. Immunocytochemistry at the light and electron microscope levels using P-glycoprotein monoclonals antibodies demonstrated the localization of the protein at the nuclear envelope of RBE4 and MLS-9 cells. Western blot analysis revealed a single band of 170-kDa in purified nuclear membranes prepared from isolated nuclei of RBE4 and MLS-9 cells. These findings indicate that P-glycoprotein is expressed at the nuclear envelope of rat brain cells and suggest a role in multidrug resistance at this subcellular site.

  16. Epitope mapping of the infectious hematopoietic necrosis virus glycoprotein by flow cytometry.

    PubMed

    Xu, Li-Ming; Liu, Miao; Zhao, Jing-Zhuang; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

    2014-10-01

    The glycoprotein of infectious hematopoietic necrosis virus was truncated to ten overlapping fragments. All fragments were displayed on the inner membrane of the Escherichia coli periplasm. After disruption of the outer membrane, spheroplasts that had anchored with the glycoprotein fragment were incubated with an anti-glycoprotein polyclonal antibody. Prey pairs were detected and quantitated by flow cytometry with all fragments but one, G2, reacting with the polyclonal antibody. The antigenicity of all ten fragments was analyzed using conventional methods, and epitopes were localized in all fragments, except for G2 and were consistent with FCM analysis. Antigenicity of purified glycoprotein fusion proteins was confirmed by western blotting and ELISA. This method provides a rapid, quantitative and simple strategy for identifying linear B cell epitopes of a given protein.

  17. Purification and characterization of a novel glycoprotein from Streptomyces sp. ZX01.

    PubMed

    Zhang, Guoqiang; Han, Lirong; Zhang, Guifeng; Zhang, Xing; Feng, Juntao

    2015-01-01

    A novel glycoprotein GP-1 with antiviral activity against plant virus was isolated from the fermentation broth of the actinomycete Streptomyces sp. ZX01. MALDI-TOF-MS proved that molecular weight of GP-1 approximately was 8.5 kDa. GP-1 was a heat-sensitive glycoprotein with decreasing antiviral activity after treated from 80 °C to 100 °C for 30 min. GP-1 contained 40.23% carbohydrate with N-linked and O-linked glycan. FT-IR and NMR spectra proved that GP-1 contained protein and carbohydrate portions with α-D-(1,6)-glucose residues. Circular dichroism revealed that GP-1 was a glycoprotein with a large unordered content. Moreover, protein sequencing was predicted by using MALDI-TOF-MS and Mascot search. These results suggested that glycoprotein GP-1 could be used as a novel natural antiviral agent in agricultural industry.

  18. Boronic Acid-Based Approach for Separation and Immobilization of Glycoproteins and Its Application in Sensing

    PubMed Central

    Wang, Xiaojin; Xia, Ning; Liu, Lin

    2013-01-01

    Glycoproteins influence a broad spectrum of biological processes including cell-cell interaction, host-pathogen interaction, or protection of proteins against proteolytic degradation. The analysis of their glyco-structures and concentration levels are increasingly important in diagnosis and proteomics. Boronic acids can covalently react with cis-diols in the oligosaccharide chains of glycoproteins to form five- or six-membered cyclic esters. Based on this interaction, boronic acid-based ligands and materials have attracted much attention in both chemistry and biology as the recognition motif for enrichment and chemo/biosensing of glycoproteins in recent years. In this work, we reviewed the progress in the separation, immobilization and detection of glycoproteins with boronic acid-functionalized materials and addressed its application in sensing. PMID:24141187

  19. P-glycoprotein inhibitory activity of two phenolic compounds, (-)-syringaresinol and tricin from Sasa borealis.

    PubMed

    Jeong, Yeon Hee; Chung, Soo Yeon; Han, Ah-Reum; Sung, Min Kyung; Jang, Dae Sik; Lee, Jun; Kwon, Youngjoo; Lee, Hwa Jeong; Seo, Eun-Kyoung

    2007-01-01

    (-)-Syringaresinol and tricin, isolated from the AcOEt-soluble extract of the whole plants of Sasa borealis (Gramineae), showed inhibitory effects on the P-glycoprotein in adriamycin-resistant human breast cancer cells, MCF-7/ADR. PMID:17256728

  20. Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.

    PubMed

    Molinari, Maurizio; Eriksson, Klara Kristin; Calanca, Verena; Galli, Carmela; Cresswell, Peter; Michalak, Marek; Helenius, Ari

    2004-01-16

    Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides. PMID:14731400

  1. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    SciTech Connect

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

    2007-09-30

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions.

  2. Glycoprotein isolated from Solanum nigrum L. kills HT-29 cells through apoptosis.

    PubMed

    Lim, Kye-Taek

    2005-01-01

    Solanum nigrum L. (SNL) has been used in folk medicine for its anti-inflammatory activity. We previously isolated glycoprotein from SNL and observed that it decreased viable HT-29 cell numbers at a low concentration (60 microg/mL). This study investigated the apoptotic signal pathway triggered by glycoprotein isolated from SNL in HT-29 cells. Treatment of HT-29 cells with SNL glycoprotein (60 microg/mL) for 4 hours resulted in a cytotoxic effect of more than 60%, compared with the control. To explain the apoptotic effects of SNL glycoprotein, we investigated its effects on 12-O-tetradecanoylphorbol 13-acetate (TPA)-stimulated protein kinase C (PKC) alpha activity and DNA-binding activity of nuclear factor (NF) kappaB in HT-29 cells, using western blot analysis and electrophoretic mobility shift assays. Results from these experiments showed that SNL glycoprotein has remarkable inhibitory effects on the activities of TPA (100 nM)-stimulated PKCalpha and NF-kappaB in HT-29 cells. They also substantiated that PKCalpha is a part of the TPA-activated upstream signal pathway of NF-kappaB, since NF-kappaB activity was inhibited by staurosporine (a PKC inhibitor) and pyrrolidine dithiocarbamate (an NF-kappaB inhibitor) in a western blot analysis. Furthermore, to verify the triggering of apoptosis by the SNL glycoprotein, we performed DNA fragmentation, nuclear staining, and protein expression assays of apoptotic-related proteins. The amount of DNA fragmentation and apoptotic cell numbers increased in a dose-dependent manner after treatment with SNL glycoprotein. Apoptosis-related protein assays demonstrated that SNL glycoprotein-induced apoptosis is associated with the regulation of bcl-2 and Bax expression. Taken together, the results of this study showed that the activation of PKCalpha, NF-kappaB, and Bax expression by SNL glycoprotein is possibly involved in the apoptotic process. Consequently, these results indicate that SNL glycoprotein causes HT-29 cell death through

  3. Postnatal changes in sialylation of glycoproteins in rat liver.

    PubMed Central

    Oda-Tamai, S; Kato, S; Akamatsu, N

    1991-01-01

    Glycoproteins containing N-linked oligosaccharides were prepared from plasma and liver microsomes of rats aged 0-5 weeks, and galactose and sialic acid content were determined. The sialic acid/galactose ratios in plasma membrane N-glycans remained at about 1 throughout the postnatal period, suggesting that most of the galactose residues are sialylated. In the same way, it was suggested that most of the galactose residues of microsomal N-glycans were sialylated at 0, 4 and 5 weeks of age, but that the degree of sialylation was lower at the other ages, with a minimum at 2 weeks. When the activities of sialyltransferase and galactosyltransferase in liver Golgi membranes were determined, age-dependent changes were found, not only in the specific activities of the enzymes, but also in the Golgi membrane content per g of liver. The activity of galactosyltransferase per g of liver increased immediately after birth, whereas that of sialyltransferase remained at a low level for 2 weeks and then increased to a constant level at 4 weeks. It is probable that this delayed increase in the activity of sialyltransferase results in the decreased sialylation of microsomal N-glycans at 1, 2 and 3 weeks. Sialyltransferase was solubilized from the liver microsomes of rats aged 2, 3 and 4 weeks and characterized. Phosphocellulose column chromatography separated the activity into two subfractions, designated transferase I and transferase II in the order of elution. The increase in total sialyltransferase activity during this period was caused mainly by an increase in transferase I. Rechromatography of each transferase from 3-week-old rats after neuraminidase treatment showed that transferase I but not transferase II contained sialic acid residue(s) and that desialylated transferase I was eluted in a similar way as transferase II. Although the apparent Km value for CMP-N-acetylneuraminic acid and the heat stability of transferase I were different from those of transferase II, the

  4. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    SciTech Connect

    Filipski, Elisabeth; Berland, Elodie; Ozturk, Narin; Guettier, Catherine; Horst, Gijsbertus T.J. van der; Lévi, Francis; and others

    2014-02-01

    The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F{sub 1} mice. A three-fold 24 h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p < 0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50 mg/kg/day i.v. × 4 days) as a single agent or combined with P-gp inhibitor PSC833 (6.25 mg/kg/day i.p. × 4 days) at ZT3 or ZT15, respectively corresponding to the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40 mg/kg/day × 4 days) and +/− PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p < 0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p < 0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ∼ 60%. In tumor-bearing mice, body weight loss was ∼ halved in the mice on irinotecan or irinotecan–PSC833 combination at ZT15 as compared to ZT3 (p < 0.001). PSC833–irinotecan at ZT15 increased tumor inhibition by ∼ 40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan. - Highlights: • Irinotecan chronotolerance and chronoefficacy change as drug was applied with PSC833. • P-glycoprotein is an important player of the toxicity and efficacy of irinotecan. • Timing should be considered if chemotherapy is performed with a MDR1 inhibitor.

  5. Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

    PubMed Central

    Mohseni, Mahsa; Samadi, Nasser; Ghanbari, Parisa; Yousefi, Bahman; Tabasinezhad, Maryam; Sharifi, Simin; Nazemiyeh, Hossein

    2016-01-01

    Objective(s): Chemoresistance remains the main causes of treatment failure and mortality in cancer patients. There is an urgent need to investigate novel approaches to improve current therapeutic modalities and increase cancer patients’ survival. Induction of drug efflux due to overexpression of P-glycoproteins is considered as an important leading cause of multidrug resistance. In this study, we investigated the role of combination treatments of docetaxel and vinblastine in overcoming P-glycoprotein mediated inhibition of apoptosis and induction of cell proliferation in human non-small cell lung carcinoma cells. Materials and Methods: Cell proliferation and apoptosis were assessed using MTT assay and DAPI staining, respectively. P-glycoprotein expression was evaluated in gene and protein levels by Real-time RT-PCR and Western blot analysis, respectively. Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1) to (15±2.6) nM and for vinblastine from (30±5.9) to (5±5.6) nM (P≤0.05). P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001). Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05). Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents. Conclusion: Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression. PMID:27114800

  6. Identification of a human immunodeficiency virus type 1 envelope glycoprotein variant resistant to cold inactivation.

    PubMed

    Kassa, Aemro; Finzi, Andrés; Pancera, Marie; Courter, Joel R; Smith, Amos B; Sodroski, Joseph

    2009-05-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability.

  7. P-glycoprotein is a major determinant of norbuprenorphine brain exposure and antinociception.

    PubMed

    Brown, Sarah M; Campbell, Scott D; Crafford, Amanda; Regina, Karen J; Holtzman, Michael J; Kharasch, Evan D

    2012-10-01

    Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of μ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(-/-) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(-/-) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(-/-) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine. PMID:22739506

  8. P-Glycoprotein Is a Major Determinant of Norbuprenorphine Brain Exposure and Antinociception

    PubMed Central

    Brown, Sarah M.; Campbell, Scott D.; Crafford, Amanda; Regina, Karen J.; Holtzman, Michael J.

    2012-01-01

    Norbuprenorphine is a major metabolite of buprenorphine and potent agonist of μ, δ, and κ opioid receptors. Compared with buprenorphine, norbuprenorphine causes minimal antinociception but greater respiratory depression. It is unknown whether the limited antinociception is caused by low efficacy or limited brain exposure. Norbuprenorphine is an in vitro substrate of the efflux transporter P-glycoprotein (Mdr1), but the role of P-glycoprotein in norbuprenorphine transport in vivo is unknown. This investigation tested the hypothesis that limited norbuprenorphine antinociception results from P-glycoprotein-mediated efflux and limited brain access. Human P-glycoprotein-mediated transport in vitro of buprenorphine, norbuprenorphine, and their respective glucuronide conjugates was assessed by using transfected cells. P-glycoprotein-mediated norbuprenorphine transport and consequences in vivo were assessed by using mdr1a(+/+) and mdr1a(−/−) mice. Antinociception was determined by hot-water tail-flick assay, and respiratory effects were determined by unrestrained whole-body plethysmography. Brain and plasma norbuprenorphine and norbuprenorphine-3-glucuronide were quantified by mass spectrometry. In vitro, the net P-glycoprotein-mediated efflux ratio for norbuprenorphine was nine, indicating significant efflux. In contrast, the efflux ratio for buprenorphine and the two glucuronide conjugates was unity, indicating absent transport. The norbuprenorphine brain/plasma concentration ratio was significantly greater in mdr1a(−/−) than mdr1a(+/+) mice. The magnitude and duration of norbuprenorphine antinociception were significantly increased in mdr1a(−/−) compared with mdr1a(+/+) mice, whereas the reduction in respiratory rate was similar. Results show that norbuprenorphine is an in vitro and in vivo substrate of P-glycoprotein. P-glycoprotein-mediated efflux influences brain access and antinociceptive, but not the respiratory, effects of norbuprenorphine. PMID

  9. Bypassing P-Glycoprotein Drug Efflux Mechanisms: Possible Applications in Pharmacoresistant Schizophrenia Therapy

    PubMed Central

    Hoosain, Famida G.; Choonara, Yahya E.; Tomar, Lomas K.; Kumar, Pradeep; Tyagi, Charu; du Toit, Lisa C.; Pillay, Viness

    2015-01-01

    The efficient noninvasive treatment of neurodegenerative disorders is often constrained by reduced permeation of therapeutic agents into the central nervous system (CNS). A vast majority of bioactive agents do not readily permeate into the brain tissue due to the existence of the blood-brain barrier (BBB) and the associated P-glycoprotein efflux transporter. The overexpression of the MDR1 P-glycoprotein has been related to the occurrence of multidrug resistance in CNS diseases. Various research outputs have focused on overcoming the P-glycoprotein drug efflux transporter, which mainly involve its inhibition or bypassing mechanisms. Studies into neurodegenerative disorders have shown that the P-glycoprotein efflux transporter plays a vital role in the progression of schizophrenia, with a noted increase in P-glycoprotein function among schizophrenic patients, thereby reducing therapeutic outcomes. In this review, we address the hypothesis that methods employed in overcoming P-glycoprotein in cancer and other disease states at the level of the BBB and intestine may be applied to schizophrenia drug delivery system design to improve clinical efficiency of drug therapies. In addition, the current review explores polymers and drug delivery systems capable of P-gp inhibition and modulation. PMID:26491671

  10. Sweating the small stuff: Glycoproteins in human sweat and their unexplored potential for microbial adhesion.

    PubMed

    Peterson, Robyn A; Gueniche, Audrey; Adam de Beaumais, Ségolène; Breton, Lionel; Dalko-Csiba, Maria; Packer, Nicolle H

    2016-03-01

    There is increasing evidence that secretory fluids such as tears, saliva and milk play an important role in protecting the human body from infection via a washing mechanism involving glycan-mediated adhesion of potential pathogens to secretory glycoproteins. Interaction of sweat with bacteria is well established as the cause of sweat-associated malodor. However, the role of sweat glycoproteins in microbial attachment has received little, if any, research interest in the past. In this review, we demonstrate how recent published studies involving high-throughput proteomic analysis have inadvertently, and fortuitously, exposed an abundance of glycoproteins in sweat, many of which have also been identified in other secretory fluids. We bring together research demonstrating microbial adhesion to these secretory glycoproteins in tears, saliva and milk and suggest a similar role of the sweat glycoproteins in mediating microbial attachment to sweat and/or skin. The contribution of glycan-mediated microbial adhesion to sweat glycoproteins, and the associated impact on sweat derived malodor and pathogenic skin infections are unchartered new research areas that we are beginning to explore. PMID:26582610

  11. Development and validation of glycoprotein-based native-subunit vaccine for fish against Aeromonas hydrophila.

    PubMed

    Çiftci, A; Onuk, E E; Çiftci, G; Fındık, A; Söğüt, M Ü; Didinen, B I; Aksoy, A; Üstünakın, K; Gülhan, T; Balta, F; Altun, S

    2016-08-01

    Aeromonas hydrophila is known to be causative agent of an infection named as Bacterial haemorrhagic septicaemia or red pest in freshwater fish. The aim of this study was to develop and validate the glycoprotein-based fish vaccine against Aeromonas hydrophila. For this aim, after identification and characterization of A. hydrophila isolates from fish farms, one A. hydrophila isolate was selected as vaccine strain. Antigenic glycoproteins of this vaccine strain were determined by Western blotting and glycan detection kit. The connection types of these glycoproteins were examined by glycoprotein differentiation kit. Two glycoproteins, molecular weights of 19 and 38 kDa, with SNA connection type were selected for use in vaccination trials. After their purification by SNA-specific lectin and size-exclusion chromatography, protection studies with purified proteins were performed. For challenge trials, four experimental fish groups were designated: Group I (with montanide), Group II (with montanide and ginseng), Group III [with Al(OH)3 ] and Group IV [with Al(OH)3 and ginseng]. The survival ratings of fish were determined, and protection was calculated as 21.56%, 29.41%, 69.83% and 78.88% in groups I, II, III and IV, respectively. In conclusion, A. hydrophila glycoproteins with Al(OH)3 and ginseng could be used as a safe and effective vaccine for fish. PMID:27144782

  12. Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

    SciTech Connect

    Kunicki, T.J.; Nurden, A.T.; Pidard, D.; Russell, N.R.; Caen, J.P.

    1981-12-01

    Washed human platelets were labeled with 125I by the lactoperoxidase-catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

  13. Bile canalicular cationic dye secretion as a model for P-glycoprotein mediated transport.

    PubMed

    Thalhammer, T; Stapf, V; Gajdzik, L; Graf, J

    1994-04-01

    This study explores properties of P-glycoprotein dependent membrane transport in rat liver with the use of acridine orange as the substrate. We studied the biliary secretion of the dye, its binding to canalicular membrane P-glycoprotein, and effects of the inhibitor cyclosporin A: acridine orange is excreted into bile together with less hydrophobic and glucuronidated metabolites. Cyclosporin A inhibited both the secretion of acridine orange and of its metabolites. In TR- animals, a rat strain that is deficient of the canalicular multi-specific organic anion transport system, non-metabolized acridine orange is the predominant species in bile and its secretion is also inhibited by cyclosporin A. Binding of acridine orange to liver P-glycoprotein was analyzed by photoaffinity labeling with azidopine, a substrate of P-glycoprotein dependent transport in multi-drug resistant tumor cells. Labeling of the immunoprecipitated P-glycoprotein was inhibited by acridine orange, verapamil, and by cyclosporin A. The results show that biliary secretion of acridine orange is highly analogous to P-glycoprotein mediated membrane drug transport in tumor cells that exhibit multi-drug resistance.

  14. Expression of P-glycoprotein in high grade osteosarcomas with special emphasis on chondroblastic subtype.

    PubMed

    Radig, K; Häckel, C; Herting, J; Oda, Y; Mittler, U; Neumann, W; Roessner, A

    1997-02-01

    The development of chemoresistance is one of the major clinical problems in the therapy of malignant bone tumors in childhood. The expression of membrane-bound P-glycoprotein turned out to be an essential factor in the evidence of resistant tumor cells. To investigate the significance of multidrug resistance in the prognosis of highly malignant osteosarcomas, the immunohistologic expression of P-glycoprotein was investigated in the tumor tissue of 52 patients under special consideration of the histologic subtype. The data were compared with the histologic regression grade in the resection specimen and correlated with clinical data. Formalin-fixed, paraffin-embedded tissue and, additionally, fresh frozen material taken from the primary biopsy were stained using monoclonal antibody JSB1. 29 (55%) of the tumors investigated were P-glycoprotein positive. Considering the response to chemotherapy, no conclusion could be drawn regarding P-glycoprotein expression, regression grade in the resection specimens, and the clinical follow-up. P-glycoprotein was detected in only 52% of the non-responders. A positive reaction was also evidenced in 59% of the patients with high chemosensitivity. A comparison of the histologic subtypes yielded a significant result in the chondroblastic osteosarcomas. 11 of 12 cases showed a strong expression of P-glycoprotein. Most of the cases were non-responders, and using Kaplan-Meier live tables, an unfavorable clinical outcome could be demonstrated. Possibly, chondroblastic tumors have a special position among osteosarcomas because of their differentiation. PMID:9065577

  15. Identification of sialylated glycoproteins in Doxorubicin-treated hepatoma cells with glycoproteomic analyses.

    PubMed

    Azuma, Kanako; Serada, Satoshi; Takamatsu, Shinji; Terao, Naoko; Takeishi, Shunsaku; Kamada, Yoshihiro; Naka, Tetsuji; Miyoshi, Eiji

    2014-11-01

    Sialylation is one of the most important types of glycosylation involved in carcinogenesis and establishment of cancer stemness. We previously showed that increased sialylation is a characteristic glycan change in cancer stem cells (CSCs) from hepatocellular carcinoma. However, the identities of glycoproteins targeted for sialylation remain unknown. In the present study, we identified glycoproteins targeted for sialylation in doxorubicin (DXR)-treated hepatocarcinoma cell line, Huh7, using glycoproteomic analyses. Since CSCs constitute a small subset of cells within carcinoma cell lines, it is difficult to identify sialylated proteins using general glycoproteomic strategies. It is known that treatment with anticancer drug can condense CSCs, we used DXR to concentrate CSCs. In DXR-treated Huh7 cells, isobaric tag for relative and absolute quantitation (iTRAQ) analysis identified 17 sialylated glycoproteins. Most of the identified glycoproteins were cancer-associated proteins. Furthermore, two proteins of approximately 70 kDa were detected using Sambucus sieboldoana agglutinin (SSA) blot analysis and identified as beta-galactosidase and alpha-2-HS-glycoprotein (fetuin-A) by SSA precipitation followed by liquid chromatography-tandem mass spectrometry analyses. Sialylation levels of fetuin-A were increased in DXR-treated Huh7 cell lysates. These changes in sialylation of glycoproteins might be involved in the establishment of cancer stemness.

  16. Lipid modification gives rise to two distinct Haloferax volcanii S-layer glycoprotein populations.

    PubMed

    Kandiba, Lina; Guan, Ziqiang; Eichler, Jerry

    2013-03-01

    The S-layer glycoprotein is the sole component of the protein shell surrounding Haloferax volcanii cells. The deduced amino acid sequence of the S-layer glycoprotein predicts the presence of a C-terminal membrane-spanning domain. However, several earlier observations, including the ability of EDTA to selectively solubilize the protein, are inconsistent with the presence of a trans-membrane sequence. In the present report, sequential solubilization of the S-layer glycoprotein by EDTA and then with detergent revealed the existence of two distinct populations of the S-layer glycoprotein. Whereas both S-layer glycoprotein populations underwent signal peptide cleavage and N-glycosylation, base hydrolysis followed by mass spectrometry revealed that a lipid, likely archaetidic acid, modified only the EDTA-solubilized version of the protein. These observations are consistent with the S-layer glycoprotein being initially synthesized as an integral membrane protein and subsequently undergoing a processing event in which the extracellular portion of the protein is separated from the membrane-spanning domain and transferred to a waiting lipid moiety.

  17. Reconstructed glycan profile for evaluation of operating status of the endoplasmic reticulum glycoprotein quality control.

    PubMed

    Iwamoto, Shogo; Isoyama, Miho; Hirano, Makoto; Yamaya, Kenta; Ito, Yukishige; Matsuo, Ichiro; Totani, Kiichiro

    2013-01-01

    Glycoprotein oligosaccharides function as tags for protein quality control in the endoplasmic reticulum (ER). Since most of proteins are glycosylated and function only after they are properly folded, glycoprotein glycan profiles in the ER might be useful to analyze various cellular status including diseases. Here, we examined whether ER glycan-processing profiles in diabetic rats and osteoporotic mice as models might have different cellular status from those of normal controls. Direct analysis of glycoprotein-processing profiles in the ER is often hampered by glycoforms that are retro-translocated to the ER from other cellular compartments. Moreover, when we focus on the mixture of glycoproteins as the processing substrates, the glycan-processing efficiencies are influenced by the aglycon states including their polypeptide folding. To overcome this problem, we reconstructed glycan profiles using ER extracts as an enzymatic source and synthetic glycoprotein mimetic having homogeneous aglycon as a substrate, resulted in disease-specific glycan profiles. To understand such differences, we also analyzed the activity, and expression level, of each glycan-related enzyme. These glycan profiles are expected to be useful indexes for operational status of the ER glycoprotein quality control, and may also give information to classify some diseases.

  18. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

    SciTech Connect

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    2011-09-16

    Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.

  19. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

    PubMed Central

    Ferris, Sean P.; Jaber, Nikita S.; Molinari, Maurizio; Arvan, Peter; Kaufman, Randal J.

    2013-01-01

    Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER. PMID:23864712

  20. Studies on a novel macrophage-specific calmodulin binding glycoprotein

    SciTech Connect

    Orlow, S.J.

    1986-01-01

    The murine macrophage-like cell line J774 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin-binding protein which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and these elicited with concanavalin A, lipopolysaccharide, proteose peptone or Bacillus Clamette Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed using a rabbit antiserum raised to the partially purified calmodulin binding protein and (/sup 125/I) calmodulin covalently crosslinked to the principal calmodulin binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of molecular weight 50,000 to 60,000 was isolated. It could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property plus its sensitivity to endoglycosidase F led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization and evidence of glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for calcium-calmodulin.

  1. Effects of hydrocortisone on pulsatile pituitary glycoprotein secretion.

    PubMed

    Samuels, M H; Luther, M; Henry, P; Ridgway, E C

    1994-01-01

    During states of stress, hypothalamic-pituitary-thyroid and hypothalamic-pituitary-gonadal function can be suppressed. One putative mediator of this stress response may be glucocorticoids, which have widespread effects on thyroid and gonadal function. To characterize dynamic pituitary glycoprotein secretion during glucocorticoid administration, 24-h TSH, LH, FSH, and alpha-subunit pulses were measured in 10 healthy young subjects on 3 occasions: 1) at baseline, 2) during infusions of 100 mg hydrocortisone (HC) over 24 h, and 3) during infusions of 300 mg HC over 24 h. These HC infusions led to serum cortisol levels similar to the endogenous cortisol levels seen in moderate and severe stress. Both HC infusions had profound rapid effects on TSH levels, decreasing TSH pulse amplitude by 60% and abolishing the nocturnal TSH surge. However, TSH pulse frequency was unaltered. In contrast, HC infusions did not change mean or pulsatile LH, FSH, or alpha-subunit secretion. These results suggest that stress levels of cortisol acutely suppress TSH secretion at the pituitary level, with little effect on the TSH pulse generator. On the other hand, the effects of stress and/or hypercortisolism on the gonadal axis may require higher cortisol levels, more prolonged exposure, or other mediators of the stress response.

  2. Synonymous codon usage pattern in glycoprotein gene of rabies virus.

    PubMed

    Morla, Sudhir; Makhija, Aditi; Kumar, Sachin

    2016-06-10

    Rabies virus (RABV) is the causative agent of a fatal nervous system ailment. The disease is zoonotic and prevalent in many developing countries. The glycoprotein (G) of RABV is the major antigenic determinant of the virus and plays a pivotal role in its neurovirulence. Various aspects of 'G' protein biology have been explored, but the factors affecting the nucleotide choice and synonymous codon usage have never been reported. In the present study, we have analyzed the relative synonymous codon usage and effective number of codons (Nc) using 132 'G' protein genes of RABV. Corresponding analysis was used to calculate major trends in codon usage. The correlation between base composition and codon usage as well as the plot between Nc and GC3 suggest that mutational pressure is the major factor that influences the codon usage in the G gene of RABV. In addition, factors like aromaticity, aliphatic index and hydropathy have shown slight correlation suggesting that natural selection also contributes to the codon usage variations of the 'G' gene. In conclusion, codon usage bias in 'G' gene of RABV is mainly by mutational pressure and natural selection. PMID:26945626

  3. [Classification models of structure - P-glycoprotein activity of drugs].

    PubMed

    Grigorev, V Yu; Solodova, S L; Polianczyk, D E; Raevsky, O A

    2016-01-01

    Thirty three classification models of substrate specificity of 177 drugs to P-glycoprotein have been created using of the linear discriminant analysis, random forest and support vector machine methods. QSAR modeling was carried out using 2 strategies. The first strategy consisted in search of all possible combinations from 1÷5 descriptors on the basis of 7 most significant molecular descriptors with clear physico-chemical interpretation. In the second case forward selection procedure up to 5 descriptors, starting from the best single descriptor was used. This strategy was applied to a set of 387 DRAGON descriptors. It was found that only one of 33 models has necessary statistical parameters. This model was designed by means of the linear discriminant analysis on the basis of a single descriptor of H-bond (ΣC(ad)). The model has good statistical characteristics as evidenced by results to both internal cross-validation, and external validation with application of 44 new chemicals. This confirms an important role of hydrogen bond in the processes connected with penetration of chemical compounds through a blood-brain barrier.

  4. [Classification models of structure - P-glycoprotein activity of drugs].

    PubMed

    Grigorev, V Yu; Solodova, S L; Polianczyk, D E; Raevsky, O A

    2016-01-01

    Thirty three classification models of substrate specificity of 177 drugs to P-glycoprotein have been created using of the linear discriminant analysis, random forest and support vector machine methods. QSAR modeling was carried out using 2 strategies. The first strategy consisted in search of all possible combinations from 1÷5 descriptors on the basis of 7 most significant molecular descriptors with clear physico-chemical interpretation. In the second case forward selection procedure up to 5 descriptors, starting from the best single descriptor was used. This strategy was applied to a set of 387 DRAGON descriptors. It was found that only one of 33 models has necessary statistical parameters. This model was designed by means of the linear discriminant analysis on the basis of a single descriptor of H-bond (ΣC(ad)). The model has good statistical characteristics as evidenced by results to both internal cross-validation, and external validation with application of 44 new chemicals. This confirms an important role of hydrogen bond in the processes connected with penetration of chemical compounds through a blood-brain barrier. PMID:27143376

  5. A monocistronic transcript for a trypanosome variant surface glycoprotein.

    PubMed Central

    Alarcon, C M; Son, H J; Hall, T; Donelson, J E

    1994-01-01

    Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage. Images PMID:8035832

  6. Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine

    PubMed Central

    BAO, LIDAO; WEI, GUOMIN; GAN, HONGMEI; REN, XIANHUA; MA, RUILIAN; WANG, YI; LV, HAIJUN

    2016-01-01

    In the present study a eukaryotic expression vector of varicella zoster virus (VZV) glycoprotein E (gE) was constructed and enabled to express in COS7 cells. Furthermore, a specific immune response against the VZV gE eukaryotic expression plasmid was induced in BALB/c mice. The VZV gE gene was amplified using polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector, pcDNA3.1. The recombinant vector was subsequently transfected into COS7 cells using a liposome transfection reagent. The recombinant protein was instantaneously expressed by the transfected cells, as detected by immunohistochemistry, and the recombinant pcDNA-VZV gE plasmid was subsequently used to immunize mice. Tissue expression levels were analyzed by reverse transcription-PCR. In addition, the levels of serum antibodies and spleen lymphocyte proliferation activity were investigated. The amplified target gene included the full-length gE gene (~2.7 kb), and the recombinant expression vector induced gE expression in COS7 cells. In addition, the expression plasmid induced sustained expression in vivo following immunization of mice. Furthermore, the plasmid was capable of inducing specific antibody production and effectively stimulating T cell proliferation. Effective humoral and cellular immunity was triggered in the mice immunized with the VZV gE eukaryotic expression vector. The results of the present study laid the foundation for future research into a VZV DNA vaccine. PMID:27168804

  7. Advances in plant-based inhibitors of P-glycoprotein.

    PubMed

    Yu, Jun; Zhou, Peng; Asenso, James; Yang, Xiao-Dan; Wang, Chun; Wei, Wei

    2016-12-01

    Multidrug resistance (MDR) has emerged as the main problem in anti-cancer therapy. Although MDR involves complex factors and processes, the main pivot is the expression of multidrug efflux pumps. P-glycoprotein (P-gp) belongs to the family of adenosine triphosphate (ATP)-binding cassette (ABC) transporters. It functions in cellular detoxification, pumping a wide range of xenobiotic compounds out of the cell. An attractive therapeutic strategy for overcoming MDR is to inhibit the transport function of P-gp and thus, increase intracellular concentration of drugs. Recently, various types of P-gp inhibitors have been found and used in experiments. However, none of them has passed clinical trials due to their high side-effects. Hence, the search for alternatives, such as plant-based P-gp inhibitors have gained attention recently. Therefore, we give an overview of the source, function, structure and mechanism of plant-based P-gp inhibitors and give more attention to cancer-related studies. These products could be the future potential drug candidates for further research as P-gp inhibitors.

  8. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

    PubMed Central

    López-Pacheco, Felipe; Pérez-Chavarría, Roberto; González-Vázquez, Juan Carlos; González-González, Everardo; Trujillo-de Santiago, Grissel; Ponce-Ponce de León, César Alejandro; Zhang, Yu Shrike; Dokmeci, Mehmet Remzi; Khademhosseini, Ali; Alvarez, Mario Moisés

    2015-01-01

    Background Current Ebola virus (EBOV) detection methods are costly and impractical for epidemic scenarios. Different immune-based assays have been reported for the detection and quantification of Ebola virus (EBOV) proteins. In particular, several monoclonal antibodies (mAbs) have been described that bind the capsid glycoprotein (GP) of EBOV GP. However, the currently available platforms for the design and production of full-length mAbs are cumbersome and costly. The use of antibody fragments, rather than full-length antibodies, might represent a cost-effective alternative for the development of diagnostic and possibly even therapeutic alternatives for EBOV. Methods/Principal Findings We report the design and expression of three recombinant anti-GP mAb fragments in Escherichia coli cultures. These fragments contained the heavy and light variable portions of the three well-studied anti-GP full-length mAbs 13C6, 13F6, and KZ52, and are consequently named scFv-13C6, scFv-13F6, and Fab-KZ52, respectively. All three fragments exhibited specific anti-GP binding activity in ELISA experiments comparable to that of full-length anti-GP antibodies (i.e., the same order of magnitude) and they are easily and economically produced in bacterial cultures. Conclusion/Significance Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications. PMID:26489048

  9. Four glycoproteins are expressed in the cat zona pellucida.

    PubMed

    Stetson, I; Avilés, M; Moros, C; García-Vázquez, F A; Gimeno, L; Torrecillas, A; Aliaga, C; Bernardo-Pisa, M V; Ballesta, J; Izquierdo-Rico, M J

    2015-04-15

    The mammalian oocyte is surrounded by a matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding and may be involved in speciation. In cat (Felis catus), this matrix is composed of at least three glycoproteins called ZP2, ZP3, and ZP4. However, recent studies have pointed to the presence of a fourth protein in several mammals (rat, human, hamster or rabbit), meaning that a reevaluation of cat ZP is needed. For this reason, the objective of this research was to analyze the protein composition of cat ZP by means of proteomic analysis. Using ZP from ovaries and oocytes, several peptides corresponding to four proteins were detected, yielding a coverage of 33.17%, 71.50%, 50.23%, and 49.64% for ZP1, ZP2, ZP3, and ZP4, respectively. Moreover, the expression of four genes was confirmed by molecular analysis. Using total RNA isolated from cat ovaries, the complementary deoxyribonucleic acids encoding cat ZP were partially amplified by reverse-transcribed polymerase chain reaction. Furthermore, ZP1 was totally amplified for the first time in this species. As far as we are aware, this is the first study that confirms the presence of four proteins in cat ZP.

  10. Dynamics of antifreeze glycoproteins in the presence of ice.

    PubMed Central

    Tsvetkova, Nelly M; Phillips, Brian L; Krishnan, Viswanathan V; Feeney, Robert E; Fink, William H; Crowe, John H; Risbud, Subhash H; Tablin, Fern; Yeh, Yin

    2002-01-01

    Antifreeze glycoproteins from the Greenland cod Boreogadus saida were dimethylated at the N-terminus (m*AFGP) and their dynamics and conformational properties were studied in the presence of ice using (13)C-NMR and FTIR spectroscopy. (13)C-NMR experiments of m*AFGP in D(2)O, in H(2)O, and of freeze-dried m*AFGP were performed as a function of temperature. Dynamic parameters ((1)H T(1 rho) and T(CH)) obtained by varying the contact time revealed notable differences in the motional properties of AFGP between the different states. AFGP/ice dynamics was dominated by fast-scale motions (nanosecond to picosecond time scale), suggesting that the relaxation is markedly affected by the protein hydration. The data suggest that AFGP adopts a similar type of three-dimensional fold both in the presence of ice and in the freeze-dried state. FTIR studies of the amide I band did not show a single prevailing secondary structure in the frozen state. The high number of conformers suggests a high flexibility, and possibly reflects the necessity to expose more ice-binding groups. The data suggest that the effect of hydration on the local mobility of AFGP and the lack of significant change in the backbone conformation in the frozen state may play a role in inhibiting the ice crystal growth. PMID:11751333

  11. The microfibril-associated glycoproteins (MAGPs) and the microfibrillar niche.

    PubMed

    Mecham, Robert P; Gibson, Mark A

    2015-09-01

    The microfibril-associated glycoproteins MAGP-1 and MAGP-2 are extracellular matrix proteins that interact with fibrillin to influence microfibril function. The two proteins are related through a 60 amino acid matrix-binding domain but their sequences differ outside of this region. A distinguishing feature of both proteins is their ability to interact with TGFβ family growth factors, Notch and Notch ligands, and multiple elastic fiber proteins. MAGP-2 can also interact with αvβ3 integrins via a RGD sequence that is not found in MAGP-1. Morpholino knockdown of MAGP-1 expression in zebrafish resulted in abnormal vessel wall architecture and altered vascular network formation. In the mouse, MAGP-1 deficiency had little effect on elastic fibers in blood vessels and lung but resulted in numerous unexpected phenotypes including bone abnormalities, hematopoietic changes, increased fat deposition, diabetes, impaired wound repair, and a bleeding diathesis. Inactivation of the gene for MAGP-2 in mice produced a neutropenia yet had minimal effects on bone or adipose homeostasis. Double knockouts had phenotypes characteristic of each individual knockout as well as several additional traits only seen when both genes are inactivated. A common mechanism underlying all of the traits associated with the knockout phenotypes is altered TGFβ signaling. This review summarizes our current understanding of the function of the MAGPs and discusses ideas related to their role in growth factor regulation. PMID:25963142

  12. An extensive endoplasmic reticulum-localised glycoprotein family in trypanosomatids

    PubMed Central

    Allison, Harriet; O’Reilly, Amanda J.; Sternberg, Jeremy; Field, Mark C.

    2015-01-01

    African trypanosomes are evolutionarily highly divergent parasitic protozoa, and as a consequence the vast majority of trypanosome membrane proteins remain uncharacterised in terms of location, trafficking or function. Here we describe a novel family of type I membrane proteins which we designate ‘invariant glycoproteins’ (IGPs). IGPs are trypanosome-restricted, with extensive, lineage-specific paralogous expansions in related taxa. In T. brucei three IGP subfamilies, IGP34, IGP40 and IGP48 are recognised; all possess a putative C-type lectin ectodomain and are ER-localised, despite lacking a classical ER-retention motif. IGPs exhibit highest expression in stumpy stage cells, suggesting roles in developmental progression, but gene silencing in mammalian infective forms suggests that each IGP subfamily is also required for normal proliferation. Detailed analysis of the IGP48 subfamily indicates a role in maintaining ER morphology, while the ER lumenal domain is necessary and sufficient for formation of both oligomeric complexes and ER retention. IGP48 is detected by antibodies from T. b. rhodesiense infected humans. We propose that the IGPs represent a trypanosomatid-specific family of ER-localised glycoproteins, with potential contributions to life cycle progression and immunity, and utilise oligomerisation as an ER retention mechanism. PMID:26167471

  13. Rabies virus glycoprotein as a carrier for anthrax protective antigen

    SciTech Connect

    Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J. . E-mail: matthias.schnell@jefferson.edu

    2006-09-30

    Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.

  14. Interaction modes and approaches to glycopeptide and glycoprotein enrichment.

    PubMed

    Chen, Chen-Chun; Su, Wan-Chih; Huang, Bao-Yu; Chen, Yu-Ju; Tai, Hwan-Ching; Obena, Rofeamor P

    2014-02-21

    Protein glycosylation has received increased attention for its critical role in cell biology and diseases. Developing new methodologies to discern phenotype-dependent glycosylation will not only elucidate the mechanistic aspects of cell signaling cascades but also accelerate biomarker discovery for disease diagnosis or prognosis. In the analytical pipeline, enrichment at either the protein or peptide level is the most critical prerequisite for analyzing heterogeneous glycan composition, linkage, site occupancy and carrier proteins. Because the critical factor for choosing a suitable enrichment method is primarily a particular technique's selectivity and affinity towards target glycoproteins/glycopeptides, it is important to fully understand the working principles for the different approaches. For mechanistic insight into the enrichment protocol, we focused on the fundamental chemical and physical processes for the commonly used approaches based on: (a) glycan/peptide physicochemical properties (hydrophilic interactions, chelation/coordination chemistry) and (b) glycan-specific recognition (lectin-based affinity, covalent bond formation by hydrazide/boronic acid). Various interaction modes, such as hydrogen bonding, van der Waals interaction, multivalency, and metal- or water-mediated stabilization, are discussed in detail. In addition, we will review the design of and modifications to such methods, hyphenated approaches, and glycoproteomic applications. Finally, we will outline challenges to existing strategies and offer novel proposals for glycoproteome enrichment. PMID:24336240

  15. Extracellular matrix glycoproteins and diffusion barriers in human astrocytic tumours.

    PubMed

    Zámecník, J; Vargová, L; Homola, A; Kodet, R; Syková, E

    2004-08-01

    The extracellular matrix (ECM) and changes in the size and geometry of the extracellular space (ECS) in tumour tissue are thought to be of critical importance in influencing the migratory abilities of tumour cells as well as the delivery of therapeutic agents into the tumour. In 21 astrocytic neoplasms, the ECM composition was investigated in situ by the immunohistochemical detection of ECM glycoproteins (tenascin, laminin, vitronectin, fibronectin, collagen types I-VI). To explain the changes in ECS size and to detect barriers to diffusion in the tumour tissue, the ECM composition, the cellularity, the density of glial fibrillary acidic protein (GFAP)-positive tumour cell processes and the proliferative activity of the tumours were compared with the size and geometry of the ECS. The ECS volume fraction and the complex of hindrances to diffusion in the ECS (i.e. the tortuosity) were revealed by the real-time iontophoretic tetramethylammonium method. Increased proliferative activity of the tumours correlated with increased ECS volume fraction and tortuosity. The tortuosity of the tumour tissue was not significantly influenced by tumour cell density. Higher tortuosity was found in low-grade astrocytomas associated with the presence of a dense net of GFAP-positive fibrillary processes of the tumour cells. The increase in tortuosity in high-grade tumours correlated with an increased accumulation of ECM molecules, particularly of tenascin. We conclude that the increased malignancy of astrocytic tumours correlates with increases in both ECS volume and ECM deposition.

  16. Platelets deficient in glycoprotein I have normal Fc receptor expression.

    PubMed

    Pfueller, S L; de Rosbo, N K; Bilston, R A

    1984-04-01

    Platelet glycoprotein I (GPI) is known to be required for the interaction of platelets with ristocetin and factor VIII:von Willebrand factor (VIII:vWf). However, its role as Fc receptor is not clear. Some studies have shown that enzymatic removal of GPI destroys the ability of platelets to react with VIII:vWf but not their ability to bind Ig G (IgG). Others have shown that IgG immune complexes which block the Fc receptor also inhibit VIII:vWf interaction with platelets. This subject has been re-examined by testing the ability of platelets with reduced amounts of GPI to aggregate and undergo the release reaction in response to stimuli which act at the platelet Fc receptor. Platelets from two patients with Bernard-Soulier syndrome, congenitally deficient in GPI, both aggregated and released 14C-serotonin normally when exposed to latex particles coated with IgG. Levels of GPI were decreased experimentally in normal platelets by treating them with chymotrypsin. Platelets treated in this manner did not aggregate or release [14C]serotonin in response to ristocetin-VIII:vWf. They did, however, both aggregate and release when incubated with heat-aggregated IgG, antigen-antibody complexes or latex particles coated with IgG. Thus the presence of GPI is not a prerequisite for platelet stimulation via the Fc receptor. PMID:6231945

  17. Respiratory syncytial virus envelope glycoprotein (G) has a novel structure.

    PubMed Central

    Satake, M; Coligan, J E; Elango, N; Norrby, E; Venkatesan, S

    1985-01-01

    Amino acid sequence of human respiratory syncytial virus envelope glycoprotein (G) was deduced from the DNA sequence of a recombinant plasmid and confirmed by limited amino acid microsequencing of purified 90K G protein. The calculated molecular mass of the protein encoded by the only long open reading frame of 298 amino acids was 32,588 daltons and was somewhat smaller than the 36K polypeptide translated in vitro from mRNA selected by this plasmid. Inspection of the sequence revealed a single hydrophobic domain of 23 amino acids capable of membrane insertion at 41 residues from the N-terminus. There was no N-terminal signal sequence and the hydrophilic N-terminal 20 residues probably represent the cytoplasmic tail of the protein. The N-terminally oriented membrane insertion was somewhat analogous to paramyxovirus hemagglutinin-neuraminidase (HN) and influenza neuraminidase (NA). The protein was moderately hydrophilic and rich in hydroxy-amino acids. It was both N- and O-glycosylated with the latter contributing significantly to the net molecular mass 90K. Images PMID:4069997

  18. Antifreeze Glycoproteins Alter the Molecular Scale Surface Morphology of Ice

    NASA Astrophysics Data System (ADS)

    Zepeda, Salvador; Orme, Christine A.; Qiu, Roger; Yeh, Yin

    2003-03-01

    Trematomas borchgrevinki live in the harsh super-cooled waters of the Antarctic. Critical to their survival are antifreeze glycoproteins (AFGPs) that further suppress the freezing temperature of their blood serum in addition to the colligative action of salts found in the ocean. These proteins also modify ice crystal growth habits as well as inhibit recrystallization in polycrystalline ice. To date many other types of antifreeze proteins have been identified in cold weather insects, plants, and other fish, but the exact mechanism is not entirely understood. The mechanism is non-colligative since only a few mg/ml are required for ice crystal growth inhibition and a non-equilibrium melting/freezing point hysteresis is observed. Atomic force microscopy (AFM) can yield a wealth of surface information that can reveal molecular scale information of biomineralization processes. We use AFM to directly probe the surface of ice crystals grown from the vapor in the pure phase and in the presence of growth inhibitors/modifiers, AFGPs. Results show that the AFGPs heavily pin the surface of ice.

  19. Pathogenesis of mucosal injury in the blind loop syndrome. Brush border enzyme activity and glycoprotein degradation.

    PubMed

    Jonas, A; Flanagan, P R; Forstner, G G

    1977-12-01

    The effect of intestinal bacterial over-growth on brush border hydrolases and brush border glycoproteins was studied in nonoperated control rats, control rats with surgically introduced jejunal self-emptying blind loops, and rats with surgically introduced jejunal self-filling blind loops. Data were analyzed from blind loop segments, segments above and below the blind loops, and three corresponding segments in the nonoperated controls. Rats with self-filling blind loops had significantly greater fat excretion than controls and exhibited significantly lower conjugated:free bile salt ratios in all three segments. Maltase, sucrase, and lactase activities were significantly reduced in homogenates and isolated brush borders from the self-filling blind loop, but alkaline phosphatase was not affected. The relative degradation rate of homogenate and brush border glycoproteins was assessed by a double-isotope technique involving the injection of d-[6-(3)H]glucosamine 3 h and d-[U-(14)C]glucosamine 19 h before sacrifice, and recorded as a (3)H:(14)C ratio. The relative degradation rate in both homogenate and brush border fractions was significantly greater in most segments from rats with self-filling blind loops. In the upper and blind loop segments from rats with self-filling blind loops, the (3)H:(14)C ratios were higher in the brush border membrane than in the corresponding homogenates, indicating that the increased rates of degradation primarily involve membrane glycoproteins. Incorporation of d-[6-(3)H]glucosamine by brush border glycoproteins was not reduced in rats with self-filling blind loops, suggesting that glycoprotein synthesis was not affected. Polyacrylamide gel electrophoresis of brush border glycoproteins from the contaminated segments indicated that the large molecular weight glycoproteins, which include many of the surface hydrolases, were degraded most rapidly. Brush border maltase, isolated by immunoprecipitation, had (3)H:(14)C ratios characteristic of

  20. Cross-linking of glycoprotein oligomers during herpes simplex virus type 1 entry.

    PubMed

    Handler, C G; Cohen, G H; Eisenberg, R J

    1996-09-01

    Herpes simplex virus (HSV) has 10 glycoproteins in its envelope. Glycoprotein B (gB), gC, gD, gH, and gL have been implicated in virus entry. We previously used chemical cross-linking to show that these five glycoproteins were close enough to each other to be cross-linked into homodimeric and hetero-oligomeric forms; hetero-oligomers of gB-gC, gC-gD, gD-gB, gH-gL, gC-gL and gD-gL were found in purified virions. To better understand the roles of these glycoproteins in viral entry, we have modified a standard HSV penetration assay to include cross-linkers. This allowed us to examine changes in associations of viral glycoproteins during the entry process. HSV-1(KOS) was adsorbed at 4 degrees C to human neuroblastoma cells (SY5Y). The temperature was raised to 37 degrees C and cells were treated with cross-linker at various times after the temperature shift. Cytoplasmic extracts were examined by Western blotting (immunoblotting) for viral glycoproteins. We found that (i) as in virus alone, the length and concentration of the cross-linking agent affected the number of specific complexes isolated; (ii) the same glycoprotein patterns found in purified virions were also present after attachment of virions to cells; and (iii) the ability to cross-link HSV glycoproteins changed as virus penetration proceeded, e.g., gB and gD complexes which were present during attachment disappeared with increasing time, and their disappearance paralleled the kinetics of penetration. However, this phenomenon appeared to be selective since it was not observed with gC oligomers. In addition, we examined the cross-linking patterns of gB and gD in null viruses K082 and KOSgD beta. Neither of these mutants, which attach but cannot penetrate, showed changes in glycoprotein cross-linking over time. We speculate that these changes are due to conformational changes which preclude cross-linking or spatial alterations which dissociate the glycoprotein interactions during the penetration events. PMID

  1. Pre-staining of glycoprotein in SDS-PAGE by the synthesis of a new hydrazide derivative.

    PubMed

    Zhou, Ayi; Zhou, Tieli; Yu, Dongdong; Shen, Yingjie; Shen, Jiayi; Zhu, Zhongxin; Jin, Litai; Zhang, Huajie; Wang, Yang

    2015-11-01

    In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins. PMID:26256282

  2. Inhibition of rhodamine 123 secretion by cyclosporin A as a model of P-glycoprotein mediated transport in liver.

    PubMed

    Stapf, V; Thalhammer, T; Huber-Huber, R; Felberbauer, F; Gajdzik, L; Graf, J

    1994-01-01

    The interaction between P-glycoprotein modulators and P-glycoprotein mediated transport was investigated using rhodamine 123 in the isolated perfused rat liver of a mutant (TR-) rat strain. TR- rats, deficient in the canalicular multispecific anion transport system, are unable to extrude organic anions (glucuronides) and therefore excrete solely unconjugated rhodamine 123 via P-glycoprotein. Cyclosporin A, a modulator of multidrug resistance in tumor cells, inhibited the biliary secretion of rhodamine 123 dose dependently in a non-competitive manner. Both cyclosporin A and rhodamine inhibited photoaffinity labeling of immunoprecipitated P-glycoprotein with azidopine, indicating binding to hepatic P-glycoprotein. Our results indicate that monitoring the biliary rhodamine 123 secretion in the isolated perfused liver of TR- rats offers a new system for testing modulators of P-glycoprotein like cyclosporin A.

  3. A fluorescence nanosensor for glycoproteins with activity based on the molecularly imprinted spatial structure of the target and boronate affinity.

    PubMed

    Zhang, Wei; Liu, Wei; Li, Ping; Xiao, Haibin; Wang, Hui; Tang, Bo

    2014-11-10

    Glycoproteins are closely associated with the occurrence of diverse diseases, and they have been used as biomarkers and therapeutic targets in clinical diagnostics. Currently, mass spectrometry has proven to be a powerful tool for glycoprotein analysis, but it is almost impossible to directly identify glycoproteins without the preparation and pretreatment of samples. Furthermore, biological samples, especially proteins, are damaged by this process. Herein, we describe a novel fluorescence nanosensor based on a molecularly imprinted spatial structure and boronate affinity that is well-suited for monitoring glycoproteins selectively. Results showed that the recognition performance of the nanosensor for glycoproteins was regulated by controlling the pH value and temperature. Moreover, the nanosensor was successfully applied to the detection of HRP in biological fluids. This study provides a facile and efficient fluorescence tool for glycoprotein detection in clinical diagnostics.

  4. Pre-staining of glycoprotein in SDS-PAGE by the synthesis of a new hydrazide derivative.

    PubMed

    Zhou, Ayi; Zhou, Tieli; Yu, Dongdong; Shen, Yingjie; Shen, Jiayi; Zhu, Zhongxin; Jin, Litai; Zhang, Huajie; Wang, Yang

    2015-11-01

    In this study, a new hydrazide derivative (UGF202) was synthesized and introduced as a highly sensitive and selective fluorescent probe to pre-stain glycoproteins in 1D and 2D SDS-PAGE. As low as 0.5-1 ng glycoproteins (transferrin, α1-acid glycoprotein, avidin) could be selectively detected, which is comparable to that of Pro-Q Emerald 300 stain, one of the most sensitive and commonly used glycoprotein staining kit. In addition, the specificity of the newly developed method was confirmed by the study of de-glycosylation, glycoproteins affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that UGF202 pre-stain can provide an alternative for the visualization of gel-separated glycoproteins.

  5. Comparison of three distinct ELLA protocols for determination of apparent affinity constants between Con A and glycoproteins.

    PubMed

    Mislovičová, D; Katrlík, J; Paulovičová, E; Gemeiner, P; Tkac, J

    2012-06-01

    A procedure for determination of apparent affinity constants K(D)(app) between Concanavalin A (Con A) and naturally d-mannose containing glycoproteins using enzyme-linked lectin assay (ELLA) is reported. Three distinct ELLA protocols are compared to each other with 3 different fitting models used (Liliom, Hill with and without a cooperativity factor). The glycoproteins were physisorbed on a highly charged polystyrene solid surface of immunoassay plates and the amount of lectin bound to the glycoproteins was determined by photometry. The interactions of Con A with five mannose-containing glycoproteins, invertase (INV), glucoamylase (GA), glucose oxidase (GOx), ovalbumin (OVA), and transferrin (TRF) were quantified with apparent affinity constant being in the range 2×10(-7) to 9×10(-6)M. The strength of interaction between Con A and glycoproteins is discussed on the basis of glycan structure/exposure on the protein backbone for each glycoprotein.

  6. Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes

    PubMed Central

    1975-01-01

    The presence in the Golgi fraction of glycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [14C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS- gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components. PMID:1202020

  7. Method of using alpha-1 acid glycoprotein on T-cells as a marker for alzheimer's disease

    SciTech Connect

    Fudenberg, H.H.

    1989-01-31

    A method is described of diagnosing a dementia of the Alzheimer's type characterized by a change in the percentage of T-cells bearing surface membrane alpha-1 acid glycoprotein which comprises providing T-cells from a subject, determining the percentage of those T cells which bear surface membrane alpha-1 acid glycoprotein, and comparing that percentage of the percentage of T cells which bear the glycoprotein in a control, whereby the dementia is diagnosed.

  8. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin

    PubMed Central

    1993-01-01

    The dystrophin-glycoprotein complex was tested for interaction with several components of the extracellular matrix as well as actin. The 156-kD dystrophin-associated glycoprotein (156-kD dystroglycan) specifically bound laminin in a calcium-dependent manner and was inhibited by NaCl (IC50 = 250 mM) but was not affected by 1,000-fold (wt/wt) excesses of lactose, IKVAV, or YIGSR peptides. Laminin binding was inhibited by heparin (IC50 = 100 micrograms/ml), suggesting that one of the heparin-binding domains of laminin is involved in binding dystroglycan while negatively charged oligosaccharide moieties on dystroglycan were found to be necessary for its laminin-binding activity. No interaction between any component of the dystrophin- glycoprotein complex and fibronectin, collagen I, collagen IV, entactin, or heparan sulfate proteoglycan was detected by 125I-protein overlay and/or extracellular matrix protein-Sepharose precipitation. In addition, laminin-Sepharose quantitatively precipitated purified dystrophin-glycoprotein complex, demonstrating that the laminin-binding site is accessible when dystroglycan is associated with the complex. Dystroglycan of nonmuscle tissues also bound laminin. However, the other proteins of the striated muscle dystrophin-glycoprotein complex appear to be absent, antigenically dissimilar or less tightly associated with dystroglycan in nonmuscle tissues. Finally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin- based cytoskeleton with the extracellular matrix. Furthermore, our results suggest that dystrophin and dystroglycan may play substantially different functional roles in nonmuscle tissues. PMID:8349731

  9. Tromantadine inhibits HSV-1 induced syncytia formation and viral glycoprotein processing

    SciTech Connect

    Ickes, D.E.

    1989-01-01

    Tromantadine inhibits a late event in Herpes Simplex Virus Type 1 (HSV-1) replication, visualized by the inhibition of both the size and number of syncytia. Tromantadine can be added at any time between 1 and 9 h post infection with complete inhibition of syncytia formation. Glycan synthesis of the viral glycoproteins, important for syncytia formation, is incomplete due to tromantadine treatment. Tromantadine does not inhibit the initiation of glycosylation, since viral glycoproteins, gX{sub t}, synthesized in the presence of tromantadine still incorporate {sup 3}H-glucosamine. Tromantadine does not inhibit the transport of t e viral glycoproteins to the cell surface, since glycoproteins B, C, and D are expressed, as demonstrated by immunofluorescence. Tromantadine inhibition of HSV-1 glycoprotein processing is demonstrated by an increase in mobility of the radioimmunoprecipitated gX{sub t}, on SDS-PAGE. The gX{sub t} of KOS, a non-syncytial strain of HSV-1, had a similar increase in mobility, suggesting that the block in glycoprotein processing is a general effect of tromantadine treatment. Fucose, which is incorporated into oligosaccharides in the medial Golgi, is incorporated into gX{sub t}, indicating that the tromantadine block in glycoprotein processing occurs after this step. Lectin binding studies and SDS-PAGE analysis of gC processed in the presence of tromantadine, gC{sub t}, indicates that it has terminal galactose residues in both N- and O-linked glycans (binds Peanut and Ricin Agglutinins, respectively). The inhibition of sialylation of N-linked glycans by tromantadine was indicated by the extent of the increase in SDS-PAGE mobility of the G protein from Vesicular Stomatitis Virus. O-glycanase digestion and SDS-PAGE analysis of gC{sub t} indicate that the O-linked disaccharide NAcGal-Galactose is present.

  10. Effects of chronic ethanol administration on hepatic glycoprotein secretion in the rat

    SciTech Connect

    Sorrell, M.F.; Nauss, J.M.; Donohue, T.M. Jr.; Tuma, D.J.

    1983-03-01

    The effects of chronic ethanol feeding on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Liver slices from rats fed ethanol for 4-5 wk showed a decreased ability to incorporate (/sup 14/C)glucosamine into medium trichloracetic acid-precipitable proteins when compared to the pair-fed controls; however, the labeling of hepatocellular glycoproteins was unaffected by chronic ethanol treatment. Immunoprecipitation of radiolabeled secretory (serum) glycoproteins with antiserum against rat serum proteins showed a similar marked inhibition in the appearance of glucosamine-labeled proteins in the medium of slices from ethanol-fed rats. Minimal effects, however, were noted in the labeling of intracellular secretory glycoproteins. Protein synthesis, as determined by measuring (/sup 14/C)leucine incorporation into medium and liver proteins, was decreased in liver slices from ethanol-fed rats as compared to the pair-fed controls. This was the case for both total proteins as well as immunoprecipitable secretory proteins, although the labeling of secretory proteins retained in the liver slices was reduced to a lesser extent than total radiolabeled hepatic proteins. When the terminal sugar, (/sup 14/C)fucose, was employed as a precursor in order to more closely focus on the final steps of hepatic glycoprotein secretion, liver slices obtained from chronic ethanol-fed rats exhibited impaired secretion of fucose-labeled proteins into the medium. When ethanol (5 or 10 mM) was added to the incubation medium containing liver slices from the ethanol-fed rats, the alterations in protein and glycoprotein synthesis and secretion caused by the chronic ethanol treatment were further potentiated. The results of this study indicate that liver slices prepared from chronic ethanol-fed rats exhibit both impaired synthesis and secretion of proteins and glycoproteins, and these defects are further potentiated by acute ethanol administration.

  11. Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain

    SciTech Connect

    Margolis, R.K.; Goossen, B.; Margolis, R.U.

    1988-05-03

    PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellium were labeled with (/sup 3/H)glucosamine, (/sup 3/H)fucose, (/sup 3/H)leucine, (/sup 3/H)ethanolamine, or sodium (/sup 35/S)sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of (/sup 3/H) glucosamine- or (/sup 3/H)fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel ectrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-l glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-..beta..-galactosidase, 40-45% of the (/sup 3/H)glucosamine of (/sup 3/H)fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of (/sup 3/H)ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence,while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in (/sup 3/H)ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.

  12. Discovery and Development of Small Molecule Allosteric Modulators of Glycoprotein Hormone Receptors

    PubMed Central

    Nataraja, Selvaraj G.; Yu, Henry N.; Palmer, Stephen S.

    2015-01-01

    Glycoprotein hormones, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) are heterodimeric proteins with a common α-subunit and hormone-specific β-subunit. These hormones are dominant regulators of reproduction and metabolic processes. Receptors for the glycoprotein hormones belong to the family of G protein-coupled receptors. FSH receptor (FSHR) and LH receptor are primarily expressed in somatic cells in ovary and testis to promote egg and sperm production in women and men, respectively. TSH receptor is expressed in thyroid cells and regulates the secretion of T3 and T4. Glycoprotein hormones bind to the large extracellular domain of the receptor and cause a conformational change in the receptor that leads to activation of more than one intracellular signaling pathway. Several small molecules have been described to activate/inhibit glycoprotein hormone receptors through allosteric sites of the receptor. Small molecule allosteric modulators have the potential to be administered orally to patients, thus improving the convenience of treatment. It has been a challenge to develop a small molecule allosteric agonist for glycoprotein hormones that can mimic the agonistic effects of the large natural ligand to activate similar signaling pathways. However, in the past few years, there have been several promising reports describing distinct chemical series with improved potency in preclinical models. In parallel, proposal of new structural model for FSHR and in silico docking studies of small molecule ligands to glycoprotein hormone receptors provide a giant leap on the understanding of the mechanism of action of the natural ligands and new chemical entities on the receptors. This review will focus on the current status of small molecule allosteric modulators of glycoprotein hormone receptors, their effects on common signaling pathways in cells, their utility for clinical application as demonstrated in preclinical models

  13. The Domains of Glycoprotein D Required To Block Apoptosis Depend on Whether Glycoprotein D Is Present in the Virions Carrying Herpes Simplex Virus 1 Genome Lacking the Gene Encoding the Glycoprotein

    PubMed Central

    Zhou, Guoying; Roizman, Bernard

    2001-01-01

    An earlier report showed that viruses lacking the open reading frames encoding glycoproteins J and D but containing the glycoprotein D in their envelopes (gD−/+ stocks) and viruses lacking both the open reading frames and the glycoproteins in their envelopes (gD−/− stocks) induce apoptosis (G. Zhou, V. Galvan, G. Campadelli-Fiume, and B. Roizman, J. Virol. 74:11782–11791, 2000). Furthermore, apoptosis was blocked by delivery in trans of genes expressing glycoprotein D or J. Whereas gD−/− stocks attach but cannot initiate productive infection, gD−/+ stocks infect cells and produce gD−/− progeny virus. The difference in the infectivity of these two stocks suggested the possibility that the requirements for blocking apoptosis may be different. To test this hypothesis, we cloned into baculoviruses the entire wild-type glycoprotein D (Bac-gD-WT), the ectodomain only (Bac-gD-A), the ectodomain and the transmembrane domain (Bac-gD-B), the ectodomain and the cytoplasmic domain without the transmembrane domain (Bac-gD-C), or the transmembrane domain and the carboxyl-terminal cytoplasmic domain (Bac-gD-D). We report the following. Apoptosis induced by gD−/+ stocks was blocked by delivery in trans of recombinant baculovirus Bac-gD-WT, Bac-gD-A, Bac-gD-B, or Bac-gD-C but not of Bac-gD. Apoptosis induced by gD−/− stocks was blocked by Bac-gD-WT or by a mixture of Bac-gD-B and Bac-gD-D but not by any baculoviruses expressing truncated glycoprotein D alone or by the mixture of Bac-gD-A and Bac-gD-D. We conclude that the requirements to block apoptosis induced by the two virus stocks are different. The gD ectodomain is sufficient to block apoptosis induced by gD, whereas both the ectodomain and the cytoplasmic domain are required to block apoptosis induced by gD−/− stocks. The results indicate that in the case of gD−/− stocks, the transmembrane domain is required either to deliver the ectodomain to the appropriate intracellular compartment or to

  14. New insight into p-glycoprotein as a drug target.

    PubMed

    Breier, Albert; Gibalova, Lenka; Seres, Mario; Barancik, Miroslav; Sulova, Zdenka

    2013-01-01

    Multidrug resistance (MDR) of cancer tissue is a phenomenon in which cancer cells exhibit reduced sensitivity to a large group of unrelated drugs with different mechanisms of pharmacological activity. Mechanisms that reduce cell sensitivity to damage induced by a variety of chemicals were found to be caused by diverse, albeit well-defined, phenotypic alterations. The molecular basis of MDR commonly involves overexpression of the plasma membrane drug efflux pump - P-glycoprotein (P-gp). This glycoprotein is an ABCB1 member of the ABC transporter family. Cells that develop MDR of this type express massive amounts of P-gp that can induce a drug resistance of more than 100 times higher than normal cells to several drugs, which are substrates of P-gp. Expression of P-gp could be inherent to cancer cells with regard to the specialized tissues from which the cells originated. This is often designated as intrinsic Pgp- mediated MDR. However, overexpression of P-gp may be induced by selection and/or adaptation of cells during exposure to anticancer drugs; this particular example is known as acquired P-gp-mediated MDR. Drugs that are potential inducers of P-gp are often substrates of this transporter. However, several substances that have been proven to not be transportable by P-gp (such as cisplatin or alltrans retinoic acid) could induce minor improvements in P-gp overexpression. It is generally accepted that the drug efflux activity of Pgp is a major cause of reduced cell sensitivity to several compounds. However, P-gp may have side effects that are independent of its drug efflux activity. Several authors have described a direct influence of P-gp on the function of proteins involved in regulatory pathways, including apoptotic progression (such as p53, caspase-3 and Pokemon). Moreover, alterations of cell regulatory pathways, including protein expression, glycosylation and phosphorylation, have been demonstrated in cells overexpressing P-gp, which may consequently induce

  15. New insight into p-glycoprotein as a drug target.

    PubMed

    Breier, Albert; Gibalova, Lenka; Seres, Mario; Barancik, Miroslav; Sulova, Zdenka

    2013-01-01

    Multidrug resistance (MDR) of cancer tissue is a phenomenon in which cancer cells exhibit reduced sensitivity to a large group of unrelated drugs with different mechanisms of pharmacological activity. Mechanisms that reduce cell sensitivity to damage induced by a variety of chemicals were found to be caused by diverse, albeit well-defined, phenotypic alterations. The molecular basis of MDR commonly involves overexpression of the plasma membrane drug efflux pump - P-glycoprotein (P-gp). This glycoprotein is an ABCB1 member of the ABC transporter family. Cells that develop MDR of this type express massive amounts of P-gp that can induce a drug resistance of more than 100 times higher than normal cells to several drugs, which are substrates of P-gp. Expression of P-gp could be inherent to cancer cells with regard to the specialized tissues from which the cells originated. This is often designated as intrinsic Pgp- mediated MDR. However, overexpression of P-gp may be induced by selection and/or adaptation of cells during exposure to anticancer drugs; this particular example is known as acquired P-gp-mediated MDR. Drugs that are potential inducers of P-gp are often substrates of this transporter. However, several substances that have been proven to not be transportable by P-gp (such as cisplatin or alltrans retinoic acid) could induce minor improvements in P-gp overexpression. It is generally accepted that the drug efflux activity of Pgp is a major cause of reduced cell sensitivity to several compounds. However, P-gp may have side effects that are independent of its drug efflux activity. Several authors have described a direct influence of P-gp on the function of proteins involved in regulatory pathways, including apoptotic progression (such as p53, caspase-3 and Pokemon). Moreover, alterations of cell regulatory pathways, including protein expression, glycosylation and phosphorylation, have been demonstrated in cells overexpressing P-gp, which may consequently induce

  16. P-glycoprotein expression in normal and reactive bone marrows.

    PubMed Central

    Hegewisch-Becker, S.; Fliegner, M.; Tsuruo, T.; Zander, A.; Zeller, W.; Hossfeld, D. K.

    1993-01-01

    The expression of mdr1 gene product P-glycoprotein (P-gp) was investigated in 53 normal and reactive bone marrows by means of immunocytochemistry, using the monoclonal antibody (mAb) C219 and the alkaline phosphatase anti-alkaline phosphatase method. In a limited number of patients, data were confirmed by using the mAb MRK16 or a polymerase chain reaction assay for mdr1 gene expression. There was no history of prior chemotherapy or any malignancy in this group. Bone marrow aspirates were obtained as part of a routine diagnostic programme in bone marrow donors or in patients presenting with a variety of diagnoses such as unexplained gammopathy, fever, anaemia, other changes in peripheral blood smear, rheumatoid arthritis, vasculitis, or urticaria pigmentosa. Morphologically the bone marrow was normal in 23 patients, a megaloblastic erythropoiesis was seen in two patients and unspecific changes were seen in 28 patients. Twenty-seven of 53 samples were found to be positive for P-gp expression with the percentage of positive cells ranging from 2%-80% (mean = 24%). With a cutoff point of 10%, five of 23 normal (22%) and 13 of 28 reactive bone marrows (46%) were considered positive for P-gp expression. There was no obvious correlation between diagnosis or age and P-gp expression. Additional staining for the early surface marker CD-34 was performed in 12 samples, with none of them revealing more than 1% positivity. Since P-gp expression has so far been described only in CD-34 positive bone marrow cells, data suggest that P-gp expression may be reinduced in CD-34 negative cells under conditions which remain to be determined. Images Figure 1 Figure 2 PMID:8094974

  17. Aldosterone-induced glycoproteins: electrophysiological-biochemical correlation.

    PubMed

    Szerlip, H M; Weisberg, L; Geering, K; Rossier, B C; Cox, M

    1988-05-01

    Aldosterone induces the synthesis of a group of glycoproteins (GP65,70) in toad urinary bladders which are potential effectors of the natriferic action of this hormone. In the present study we have confirmed that aldosterone produces a two-phase electrophysiological response. During the early phase (less than 3 h) short-circuit current and transepithelial conductance increase in parallel, while during the late phase (greater than 3 h) short-circuit current continues to increase without any further change in conductance. By biosynthetically labeling aldosterone-treated toad bladders with [35S]methionine either during the early (h 0-2 or 1-3) or the late (h 4-6 or 7-9) phases of the natriferic response, we have demonstrated that GP65,70 is synthesized as a late effect of aldosterone. Since synthesis of GP65,70 occurs at a time when the electromotive force of the Na+ pump is increasing, and since GP65,70 biochemically resembles the beta subunit of Na+/K+-ATPase, studies were undertaken to examine whether GP65,70 is the beta subunit. Purified amphibian renal beta subunit was analyzed by two-dimensional polyacrylamide gel electrophoresis and was found to have an isoelectric point and Mr value similar to those of GP65,70. However, when nitrocellulose blots containing wheat germ agglutinin-purified proteins from aldosterone-treated bladders were stained with monospecific polyclonal antibodies developed against the beta subunit, GP65,70 was not recognized, whereas a group of slightly more acidic proteins of similar Mr were recognized. Thus, GP65,70 is not the beta subunit of Na+/Ka+-ATPase. Further studies are needed to determine the cellular function of GP65,70. PMID:2835098

  18. Synthesis and P-glycoprotein induction activity of colupulone analogs.

    PubMed

    Bharate, Jaideep B; Batarseh, Yazan S; Wani, Abubakar; Sharma, Sadhana; Vishwakarma, Ram A; Kaddoumi, Amal; Kumar, Ajay; Bharate, Sandip B

    2015-05-21

    Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 μM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 μM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol displayed a significant increase (29%) in Aβ transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent.

  19. Multiple Drug Transport Pathways through Human P-Glycoprotein.

    PubMed

    McCormick, James W; Vogel, Pia D; Wise, John G

    2015-07-21

    P-Glycoprotein (P-gp) is a plasma membrane efflux pump that is commonly associated with therapy resistances in cancers and infectious diseases. P-gp can lower the intracellular concentrations of many drugs to subtherapeutic levels by translocating them out of the cell. Because of the broad range of substrates transported by P-gp, overexpression of P-gp causes multidrug resistance. We reported previously on dynamic transitions of P-gp as it moved through conformations based on crystal structures of homologous ABCB1 proteins using in silico targeted molecular dynamics techniques. We expanded these studies here by docking transport substrates to drug binding sites of P-gp in conformations open to the cytoplasm, followed by cycling the pump through conformations that opened to the extracellular space. We observed reproducible transport of two substrates, daunorubicin and verapamil, by an average of 11-12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methylpyrophosphate, a ligand that should not be transported by P-gp, did not show this movement through P-gp. Drug binding to either of two subsites on P-gp appeared to determine the initial pathway used for drug movement through the membrane. The specific side-chain interactions with drugs within each pathway seemed to be, at least in part, stochastic. The docking and transport properties of a P-gp inhibitor, tariquidar, were also studied. A mechanism of inhibition by tariquidar that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp is presented.

  20. Molecular insight into conformational transmission of human P-glycoprotein

    SciTech Connect

    Chang, Shan-Yan; Liu, Fu-Feng E-mail: ysun@tju.edu.cn; Dong, Xiao-Yan; Sun, Yan E-mail: ysun@tju.edu.cn

    2013-12-14

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  1. Molecular insight into conformational transmission of human P-glycoprotein

    NASA Astrophysics Data System (ADS)

    Chang, Shan-Yan; Liu, Fu-Feng; Dong, Xiao-Yan; Sun, Yan

    2013-12-01

    P-glycoprotein (P-gp), a kind of ATP-binding cassette transporter, can export candidates through a channel at the two transmembrane domains (TMDs) across the cell membranes using the energy released from ATP hydrolysis at the two nucleotide-binding domains (NBDs). Considerable evidence has indicated that human P-gp undergoes large-scale conformational changes to export a wide variety of anti-cancer drugs out of the cancer cells. However, molecular mechanism of the conformational transmission of human P-gp from the NBDs to the TMDs is still unclear. Herein, targeted molecular dynamics simulations were performed to explore the atomic detail of the conformational transmission of human P-gp. It is confirmed that the conformational transition from the inward- to outward-facing is initiated by the movement of the NBDs. It is found that the two NBDs move both on the two directions (x and y). The movement on the x direction leads to the closure of the NBDs, while the movement on the y direction adjusts the conformations of the NBDs to form the correct ATP binding pockets. Six key segments (KSs) protruding from the TMDs to interact with the NBDs are identified. The relative movement of the KSs along the y axis driven by the NBDs can be transmitted through α-helices to the rest of the TMDs, rendering the TMDs to open towards periplasm in the outward-facing conformation. Twenty eight key residue pairs are identified to participate in the interaction network that contributes to the conformational transmission from the NBDs to the TMDs of human P-gp. In addition, 9 key residues in each NBD are also identified. The studies have thus provided clear insight into the conformational transmission from the NBDs to the TMDs in human P-gp.

  2. Synthesis and P-glycoprotein induction activity of colupulone analogs.

    PubMed

    Bharate, Jaideep B; Batarseh, Yazan S; Wani, Abubakar; Sharma, Sadhana; Vishwakarma, Ram A; Kaddoumi, Amal; Kumar, Ajay; Bharate, Sandip B

    2015-05-21

    Brain amyloid-beta (Aβ) plaques are one of the primary hallmarks associated with Alzheimer's disease (AD) pathology. Efflux pump proteins located at the blood-brain barrier (BBB) have been reported to play an important role in the clearance of brain Aβ, among which the P-glycoprotein (P-gp) efflux transporter pump has been shown to play a crucial role. Thus, P-gp has been considered as a potential therapeutic target for treatment of AD. Colupulone, a prenylated phloroglucinol isolated from Humulus lupulus, is known to activate pregnane-X-receptor (PXR), which is a nuclear receptor controlling P-gp expression. In the present work, we aimed to synthesize and identify analogs of colupulone that are potent P-gp inducer(s) with an ability to enhance Aβ transport across the BBB. A series of colupulone analogs were synthesized by modifications at both prenyl as well as acyl domains. All compounds were screened for P-gp induction activity using a rhodamine 123 based efflux assay in the P-gp overexpressing human adenocarcinoma LS-180 cells, wherein all compounds showed significant P-gp induction activity at 5 μM. In the western blot studies in LS-180 cells, compounds 3k and 5f were able to induce P-gp as well as LRP1 at 1 μM. The effect of compounds on the Aβ uptake and transport was then evaluated. Among all tested compounds, diprenylated acyl phloroglucinol displayed a significant increase (29%) in Aβ transport across bEnd3 cells grown on inserts as a BBB model. The results presented here suggest the potential of this scaffold to enhance clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent. PMID:25875530

  3. Toremifene interacts with and destabilizes the Ebola virus glycoprotein.

    PubMed

    Zhao, Yuguang; Ren, Jingshan; Harlos, Karl; Jones, Daniel M; Zeltina, Antra; Bowden, Thomas A; Padilla-Parra, Sergi; Fry, Elizabeth E; Stuart, David I

    2016-07-01

    Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits), which is solely responsible for host cell attachment, endosomal entry and membrane fusion. GP is thus a primary target for the development of antiviral drugs. Here we report the first, to our knowledge, unliganded structure of EBOV GP, and high-resolution complexes of GP with the anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered. Unexpectedly, both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the three-fold axis. Protein–drug interactions with both GP1 and GP2 are predominately hydrophobic. Residues lining the binding site are highly conserved among filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in the protein melting temperature after toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. These results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, thereby preventing fusion between the viral and endosome membranes. Thus, these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs. PMID:27362232

  4. Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.

    PubMed Central

    Hays, L M; Feeney, R E; Crowe, L M; Crowe, J H; Oliver, A E

    1996-01-01

    Antifreeze glycoproteins (AFGPs), found in the blood of polar fish at concentrations as high as 35 g/liter, are known to prevent ice crystal growth and depress the freezing temperature of the blood. Previously, Rubinsky et al. [Rubinsky, B., Mattioli, M., Arav, A., Barboni, B. & Fletcher, G. L. (1992) Am. J. Physiol. 262, R542-R545] provided evidence that AFGPs block ion fluxes across membranes during cooling, an effect that they ascribed to interactions with ion channels. We investigated the effects of AFGPs on the leakage of a trapped marker from liposomes during chilling. As these liposomes are cooled through the transition temperature, they leak approximately 50% of their contents. Addition of less than 1 mg/ml of AFGP prevents up to 100% of this leakage, both during chilling and warming through the phase transition. This is a general effect that we show here applies to liposomes composed of phospholipids with transition temperatures ranging from 12 degrees C to 41 degrees C. Because these results were obtained with liposomes composed of phospholipids alone, we conclude that the stabilizing effects of AFGPs on intact cells during chilling reported by Rubinsky et al. may be due to a nonspecific effect on the lipid components of native membranes. There are other proteins that prevent leakage, but only under specialized conditions. For instance, antifreeze proteins, bovine serum albumin, and ovomucoid all either have no effect or actually induce leakage. Following precipitation with acetone, all three proteins inhibited leakage, although not to the extent seen with AFGPs. Alternatively, there are proteins such as ovotransferrin that have no effect on leakage, either before or after acetone precipitation. PMID:8692905

  5. Interaction of the P-Glycoprotein Multidrug Transporter with Sterols.

    PubMed

    Clay, Adam T; Lu, Peihua; Sharom, Frances J

    2015-11-01

    The ABC transporter P-glycoprotein (Pgp, ABCB1) actively exports structurally diverse substrates from within the lipid bilayer, leading to multidrug resistance. Many aspects of Pgp function are altered by the phospholipid environment, but its interactions with sterols remain enigmatic. In this work, the functional interaction between purified Pgp and various sterols was investigated in detergent solution and proteoliposomes. Fluorescence studies showed that dehydroergosterol, cholestatrienol, and NBD-cholesterol interact intimately with Pgp, resulting in both quenching of protein Trp fluorescence and enhancement of sterol fluorescence. Kd values indicated binding affinities in the range of 3-9 μM. Collisional quenching experiments showed that Pgp-bound NBD-cholesterol was protected from the external milieu, resonance energy transfer was observed between Pgp Trp residues and the sterol, and the fluorescence emission of bound sterol was enhanced. These observations suggested an intimate interaction of bound sterols with the transporter at a protected nonpolar site. Cholesterol hemisuccinate altered the thermal unfolding of Pgp and greatly stabilized its basal ATPase activity in both a detergent solution and reconstituted proteoliposomes of certain phospholipids. Other sterols, including dehydroergosterol, did not stabilize the basal ATPase activity of detergent-solubilized Pgp, which suggests that this is not a generalized sterol effect. The phospholipid composition and cholesterol hemisuccinate content of Pgp proteoliposomes altered the basal ATPase and drug transport cycles differently. Sterols may interact with Pgp and modulate its structure and function by occupying part of the drug-binding pocket or by binding to putative consensus cholesterol-binding (CRAC/CARC) motifs located within the transmembrane domains.

  6. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition.

    PubMed

    Filipski, Elisabeth; Berland, Elodie; Ozturk, Narin; Guettier, Catherine; van der Horst, Gijsbertus T J; Lévi, Francis; Okyar, Alper

    2014-02-01

    The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F1 mice. A three-fold 24h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p<0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50mg/kg/day i.v.×4days) as a single agent or combined with P-gp inhibitor PSC833 (6.25mg/kg/day i.p.×4 days) at ZT3 or ZT15, respectively corresponding to the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40mg/kg/day×4days) and +/-PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p<0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p<0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ~60%. In tumor-bearing mice, body weight loss was ~halved in the mice on irinotecan or irinotecan-PSC833 combination at ZT15 as compared to ZT3 (p<0.001). PSC833-irinotecan at ZT15 increased tumor inhibition by ~40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan.

  7. Pneumocystis carinii glycoprotein A binds macrophage mannose receptors.

    PubMed Central

    O'Riordan, D M; Standing, J E; Limper, A H

    1995-01-01

    Pneumocystis carinii causes life-threatening pneumonia in patients with impaired immunity. Recent studies suggest that alveolar macrophages interact with P. carinii through macrophage mannose receptors. However, the ligand(s) on P. carinii that is recognized by these receptors has not been fully defined. P. carinii contains a major mannose-rich surface antigen complex termed glycoprotein A (gpA). It was therefore hypothesized that gpA binds directly to macrophage mannose receptors and mediates organism attachment to these phagocytes. To assess this, gpA was purified from P. carinii by continuous-elution gel electrophoresis. 125I-labeled gpA bound to alveolar macrophages in a saturable fashion. In addition, gpA binding was substantially inhibited by both alpha-mannan and EDTA, further suggesting that gpA interacts with macrophage mannose receptors. Macrophage membrane proteins capable of binding to gpA were isolated with a gpA-Sepharose column. A 165-kDa membrane-associated protein was specifically eluted from the gpA-Sepharose column with EDTA (20 mM). This protein was identified as the macrophage mannose receptor by immunoprecipitation with a polyclonal anti-mannose receptor antiserum. To further investigate the role of gpA in P. carinii-macrophage interactions, 51Cr-labeled P. carinii cells were incubated with macrophages in the presence of increasing concentrations of soluble gpA, and organism attachment was quantified. Soluble gpA (2.5 mg/dl) competitively inhibited P. carinii attachment to alveolar macrophages by 51.3% +/- 3.7% (P = 0.01). Our findings demonstrate that gpA present on P. carinii interacts directly with mannose receptors, thereby mediating organism attachment to alveolar macrophages. PMID:7868247

  8. Purification and structural characterization of herpes simplex virus glycoprotein C

    SciTech Connect

    Kikuchi, G.E.; Baker, S.A.; Merajver, S.D.; Coligan, J.E.; Levine, M.; Glorioso, J.C.; Nairn, R.

    1987-01-27

    Purification of herpes simplex virus glycoprotein C (gC) in microgram amounts yielded sufficient material for an analysis of its secondary structure. Purification was facilitated by using the mutant virus gC-3, which bears a point mutation that interrupts the putative hydrophobic membrane anchor sequence, causing the secretion of gC-3 protein into the cell culture medium. gC-3 protein was purified by size fractionation of concentrated culture medium from infected cells on a gel filtration column of Sephacryl S-200, followed by immunoaffinity chromatography on a column constructed of gC-specific monoclonal antibodies cross-linked to a protein A-Sepharose CL-4B matrix. Purified gC-3 had a molecular weight of 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size expected for gC, was reactive with gC-specific monoclonal antibodies in protein immunoblots, and contained amino acid sequences characteristic of gC as determined by radiochemical amino acid microsequence analyses. Polyclonal antisera obtained from a rabbit immunized with gC-3 reacted with wild-type gC in immunoprecipitation, enzyme immunoassay, and immunoelectroblot (western blot) assays. Deglycosylation by treatment with trifluoromethanesulfonic acid reduced the molecular weight of gC-3 by approximately 35%. Analyses of both native and deglycosylated gC-3 by Raman spectroscopy showed that the native molecule consists of about 17%..cap alpha..-helix, 24% ..beta..-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% ..cap alpha..-helix, 10% ..beta..-sheet, 81% disordered structures. These data were in good agreement with the 11% ..cap alpha..-helix, 18% ..beta..-sheet, 61% ..beta..-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3.

  9. Alterations of intestinal glycoprotein hydrolases in congenital diabetes

    SciTech Connect

    Najjar, S.M.

    1989-01-01

    The diabetic BioBreed (BB{sub d}) rat was used for the study of the molecular structure of intestinal brush border sucrase-{alpha}-dextrinase (SD) and aminooligopeptidase (AOP) in diabetes mellitus. The specific catalytic activity of S-D and AOP in the BB{sub d} rat is normal. However, solid-phase radioimmunoassay revealed loss of some antigenic determinants in the BB{sub d} rat. S-D and AOP migrated abnormally on 6% SDS-gel electrophoresis in the BB{sub d} rat. S was larger (+5 kDa), D was either smaller (-5 kDa) or unaltered, and AOP was smaller (-5 kDa) in the BB{sub d} than in the normal Wistar. The structural abnormalities were independent of hyperglycemia or ketoacidosis and restored to normal by daily insulin treatment (NPH, 3-4 units/rat) for two to three weeks. Newly-synthesized brush border hydrolases were examined after 6 hours of intraperitoneal injection of ({sup 35}S) methionine (2 mCi) and found to be altered, suggesting that structural abnormality appeared acutely during intracellular synthesis rather than being due to slow extracellular modifications such as non-enzymatic glycosylation. Deglycosylation of brush border proteins by trifluoromethanesulfonic acid resulted in an apoprotein with normal electrophoretic migration in BB{sub d}, indicating that the alteration was due to the carbohydrates component of the glycoprotein. Pulse-chase studies with ({sup 35}S) methionine were consistent with normal protein an co-translational and initial N-linked carbohydrate assembly in association with the endoplasmic reticulum in BB{sub d}. However, the post-translational maturation of N-linked and addition of 0-linked carbohydrate chains in Golgi were prolonged, and produced a larger single-chain precursor of S-D in BB{sub d} than normal.

  10. Thyroid Hormone and P-Glycoprotein in Tumor Cells

    PubMed Central

    Davis, Paul J.; Lin, Hung-Yun; Sudha, Thangirala; Mousa, Shaker A.

    2015-01-01

    P-glycoprotein (P-gp; multidrug resistance pump 1, MDR1; ABCB1) is a plasma membrane efflux pump that when activated in cancer cells exports chemotherapeutic agents. Transcription of the P-gp gene (MDR1) and activity of the P-gp protein are known to be affected by thyroid hormone. A cell surface receptor for thyroid hormone on integrin αvβ3 also binds tetraiodothyroacetic acid (tetrac), a derivative of L-thyroxine (T4) that blocks nongenomic actions of T4 and of 3,5,3′-triiodo-L-thyronine (T3) at αvβ3. Covalently bound to a nanoparticle, tetrac as nanotetrac acts at the integrin to increase intracellular residence time of chemotherapeutic agents such as doxorubicin and etoposide that are substrates of P-gp. This action chemosensitizes cancer cells. In this review, we examine possible molecular mechanisms for the inhibitory effect of nanotetrac on P-gp activity. Mechanisms for consideration include cancer cell acidification via action of tetrac/nanotetrac on the Na+/H+ exchanger (NHE1) and hormone analogue effects on calmodulin-dependent processes and on interactions of P-gp with epidermal growth factor (EGF) and osteopontin (OPN), apparently via αvβ3. Intracellular acidification and decreased H+ efflux induced by tetrac/nanotetrac via NHE1 is the most attractive explanation for the actions on P-gp and consequent increase in cancer cell retention of chemotherapeutic agent-ligands of MDR1 protein. PMID:25866761

  11. On-line identification of P-glycoprotein substrates by monitoring of extracellular acidification and respiration rates in living cells.

    PubMed

    Seeland, Swen; Treiber, Alexander; Hafner, Mathias; Huwyler, Jörg

    2011-07-01

    The influence of P-glycoprotein (ABCB1) in drug resistance as well as drug absorption and disposition is an important factor to be considered during the development of new drugs. Thus, the early identification and exclusion of compounds showing a high affinity towards P-glycoprotein can help to select drug candidates. The aim of our study was to implement a label-free assay for the identification of P-glycoprotein substrates in living cells. For this approach, a multiparametric, chip-based sensor system was used to determine extracellular acidification, cell respiration and adhesion upon stimulation with P-glycoprotein substrates. Using L-MDR1 cells, a human P-glycoprotein overexpressing cell line, the influence of P-glycoprotein activity was determined for seven different compounds, demonstrating the applicability of the system for P-glycoprotein substrate identification. Effects were concentration dependent, as shown for the P-glycoprotein substrate verapamil, and were associated with cellular acidification and respiration. P-glycoprotein ATPase activation by verapamil could be described by a Michaelis-Menten type kinetic profile showing saturation at high substrate concentrations. The Michaelis-Menten constants K(M) were determined to be 0.92μM (calculated based on extracellular acidification) and 4.9μM (calculated based on cellular respiration). Control experiments using 100nM of the P-glycoprotein inhibitor elacridar indicated that the observed effects were related to P-glycoprotein ATPase activity. In contrast, wild-type LLC-PK1 cells not expressing P-glycoprotein were not responsive towards stimulation with different P-glycoprotein substrates. Summarizing these findings, the used microsensor system is a generic system suitable for the identification of P-glycoprotein substrates. In contrast to biochemical P-glycoprotein assays, activation of the drug efflux pump can be monitored on-line in living cells to identify P-glycoprotein substrates and to study the

  12. Envelope glycoproteins of HIV-1, HIV-2, and SIV purified with Galanthus nivalis agglutinin induce strong immune responses.

    PubMed

    Gilljam, G

    1993-05-01

    Lectin affinity chromatography was used to purify in a single step the envelope glycoproteins of HIV-1, HIV-2, and SIV. Envelope glycoproteins carry the major determinants essential for protection by the humoral immune response. The purification of these proteins has previously been a laborious procedure. The glycoproteins were purified by a one-step procedure to a high level of purity by using Galanthus nivalis agglutinin (GNA). The purified glycoprotein had CD4-binding and antigenic reactivities. Strong immune responses to envelope proteins and peptides were seen in mice and primates after immunization with these preparations.

  13. A major proportion of N-glycoproteins are transiently glucosylated in the endoplasmic reticulum

    SciTech Connect

    Ganan, S.; Cazzulo, J.J.; Parodi, A.J. )

    1991-03-26

    N-Linked, high-mannose-type oligosaccharides lacking glucose residues may be transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum of mammalian, plant, fungal, and protozoan cells. The products formed have been identified as N-linked Glc{sub 1}Man{sub 5-9}GlcNAc{sub 2} and glucosidase II is apparently the enzyme responsible for the in vivo deglucosylation of the compounds. As newly glucosylated glycoproteins are immediately deglucosylated, it is unknown whether transient glucosylation involves all or nearly all N-linked glycoproteins or if, on the contrary, it only affects a minor proportion of them. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated, cells of the trypanosomatid protozoan Trypanosoma cruzi (a parasite transferring Man{sub 9}GlcNAc{sub 2} in protein N-glycosylation) were grown in the presence of ({sup 14}C)glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and castanospermine that were more than 1,000-fold higher than those required to produce a 50% inhibition of the T. cruzi enzyme. No evidence for the presence of an endomannosidase yielding GlcMan from the glucosylated compounds was obtained. As the average number of N-linked oligosaccharides per molecule in glycoproteins is higher than one, these results indicate that more than 52-33% of total glycoproteins are glucosylated and that transient glucosylation is a major event in the normal processing of glycoproteins.

  14. Membrane Glycoproteins Associated with Breast Tumor Cell Progression Identified by a Lectin Affinity Approach

    PubMed Central

    Wang, Yanfei; Ao, Xiaoping; Vuong, Huy; Konanur, Meghana; Miller, Fred R.; Goodison, Steve; Lubman, David M.

    2008-01-01

    The membrane glycoprotein component of the cellular proteome represents a promising source for potential disease biomarkers and therapeutic targets. Here we describe the development of a method that facilitates the analysis of membrane glycoproteins and apply it to the differential analysis of breast tumor cells with distinct malignant phenotypes. The approach combines two membrane extraction procedures, and enrichment using ConA and WGA lectin affinity columns, prior to digestion and analysis by LC–MS/MS. The glycoproteins are identified and quantified by spectral counting. Although the distribution of glycoprotein expression as a function of MW and pI was very similar between the two related cell lines tested, the approach enabled the identification of several distinct membrane glycoproteins with an expression index correlated with either a precancerous (MCF10AT1), or a malignant, metastatic cellular phenotype (MCF10CA1a). Among the proteins associated with the malignant phenotype, Gamma-glutamyl hydrolase, CD44, Galectin-3-binding protein, and Syndecan-1 protein have been reported as potential biomarkers of breast cancer. PMID:18729497

  15. Haemonchus contortus P-glycoprotein-2: in situ localisation and characterisation of macrocyclic lactone transport.

    PubMed

    Godoy, Pablo; Lian, Jing; Beech, Robin N; Prichard, Roger K

    2015-01-01

    Haemonchus contortus is a veterinary nematode that infects small ruminants, causing serious decreases in animal production worldwide. Effective control through anthelmintic treatment has been compromised by the development of resistance to these drugs, including the macrocyclic lactones. The mechanisms of resistance in H. contortus have yet to be established but may involve efflux of the macrocyclic lactones by nematode ATP-binding-cassette transporters such as P-glycoproteins. Here we report the expression and functional activity of H. contortus P-glycoprotein 2 expressed in mammalian cells and characterise its interaction with the macrocyclic lactones, ivermectin, abamectin and moxidectin. The ability of H. contortus P-glycoprotein 2 to transport different fluorophore substrates was markedly inhibited by ivermectin and abamectin in a dose-dependent and saturable way. The profile of transport inhibition by moxidectin was markedly different. H. contortus P-glycoprotein 2 was expressed in the pharynx, the first portion of the worm's intestine and perhaps in adjacent nervous tissue, suggesting a role for this gene in regulating the uptake of avermectins and in protecting nematode tissues from the effects of macrocyclic lactone anthelmintic drugs. H. contortus P-glycoprotein 2 may thus contribute to resistance to these drugs in H. contortus.

  16. Archaeal S-layer glycoproteins: post-translational modification in the face of extremes

    PubMed Central

    Kandiba, Lina; Eichler, Jerry

    2014-01-01

    Corresponding to the sole or basic component of the surface (S)-layer surrounding the archaeal cell in most known cases, S-layer glycoproteins are in direct contact with the harsh environments that characterize niches where Archaea can thrive. Accordingly, early work examining archaeal S-layer glycoproteins focused on identifying those properties that allow members of this group of proteins to maintain their structural integrity in the face of extremes of temperature, pH, and salinity, as well as other physical challenges. However, with expansion of the list of archaeal strains serving as model systems, as well as growth in the number of molecular tools available for the manipulation of these strains, studies on archaeal S-layer glycoproteins are currently more likely to consider the various post-translational modifications these polypeptides undergo. For instance, archaeal S-layer glycoproteins can undergo proteolytic cleavage, both N- and O-glycosylation, lipid-modification and oligomerization. In this mini-review, recent findings related to the post-translational modification of archaeal S-layer glycoproteins are considered. PMID:25505464

  17. [Diagnostic value of the determination of serum alpha2-HS-glycoprotein].

    PubMed

    Kalabay, L; Cseh, K; Jakab, L; Pozsonyi, T; Jakab, L; Benedek, S; Fekete, S; Telegdy, L

    1992-06-21

    Opsonic glycoprotein, alpha 2-HS-glycoprotein concentration was studied in the serum of 753 patients with various hematological, malignant, immunological, metabolic, endocrine and liver diseases and 68 healthy controls. Decreased serum alpha 2-HS-glycoprotein levels were detected in patients with acute leukemias, chronic granulocyte and myelomonocyte leukemias, lymphomas, myelofibrosis, multiple myeloma, metastatizing solid tumors, systemic lupus erythematosus, rheumatoid arthritis, acute alcoholic hepatitis, fatty liver, chronic active hepatitis, liver cirrhosis, acute and chronic pancreatitis, and Crohn's disease. Elevated levels were measured in patients with B and NANB/C hepatitis. Further decreased levels were observed in some groups with secondary infections. Serum alpha 2-HS-glycoprotein levels are affected by many factors, influencing the synthesis and elimination of the protein. The detection of serum alpha 2-HS-glycoprotein concentration has no specific diagnostic value as a marker for tumors or other diseases, however, its determination can be useful for the assessment of a non-specific regulator of the host defence.

  18. Serum alpha 2-HS glycoprotein concentration in patients with hematological malignancies. A follow-up study.

    PubMed

    Kalabay, L; Cseh, K; Benedek, S; Fekete, S; Masszi, T; Herjeczki, K; Pozsonyi, T; Jakab, L; Jakab, L

    1991-11-01

    We observed significantly reduced serum alpha 2-HS glycoprotein concentrations in patients with acute lymphocytic, acute nonlymphocytic, chronic granulocytic and chronic myelomonocytic leukemias, Hodgkin's and non-Hodgkin's lymphomas, myelofibrosis, and multiple myeloma, but not in patients with chronic lymphocytic leukemia and polycythemia vera, as compared with healthy controls. We followed the serum level of the protein for 18 months. Patients with infectious complications, those receiving cytostatic treatment, and those in the preterminal period had further reduced serum alpha 2-HS glycoprotein levels. The reduction of serum alpha 2-HS glycoprotein concentration was primarily due to decreased production caused by infiltration of the liver, a hepatotoxic effect of cytostatic treatment, and, to a lesser degree, to increased consumption. We found statistically significant negative correlations between serum alpha 2-HS glycoprotein concentration and erythrocyte sedimentation rate, serum aspartate aminotransferase and alkaline phosphatase activities, and IgG and IgM concentrations. The determination of the alpha 2-HS glycoprotein concentration is useful for the assessment and follow-up of the clinical status and therapy of patients with hematological malignancies and also has prognostic significance.

  19. Confident assignment of site-specific glycosylation in complex glycoproteins in a single step.

    PubMed

    Khatri, Kshitij; Staples, Gregory O; Leymarie, Nancy; Leon, Deborah R; Turiák, Lilla; Huang, Yu; Yip, Shun; Hu, Han; Heckendorf, Christian F; Zaia, Joseph

    2014-10-01

    A glycoprotein may contain several sites of glycosylation, each of which is heterogeneous. As a consequence of glycoform diversity and signal suppression from nonglycosylated peptides that ionize more efficiently, typical reversed-phase LC-MS and bottom-up proteomics database searching workflows do not perform well for identification of site-specific glycosylation for complex glycoproteins. We present an LC-MS system for enrichment, separation, and analysis of glycopeptides from complex glycoproteins (>4 N-glycosylation sequons) in a single step. This system uses an online HILIC enrichment trap prior to reversed-phase C18-MS analysis. We demonstrated the effectiveness of the system using a set of glycoproteins including human transferrin (2 sequons), human alpha-1-acid glycoprotein (5 sequons), and influenza A virus hemagglutinin (9 sequons). The online enrichment renders glycopeptides the most abundant ions detected, thereby facilitating the generation of high-quality data-dependent tandem mass spectra. The tandem mass spectra exhibited product ions from both glycan and peptide backbone dissociation for a majority of the glycopeptides tested using collisionally activated dissociation that served to confidently assign site-specific glycosylation. We demonstrated the value of our system to define site-specific glycosylation using a hemagglutinin containing 9 N-glycosylation sequons from a single HILIC-C18-MS acquisition.

  20. Identification of glycoproteins associated with HIV latently infected cells using quantitative glycoproteomics

    PubMed Central

    Yang, Weiming; Jackson, Brooks; Zhang, Hui

    2016-01-01

    HIV infection is not curable due to viral latency. Compelling reports suggest that there is a distinct profile of surface proteins that can be used for targeting latently infected cells. We have recently reported that glycoproteins were differentially secreted from HIV latently infected ACH-2 cells compared to the parental A3.01 cells. This finding suggests that glycophenotype might be different in these two cell lines. To determine the difference, the ACH-2 and A3.01 cell lines were subjected to a glycoproteomic analysis. A total number of 940 unique N-linked glycosite-containing peptides from 515 glycoproteins were identified. Among the glycoproteins, 365 and 104 were annotated as cell surface and membrane-associated proteins, respectively. Quantitative LC-MS/MS analysis revealed a change of 236 glycosite-containing peptides from 172 glycoproteins between the two cell lines without reactivation. Bioinformatic analysis suggests that cell adhesion, immune response, glycoprotein metabolic process, cell motion, and cell activation were associated with the changed proteins. After reactivation of latency, changes in glycosite-containing peptides were observed in both cell lines. The changed proteins suggest that cell migration, response to wounding and immune response might be impaired in reactivated latently infected cells. Glycoproteomics merits future application using primary cells to discover reveal mechanisms in HIV pathogenesis. PMID:27195445

  1. Study on the extraction and purification of glycoprotein from the yellow seahorse, Hippocampus kuda Bleeker.

    PubMed

    Su, Yuting; Xu, Yongjian

    2015-07-01

    The optimum parameters of extraction for glycoprotein from seahorse were examined and determined by Box-Behnken combined with ultrasonic extraction technology. Column chromatography of glycoprotein was used for further purification. The optimal extraction conditions of seahorse glycoprotein were extracting time 4.3 h, salt concentration 0.08 mol/L, extracting temperature 73°C, raw material, and water ratio 1:6. At the optimal conditions, the yield of saccharide reached to 1.123%, and the yield of protein reached to 5.898%. For purifying the crude glycoprotein, the stage renounces of DEAE-52 column chromatography were done, respectively, with 0.05, 0.1, 0.5 mol/L NaHCO3 solution, and further purification was done with Sephadex G-100 column chromatography. Finally, two pieces of seahorse glycoprotein were obtained by the column chromatography, that is, HG-11 and HG-21. The saccharide content was 56.7975% and 39.479%, the protein content was 30.5475% and 51.747%, respectively.

  2. Immunological responses to envelope glycoprotein 120 from subtypes of human immunodeficiency virus type 1.

    PubMed

    Gilljam, G; Svensson, A; Ekström, A; Wahren, B

    1999-07-01

    The outer envelope glycoprotein (gp120) from subtypes A-E of HIV-1 was purified using a specific high mannose-binding lectin, Galanthus nivalis agglutinin. All isolates were grown in peripheral blood lymphocyte cells in order to avoid selection in cell lines. A comparison of the reactivities of the envelope proteins was made using sera from patients infected with the different subtypes. In this study, the B and C subtype envelope glycoproteins showed the strongest immunological reactivity, when reacted with sera from patients infected with the same subtype of virus. On the other hand, sera of patients infected with subtype A or C virus had the strongest and broadest reactivities, to envelope glycoproteins of many subtypes. The purified gp120 proteins from all five subtypes stimulated mononuclear cells from HIV-1 (subtype B)-infected patients, indicating conserved T cell-activating epitopes. The immunological reactivities indicate that strong antigenicity does not always predict the broadest immunogenicity of an envelope glycoprotein. Glycoprotein 120 from foreign subtypes may serve to induce strong cross-reactive immune responses.

  3. HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

    PubMed

    Albecka, Anna; Laine, Romain F; Janssen, Anne F J; Kaminski, Clemens F; Crump, Colin M

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

  4. Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation

    SciTech Connect

    Piwnica-Worms, D.; Vallabhaneni, V.R.; Kronauge, J.F.

    1995-09-26

    Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an {approximately}170 integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis(2-methoxyisobutyl isonitrile) technetium(I) (Tc-SESTAMIBI), a {gamma}-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (T{sub 1/2} {approx} 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. 70 refs., 7 figs., 2 tabs.

  5. Recognition of glycoprotein peroxidase via Con A-carrying self-assembly layer on gold.

    PubMed

    Liu, Songqin; Wang, Kewei; Du, Dan; Sun, Yueming; He, Lin

    2007-07-01

    We have successfully fabricated a self-assembled layer of concanavalin A (Con A) on a gold surface for recognition of glycoproteins. The type IV Con A is covalently bound to 11-mercaptoundecanoic acid (MUA) on gold with a 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) linkage. The binding interaction between glycoproteins and self-assembled Con A is studied using horseradish peroxidase (HRP) as a model glycoprotein. Voltammetric, electrochemical impedance studies, and photometric activity measurements show the presence of both specific and nonspecific bindings of HRP to the Con A interface. The specific binding is attributed to the Con A-sugar interaction where Con A selectively recognizes the glycosylation sites of HRP. The catalytic current of the HRP-loaded electrode, because of catalytic oxidation of thionine in the presence of hydrogen peroxide (H2O2), is found to be proportional to the HRP concentrations in the incubation solution. A linear correlation coefficient of 0.993 was obtained over a wide HRP concentration range of 12.5 microg/mL to 1 mg/mL. The approach described in this study provides a simple yet selective means to immobilize glycoproteins on a solid support. The specific binding achieved is desirable in biosensor fabrication, glycoprotein separation, recognition, and purification as well as in drug-releasing systems.

  6. Defence sugarcane glycoproteins disorganize microtubules and prevent nuclear polarization and germination of Sporisorium scitamineum teliospores.

    PubMed

    Sánchez-Elordi, Elena; Baluška, František; Echevarría, Clara; Vicente, Carlos; Legaz, M Estrella

    2016-08-01

    Microtubules (MTs) are involved in the germination of Sporisorium scitamineum teliospores. Resistant varieties of sugar cane plants produce defence glycoproteins that prevent the infection of the plants by the filamentous fungi Sporisorium scitamineum. Here, we show that a fraction of these glycoproteins prevents the correct arrangement of MTs and causes nuclear fragmentation defects. As a result, nuclei cannot correctly migrate through the growing hyphae, causing germinative failure. Arginase activity contained in defence glycoproteins is already described for preventing fungal germination. Now, its enzymatically active form is presented as a link between the defensive capacity of glycoproteins and the MT disorganization in fungal cells. Active arginase is produced in healthy and resistant plants; conversely, it is not detected in the juice from susceptible varieties, which explains why MT depolarization, nuclear disorganization as well as germination of teliospores are not significantly affected by glycoproteins from non-resistant plants. Our results also suggest that susceptible plants try to increase their levels of arginase after detecting the presence of the pathogen. However, this signal comes "too late" and such defensive mechanism fails. PMID:27372179

  7. Characterization of Murine Brain Membrane Glycoproteins by Detergent Assisted Lectin Affinity Chromatography (DALAC)

    PubMed Central

    Wei, Xin; Dulberger, Charles; Li, Lingjun

    2010-01-01

    Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization has been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that under certain concentrations, detergents can minimize the nonspecific bindings and facilitate the elution of hydrophobic glycoproteins. With the Detergent Assisted Lectin Affinity Chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. 699 proteins were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome. PMID:20700909

  8. P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

    PubMed

    Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

    2010-03-01

    We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. PMID:20004641

  9. Envelope glycoproteins of human immunodeficiency virus type 1: profound influences on immune functions.

    PubMed Central

    Chirmule, N; Pahwa, S

    1996-01-01

    Infection by human immunodeficiency virus type 1 (HIV-1) leads to progressive destruction of the CD4+ T-cell subset, resulting in immune deficiency and AIDS. The specific binding of the viral external envelope glycoprotein of HIV-1, gp120, to the CD4 molecules initiates viral entry. In the past few years, several studies have indicated that the interaction of HIV-1 envelope glycoprotein with cells and molecules of the immune system leads to pleiotropic biological effects on immune functions, which include effects on differentiation of CD34+ lymphoid progenitor cells and thymocytes, aberrant activation and cytokine secretion patterns of mature T cells, induction of apoptosis, B-cell hyperactivity, inhibition of T-cell dependent B-cell differentiation, modulation of macrophage functions, interactions with components of complement, and effects on neuronal cells. The amino acid sequence homologies of the envelope glycoproteins with several cellular proteins have suggested that molecular mimicry may play a role in the pathogenesis of the disease. This review summarizes work done by several investigators demonstrating the profound biological effects of envelope glycoproteins of HIV-1 on immune system cells. Extensive studies have also been done on interactions of the viral envelope proteins with components of the immune system which may be important for eliciting a "protective immune response." Understanding the influences of HIV-1 envelope glycoproteins on the immune system may provide valuable insights into HIV-1 disease pathogenesis and carries implications for the trials of HIV-1 envelope protein vaccines and immunotherapeutics. PMID:8801439

  10. The transmembrane protein of HIV-1 primary isolates modulates cell surface expression of their envelope glycoproteins.

    PubMed

    Lebigot, S; Roingeard, P; Thibault, G; Lemiale, F; Verrier, B; Barin, F; Brand, D

    2001-11-10

    We have recently shown that the level of cell surface expression of envelope glycoproteins derived from various human immunodeficiency virus type 1 (HIV-1) primary isolates (PI) was lower than those of envelope glycoproteins derived from T-cell laboratory-adapted (TCLA) HIV-1 (D. Brand et al., 2000, Virology 271, 350-362). We investigated this phenomenon by comparing the cell surface expression of chimeric envelope glycoproteins constructed by swapping the gp120 surface and gp41 transmembrane glycoproteins of the TCLA HIV-1MN and the PI HIV-1(133), HIV-1G365, or HIV-1EFRA. We found that each chimeric envelope construct had a cell surface-specific pattern of expression similar to that of the parental envelope glycoproteins corresponding to the gp41. Thus, the difference in cell surface expression observed between TCLA viruses and various PI is probably due to a signal located in gp41. Identification of this signal may be important for the design of PI envelope-derived immunogens and may increase our understanding of the mechanisms by which HIV-1 escapes from the immune system.

  11. Ethanol-induced impairment of hepatic glycoprotein secretion in the isolated rat liver perfusion model

    SciTech Connect

    Volentine, G.D.; Ogden, K.A.; Tuma, D.J.; Sorrell, M.F.

    1987-05-01

    The authors have previously shown that acute administration of ethanol inhibits hepatic glycoprotein secretion in vivo. This ethanol-induced effect appears to be mediated by its reactive metabolite, acetaldehyde. Since hormonal influences and vascular changes can not be controlled in vivo during ethanol administration, they investigated the effect of ethanol in the isolated perfused liver model. Rat liver from fed animals was perfused with oxygenated KRB at 3 ml/min/g liver for 4 hrs. Since ethanol inhibits proteins synthesis in vitro, protein acceptor pool size was equalized in both ethanol and control perfused livers with 1 mM cycloheximide. /sup 3/H-glucosamine was used to label hepatic secretory glycoproteins in the perfusate. Colchicine, a known inhibitor of protein secretion, impaired the secretion of labeled glycoproteins with a concomitant retention of these export proteins in the liver; therefore, confirming the authors secretory model. Ethanol (50 mM) inhibited the appearance of glucosamine-labeled glycoproteins by 60% into the perfusate as compared to control livers. Pretreatment of animals with cyanamide (an aldehyde dehydrogenase inhibitor) further potentiated this effect of ethanol in the isolated perfused liver. These data suggest that ethanol inhibits hepatic glycoprotein secretion in the isolated liver perfusion model, and this ethanol-induced impairment appears to be mediated by acetaldehyde.

  12. Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G

    PubMed Central

    Baquero, Eduard; Buonocore, Linda; Rose, John K.; Bressanelli, Stéphane; Gaudin, Yves; Albertini, Aurélie A.

    2012-01-01

    Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV Gth) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV Gth obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal. PMID:22949203

  13. Glucocorticoid-regulated glycoprotein maturation in wild-type and mutant rat cell lines

    PubMed Central

    1986-01-01

    Glucocorticoid hormones can regulate the posttranslational maturation of mouse mammary tumor virus (MTV) precursor polyproteins in M1.54, a stably infected rat hepatoma cell line. We have used complement- mediated cytolysis to recover variants of M1.54 that fail to express MTV cell surface glycoproteins in a hormone-regulated manner (Firestone, G.L., and K.R. Yamamoto, 1983, Mol. Cell. Biol., 3:149- 160). One such clonal isolate, CR4, is similar to wild-type with respect to synthesis of MTV mRNAs, production of the MTV glycoprotein precursor (gPr74env) and a glycosylated maturation product (gp51), and hormone-induced processing of two MTV phosphoproteins. In contrast, three viral cell surface glycoproteins (gp78, gp70, and gp32) and one extracellular species (gp70s), which derive from gPr74env in glucocorticoid-treated wild-type cells, fail to appear in CR4. CR4 showed no apparent alterations in proliferation rate, cell shape, or expression of total functional mRNA and bulk glycoproteins. We conclude that the genetic lesion in CR4 defines a highly selective hormone- regulated glycoprotein maturation pathway that alters the fate of a restricted subset of precursor species. PMID:3023398

  14. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    PubMed Central

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  15. Differentially-expressed glycoproteins in Locusta migratoria hemolymph infected with Metarhizium anisopliae.

    PubMed

    Wang, Chutao; Cao, Yueqing; Wang, Zhongkang; Yin, Youping; Peng, Guoxiong; Li, Zhenlun; Zhao, Hua; Xia, Yuxian

    2007-11-01

    Glycoproteins play important roles in insect physiology. Infection with pathogen always results in the differential expression of some glycoproteins, which may be involved in host-pathogen interactions. In this report, differentially-expressed glycoproteins from the hemolymph of locusts infected with Metarhizium anisopliae were analyzed by two-dimensional electrophoresis (2-DE) and PDQuest software. The results showed that 13 spots were differentially expressed, of which nine spots were upregulated and four were downregulated. Using MS/MS with de novo sequencing and NCBI database searches, three upregulated proteins were identified as locust transferrin, apolipoprotein precursor, and hexameric storage protein 3. These proteins have been reported to be involved in the insect innate immune response to microbial challenge. Due to the limited available genome information and protein sequences of locusts, the possible functions of the other 10 differentially-expressed spots remain unknown. PMID:17658547

  16. An efficient platform for screening expression and crystallization of glycoproteins produced in human cells

    PubMed Central

    Lee, Jeffrey E.; Fusco, Marnie L.; Saphire, Erica Ollmann

    2010-01-01

    Glycoproteins mediate multiple, diverse and critical cellular functions, that are desirable to explore by structural analysis. However, structure determination of these molecules has been hindered by difficulties expressing milligram quantities of stable, homogeneous protein and in determining, which modifications will yield samples amenable to structural studies. We describe a platform proven effective for rapidly screening expression and crystallization of challenging glycoprotein targets produced in mammalian cells. Here, multiple glycoprotein constructs are produced in parallel by transient expression of adherent human embryonic kidney (HEK) 293T cells and subsequently screened in small quantities for crystallization by microfluidic free interface diffusion. As a result, recombinant proteins are produced and processed in a native, mammalian environment and crystallization screening can be accomplished with as little as 65 μg of protein. Moreover, large numbers of constructs can be screened for expression and crystallization and scaled up for structural studies in a matter of five weeks. PMID:19373230

  17. Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells.

    PubMed

    Strenge, K; Schauer, R; Kelm, S

    1999-02-01

    The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG. PMID:10037148

  18. P-glycoprotein expression in canine mammary gland tumours related with myoepithelial cells.

    PubMed

    Kim, N-H; Hwang, Y-H; Im, K-S; Kim, J-H; Chon, S-K; Kim, H-Y; Sur, J-H

    2012-12-01

    P-glycoprotein is influential in chemotherapy-resistance in numerous cancers and has been widely studied in human breast cancer research, but is less studied in canine mammary gland tumour (MGT). The study was to evaluate P-glycoprotein expression and its localisations related with prognostic factors with monoclonal antibody C219, by immunohistochemistry (IHC) of 68 cases of canine malignant (n=54) and benign (n=14) MGT. Additional immunofluorescence (IF) and reverse transcriptase-polymerase chain reaction (RT-PCR) were also performed. There was a novel finding that P-glycoprotein expression with C219 localised at two different cell types: epithelial and myoepithelial cells. Myoepithelial localised tumours were 5 benign (35.5%) and 21 malignant (63.6%), while epithelial localised tumours were 12 cases, all malignant (36.5%). Unlike conventional belief, semi-quantitative evaluation of IHC intensity scores of C219 expression in malignant MGT was related with favourable histopathological parameters. PMID:22554937

  19. Temporal pattern of incorporation of /sup 3/H precursors into pituitary glycoproteins and their subsequent release

    SciTech Connect

    Grotjan, H.E. Jr.

    1982-04-01

    The temporal pattern of incorporation of various /sup 3/H precursors into glycoproteins by rat anterior pituitaries incubated in vitro and the release of /sup 3/H-glycoproteins was examined. (/sup 3/H)Leucine incorporation was linear with respect to time and (/sup 3/H)leucine-containing macromolecules appeared in the media in about 1 hr. The temporal pattern of (/sup 3/H)mannose incorporation and release was similar. (/sup 3/H)Galactose and (/sup 3/H)fucose were incorporated after apparent time of delays of approximately 15 min and soon thereafter (20-25 min) appeared in the medium in /sup 3/H-glycoproteins. Thus, these precursors appear to be added as terminal residues. (/sup 3/H)Glucosamine exhibited a pattern intermediate between (/sup 3/H)leucine and (/sup 3/H)fucose whereas (/sup 3/H)GlcNAc appeared to be incorporated as a terminal residue.

  20. Mason-Pfizer monkey virus: analysis and localization of virion proteins and glycoproteins.

    PubMed Central

    Schochetman, G; Kortright, K; Schlom, J

    1975-01-01

    The polypeptide composition of Mason-Pfizer monkey virus was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six major polypeptides of molecular weights 68,000, 27,000, 20,000, 14,000, 12,000, and 10,000 were resolved regardless of the cell type (i.e., two human and two rhesus) in which the virus was grown. Protein gp68 (68,000) represented the major virus glycoprotein and protein gp20 (20,000) represented a minor glycoprotein of the virion, again regardless of the cell type of origin of the virus. Protein gp68 appears to be located on the outer surface of the viral envelope, as demonstrated by lactoperoxidase catalyzed iodination of intact virions. Additional glycoproteins were shown to be virion associated; their presence depended, however, on the cell type in which the virus was propagated. PMID:810603

  1. Crystal structure of the prefusion surface glycoprotein of the prototypic arenavirus LCMV

    PubMed Central

    Sullivan, Brian M.; Legrand, Pierre; Zandonatti, Michelle A.; Robinson, James E.; Garry, Robert F.; Rey, Félix A.; Oldstone, Michael B.; Saphire, Erica Ollmann

    2016-01-01

    Arenaviruses exist worldwide and can cause hemorrhagic fever and neurologic disease. A single glycoprotein is expressed on the viral surface that mediates entry into target cells. This glycoprotein, termed GPC, contains a membrane-associated signal peptide, a receptor-binding subunit termed GP1 and a fusion-mediating subunit termed GP2. Although GPC is a critical target of antibodies and vaccines, the structure of the metastable GP1-GP2 prefusion complex has remained elusive for all arenaviruses. Here we describe the crystal structure of the fully glycosylated, prefusion GP1-GP2 complex of the prototypic arenavirus LCMV at 3.5Å. This structure reveals the conformational changes that the arenavirus glycoprotein must undergo to cause fusion, and illustrates the fusion regions and potential oligomeric states. PMID:27111888

  2. P-glycoprotein expression in primary breast cancer detected by immunocytochemistry with two monoclonal antibodies.

    PubMed Central

    Wishart, G. C.; Plumb, J. A.; Going, J. J.; McNicol, A. M.; McArdle, C. S.; Tsuruo, T.; Kaye, S. B.

    1990-01-01

    We have investigated P-glycoprotein (P-gp) expression in samples of primary breast cancer from 29 patients before therapy. We employed immunohistochemical techniques using two monoclonal antibodies (C219 and MRK16) and an indirect alkaline phosphatase method. Heterogeneous expression in epithelial cells was detected with both C219 (21 of 29) and MRK16 (16 of 29). A surprising finding was P-glycoprotein expression in stromal cells with both C219 (26 of 29) and MRK16 (12 of 29). Our results suggest that significant levels of P-glycoprotein expression may be present in breast cancer before exposure to drugs associated with multidrug resistance. Images Figure 1 PMID:1978783

  3. The effect of castanospermine on the synthesis of synaptic glycoproteins by rat brain slices.

    PubMed

    Howes, S; Bissoon, N; Ito, M; Beesley, P W; Gurd, J W

    1990-03-01

    Slices were prepared from rat forebrains and the incorporation of [3H]mannose and [35S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [3H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man8GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc3Man9GlcNAc2 accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [3H]mannose or [35S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [3H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [35S] methionine-labeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes. PMID:2195374

  4. The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins.

    PubMed

    Elbein, A D; Legler, G; Tlusty, A; McDowell, W; Schwarz, R

    1984-12-01

    Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species. PMID:6240228

  5. Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

    PubMed

    Li, Y; Drone, C; Sat, E; Ghosh, H P

    1993-07-01

    The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.

  6. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles.

    PubMed

    Freitag, Tanja C; Maisner, Andrea

    2016-03-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  7. Nephrotoxic potency of antisera to three rat glomerular basement membrane glycoproteins.

    PubMed Central

    Devulder, B; Bardos, P; Plouvier, B; Martin, J C; Muh, J P; Tacquet, A

    1978-01-01

    In a previous article, we cited studies which have allowed us to isolate diverse glycoproteins of the rat glomerular basement membrane (GMB) and to study their biochemical structures and antigenicity. This present study attempts to examine, using the heterologous nephrotoxic nephritis model (Masugi's nephritis) the nephrotoxicity of immune sera prepared from three of these glycoproteins: one fairly rich in collagen-like structures (A3), another lacking collagen-like structures (A1), and a third of intermediate composition (A2). The results obtained are discussed in relation to those already published concerning the nature of the GBM antigen(s) responsible for the nephrotoxicity of the sera. PMID:357054

  8. Isolation and partial chemical characterization of a 64,000-dalton glycoprotein of human cytomegalovirus

    SciTech Connect

    Clark, B.R.; Zaia, J.A.; Balce-Directo, L.; Ting, Y.P.

    1984-01-01

    A guanidinium chloride extract of (/sup 3/H)glucosamine- and (/sup 35/S)methionine-labeled virions plus dense bodies of human cytomegalovirus (Towne) was separated by reverse-phase high-pressure liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eluate revealed the major peak to be a glycoprotein with a relative mass of 64,000. This glycoprotein (HCMVgp64) was characterized by amino acid analysis and a high-pressure liquid chromatographic map of its tryptic peptides.

  9. Quantitative characterization of glycoproteins in neurodegenerative disorders using iTRAQ.

    PubMed

    Shi, Min; Hwang, Hyejin; Zhang, Jing

    2013-01-01

    Aberrant protein glycosylation has been recognized to be associated with many neurodegenerative disorders, including Alzheimer and Parkinson disease. Using mass spectrometry-based technologies to catalog and quantify glycoproteins in these diseases is expected to provide insight into not only the biochemical pathogenesis of neurodegeneration but also the biomarker discovery. This chapter describes a multidisciplinary approach to accomplish the goal of glycoprotein identification and quantification in human brain tissue and cerebrospinal fluid, which includes sample preparation, isobaric tag labeling of digested peptides, glycopeptide enrichment using hydrazide chemistry, protein/peptide identification and quantification by liquid chromatography-based high-resolution tandem mass spectrometry, as well as bioinformatic data processing. PMID:23296538

  10. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

    PubMed

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain.

  11. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    SciTech Connect

    Vogler, Meike; Dickens, David; Dyer, Martin J.S.; Owen, Andrew; Pirmohamed, Munir; Cohen, Gerald M.

    2011-05-06

    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  12. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

    SciTech Connect

    Follis, Kathryn E.; York, Joanne; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2006-07-05

    The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.

  13. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

    PubMed

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain. PMID:27000241

  14. Determination of N-linked glycosylation in viral glycoproteins by negative ion mass spectrometry and ion mobility

    PubMed Central

    Bitto, David; Harvey, David J.; Halldorsson, Steinar; Doores, Katie J.; Pritchard, Laura K.; Huiskonen, Juha T.; Bowden, Thomas A.; Crispin, Max

    2016-01-01

    Summary Glycan analysis of virion-derived glycoproteins is challenging due to the difficulties in glycoprotein isolation and low sample abundance. Here, we describe how ion mobility mass spectrometry can be used to obtain spectra from virion samples. We also describe how negative ion fragmentation of glycans can be used to probe structural features of virion glycans. PMID:26169737

  15. Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

    PubMed

    McClements, W L; Armstrong, M E; Keys, R D; Liu, M A

    1996-10-15

    DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

  16. Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    SciTech Connect

    Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

    1986-10-01

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.

  17. Progesterone regulates the expression and activity of two mouse isoforms of the glycoprotein folding sensor UDP-Glc: glycoprotein glucosyltransferase (UGGT).

    PubMed

    Prados, María B; Caramelo, Julio J; Miranda, Silvia E

    2013-12-01

    UDP-Glucose:glycoprotein glucosyltransferase (UGGT) is a central component of the endoplasmic reticulum (ER) glycoprotein-folding quality control system, which prevents the exit of partially folded species. UGGT activity can be regulated by the accumulation of misfolded proteins in the ER, a stimulus that triggers a complex signaling pathway known as unfolded protein response (UPR) which is closely associated with inflammation and disease. In this work, we investigated the effect of progesterone (P4) on the expression and activity of UGGT in a mouse hybridoma. We detected the expression of two UGGT isoforms, UGGT1 and UGGT2, and demonstrated that both isoforms are active in these cells. Interestingly, the expression of each isoform is regulated by high physiological P4 concentrations. This work provides the first evidence of a hormonal regulation of UGGT isoform expression and activity, which might influence the glycoprotein quality control mechanism. These findings could contribute to the study of pathologies triggered by the accumulation of misfolded proteins.

  18. Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane.

    PubMed

    Hwangbo, S; Azuma, N; Kurisaki, J; Kanno, C

    1997-09-01

    A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.

  19. Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

    PubMed Central

    Rauh, I; Weiland, F; Fehler, F; Keil, G M; Mettenleiter, T C

    1991-01-01

    The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is

  20. Identification of key structural characteristics of Schisandra chinensis lignans involved in P-glycoprotein inhibition.

    PubMed

    Slanina, Jiří; Páchniková, Gabriela; Carnecká, Martina; Porubová Koubíková, Ludmila; Adámková, Lenka; Humpa, Otakar; Smejkal, Karel; Slaninová, Iva

    2014-10-24

    The aim of the present study was to determine the structural requirements for dibenzocyclooctadiene lignans essential for P-glycoprotein inhibition. Altogether 15 structurally related lignans isolated from Schisandra chinensis or prepared by modification of their backbone were investigated, including three pairs of enantiomers. P-Glycoprotein inhibition was quantified using a doxorubicin accumulation assay in human promyelotic leukemia HL60/MDR cells overexpressing P-glycoprotein. A preliminary quantitative structure-activity relationship analysis revealed three main structural features involved in P-glycoprotein inhibition: a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group. The most effective inhibitors, (-)-gomisin N (1) and (+)-deoxyschizandrin [(+)-2], were selected for further evaluation of their effects. Both these lignans restored the cytotoxic effect of doxorubicin in HL60/MDR cells and when combined with a subtoxic concentration of this compound increased the proportion of G2/M cells significantly, which is a usual response to treatment with this anticancer drug.

  1. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  2. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  3. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  4. Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015.

    PubMed

    Kim, Dae-Won; Kim, You-Jin; Park, Sung Han; Yun, Mi-Ran; Yang, Jeong-Sun; Kang, Hae Ji; Han, Young Woo; Lee, Han Saem; Kim, Heui Man; Kim, Hak; Kim, A-Reum; Heo, Deok Rim; Kim, Su Jin; Jeon, Jun Ho; Park, Deokbum; Kim, Joo Ae; Cheong, Hyang-Min; Nam, Jeong-Gu; Kim, Kisoon; Kim, Sung Soon

    2016-01-01

    An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

  5. Identification of a glycoprotein ligand for E-selectin on mouse myeloid cells

    PubMed Central

    1993-01-01

    E-selectin is an inducible endothelial cell adhesion molecule for neutrophils which functions as a Ca(2+)-dependent lectin. Using a recombinant, antibody-like form of mouse E-selectin, we have searched for glycoprotein ligands on mouse neutrophils and the neutrophil progenitor cell line 32D cl 3. We have identified a 150-kD glycoprotein as the only protein which could be affinity-isolated with soluble E- selectin from [35S]methionine/[35S]cysteine-labeled 32D cl 3 cells. Binding of this protein was strictly Ca(2+)-dependent, was blocked by a cell adhesion-blocking mAb against mouse E-selectin, and required the presence of sialic acid on the 150-kD ligand. This glycoprotein was also affinity-isolated from mature neutrophils, in addition to a minor component at 250 kD, but could not be isolated from several other non- myeloid cell lines. The 150-kD glycoprotein was the only protein from 32D cl 3 cells, which was detectable by silver-staining after a one- step affinity-isolation. PMID:7682218

  6. Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes

    PubMed Central

    Crill, Wayne D.; Chang, Gwong-Jen J.

    2004-01-01

    The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231. PMID:15564505

  7. Averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using Jsubtomo.

    PubMed

    Huiskonen, Juha T; Parsy, Marie-Laure; Li, Sai; Bitto, David; Renner, Max; Bowden, Thomas A

    2014-01-01

    Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein 'spikes' is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health. PMID:25350719

  8. Alpha 1-acid glycoprotein has immunomodulatory effects in neonatal swine adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha 1-acid glycoprotein (AGP) is the most abundant protein in serum of neonatal swine. This protein functions as an immunomodulator in the pig. Recent work has demonstrated that adipose tissue can express AGP mRNA, as well as numerous cytokine mRNA. The present study was designed to determine i...

  9. Support studies for installing the phosphodiester residues of the Thy-1 glycoprotein membrane anchor.

    PubMed

    Campbell, A S; Fraser-Reid, B

    1994-11-01

    Support studies for late-stage installation of the three different types of phosphodiesters found in the rat brain Thy-1 glycoprotein membrane anchor are described. The strategy is geared towards optimizing convergency and the development of chemoselective procedures including deprotection, phosphorylation, esterification and cysteinylation has been investigated. Some of these procedures are being designed for oligosaccharides containing several unprotected hydroxy groups.

  10. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha 1-acid glycoprotein (AGP, ORM-1) is a highly glycosylated mammalian acute phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute phase protein in this ...

  11. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

    SciTech Connect

    Backovic, Marija; Longnecker, Richard; Jardetzky, Theodore S

    2009-03-16

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.

  12. A high boronate avidity monolithic capillary for the selective enrichment of trace glycoproteins.

    PubMed

    Li, Daojin; Li, Yang; Li, Xinglin; Bie, Zijun; Pan, Xianghua; Zhang, Qian; Liu, Zhen

    2015-03-01

    Boronate affinity materials, as effective sample enrichment sorbents for glycoproteomic analysis, have attracted increasing attention in recent years. However, most of boronate affinity materials suffer from an apparent limitation, limited binding strength. As a result, extraction of glycoproteins of trace concentration is rather difficult or impossible. In this study, we present a high boronate avidity monolithic capillary. Branched polyethyleneimine (PEI) was used as a scaffold to amplify the number of boronic acid moieties. While 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA), which exhibited ultrahigh affinity toward cis-diol-containing compounds, was employed as an affinity ligand. Due to the PEI-assisted synergistic multivalent binding, the monolithic column exhibited high boronate avidity toward glycoproteins, with binding constants of 10(-6)-10(-7)M. Such binding strength was the highest among already reported boronic acid-functionalized materials that can be used for glycoproteomic analysis. Besides, the boronate avidity monolithic column exhibited one additional beneficial feature, lowered binding pH (≥6.5). These features greatly favored the selective enrichment of trace glycoproteins from real samples. The feasibility for practical applications was demonstrated with the selective enrichment of trace glycoproteins in human saliva. As compared with other boronate avidity/affinity materials, the boronate avidity monolithic capillary exhibited the best performance.

  13. Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins.

    PubMed

    Cova, Marta; Oliveira-Silva, Rui; Ferreira, José Alexandre; Ferreira, Rita; Amado, Francisco; Daniel-da-Silva, Ana Luísa; Vitorino, Rui

    2015-01-01

    Protein post-translational modifications (PTMs) have increasingly become a research field of incredible importance to fully understand the regulation of biological processes in health and disease. Among PTMs, glycosylation is one of the most studied for which contributed the development and improvement of enrichment techniques. Nowadays, glycoprotein enrichment methods are based on lectin affinity, covalent interactions, and hydrophilic interaction liquid chromatography (HILIC). Nonetheless, the nanotechnology era has fetched new methods to enrich glycoproteins from complex samples as human biological fluids. For instance, magnetic nanoparticles (MNPs) are being used as an interesting enrichment approach allowing a better characterization of glycoproteins and glycopeptides.In this chapter, we describe an enrichment method based on MNPs functionalized with lectins (Concavalin A, wheat germ agglutinin, and Maackia amurensis lectin) to enrich specific sets of glycoproteins from biological fluids. Moreover, it is proposed a bioinformatic strategy to deal with data retrieved from mass spectrometry analysis of enriched samples aiming the identification of relevant biological processes modulated by a given stimuli and, ultimately, of new biomarkers for disease screening/management.

  14. Intracellular transport and stability of varicella-zoster virus glycoprotein K

    SciTech Connect

    Hall, Susan L.; Govero, Jennifer L.; Heineman, Thomas C. . E-mail: heinemtc@slu.edu

    2007-02-20

    VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established that gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.

  15. Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production

    PubMed Central

    El Najjar, Farah; Schmitt, Anthony P.; Dutch, Rebecca Ellis

    2014-01-01

    Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. PMID:25105277

  16. Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

    PubMed

    Kobayashi, Kenta; Kato, Shigeki; Inoue, Ken-Ichi; Takada, Masahiko; Kobayashi, Kazuto

    2016-01-01

    A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders.

  17. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion

    PubMed Central

    Garcia, Natalie K.; Lee, Kelly K.

    2016-01-01

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system. PMID:26761026

  18. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

    PubMed

    Loo, T W; Clarke, D M

    1997-01-10

    There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

  19. Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these i...

  20. Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

    PubMed Central

    Huiskonen, Juha T.; Parsy, Marie-Laure; Li, Sai; Bitto, David; Renner, Max; Bowden, Thomas A.

    2014-01-01

    Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein ‘spikes’ is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health. PMID:25350719

  1. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  2. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion.

    PubMed

    Garcia, Natalie K; Lee, Kelly K

    2016-01-11

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

  3. P-glycoprotein regulates trafficking of CD8(+) T cells to the brain parenchyma.

    PubMed

    Kooij, Gijs; Kroon, Jeffrey; Paul, Debayon; Reijerkerk, Arie; Geerts, Dirk; van der Pol, Susanne M A; van Het Hof, Bert; Drexhage, Joost A; van Vliet, Sandra J; Hekking, Liesbeth H P; van Buul, Jaap D; Pachter, Joel S; de Vries, Helga E

    2014-05-01

    The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

  4. Structure–Function Relationships of Glycoprotein Hormones and Their Subunits’ Ancestors

    PubMed Central

    Cahoreau, Claire; Klett, Danièle; Combarnous, Yves

    2015-01-01

    Glycoprotein hormones (GPHs) are the most complex molecules with hormonal activity. They exist only in vertebrates but the genes encoding their subunits’ ancestors are found in most vertebrate and invertebrate species although their roles are still unknown. In the present report, we review the available structural and functional data concerning GPHs and their subunits’ ancestors. PMID:25767463

  5. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  6. Glycoprotein Biochemistry (Biosynthesis)--A Vehicle for Teaching Many Aspects of Biochemistry and Molecular Biology.

    ERIC Educational Resources Information Center

    Cole, Clair R.; Smith, Christopher A.

    1990-01-01

    Information about the biosynthesis of the carbohydrate portions or glycans of glycoproteins is presented. The teaching of glycosylation can be used to develop and emphasize many general aspects of biosynthesis, in addition to explaining specific biochemical and molecular biological features associated with producing the oligosaccharide portions of…

  7. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  8. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  9. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  10. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  11. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  12. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  13. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

    PubMed

    Gardner, Thomas J; Tortorella, Domenico

    2016-09-01

    The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

  14. Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus.

    PubMed

    Coleman, Stewart; Hornig, Julia; Maddux, Sarah; Choi, K Yeon; McGregor, Alistair

    2015-01-01

    Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene

  15. Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus

    PubMed Central

    Maddux, Sarah; Choi, K. Yeon; McGregor, Alistair

    2015-01-01

    Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene

  16. Blood-brain barrier P-glycoprotein function in Alzheimer's disease.

    PubMed

    van Assema, Daniëlle M E; Lubberink, Mark; Bauer, Martin; van der Flier, Wiesje M; Schuit, Robert C; Windhorst, Albert D; Comans, Emile F I; Hoetjes, Nikie J; Tolboom, Nelleke; Langer, Oliver; Müller, Markus; Scheltens, Philip; Lammertsma, Adriaan A; van Berckel, Bart N M

    2012-01-01

    A major pathological hallmark of Alzheimer's disease is accumulation of amyloid-β in senile plaques in the brain. Evidence is accumulating that decreased clearance of amyloid-β from the brain may lead to these elevated amyloid-β levels. One of the clearance pathways of amyloid-β is transport across the blood-brain barrier via efflux transporters. P-glycoprotein, an efflux pump highly expressed at the endothelial cells of the blood-brain barrier, has been shown to transport amyloid-β. P-glycoprotein function can be assessed in vivo using (R)-[(11)C]verapamil and positron emission tomography. The aim of this study was to assess blood-brain barrier P-glycoprotein function in patients with Alzheimer's disease compared with age-matched healthy controls using (R)-[(11)C]verapamil and positron emission tomography. In 13 patients with Alzheimer's disease (age 65 ± 7 years, Mini-Mental State Examination 23 ± 3), global (R)-[(11)C]verapamil binding potential values were increased significantly (P = 0.001) compared with 14 healthy controls (aged 62 ± 4 years, Mini-Mental State Examination 30 ± 1). Global (R)-[(11)C]verapamil binding potential values were 2.18 ± 0.25 for patients with Alzheimer's disease and 1.77 ± 0.41 for healthy controls. In patients with Alzheimer's disease, higher (R)-[(11)C]verapamil binding potential values were found for frontal, parietal, temporal and occipital cortices, and posterior and anterior cingulate. No significant differences between groups were found for medial temporal lobe and cerebellum. These data show altered kinetics of (R)-[(11)C]verapamil in Alzheimer's disease, similar to alterations seen in studies where P-glycoprotein is blocked by a pharmacological agent. As such, these data indicate that P-glycoprotein function is decreased in patients with Alzheimer's disease. This is the first direct evidence that the P-glycoprotein transporter at the blood-brain barrier is compromised in sporadic

  17. Inhibition of Arenavirus Infection by a Glycoprotein-Derived Peptide with a Novel Mechanism

    PubMed Central

    Spence, Jennifer S.; Melnik, Lilia I.; Badani, Hussain; Wimley, William C.

    2014-01-01

    ABSTRACT The family Arenaviridae includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC50) for the peptide is 7 μM, with complete inhibition of viral plaque formation at approximately 20 μM, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection. IMPORTANCE Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic

  18. Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate.

    PubMed

    Mbemba, E; Gluckman, J C; Gattegno, L

    1994-02-01

    Envelope glycoproteins of human immunodeficiency viruses (HIV-1 and HIV-2) can interact with high-mannose glycans and with the mannosyl or N-acetylglucosaminyl core of complex-type oligosaccharidic structures. HIV-1 glycoproteins also specifically bind sulphated polysaccharides such as dextran sulphate (DS) and heparin. Here, we show that the latter property is shared by HIV-2 recombinant gp140 (rgp140) precursor glycoprotein. Binding of rgp140 and of corresponding rgp160 of HIV-1 to heparin- and DS-substituted (sulphated dextran beads; SDB) affinity matrices was inhibited by the soluble specific ligand and also by fetuin, asialofetuin or the anionic simple carbohydrate derivative mannose-6-phosphate (M6P). Interaction of HIV-1 rgp120 subunit with the two affinity matrices was also inhibited by M6P, but only rgp120 binding to heparin-agarose, and not that to SDB, was affected by fetuin and asialofetuin. These results suggest that HIV-1 and HIV-2 envelope glycoproteins presumably display different sulphated polysaccharide and carbohydrate recognition sites. Some of these may be common or in close proximity: with respect to rgp160, for example, the sites may be common on the gp41 moiety and/or in a region of gp120 which would be more accessible when expressed on rgp160 than on processed gp120, while they may be distinct on the cleaved gp120 subunit. Finally, because M6P is a marker of lysosomal enzymes, we verified that HIV-1 and HIV-2 envelope glycoproteins could specifically bind in a M6P-inhibitable manner to a representative lysosomal enzyme, bovine liver beta-glucuronidase coupled to agarose, suggesting that they may possibly interfere with lysosomal enzyme sorting in HIV-infected cells.

  19. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  20. Emergence of an Ancestral Glycoprotein Hormone in the Pituitary of the Sea Lamprey, a Basal Vertebrate.

    PubMed

    Sower, Stacia A; Decatur, Wayne A; Hausken, Krist N; Marquis, Timothy J; Barton, Shannon L; Gargan, James; Freamat, Mihael; Wilmot, Michael; Hollander, Lian; Hall, Jeffrey A; Nozaki, Masumi; Shpilman, Michal; Levavi-Sivan, Berta

    2015-08-01

    The gnathostome (jawed vertebrates) classical pituitary glycoprotein hormones, FSH, LH, and TSH, consist of a common α-subunit (GpA1) and unique β-subunits (Gpβ1, -2, and -3), whereas a recently identified pituitary glycoprotein hormone, thyrostimulin, consists of GpA2 and GpB5. This paper reports the identification, expression, and function of an ancestral, nonclassical, pituitary heterodimeric glycoprotein hormone (GpH) consisting of the thyrostimulin A2 subunit with the classical β-subunit in the sea lamprey, Petromyzon marinus, a jawless basal vertebrate. Lamprey (l) GpA2, and lGpHβ were shown to form a heterodimer by coimmunoprecipitation of lGpA2 with FLAG-tagged lGpHβ after the overexpression in transiently transfected COS7 cells using a bipromoter vector. Dual-label fluorescent in situ hybridization and immunohistochemistry showed the coexpression of individual subunits in the proximal pars distalis of the pituitary. GnRH-III (1μΜ) significantly increased the expression of lGpHβ and lGpA2 in in vitro pituitary culture. Recombinant lamprey GpH was constructed by tethering the N terminal of lGpA2 to the C terminal of lGpHβ with a linker region composed of six histidine residues followed by three glycine-serine repeats. This recombinant lamprey GpH activated the lamprey glycoprotein hormone receptor I as measured by increased cAMP/luciferase activity. These data are the first to demonstrate a functional, unique glycoprotein heterodimer that is not found in any other vertebrate. These data suggest an intermediate stage of the structure-function of the gonadotropin/thyroid-stimulating hormone in a basal vertebrate, leading to the emergence of the highly specialized gonadotropin hormones and thyroid stimulating hormones in gnathostomes.

  1. Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein

    SciTech Connect

    Lee, Jeffrey E.; Fusco, Marnie L.; Abelson, Dafna M.; Hessell, Ann J.; Burton, Dennis R.; Saphire, Erica Ollmann

    2009-11-01

    Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing of the trimeric ebolavirus glycoprotein are described. The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008 ▶), Nature (London), 454, 177–182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the

  2. Analysis of the apparent biphasic axonal transport kinetics of fucosylated glycoproteins

    SciTech Connect

    Goodrum, J.F.; Morell, P.

    1984-07-01

    Following intraocular injection of (/sup 3/H)fucose, the accumulation of transported radioactivity arriving at the superior colliculus peaks within a few hours and decays with a time course of hours. Then, over a period of several days, radioactivity again accumulates at the superior colliculus and then decays with a half-life of days. Such data have been interpreted as evidence for both a group of rapidly released, rapidly transported glycoproteins (first peak) and a group of slowly released but rapidly transported glycoproteins (second peak). This supposition was investigated by studying in more detail the metabolism of some individual fucosylated proteins in both the retina and superior colliculus. It was noted that much of the radioactivity incorporated in fucosylated glycoproteins at the retina was rapidly metabolized, while the remainder of the fucosylated moieties had a metabolic half-life on the order of days. In other experiments (/sup 35/S)methionine was injected intraocularly, the metabolism in the retina was examined and a study was made of the kinetics of transport to the superior colliculus of the peptide backbone of these same individual proteins. In contrast to the two waves of accumulation of radioactivity from (/sup 3/H)fucose, accumulation of radioactivity of the peptide backbone of the same glycoproteins was monophasic. The author's explanation of these data involves the presence of two types of fucose moieties on the peptides. One group of fucose moieties is labile and is lost from the peptide backbone over a period of hours. Other fucose moieties are approximately as metabolically stable as the peptide backbones to which they are attached. The actual peptide backbones of the glycoproteins are committed to rapid transport over a period of several days.

  3. Feasibility and challenges in the development of immunocontraceptive vaccine based on zona pellucida glycoproteins.

    PubMed

    Choudhury, Sangeeta; Srivastava, Neelu; Narwal, P S; Rath, Archana; Jaiswal, Sonika; Gupta, Satish K

    2007-01-01

    The zona pellucida (ZP) glycoproteins play a crucial role during fertilization and thus are considered as important target antigens for the development of immunocontraceptive vaccines aiming to inhibit fertility at a pre-fertilization stage. In order to evaluate the immunocontraceptive potential of ZP glycoproteins, bonnet monkey (Macaca radiata) ZP2, ZP3 and ZP4 have been cloned and expressed using either E. coli or baculovirus expression systems. Active immunization studies with the recombinant ZP glycoproteins in female baboons (Papio anubis) and bonnet monkeys revealed curtailment of fertility. In order to minimize the ovarian pathology, synthetic peptides corresponding to B cell epitopes that are devoid of 'oophoritogenic' T cell epitopes were designed and their in vitro immunocontraceptive potential explored. There are several issues that need to be addressed before ZP glycoproteins based immunocontraceptive vaccines become feasible for use in humans. Nonetheless, the utility of such a vaccine is imminent for controlling wild life population. In this direction, active immunization of female non-descript dogs with recombinant canine ZP3 conjugated to diphtheria toxoid led to curtailment of fertility. Further, canine ZP3 has also been expressed in insect cells as a fusion protein with rabies virus glycoprotein G (RV-G), an antigen that is involved in providing protection against rabies. The immunogenicity of such a recombinant protein and its potential to curtail fertility was explored both in female mice and dogs. Simultaneously, DNA vaccine encoding canine ZP3 and RV-G have been made and evaluated for their immunogenicity. The results obtained so far, current shortcomings and the possible ways to circumvent these have been discussed in the present manuscript. PMID:17566293

  4. Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

    PubMed

    Emerson, W A; Kornfeld, S

    1976-04-20

    The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule. PMID:1268191

  5. Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

    PubMed Central

    Burke, David; Keegstra, Kenneth

    1979-01-01

    Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways. PMID:430605

  6. P-glycoprotein alters blood–brain barrier penetration of antiepileptic drugs in rats with medically intractable epilepsy

    PubMed Central

    Ma, Aimei; Wang, Cuicui; Chen, Yinghui; Yuan, Weien

    2013-01-01

    P-glycoprotein is one of the earliest known multidrug transporters and plays an important role in resistance to chemotherapeutic drugs. In this study, we detected levels of P-glycoprotein and its mRNA expression in a rat brain model of medically intractable epilepsy established by amygdala kindling and drug selection. We investigated whether inhibition of P-glycoprotein affects the concentration of antiepileptic drugs in cortical extracellular fluid. We found that levels of P-glycoprotein and its mRNA expression were upregulated in epileptic cerebral tissue compared with cerebral tissue from normal rats. The concentrations of two antiepileptic drugs, carbamazepine and phenytoin, were very low in the cortical extracellular fluid of rats with medically intractable epilepsy, and were restored after blockade of P-glycoprotein by verapamil. These results show that increased P-glycoprotein levels alter the ability of carbamazepine and phenytoin to penetrate the blood–brain barrier and reduce the concentrations of these agents in extracellular cortical fluid. High P-glycoprotein levels may be involved in resistance to antiepileptic drugs in medically intractable epilepsy. PMID:24348021

  7. Glucose persistence on high-mannose oligosaccharides selectively inhibits the macroautophagic sequestration of N-linked glycoproteins.

    PubMed Central

    Ogier-Denis, E; Bauvy, C; Cluzeaud, F; Vandewalle, A; Codogno, P

    2000-01-01

    The macroautophagic-lysosomal pathway is a bulk degradative process for cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway. PMID:10642502

  8. Simple boric acid-based fluorescent focusing for sensing of glucose and glycoprotein via multipath moving supramolecular boundary electrophoresis chip.

    PubMed

    Dong, Jingyu; Li, Si; Wang, Houyu; Meng, Qinghua; Fan, Liuyin; Xie, Haiyang; Cao, Chengxi; Zhang, Weibing

    2013-06-18

    Boric acid-based fluorescent complex probe of BBV-HPTS (boronic acid-based benzyl viologen (BBV) and hydroxypyrene trisulfonic acid trisodium salt (HPTS)) was rarely used for sensitive sensing of saccharide (especially glycoprotein) via electrophoresis. We proposed a novel model of moving supramolecular boundary (MSB) formed with monosaccharide or glycoprotein in microcolumn and the complex probe of BBV-HPTS in the cathodic injection tube, developed a method of MSB fluorescent focusing for sensitive recognition of monosaccharide and glycoprotein, and designed a special multipath capillary electrophoresis (CE) chip for relative experiments. As a proof of concept, glucose and hemoglobin A1c (HbA1c) were respectively used as the mode saccharide and glycoprotein for the relevant demonstration. The experiments revealed that (i) the complex of BBV-HPTS could interact with free glucose or bound one in glycoprotein; (ii) the fluorescent signal was a function of glucose or glycoprotein content approximately; and (iii) interestingly the fluorescent band motion was dependent on glucose content. The developed method had the following merits: (i) low cost; (ii) low limit of detection (down to 1.39 pg/mL for glucose and 2.0 pg per capillary HbA1c); and (iii) high throughput (up to 12 runs or more per patch) and speed (less than 5 min). The developed method has potential use for sensitive monitoring of monosaccharide and glycoprotein in biomedical samples.

  9. Benzoboroxole-functionalized magnetic core/shell microspheres for highly specific enrichment of glycoproteins under physiological conditions.

    PubMed

    Zhang, Yuting; Ma, Wanfu; Li, Dian; Yu, Meng; Guo, Jia; Wang, Changchun

    2014-04-01

    Efficient enrichment of specific glycoproteins from complex biological samples is of great importance towards the discovery of disease biomarkers in biological systems. Recently, phenylboronic acid-based functional materials have been widely used for enrichment of glycoproteins. However, such enrichment was mainly carried out under alkaline conditions, which is different to the status of glycoproteins in neutral physiological conditions and may cause some unpredictable degradation. In this study, on-demand neutral enrichment of glycoproteins from crude biological samples is accomplished by utilizing the reversible interaction between the cis-diols of glycoproteins and benzoboroxole-functionalized magnetic composite microspheres (Fe3O4/PAA-AOPB). The Fe3O4/PAA-AOPB composite microspheres are deliberately designed and constructed with a high-magnetic-response magnetic supraparticle (MSP) core and a crosslinked poly(acrylic acid) (PAA) shell anchoring abundant benzoboroxole functional groups on the surface. These nanocomposites possessed many merits, such as large enrichment capacity (93.9 mg/g, protein/beads), low non-specific adsorption, quick enrichment process (10 min) and magnetic separation speed (20 s), and high recovery efficiency. Furthermore, the as-prepared Fe3O4/PAA-AOPB microspheres display high selectivity to glycoproteins even in the E. coli lysate or fetal bovine serum, showing great potential in the identify of low-abundance glycoproteins as biomarkers in real complex biological systems for clinical diagnoses.

  10. Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα

    PubMed Central

    Gitz, Eelo; Koopman, Charlotte D.; Giannas, Alèkos; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan-Willem N.; Gerritsen, Hans C.; Urbanus, Rolf T.

    2013-01-01

    Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s−1 induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s−1. Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation. PMID:23753027

  11. Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone.

    PubMed

    Aussedat, Baptiste; Fasching, Bernhard; Johnston, Eric; Sane, Neeraj; Nagorny, Pavel; Danishefsky, Samuel J

    2012-02-22

    Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.

  12. Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

    PubMed

    Zeinyeh, Waël; Mahiout, Zahia; Radix, Sylvie; Lomberget, Thierry; Dumoulin, Axel; Barret, Roland; Grenot, Catherine; Rocheblave, Luc; Matera, Eva-Laure; Dumontet, Charles; Walchshofer, Nadia

    2012-10-01

    Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

  13. Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Chi, Lisha; Ruan, Dandan; Xuan, Yuanhu; Cong, Weitao; Jin, Litai

    2014-06-01

    A fluorescent detection method for glycoproteins in SDS-PAGE by using 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH) was developed in this study. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be specifically detected by the BH staining method, which is twofold more sensitive than that of the most commonly used Pro-Q Emerald 488 glycoprotein stain. Furthermore, the specificity of the newly developed stain for glycoproteins was demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that BH stain may provide new choices for convenient, sensitive, specific and economic visualization of gel-separated glycoproteins. PMID:24712021

  14. Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Chi, Lisha; Ruan, Dandan; Xuan, Yuanhu; Cong, Weitao; Jin, Litai

    2014-06-01

    A fluorescent detection method for glycoproteins in SDS-PAGE by using 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH) was developed in this study. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be specifically detected by the BH staining method, which is twofold more sensitive than that of the most commonly used Pro-Q Emerald 488 glycoprotein stain. Furthermore, the specificity of the newly developed stain for glycoproteins was demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that BH stain may provide new choices for convenient, sensitive, specific and economic visualization of gel-separated glycoproteins.

  15. Human platelet glycoprotein V: characterization of the polypeptide and the related Ib-V-IX receptor system of adhesive, leucine-rich glycoproteins.

    PubMed Central

    Hickey, M J; Hagen, F S; Yagi, M; Roth, G J

    1993-01-01

    Human platelet glycoprotein (GP) V (M(r) 83,300), whose primary structure is reported here, is a part of the Ib-V-IX system of surface glycoproteins (GPs Ib alpha, Ib beta, V, IX) that constitute the receptor for von Willebrand factor (vWf) and mediate the adhesion of platelets to injured vascular surfaces in the arterial circulation, a critical initiating event in hemostasis. System members share physical associations, leucine-rich glycoprotein (LRG) structures, and a congenital deficiency state, Bernard-Soulier syndrome. With PCR techniques and platelet cDNA templates, 1.4 kb of GP V cDNA sequence was obtained that encodes 469 GP V amino acids. A genomic 3.5-kb BamHI fragment was then isolated that includes 3.46 kb of GP V cDNA sequence: the 1.7-kb open reading frame plus 2 bases of the 5' and 1.8 kb of the 3' untranslated regions. Northern blot analysis reveals three GP V platelet transcripts of 3.8, 4.2, and 5.2 kb. A 16-amino acid signal peptide is present. Mature GP V is a 544-amino acid transmembrane protein with a 504-amino acid extracellular domain that encompasses a set of 15 tandem LRG repeats in a "flank-LRG center-flank" array [Roth, G. J. (1991) Blood 77, 5-19] along with eight putative N-linked glycosylation sites and cleavage sites for thrombin and calpain. GP V is a transmembrane, adhesive LRG protein that plays an undefined, but potentially critical, role in the expression and/or function of the Ib-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries. Images Fig. 2 PMID:7690959

  16. Avian serum. cap alpha. /sub 1/-glycoprotein, hemopexin, differing significantly in both amino acid and carbohydrate composition from mammalian (. beta. -glycoprotein) counter parts

    SciTech Connect

    Goldfarb, V.; Trimble, R.B.; Falco, M.D.; Liem, H.H.; Metcalfe, S.A.; Wellner, D.; Muller-Eberhard, U.

    1986-10-21

    The physicochemical characteristics of chicken hemopexin, which can be isolated by heme-agarose affinity chromatography, is compared with representative mammalian hemopexins of rat, rabbit, and human. The avian polypeptide chain appears to be slightly longer (52 kDa) than the human, rat, or rabbit forms (49 kDa), and also the glycoprotein differs from the mammalian hemopexins in being an ..cap alpha../sub 1/-glycoprotein instead of a ..beta../sub 1/-glycoprotein. The distinct electrophoretic mobility probably arises from significant differences in the amino acid composition of the chicken form, which, although lower in serine and particularly in lysine, has a much higher glutamine/glutamate and agrinine content, and also a higher proline, glycine, and histidine content, than the mammalian hemopexins. Compositional analyses and /sup 125/I concanavalin A and /sup 125/I wheat germ agglutinin binding suggest that chicken hemopexin has a mixture of three fucose-free N-linked bi- and triantennary oligosaccharides. In contrast, human hemopexin has give N-linked oligosaccharides and an additional O-linked glycan blocking the N-terminal threonine residue, while the rabbit form has four N-linked oligosaccharides. In keeping with the finding of a simpler carbohydrate structure, the avian hemopexin shows only a single band on polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions, whereas the hemopexins of the three mammalian species tested show several bands. In contrast, the isoelectric focusing pattern of chicken hemopexin is very complex, revealing at least nine bands between pH 4.0 and pH band 5.0, while the other hemopexins show a broad smear of multiple ill-defined bands in the same region.Results indicate the hemopexin of avians differs substantially from the hemopexins of mammals, which show a notable similarity with regard to carbohydrate structure and amino acid composition.

  17. Properties of potato lectin and the nature of its glycoprotein linkages

    PubMed Central

    Allen, Anthony K.; Desai, Nila N.; Neuberger, Albert; Creeth, J. Michael

    1978-01-01

    1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer–dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000–34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the β-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although α-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of β-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single α-galactopyranoside residues that can be removed by the action of α-galactosidase from coffee beans but not by a β-galactosidase. This is the first report of an α-galactoside linkage to serine. The effect of α-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of β-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues

  18. Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

    PubMed

    Zhou, Xuan; Hong, Guo-Ying; Huang, Bin-Bin; Duan, Yuan-Meng; Shen, Jia-Yi; Ni, Mao-Wei; Cong, Wei-Tao; Jin, Li-Tai

    2014-05-01

    An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins. PMID:24591039

  19. Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum.

    PubMed Central

    Trombetta, E S; Helenius, A

    1999-01-01

    UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle. We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP. This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences. It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER. PMID:10369669

  20. On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins.

    PubMed

    Barroso, Begona; Dijkstra, Rene; Geerts, Marlieke; Lagerwerf, Fija; van Veelen, Peter; de Ru, Arnoud

    2002-01-01

    An on-line high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is described for the rapid characterization of any type of oligosaccharide released from glycoproteins. The procedure can be applied without further manipulation to fractions collected from a high-performance anion-exchange chromatography-pulse amperometric detection (HPAEC-PAD) system commonly used for glycosylation mapping of glycoproteins, or to a pool of oligosaccharides directly released from glycoproteins. The system consists of a porous graphitized high-performance chromatography column (Hypercarb) coupled to a quadrupole time-of-flight (TOF) mass spectrometer. Oligosaccharides are eluted from the column with a gradient of ammonium acetate/acetonitrile and directly identified following in-source fragmentation. Some applications of the method are presented, as well as information about the spectra and fragmentation behavior observed for N- and O-linked oligosaccharides released from some recombinant glycoproteins. Low femtomole limits of detection are achieved using proper miniaturization. PMID:12112260

  1. A selective method for sequential splitting of O- and N-linked glycans from N,O-glycoproteins.

    PubMed

    Likhosherstov, L M; Novikova, O S; Derevitskaya, V A; Kochetkov, N K

    1990-05-15

    O-Linked oligosaccharides from N,O-glycoproteins were selectively split off by treatment with alkaline sodium borohydride in the presence of cadmium salt. The side reaction of reductive cleavage of N-glycosylamide and peptide bonds, observed under standard conditions of splitting of O-linked chains (M NaBH4 and 50mM NaOH, 16 h, 50 degrees), was inhibited by addition of 50-10 mM cadium acetate and 5-10mM EDTA.Na4, as shown by treatment of model compounds and several glycoproteins (ovomucoid, group-specific glycoproteins H and B, fetuin, and asialofetuin). This treatment, in combination with the previously developed procedure for the release of the N-linked oligosaccharide chains by lithium borohydride, allows a sequential, selective cleavage of O-, and then N-linked oligosaccharides from N,O-glycoproteins by chemical methods.

  2. Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells

    SciTech Connect

    Owens, R.J.; Compans, R.W.

    1989-02-01

    Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. The authors have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, /sup 125/I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.

  3. Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses.

    PubMed

    Zimmer, Gert; Locher, Samira; Berger Rentsch, Marianne; Halbherr, Stefan J

    2014-08-01

    Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

  4. Effects of carbohydrate precursors of glycoproteins on retention performance of a brightness discrimination task in rats.

    PubMed

    Popov, N; Hecker, C; Riechert, U; Matthies, H

    1985-01-01

    The influence of intraventricularly applied carbohydrate precursors of glycoproteins on acquisition and retention of a brightness discrimination task in rats was tested. The injection of 0.8 mumoles L-fucose/animal, N-acetylneuraminic acid or D-galactosamine as well as 2.4 mumoles/animal D-galactose or D-glucosamine 30 min before starting the behavioural experiments significantly improved the retention performance of the acquired behaviour. The intraventricular application of 2.4 mumoles D-mannose/animal had no influence on the behavioural parameters tested. The results are discussed in the light of an activation of glycoprotein formation in brain tissue mainly by carbohydrates which occupy terminal positions in the oligosaccharide chains.

  5. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology.

    PubMed

    Chua, Chong Long; Sam, I-Ching; Chan, Yoke Fun

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection. PMID:27233260

  6. Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carteri.

    PubMed

    Wenzl, S; Thym, D; Sumper, M

    1984-04-01

    We report the chemical characterization of the highly sulphated glycoprotein SSG 185 from Volvox carteri. SSG 185 is a hydroxyproline-containing, extracellular glycoprotein. The sulphate residues are clustered within the parent saccharide structure of SSG 185, since on mercaptolysis all the sulphate residues are recovered in a small saccharide fragment containing mannose, arabinose and sulphate (in a molar ratio of 112). SSG 185 is a short-lived molecule, serving as a precursor for a high mol. wt. component of the extracellular matrix. Synthesis of SSG 185 is developmentally controlled. Different SSG 185 variants, with unknown modifications in the sulphated saccharide fragment, are synthesized at different developmental stages or under the influence of the sexual inducer. These modifications remain conserved in the aggregated state of SSG 185, indicating the development-dependent modification of the extracellular matrix. PMID:16453512

  7. A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms.

    PubMed

    Machiah, Deepa K; Girish, K S; Gowda, T Veerabasappa

    2006-06-01

    Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis.

  8. Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers.

    PubMed

    Zoenen, Maxime; Urizar, Eneko; Swillens, Stéphane; Vassart, Gilbert; Costagliola, Sabine

    2012-01-01

    Glycoprotein hormone receptors show strong negative cooperativity. As a consequence, at physiological hormone concentrations, a single agonist binds to a receptor dimer. Here we present evidence that constitutively active receptors lose cooperative allosteric regulation in direct relation with their basal activity. The most constitutive mutants lost nearly all cooperativity and showed an increase of initial tracer binding, reflecting the ability of each protomer to bind with equal affinity. Allosteric interaction between the protomers takes place at the transmembrane domain. The allosteric message resulting from hormone binding to the ectodomain of one protomer travels 'downward' to its transmembrane domain, before affecting the transmembrane domain of the other protomer. This results in transmission of an 'upward' message lowering the binding affinity of the ectodomain of the second protomer. Our results demonstrate a direct relation between the conformational changes associated with activation of the transmembrane domain and the allosteric behaviour of glycoprotein hormone receptors dimers.

  9. Fibronectin is a binding partner for the myelin-associated glycoprotein (siglec-4a).

    PubMed

    Strenge, K; Brossmer, R; Ihrig, P; Schauer, R; Kelm, S

    2001-06-22

    The myelin-associated glycoprotein (MAG) mediates cell-cell interactions between myelinating glial cells and neurons. Here we describe the extracellular matrix glycoprotein fibronectin as a binding partner of MAG. It has been identified by affinity precipitation with MAG-Fc from NG108-15 cells and by microsequencing of two peptides derived from a 210-kDa protein band. Western blot analysis showed that fibronectin is also present in MAG binding partners isolated from N(2)A (murine neuroblastoma) cells, rat brain and rat spinal cord. Different fibronectin isoforms have been isolated from brains of young and adult rats, indicating that the expression of MAG binding fibronectin changes during development. PMID:11423128

  10. [Comparative studies of sera from cattle with complete leukemia virus and glycoprotein antigens].

    PubMed

    Mateva, V; Vasileva, L

    1980-01-01

    One hundred cattle serums were investigated by the AGTD-test with two antigens: an antigen produced by the whole virus and an antigen containing glycoproteins. Of all serums studied 44 showed a specific precipitation in case the glycoprotein antigen was used. In case the antigen from the whole virus was used 41 serums showed a specific precipitation line, while in 3 of the serums two precipitation lines were observed. Fifty six serums proved negative, containing no antibodies against bovine leucosis virus, after antigens were used. In 2 of the serums non specific precipitation lines were obtained when the antigen from whole virus was used. the precipitation lines produced by both antigenes did not differ in intensity and time of manifestation. PMID:6251597

  11. Glycoprotein IIb/IIIa Receptor Inhibitors During Primary Angioplasty for Acute Myocardial Infarction.

    PubMed

    Gruberg; Lansky; Dangas; Stone

    1999-12-01

    Platelet glycoprotein IIb/IIIa receptors are the final common pathway leading to platelet aggregation and coronary thrombosis during acute myocardial infarction (AMI). Therefore, they are ideal candidates for pharmacologic intervention. The recent development of glycoprotein IIb/IIIa receptor antagonists has led to several studies that have shown the benefits and efficacy of these agents in the treatment of acute coronary syndromes and in the setting of percutaneous intervention. To date, six published trials have examined the safety and efficacy of intravenous abciximab, a mouse/human chimeric version of the 7E3 antibody, as an adjunct to primary mechanical reperfusion in patients with AMI. In this article, we review these trials, as well as new studies currently underway that will provide further information on the long-term benefits of combining these pharmacologic agents and stenting in the treatment of AMI.

  12. Skeletal receptors for steroid-family regulating glycoprotein hormones: A multilevel, integrated physiological control system.

    PubMed

    Blair, Harry C; Robinson, Lisa J; Sun, Li; Isales, Carlos; Davies, Terry F; Zaidi, Mone

    2011-12-01

    Pituitary glycoprotein hormone receptors, including ACTH-R, TSH-R, and FSH-R, occur in bone. Their skeletal expression reflects that central endocrine control is evolutionarily recent. ACTH receptors, in osteoblasts or the adrenal cortex, drive VEGF synthesis. VEGF is essential to maintain vasculature. In bone, ACTH suppression by glucocorticoids can cause osteonecrosis. TSH receptors occur on osteoblasts and osteoclasts, in both cases reducing activity. Thus, TSH directly reduces skeletal turnover, consistent with evolutionary adaptation to stress. FSH receptors accelerate bone resorption, whereas estrogen promotes bone formation, the forces usually balancing. With ovarian failure, low estrogen with high FSH causes rapid bone loss. The skeletal FSH effect in the menopause seems paradoxical, but it is a logical adaptation in lactation, where prolonged FSH elevation also occurs. In addition to receptors, there is some synthesis of pituitary glycoproteins at distributed sites; this is not well studied, but it may further modify the paradigm of central endocrine regulation.

  13. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates.

    PubMed

    Wang, Yang; Rowley, Kirk J; Booth, Brian J; Sloan, Susan E; Ambrosino, Donna M; Babcock, Gregory J

    2011-08-01

    Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization.

  14. Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets.

    PubMed

    Artemenko, Elena O; Yakimenko, Alena O; Pichugin, Alexey V; Ataullakhanov, Fazly I; Panteleev, Mikhail A

    2016-02-15

    In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbβ3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.

  15. Fast and efficient online release of N-glycans from glycoproteins facilitating liquid chromatography-tandem mass spectrometry glycomic profiling.

    PubMed

    Jmeian, Yazen; Hammad, Loubna A; Mechref, Yehia

    2012-10-16

    A novel online enzyme reactor incorporating peptide-N-glycosidase F (PNGase F) on a monolithic polymer support has been developed to allow the rapid simultaneous release of both neutral and acidic N-linked glycans from glycoproteins. The PNGase F monolithic reactor was fabricated in a fused silica using glycidyl methacrylate-co-ethylene dimethacrylate polymer. The reactor was coupled to a C8 trap and a porous graphitic carbon (PGC) HPLC-chip. This arrangement was interfaced to an ion trap mass spectrometer for liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The performance of the PNGase F reactor was optimized using the MS signal for the disialylated biantennary N-glycan derived from fetuin. Optimum conditions for glycan release were attained at room temperature using a loading flow rate of 2 μL/min and a reaction time of 6 min. The loading capacity of the reactor was determined to be around 2 pmol of glycoprotein. The online digestion and MS characterization experiments resulted in sensitivities as high as 100 fmol of glycoprotein and 0.1 μL of human blood serum. The enzyme reactor activity was also shown to remain stable after 1 month of continuous use. Both small and large glycoproteins as well as glycoproteins containing high-mannose glycans, fucolsylated glycans, sialylated glycans, and hybrid structures were studied. The model glycoproteins included ribonuclease B, fetuin, α(1)-acid glycoprotein, immunoglobulin, and thyroglobulin. All N-glycans associated with these model glycoproteins were detected using the online PNGase F reactor setup.

  16. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  17. Studying the Impact of Presence of Alpha Acid Glycoprotein and Protein Glycoprotein in Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate in the State of Qatar

    PubMed Central

    Al-Dewik, Nader I.; Jewell, Andrew P.; Yassin, Mohammed A.; Morsi, Hisham M.

    2015-01-01

    Despite the efficacy of imatinib mesylate (IM) in treating chronic myeloid leukemia (CML), there is a high degree of resistance. Alpha- 1-acid glycoprotein may reduce drug efficacy through its ability to interact with IM and blocks it from reaching its target, while protein glycoprotein (PGP) may reduce the intracellular concentration of the drug via an active pump mechanism. We thus investigated the correlation between AGP and PGP levels and the resistance/response to treatment. A total of 26 CML patients were investigated for AGP and PGP levels at diagnosis and during treatment. There was no significant difference or correlation between AGP levels and the different groups of patients. There was also no significant difference in the fluorescence intensities of PGP levels among the different patient groups. The resistance observed in our CML patient population could not be correlated with AGP and PGP levels. There was no significant pattern of AGP and PGP expression, irrespective of the response or resistance to treatment. PMID:26640393

  18. Monospecific and common glycoprotein ligands for E- and P-selectin on myeloid cells

    PubMed Central

    1994-01-01

    E- and P-selectin are inducible cell adhesion molecules on endothelial cells, which function as Ca(2+)-dependent lectins and mediate the binding of neutrophils and monocytes. We have recently identified a 150- kD glycoprotein ligand for E-selectin on mouse myeloid cells, using a recombinant antibody-like form of mouse E-selectin. Here, we report that this ligand does not bind to an analogous P-selectin fusion protein. Instead, the chimeric P-selectin-IgG protein recognizes a 160- kD glycoprotein on the mouse neutrophil progenitor 32D cl 3, on mature mouse neutrophils and on human HL60 cells. The binding is Ca(2+)- dependent and requires the presence of sialic acid on the ligand. This P-selectin-ligand is not recognized by E-selectin. Removal of N-linked carbohydrate side chains from the 150-kD and the 160-kD monospecific selectin ligands abolishes the binding of both ligands to the respective selectin. Treatment of HL60 cells with Peptide: N- glycosidase F inhibited cell binding to P- and E-selectin. In addition, glycoproteins of 230 and 130 kD were found on mature mouse neutrophils, which bound both to E- and P-selectin in a Ca(2+)-dependent fashion. The signals detected for these ligands were 15-20-fold weaker than those for the monospecific ligands. Both proteins were heavily sialylated and selectin-binding was blocked by removal of sialic acid, but not by removal of N-linked carbohydrates. Our data reveal that E- and P-selectin recognize two categories of glycoprotein ligands: one type requires N-linked carbohydrates for binding and is monospecific for each of the two selectins and the other type binds independent of N- linked carbohydrates and is common for both endothelial selectins. PMID:7512971

  19. Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus

    USGS Publications Warehouse

    Huang, C.; Chien, M.S.; Landolt, M.L.; Batts, W.; Winton, J.

    1996-01-01

    Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.

  20. The "lecithotrophic" sea urchin Heliocidaris erythrogramma lacks typical yolk platelets and yolk glycoproteins.

    PubMed

    Scott, L B; Lennarz, W J; Raff, R A; Wray, G A

    1990-03-01

    The sea urchin Heliocidaris tuberculata undergoes typical development, forming an echinoid pluteus larva, whereas H. erythrogramma undergoes direct development via a highly modified, nonfeeding larva. Using a polyclonal antibody prepared against yolk glycoproteins from the typical developer Stronglyocentrotus purpuratus, we found that H. tuberculata contains cross-reactive proteins in abundance, but H. erythrogramma does not. In addition, we used immunoelectron microscopy to demonstrate that unfertilized eggs of H. tuberculata contain yolk platelets, but those of H. erythrogramma do not.

  1. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells.

    PubMed

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2014-03-01

    Glycosylation is an important attribute of baculovirus-insect cell expression systems, but some insect cell lines produce core α1,3-fucosylated N-glycans, which are highly immunogenic and render recombinant glycoproteins unsuitable for human use. To address this problem, we exploited a bacterial enzyme, guanosine-5'-diphospho (GDP)-4-dehydro-6-deoxy-d-mannose reductase (Rmd), which consumes the GDP-l-fucose precursor. We expected this enzyme to block glycoprotein fucosylation by blocking the production of GDP-l-fucose, the donor substrate required for this process. Initially, we engineered two different insect cell lines to constitutively express Rmd and isolated subclones with fucosylation-negative phenotypes. However, we found the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that this host cell engineering approach is ineffective in insect systems. Thus, we constructed a baculovirus vector designed to express Rmd immediately after infection and facilitate the insertion of genes encoding any glycoprotein of interest for expression later after infection. We used this vector to produce a daughter encoding rituximab and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a nonfucosylated form of this therapeutic antibody. These results indicate that our Rmd(+) baculoviral vector can be used to solve the immunogenic core α1,3-fucosylation problem associated with the baculovirus-insect cell system. In conjunction with existing glycoengineered insect cell lines, this vector extends the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production. This new vector also extends the utility of the baculovirus-insect cell system to include the production of recombinant antibodies with enhanced effector functions, due to its ability to block core α1,6-fucosylation.

  2. Cloning, sequence, and expression of the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus

    SciTech Connect

    Feyereisen-Koener, J.M.

    1987-01-01

    Double-stranded cDNA was prepared from infectious hematopoietic necrosis virus mRNA and cloned into the plasmid vector pUC8. A coprotein (G-protein) of infectious hematopoietic necrosis virus was selected by hybridization to a /sup 32/P-labeled probe. The restriction map and nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined using this full-length cDNA clone.

  3. Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat

    SciTech Connect

    Clermont, Y.; Tang, X.M.

    1985-09-01

    During steps 1-7 of spermiogenesis the Golgi apparatus contributes to the formation of the acrosomic system which develops at the surface of the nucleus. Later, in step 8, the Golgi apparatus detaches from the acrosome and remains suspended in the elongated cytoplasm until it degenerates during step 16. Using /sup 3/H-fucose as a tracer and the radioautographic technique, we observed that the Golgi apparatus incorporates the tracer and delivers the labeled glycoproteins to the developing acrosomic system during steps 1-7 of spermiogenesis, to multivesicular bodies during steps 1-9, and to the remaining cytoplasm and plasma membrane during steps 1-15. Throughout these steps of spermiogenesis the Golgi apparatus does not show major changes in structure; it is composed of a cortex made up of connected stacks of saccules and a medulla showing a loose aggregate of vesicular profiles. Glycoprotein synthesis in this Golgi apparatus, before and after it contributes lysosomal glycoproteins to the growing acrosomic system, was quantitatively assessed in electron microscope EM radioautographs of tissue sections from animals sacrificed at 1, 4, 8, and 24 h of 3H-fucose injection. The incorporation of the labeled sugar was found to remain quantitatively similar during steps 1-15 of spermiogenesis, and therefore, no shift in glycoprotein synthesis took place following separation of the Golgi apparatus from the acrosomic system. Throughout these steps, fucose molecules are first incorporated in the cortex of the organelle and subsequently transported to the medulla, where they temporarily accumulate before being delivered, depending on the step of spermiogenesis, to the acrosomic system, to the multivesicular bodies, and also, presumably, to the plasma membrane.

  4. Immunogenic glycoproteins of laboratory and vaccine strains of Varicella-Zoster virus.

    PubMed Central

    Grose, C; Edmond, B J; Friedrichs, W E

    1981-01-01

    High-titered antisera were prepared in guinea pigs and rabbits against two strains of varicella-zoster virus (VZV): VZV-32, a low-passage laboratory strain, and VZV-Oka, a vaccine strain attenuated by passage in both human and guinea pig embryo cells. When the animal VZV-immune sera, as well as a human zoster serum, were used to precipitate radiolabeled glycoproteins from VZV-infected cells and the immune precipitates were analyzed by polyacrylamide gel electrophoresis and fluorography, it was observed that cell cultures infected with either strain had similar electrophoretic profiles containing major glycoproteins of approximate molecular weights 62,000, 98,000, and 118,000. A prominent high-molecular-weight (approximately 150,000) nonglycosylated polypeptide was identified in both strains also. These determinants were demonstrable by both indirect (staphylococcal protein A-antibody adsorbent) and direct immunoprecipitation, as long as VZV-immune sera with an antibody titer greater than or equal to 1:128 were used. Further analysis of individual caviid VZV antisera demonstrated some heterogeneity which appeared to be related to the method of immunization rather than the level of virus-specific antibody. VZV extracts emulsified with complete Freund adjuvant elicited an antibody response to all major immunogenic viral glycoproteins, whereas guinea pigs inoculated with virus alone during the primary immunization initially produced VZV antibody which failed to precipitate the highest-molecular-weight glycoprotein (gp118). Thus, Freund-type adjuvants promoted the maturation of the humoral immune response after VZV immunization in outbred guinea pigs. Images PMID:6262245

  5. Hantavirus Gn and Gc Glycoproteins Self-Assemble into Virus-Like Particles

    PubMed Central

    Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L.; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves

    2014-01-01

    How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera. PMID:24335294

  6. Castanospermine inhibits glucosidase I and glycoprotein secretion in human hepatoma cells.

    PubMed Central

    Sasak, V W; Ordovas, J M; Elbein, A D; Berninger, R W

    1985-01-01

    We studied the effect of the plant alkaloid castanospermine on the biosynthesis and secretion of human hepatoma glycoproteins. The HepG-2 cells, grown in the presence or absence of the alkaloid, were labelled with [2-3H]mannose and then the labelled glycopeptides were prepared by Pronase digestion. This material was analysed by gel filtration on Bio-Gel P-4 before and after treatment with endo-beta-N-acetylglucosaminidase H. Castanospermine caused an accumulation of high-mannose oligosaccharides, by 70-75% over control. The major accumulated product, which could also be labelled with [3H]galactose and was only partially susceptible to alpha-mannosidase digestion, was identified by h.p.l.c. as a Glc3Man9GlcNAc. Thus the alkaloid inhibits glucosidase I in the human hepatoma cells. Analysis of total glycoproteins secreted by the cells into the medium revealed the presence of only complex oligosaccharides in both control and treated cultures, and the amount of the oligosaccharides labelled with radioactive mannose, galactose or N-acetylmannosamine, secreted by treated cells, was decreased by about 60%. The rate of secretion of total protein labelled with [35S]methionine and precipitated from the medium with trichloroacetic acid was inhibited by up to 40% in the presence of castanospermine. Pulse-chase studies utilizing [35S]methionine labelling were performed to study the effect of the alkaloid on secretion of individual plasma proteins. Immunoprecipitation at different chase times with monospecific antisera showed that castanospermine markedly decreased the secretion rates of alpha 1-antitrypsin, caeruloplasmin and, to a lesser extent, that of antithrombin-III. Secretions of apolipoprotein E, a glycoprotein containing only O-linked oligosaccharide(s), and albumin, a non-glycosylated protein, were not affected by the drug. It is suggested that castanospermine inhibits secretion of at least some glycoproteins containing N-linked oligosaccharides, owing to the inhibition

  7. Aspirin resistance in cerebrovascular disease and the role of glycoprotein IIIa polymorphism in Turkish stroke patients.

    PubMed

    Derle, Eda; Öcal, Ruhsen; Kibaroğlu, Seda; Çelikkol, Ceyda; Bayraktar, Nilüfer; Verdi, Hasibe; Ataç, Belgin F; Can, Ufuk

    2016-03-01

    Aspirin resistance occurs in 5-45% of high-risk patients, with various mechanisms proposed for its development. This study aimed to determine the relationships among aspirin resistance, aspirin dosage, type of aspirin and glycoprotein IIIa P1A1/A2 polymorphism in patients with vascular risk factors. Two hundred and eight (75 symptomatic, 133 asymptomatic) patients with vascular risk factors who were using aspirin for primary or secondary prevention were prospectively included. The symptomatic group was further classified into two groups according to aspirin use at the time of stroke. Aspirin resistance was measured by the PFA-100 system (collagen/epinephrine cartridge) and glycoprotein IIIa P1A1/A2 polymorphism was determined by PCR. The overall prevalence of aspirin resistance was 32.2%. The mean age of patients with aspirin resistance was significantly higher than that in those who did not have resistance (P = 0.009). The prevalence of aspirin resistance was similar for the symptomatic and asymptomatic under aspirin therapy groups. The resistance rate was found to be highest with 100 mg enteric-coated preparation use (39.3%). Increasing the aspirin dosage and/or shifting to uncoated preparations caused a change in aspirin sensitivity of 36-60%. Repeated measurements showed development of aspirin resistance in 14% of patients who were sensitive to aspirin in previous measurements. Glycoprotein IIIaP1A1/A2 polymorphism, aspirin resistance and development of atherothrombotic stroke were not significantly related. The effect of aspirin can change by time, dosage and type of preparation used. There are no relationships among glycoprotein IIIa P1A1/A2 polymorphism, aspirin resistance and development of atherothrombotic stroke.

  8. Mass spectrometry signal amplification for ultrasensitive glycoprotein detection using gold nanoparticle as mass tag combined with boronic acid based isolation strategy.

    PubMed

    Liu, Minbo; Zhang, Lijuan; Xu, Yawei; Yang, Pengyuan; Lu, Haojie

    2013-07-25

    We describe a novel method for rapid and ultrasensitive detection of intact glycoproteins without enzymatic pretreatment which was commonly used in proteomic research. This method is based on using gold nanoparticle (AuNP) as signal tag in laser desorption/ionization mass spectrometry (LDI-MS) analysis combined with boronic acid assisted isolation strategy. Briefly speaking, target glycoproteins were firstly isolated from sample solution with boronic acid functionalized magnetic microparticles, and then the surface modified gold nanoparticles were added to covalently bind to the glycoproteins. After that, these AuNP tagged glycoproteins were eluted from magnetic microparticles and applied to LDI-MS analysis. The mass signal of AuNP rather than that of glycoprotein was detected and recorded in this strategy. Through data processing of different standard glycoproteins, we have demonstrated that the signal of AuNP could be used to quantitatively represent glycoprotein. This method allows femtomolar detection of intact glycoproteins. We believe that the successful validation of this method on three different kinds of glycoproteins suggests the potential use for tracking trace amount of target glycoproteins in real biological samples in the near future.

  9. Comparison of N-linked Glycoproteins in Human Whole Saliva, Parotid, Submandibular, and Sublingual Glandular Secretions Identified using Hydrazide Chemistry and Mass Spectrometry.

    PubMed

    Ramachandran, Prasanna; Boontheung, Pinmanee; Pang, Eric; Yan, Weihong; Wong, David T; Loo, Joseph A

    2008-12-01

    INTRODUCTION: Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids. MATERIALS AND METHODS: We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography-tandem mass spectrometry was used to identify the salivary N-glycoproteins. RESULTS: A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions. CONCLUSIONS: Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.

  10. LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.

    PubMed

    Song, Ehwang; Zhu, Rui; Hammoud, Zane T; Mechref, Yehia

    2014-11-01

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC-MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC-ESI-MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts. PMID:25134008

  11. LC–MS/MS Quantitation of Esophagus Disease Blood Serum Glycoproteins by Enrichment with Hydrazide Chemistry and Lectin Affinity Chromatography

    PubMed Central

    2015-01-01

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC–MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC–ESI–MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts. PMID:25134008

  12. Role of P-glycoprotein in refractoriness of seizures to antiepileptic drugs in Lennox-Gastaut syndrome.

    PubMed

    Kumar, Achal; Tripathi, Deepak; Paliwal, Vimal Kumar; Neyaz, Zafar; Agarwal, Vikas

    2015-02-01

    Mechanism of seizure refractoriness to antiepileptic drugs in children with Lennox-Gastaut syndrome is not known. Efflux of antiepileptic drugs due to increased expression/function of P-glycoprotein, a multidrug efflux transporter protein on the cell surface is a proposed mechanism. The authors studied the expression/function of P-glycoprotein on peripheral blood mononuclear cells of 29 children with Lennox-Gastaut syndrome, 23 children with other epilepsies, and 19 healthy children. The authors found a higher P-glycoprotein expression/function in Lennox-Gastaut syndrome, a higher percent positive cells as compared to children with other epilepsy (P < 0.001) and to healthy controls (P = 0.012), higher P-glycoprotein expression as compared to healthy controls (P = 0.003), a higher total P-glycoprotein expression (relative florescence intensity × percent positive cells) as compared to children with other epilepsies (P < 0.001) and healthy controls (P < 0.001), and a higher P-glycoprotein function as compared to children with other epilepsies (P = 0.001) and healthy controls (P = 0.002). These findings may explain seizure refractoriness to anti-epileptic drugs in Lennox-Gastaut syndome.

  13. Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

    PubMed

    Mabashi-Asazuma, Hideaki; Shi, Xianzong; Geisler, Christoph; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2013-02-01

    Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

  14. LC-MS/MS quantitation of esophagus disease blood serum glycoproteins by enrichment with hydrazide chemistry and lectin affinity chromatography.

    PubMed

    Song, Ehwang; Zhu, Rui; Hammoud, Zane T; Mechref, Yehia

    2014-11-01

    Changes in glycosylation have been shown to have a profound correlation with development/malignancy in many cancer types. Currently, two major enrichment techniques have been widely applied in glycoproteomics, namely, lectin affinity chromatography (LAC)-based and hydrazide chemistry (HC)-based enrichments. Here we report the LC-MS/MS quantitative analyses of human blood serum glycoproteins and glycopeptides associated with esophageal diseases by LAC- and HC-based enrichment. The separate and complementary qualitative and quantitative data analyses of protein glycosylation were performed using both enrichment techniques. Chemometric and statistical evaluations, PCA plots, or ANOVA test, respectively, were employed to determine and confirm candidate cancer-associated glycoprotein/glycopeptide biomarkers. Out of 139, 59 common glycoproteins (42% overlap) were observed in both enrichment techniques. This overlap is very similar to previously published studies. The quantitation and evaluation of significantly changed glycoproteins/glycopeptides are complementary between LAC and HC enrichments. LC-ESI-MS/MS analyses indicated that 7 glycoproteins enriched by LAC and 11 glycoproteins enriched by HC showed significantly different abundances between disease-free and disease cohorts. Multiple reaction monitoring quantitation resulted in 13 glycopeptides by LAC enrichment and 10 glycosylation sites by HC enrichment to be statistically different among disease cohorts.

  15. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes▿†

    PubMed Central

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D.

    2011-01-01

    Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-Å crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen. PMID:21613394

  16. Endoplasmic reticulum-associated N-glycan degradation of cold-upregulated glycoproteins in response to chilling stress in Arabidopsis.

    PubMed

    Ma, Jun; Wang, Dinghe; She, Jessica; Li, Jianming; Zhu, Jian-Kang; She, Yi-Min

    2016-10-01

    N-glycosylation has a great impact on glycoprotein structure, conformation, stability, solubility, immunogenicity and enzyme activity. Structural characterization of N-glycoproteome has been challenging but can provide insights into the extent of protein folding and surface topology. We describe a highly sensitive proteomics method for large-scale identification and quantification of glycoproteins in Arabidopsis through (15) N-metabolic labeling, selective enrichment of glycopeptides, data-dependent MS/MS analysis and automated database searching. In-house databases of Arabidopsis glycoproteins and glycopeptides containing Asn-X-Ser/Thr/Cys motifs were constructed by reducing 20% and 90% of the public database size, respectively, to enable a rapid analysis of large datasets for comprehensive identification and quantification of glycoproteins and heterogeneous N-glycans in a complex mixture. Proteome-wide analysis identified c. 100 stress-related N-glycoproteins, of which the endoplasmic reticulum (ER) resident proteins were examined to be up-regulated. Quantitative measurements provided a molecular signature specific to glycoproteins for determining the degree of plant stress at low temperature. Structural N-glycoproteomics following time-course cold treatments revealed the stress-responsive degradation of high-mannose type N-glycans in ER in response to chilling stress, which may aid in elucidating the cellular mechanisms of protein relocation, transport, trafficking, misfolding and degradation under stress conditions. PMID:27558752

  17. Glycoproteins in human parotid saliva assessed by lectin probes after resolution by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

    PubMed

    Carpenter, G H; Proctor, G B; Pankhurst, C L; Linden, R W; Shori, D K; Zhang, X S

    1996-01-01

    Human parotid salivary glycoproteins separated by gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose have been investigated using a battery of biotinylated lectin probes of characterized sugar specificity. Lectin binding, detected on blots using avidin-biotin complex (ABC) and a chemiluminescence generating substrate, was recorded on photographic film and compared with the original fluorescein isothiocyanate (FITC) stained blots or with Coomassie Brilliant Blue R-250-stained gels run in parallel. A number of glycoprotein bands which were undetected by protein stains or the periodic acid Schiff reaction were revealed by lectins. Binding by lectins from Concanavalia ensiformis, Lens culinaris, Limax flavus, Phaseolus vulgaris, Ricinus communis, Triticum vulgaris, Lotus tetragonobulus and Ulex europaeus indicated that sialylated and fucosylated triantennary and bisected, N-linked complex sugar chains were present on many glycoproteins in addition to the major glycosylated proline-rich glycoprotein (GI). Binding with lectins from Arachis hypogaea and Dolichos biflorus indicated that the O-linked sugar chains were confined to the alpha-heavy chain of Ig A. Comparison of lectin binding in samples from five healthy individuals revealed differences in a number of glycoproteins in addition to the previously characterized G1 and CON 1/CON 2 polymorphisms and demonstrated that the H blood group antigen was expressed mainly on G1 in parotid saliva. This study will be used as a basis upon which to study salivary glycoproteins in diseases affecting parotid glands.

  18. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D.

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  19. The impact of UVB radiation on the glycoprotein glue of orb-weaving spider capture thread.

    PubMed

    Stellwagen, Sarah D; Opell, Brent D; Clouse, Mary E

    2015-09-01

    Many spider orb-webs are exposed to sunlight and the potentially damaging effects of ultraviolet B (UVB) radiation. We examined the effect of UVB on the viscoelastic glycoprotein core of glue droplets deposited on the prey capture threads of these webs, hypothesizing that webs built by species that occupy sunny habitats are less susceptible to UVB damage than are webs built by species that prefer shaded forest habitats or by nocturnal species. Threads were tested shortly after being collected in the early morning and after being exposed to UVB energy equivalent to a day of summer sun and three times this amount. Droplets kept in a dark chamber allowed us to evaluate post-production changes. Droplet volume was unaffected by treatments, indicating that UVB did not damage the hygroscopic compounds in the aqueous layer that covers droplets. UVB exposure did not affect energies of droplet extension for species from exposed and partially to mostly shaded habitats (Argiope aurantia, Leucauge venusta and Verrucosa arenata). However, UVB exposure reduced the energy of droplet extension in Micrathena gracilis from shaded forests and Neoscona crucifera, which forages at night. Only in L. venusta did the energy of droplet extension increase after the dark treatment, suggesting endogenous molecular alignment. This study adds UVB irradiation to the list of factors (humidity, temperature and strain rate) known to affect the performance of spider glycoprotein glue, factors that must be more fully understood if adhesives that mimic spider glycoprotein glue are to be produced. PMID:26333924

  20. Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

    PubMed

    Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

    2016-07-01

    Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection. PMID:27068162

  1. The N-glycan Glycoprotein Deglycosylation Complex (Gpd) from Capnocytophaga canimorsus Deglycosylates Human IgG

    PubMed Central

    Renzi, Francesco; Manfredi, Pablo; Mally, Manuela; Moes, Suzette; Jenö, Paul; Cornelis, Guy R.

    2011-01-01

    C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases. PMID:21738475

  2. Intrinsic membrane glycoproteins with cytosol-oriented sugars in the endoplasmic reticulum

    SciTech Connect

    Abeijon, C.; Hirschberg, C.B.

    1988-02-01

    The authors have examined the topography of N-acetylglucosamine-terminating glycoproteins in membranes from rat liver smooth and rough endoplasmic reticulum (SER and RER). It was found that some of these glycoproteins are intrinsic membrane proteins with their sugars facing the cytosolic rather than the luminal side. This conclusion was reached by using vesicles from the SER and RER that were sealed and of the same topographical orientation as in vivo. These vesicles were incubated with UDP-(/sup 14/C)galactose (which does not enter the vesicles) and saturating amounts of soluble galactosyltransferase from milk, an enzyme that does not penetrate the lumen of the vesicles and that specifically adds galactose to terminal N-acetylglucosamine in a ..beta..1-4 linkage. Radioactive galactose was mainly transferred to SER proteins of apparent molecular mass 56 and 110 kDa and to a lesser extent RER and SER proteins of apparent molecular mass 46 and 72 kDa. These proteins are intrinsic membrane proteins, based on the inability of sodium carbonate at pH 11.5 to remove them from the membranes. Studies with peptide N-glycosidase F and chemical ..beta..-elimination showed that the 56-kDa protein of the SER vesicles contained terminal N-acetylglucosamine in an O-linkage to the protein. The above results suggest that some sugars of glycoproteins in the endoplasmic reticulum may attain their final orientation in the membrane by mechanisms yet to be determined.

  3. Lactobacillus plantarum L67 glycoprotein protects against cadmium chloride toxicity in RAW 264.7 cells.

    PubMed

    Song, Sooyeon; Oh, Sejong; Lim, Kye-Taek

    2016-03-01

    The food and water we consume may be contaminated with a range of chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and mercury by accumulation through the food chain. Cadmium is known to be one of the major components in cigarette smoke and can cause lesions in many organs. Some lactobacilli can bind and remove heavy metals such as cadmium, lead, and copper. However, the mechanisms of cadmium toxicity and inhibition by probiotics are not clear. In this study, we demonstrated that glycoprotein (18 kDa) isolated from Lactobacillus plantarum L67 protected RAW 264.7 cells from expression of inflammation-related factors stimulated by cadmium chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium using the MTT assay and intracellular Ca(2+) using fluorescence, and assessed activities of activator protein kinase C (PKC-α), inducible nitric oxide synthase, activator protein (AP)-1, and mitogen-activated protein kinases using immunoblot. Our results indicated that glycoprotein isolated from L. plantarum L67 inhibited intracellular Ca(2+) mobilization. It also significantly suppressed inflammatory factors such as AP-1 (c-Jun and c-Fos), mitogen-activated protein kinases (ERK, JNK, and p38), and inducible nitric oxide synthase. Our findings suggest that the 24-kDa glycoprotein isolated from L. plantarum L67 might be used as a food component for protection of inflammation caused by cadmium ion. PMID:26774722

  4. Preparation of a boronate-functionalized affinity hybrid monolith for specific capture of glycoproteins.

    PubMed

    Yang, F; Mao, J; He, X W; Chen, L X; Zhang, Y K

    2013-06-01

    A novel strategy for preparation of a boronate affinity hybrid monolith was developed using a Cu(I)-catalyzed 1,3-dipolar azide-alkyne cycloaddition (CuAAC) reaction of an alkyne-boronate ligand with an azide-functionalized monolithic intermediate. An azide-functionalized hybrid monolith was first synthesized via a single-step procedure to provide reactive sites for click chemistry; then the alkyne-boronate ligands were covalently immobilized on the azide-functionalized hybrid monolith via an in-column CuAAC reaction to form a boronate affinity hybrid monolith under mild conditions. The boronate affinity monolith was characterized and evaluated by means of elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The boronate affinity hybrid monolith exhibited excellent specificity toward nucleosides and glycoproteins, which were chosen as test cis-diol-containing compounds under neutral conditions. The binding capacity of the monolith for the glycoprotein ovalbumin was 2.36 mg · g(-1) at pH 7.0. The practicability of the boronate affinity hybrid monolithic material was demonstrated by specific capture of the glycoproteins ovalbumin and ovotransferrin from an egg sample.

  5. A Particle-Associated Glycoprotein Signal Peptide Essential for Virus Maturation and Infectivity

    PubMed Central

    Lindemann, Dirk; Pietschmann, Thomas; Picard-Maureau, Marcus; Berg, Angelika; Heinkelein, Martin; Thurow, Jana; Knaus, Petra; Zentgraf, Hanswalter; Rethwilm, Axel

    2001-01-01

    Signal peptides (SP) are key determinants for targeting glycoproteins to the secretory pathway. Here we describe the involvement in particle maturation as an additional function of a viral glycoprotein SP. The SP of foamy virus (FV) envelope glycoprotein is predicted to be unusually long. Using an SP-specific antiserum, we demonstrate that its proteolytic removal occurs posttranslationally by a cellular protease and that the major N-terminal cleavage product, gp18, is found in purified viral particles. Analysis of mutants in proposed signal peptidase cleavage positions and N-glycosylation sites revealed an SP about 148 amino acids (aa) in length. FV particle release from infected cells requires the presence of cognate envelope protein and cleavage of its SP sequence. An N-terminal 15-aa SP domain with two conserved tryptophan residues was found to be essential for the egress of FV particles. While the SP N terminus was found to mediate the specificity of FV Env to interact with FV capsids, it was dispensable for Env targeting to the secretory pathway and FV envelope-mediated infectivity of murine leukemia virus pseudotypes. PMID:11390578

  6. The impact of UVB radiation on the glycoprotein glue of orb-weaving spider capture thread.

    PubMed

    Stellwagen, Sarah D; Opell, Brent D; Clouse, Mary E

    2015-09-01

    Many spider orb-webs are exposed to sunlight and the potentially damaging effects of ultraviolet B (UVB) radiation. We examined the effect of UVB on the viscoelastic glycoprotein core of glue droplets deposited on the prey capture threads of these webs, hypothesizing that webs built by species that occupy sunny habitats are less susceptible to UVB damage than are webs built by species that prefer shaded forest habitats or by nocturnal species. Threads were tested shortly after being collected in the early morning and after being exposed to UVB energy equivalent to a day of summer sun and three times this amount. Droplets kept in a dark chamber allowed us to evaluate post-production changes. Droplet volume was unaffected by treatments, indicating that UVB did not damage the hygroscopic compounds in the aqueous layer that covers droplets. UVB exposure did not affect energies of droplet extension for species from exposed and partially to mostly shaded habitats (Argiope aurantia, Leucauge venusta and Verrucosa arenata). However, UVB exposure reduced the energy of droplet extension in Micrathena gracilis from shaded forests and Neoscona crucifera, which forages at night. Only in L. venusta did the energy of droplet extension increase after the dark treatment, suggesting endogenous molecular alignment. This study adds UVB irradiation to the list of factors (humidity, temperature and strain rate) known to affect the performance of spider glycoprotein glue, factors that must be more fully understood if adhesives that mimic spider glycoprotein glue are to be produced.

  7. Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

    SciTech Connect

    Dalton, J.P.; Strand, M.; Mangold, B.L.; Dean, D.A.

    1986-06-15

    The humoral immune response of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one-and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with sulfur-35 methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by the sera of infected mice. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.

  8. Mannosidase IA is in Quality Control Vesicles and Participates in Glycoprotein Targeting to ERAD.

    PubMed

    Ogen-Shtern, Navit; Avezov, Edward; Shenkman, Marina; Benyair, Ron; Lederkremer, Gerardo Z

    2016-08-14

    Endoplasmic reticulum-associated degradation (ERAD) of a misfolded glycoprotein in mammalian cells requires the removal of 3-4 alpha 1,2 linked mannose residues from its N-glycans. The trimming and recognition processes are ascribed to ER Mannosidase I, the ER-degradation enhancing mannosidase-like proteins (EDEMs), and the lectins OS-9 and XTP3-B, all residing in the ER, the ER-derived quality control compartment (ERQC), or quality control vesicles (QCVs). Folded glycoproteins with untrimmed glycans are transported from the ER to the Golgi complex, where they are substrates of other alpha 1,2 mannosidases, IA, IB, and IC. The apparent redundancy of these enzymes has been puzzling for many years. We have now determined that, surprisingly, mannosidase IA is not located in the Golgi but resides in QCVs. We had recently described this type of vesicles, which carry ER α1,2 mannosidase I (ERManI). We show that the overexpression of alpha class I α1,2 mannosidase IA (ManIA) significantly enhances the degradation of ERAD substrates and its knockdown stabilizes it. Our results indicate that ManIA trims mannose residues from Man9GlcNAc2 down to Man5GlcNAc2, acting in parallel with ERManI and the EDEMs, and targeting misfolded glycoproteins to ERAD.

  9. Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase

    PubMed Central

    Shi, Xiaohong; Botting, Catherine H.; Li, Ping; Niglas, Mark; Brennan, Benjamin; Shirran, Sally L.; Szemiel, Agnieszka M.; Elliott, Richard M.

    2016-01-01

    The M genome segment of Bunyamwera virus (BUNV)—the prototype of both the Bunyaviridae family and the Orthobunyavirus genus—encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SPNSm and NSm domain V as SPGc. Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SPGc) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17–312 or nearby residues; NSm, 332–477; and Gc, 478–1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies. PMID:27439867

  10. Malonic acid suppresses mucin-type O-glycan degradation during hydrazine treatment of glycoproteins.

    PubMed

    Goso, Yukinobu

    2016-03-01

    Hydrazine treatment is frequently used for releasing mucin-type O-glycans (O-glycans) from glycoproteins because the method provides O-glycans that retain a reducible GalNAc at their reducing end, which is available for fluorescent labeling. However, many O-glycans are degraded by "peeling" during this treatment. In the current study, it was found that malonic acid suppressed O-glycan degradation during hydrazine treatment of bovine fetuin or porcine gastric mucin in both the gas and liquid phases. This is paradoxical because the release of O-glycans from glycoproteins occurs under alkaline conditions. However, malonic acid seems to prevent the degradation through its acidic property given that other weak acids also prevented the degradation. Accordingly, disodium malonate did not suppress O-glycan degradation. Application of this method to rat gastric mucin demonstrated that the majority of the major O-glycans obtained in the presence of malonic acid were intact, whereas those obtained in the absence of malonic acid were degraded. These results suggest that hydrazine treatment in the presence of malonic acid would allow glycomic analysis of native mucin glycoproteins.

  11. Evaluation of genipin on human cytochrome P450 isoenzymes and P-glycoprotein in vitro.

    PubMed

    Gao, Li-Na; Zhang, Ye; Cui, Yuan-Lu; Yan, Kuo

    2014-10-01

    Genipin is obtained from the fruit of Gardenia jasminoides Ellis and acts as an herbal medicine or functional food in East Asia. In addition to produce natural colorant, it possesses widely antiinflammatory, antithrombotic, antidepressive and anticarcinogenic activities. However, little research focuses on the potential of genipin for drug-drug interactions. In this study, effects of genipin on mRNA and protein expression of cytochrome P450 (CYP) 2C19, CYP2D6 and CYP3A4 were detected by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) and Western blot, respectively, in human hepatoma HepG2 cells. Enzyme activities of which were detected by luminogenic CYP assay in vitro. Moreover, effect of genipin on P-glycoprotein expression was analyzed by Western blot. Results showed that genipin possessed a significant induction on CYP2D6 and a remarkable inhibition on CYP2C19 and CYP3A4 not only from the expression of mRNA and protein (P<0.05 or P<0.01), but the level of enzyme activity. Moreover, a concentration-dependent induction of genipin on P-glycoprotein expression was observed. In conclusion, caution should be exercised with respect to the induction or inhibition of genipin on CYP isoenzymes and the strong induction on P-glycoprotein. PMID:25073096

  12. Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors

    PubMed Central

    Olejnik, Beata; Jarząb, Anna; Kratz, Ewa M.; Zimmer, Mariusz; Gamian, Andrzej; Ferens-Sieczkowska, Mirosława

    2015-01-01

    The impact of seminal plasma components on the fertilization outcomes in humans is still under question. The increasing number of couples facing problems with conception raises the need for predictive biomarkers. Detailed understanding of the molecular mechanisms accompanying fertilization remains another challenge. Carbohydrate–protein recognition may be of key importance in this complex field. In this study, we analyzed the unique glycosylation pattern of seminal plasma proteins, the display of high-mannose and hybrid-type oligosaccharides, by means of their reactivity with mannose-specific Galanthus nivalis lectin. Normozoospermic infertile subjects presented decreased amounts of lectin-reactive glycoepitopes compared to fertile donors and infertile patients with abnormal semen parameters. Glycoproteins containing unveiled mannose were isolated in affinity chromatography, and 17 glycoproteins were identified in liquid chromatography-tandem mass spectrometry with electrospray ionization. The N-glycome of the isolated glycoproteins was examined in matrix-assisted laser desorption ionization mass spectrometry. Eleven out of 27 identified oligosaccharides expressed terminal mannose residues, responsible for lectin binding. We suggest that lowered content of high-mannose and hybrid type glycans in normozoospermic infertile patients may be associated with impaired sperm protection from preterm capacitation and should be considered in the search for new infertility markers. PMID:26147424

  13. Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation

    PubMed Central

    Frendo, Jean-Louis; Olivier, Delphine; Cheynet, Valérie; Blond, Jean-Luc; Bouton, Olivier; Vidaud, Michel; Rabreau, Michèle; Evain-Brion, Danièle; Mallet, François

    2003-01-01

    We recently demonstrated that the product of the HERV-W env gene, a retroviral envelope protein also dubbed syncytin, is a highly fusogenic membrane glycoprotein inducing the formation of syncytia on interaction with the type D mammalian retrovirus receptor. In addition, the detection of HERV-W Env protein (Env-W) expression in placental tissue sections led us to propose a role for this fusogenic glycoprotein in placenta formation. To evaluate this hypothesis, we analyzed the involvement of Env-W in the differentiation of primary cultures of human villous cytotrophoblasts that spontaneously differentiate by cell fusion into syncytiotrophoblasts in vitro. First, we observed that HERV-W env mRNA and glycoprotein expression are colinear with primary cytotrophoblast differentiation and with expression of human chorionic gonadotropin (hCG), a marker of syncytiotrophoblast formation. Second, we observed that in vitro stimulation of trophoblast cell fusion and differentiation by cyclic AMP is also associated with a concomitant increase in HERV-W env and hCG mRNA and protein expression. Finally, by using specific antisense oligonucleotides, we demonstrated that inhibition of Env-W protein expression leads to a decrease of trophoblast fusion and differentiation, with the secretion of hCG in culture medium of antisense oligonucleotide-treated cells being decreased by fivefold. Taken together, these results strongly support a direct role for Env-W in human trophoblast cell fusion and differentiation. PMID:12724415

  14. Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins.

    PubMed

    Prater, Bradley D; Anumula, Kalyan R; Hutchins, Jeff T

    2007-10-15

    A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 min. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobulin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.

  15. Crystal Structure of Glycoprotein C from a Hantavirus in the Post-fusion Conformation

    PubMed Central

    Willensky, Shmuel; Bignon, Eduardo A.; Tischler, Nicole D.; Dessau, Moshe

    2016-01-01

    Hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. Like other members in the Bunyaviridae family their M segment encodes two glycoproteins, GN and GC, which are responsible for the early events of infection. Hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced pH of the endosome. Unlike phleboviruses (e.g. Rift valley fever virus), that have an icosahedral glycoprotein envelope, hantaviruses display a pleomorphic virion morphology as GN and GC assemble into spikes with apparent four-fold symmetry organized in a grid-like pattern on the viral membrane. Here we present the crystal structure of glycoprotein C (GC) from Puumala virus (PUUV), a representative member of the Hantavirus genus. The crystal structure shows GC as the membrane fusion effector of PUUV and it presents a class II membrane fusion protein fold. Furthermore, GC was crystallized in its post-fusion trimeric conformation that until now had been observed only in Flavi- and Togaviridae family members. The PUUV GC structure together with our functional data provides intriguing evolutionary and mechanistic insights into class II membrane fusion proteins and reveals new targets for membrane fusion inhibitors against these important pathogens. PMID:27783673

  16. Microchip capillary gel electrophoresis of multiply PEGylated high-molecular-mass glycoproteins.

    PubMed

    Seyfried, Birgit K; Marchetti-Deschmann, Martina; Siekmann, Jürgen; Bossard, Mary J; Scheiflinger, Friedrich; Turecek, Peter L; Allmaier, Günter

    2012-05-01

    PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation ("MCGE fingerprinting") of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range.

  17. Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

    PubMed Central

    Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

    2015-01-01

    Objective Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Methods Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. Results A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. Conclusion A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC. PMID:26487969

  18. Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms

    PubMed Central

    Hogue, Ian B.; Scherer, Julian

    2016-01-01

    ABSTRACT Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway. PMID:27273828

  19. Glycosylation and sulphation of colonic mucus glycoproteins in patients with ulcerative colitis and in healthy subjects.

    PubMed Central

    Morita, H; Kettlewell, M G; Jewell, D P; Kent, P W

    1993-01-01

    Studies have been made of mucus glycoprotein biosynthesis in different regions of the lower gastrointestinal tract in normal patients and those with ulcerative colitis (UC), active or inactive, by means of 3H-glucosamine (3H-GlcNH2)--35S-sulphate double labelling of epithelial biopsy specimens under culture conditions. The time based rate of 3H-GlcNH2 labelling of mucus in rectal tissue was similar to that in active or inactive UC whereas the rate of 35SO4(2) labelling was significantly increased in active disease. The 3H specific activities measuring the amount of isotopic incorporation into surface and tissue mucus glycoproteins were increased in patients with active UC compared with normal or inactive subjects. The 35S specific activities did not differ significantly between patients with active UC and those in remission. In the rectum, glycosylation of mucus glycoproteins decreases with the increasing age of the patient. Regional differences in 3H-labelling of mucus components are reported for ascending colon, transverse colon, sigmoid colon, and rectum. Sulphation (35S-labelling) was higher in all parts of the colon in left sided UC. Results point to accelerated glycosylation of core proteins in the active phase of UC. PMID:8344580

  20. Clearance and binding of radiolabeled glycoproteins by cells of the murine mononuclear phagocyte system

    SciTech Connect

    Imber, M.J.

    1982-01-01

    The clearance and binding of radiolabeled lactoferrin and fast ..cap alpha../sub 2/-macroglobulin were studied. Both glycoproteins cleared rapidly following intravenous injection in mice, and both bound specifically to discrete receptors on murine peritoneal macrophages. The simultaneous presence of excess, unlabeled ligands specific for receptors recognizing terminal fucose, mannose, N-acetylglucosamine or galactose residues did not inhibit the clearance or binding of either lactoferrin or fast-..cap alpha../sub 2/M. The clearance and binding of enzymatically defucosylated lactoferrin was indistinguishable from native lactoferrin, indicating that terminal ..cap alpha..(1-3)-linked fucose on lactoferrin is not necessary for receptor recognition. The clearance and binding of two fast -..cap alpha../sub 2/M forms, ..cap alpha../sub 2/M-trypsin and ..cap alpha../sub 2/M-MeNH/sub 2/ cross compete with each other. Saturation binding studies indicated that the total binding of mannosyl -BSA, fusocyl-BSA, and N-acetylglucosaminyl-BSA to macrophages activated by BCG was approximately 15% of the levels observed with inflammatory macrophages elicited by thioglycollate broth. Cross-competition binding studies demonstrated a common surface receptor mediated binding of all three neoglycoprotein ligands and was identical to the receptor on mononuclear phagocytes that binds mannosyl- and N-acetylglucosaminyl-terminated glycoproteins. These results suggest that difference between discrete states of macrophage function may be correlated with selective changes in levels of the surface receptor for mannose-containing glycoproteins.

  1. Lactobacillus plantarum L67 glycoprotein protects against cadmium chloride toxicity in RAW 264.7 cells.

    PubMed

    Song, Sooyeon; Oh, Sejong; Lim, Kye-Taek

    2016-03-01

    The food and water we consume may be contaminated with a range of chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and mercury by accumulation through the food chain. Cadmium is known to be one of the major components in cigarette smoke and can cause lesions in many organs. Some lactobacilli can bind and remove heavy metals such as cadmium, lead, and copper. However, the mechanisms of cadmium toxicity and inhibition by probiotics are not clear. In this study, we demonstrated that glycoprotein (18 kDa) isolated from Lactobacillus plantarum L67 protected RAW 264.7 cells from expression of inflammation-related factors stimulated by cadmium chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium using the MTT assay and intracellular Ca(2+) using fluorescence, and assessed activities of activator protein kinase C (PKC-α), inducible nitric oxide synthase, activator protein (AP)-1, and mitogen-activated protein kinases using immunoblot. Our results indicated that glycoprotein isolated from L. plantarum L67 inhibited intracellular Ca(2+) mobilization. It also significantly suppressed inflammatory factors such as AP-1 (c-Jun and c-Fos), mitogen-activated protein kinases (ERK, JNK, and p38), and inducible nitric oxide synthase. Our findings suggest that the 24-kDa glycoprotein isolated from L. plantarum L67 might be used as a food component for protection of inflammation caused by cadmium ion.

  2. Expression of the rhesus glycoproteins, ammonia transporter family members, RHCG and RHBG in male reproductive organs.

    PubMed

    Lee, Hyun-Wook; Verlander, Jill W; Handlogten, Mary E; Han, Ki-Hwan; Cooke, Paul S; Weiner, I David

    2013-09-01

    The rhesus glycoproteins, Rh B glycoprotein (RHBG) and Rh C glycoprotein (RHCG), are recently identified ammonia transporters. Rhcg expression is necessary for normal male fertility, but its specific cellular expression is unknown, and Rhbg has not been reported to be expressed in the male reproductive tract. This study sought to determine the specific cellular expression of Rhcg, to determine whether Rhbg is expressed in the male reproductive tract, and, if so, to determine which cells express Rhbg using real-time RT-PCR, immunoblot analysis, and immunohistochemistry. Both Rhbg and Rhcg were expressed throughout the male reproductive tract. In the testis, high levels of Rhbg were expressed in Leydig cells, and Rhcg was expressed in spermatids during the later stages of their maturation (steps 13-16) in stages I-VIII of the seminiferous epithelium cycle. In the epididymis, basolateral Rhbg was present in narrow cells in the initial segment, in principal cells in the upper corpus, and in clear cells throughout the epididymis. Apical Rhcg immunolabel was present in principal cells in the caput and upper corpus epididymidis and in clear cells in the middle and lower corpus and cauda epididymidis. In the vas deferens, apical Rhcg immunolabel and basolateral Rhbg immunolabel were present in some principal cells and colocalized with H(+)-ATPase immunolabel. We conclude that both Rhbg and Rhcg are highly expressed in specific cells in the male reproductive tract where they can contribute to multiple components of male fertility.

  3. Characterization of glycoproteins in pancreatic cyst fluid using a high performance multiple lectin affinity chromatography platform

    PubMed Central

    Gbormittah, Francisca Owusu; Haab, Brian B.; Partyka, Katie; Garcia-Ott, Carolina; Hancapie, Marina; Hancock, William S.

    2014-01-01

    Currently, pancreatic cancer is the fourth cause of cancer death. In 2013, it is estimated that approximately 38,460 people will die of pancreatic cancer. Early detection of malignant cyst (pancreatic cancer precursor) is necessary to help prevent late diagnosis of the tumor. In this study, we characterized glycoproteins and non-glycoproteins on pooled mucinous (n=10) and non-mucinous (n=10) pancreatic cyst fluid to identify ‘proteins of interest’ to differentiate between mucinous cyst from non-mucinous cyst and investigate these proteins as potential biomarker targets. An automated multi-lectin affinity chromatography (M-LAC) platform was utilized for glycoprotein enrichment followed by nano-LC-MS/MS analysis. Spectral count quantitation allowed for the identification of proteins with significant differential levels in mucinous cysts from non-mucinous cysts of which one protein (periostin) was confirmed via immunoblotting. To exhaustively evaluate differentially expressed proteins, we used a number of proteomic tools including; gene ontology classification, pathway and network analysis, Novoseek data mining and chromosome gene mapping. Utilization of complementary proteomic tools, revealed that several of the proteins such as mucin 6 (MUC6), bile salt-activated lipase (CEL) and pyruvate kinase lysozyme M1/M2 with significant differential expression have strong association with pancreatic cancer. Further, chromosome gene mapping demonstrated co-expressions and co-localization of some proteins of interest including 14-3-3 protein epsilon (YWHAE), pigment epithelium derived factor (SERPINF1) and oncogene p53. PMID:24303806

  4. A UDP-glucose:glycoprotein glucose-1-phosphotransferase in embryonic chicken neural retina

    SciTech Connect

    Koro, L.A.; Marchase, R.B.

    1982-12-01

    A subclass of cell-surface glycoproteins from embryonic chicken neural retina contains a high mannose-type oligosaccharide that terminates with glucose linked via a phosphodiester bond to penultimate mannose. This unusual oligosaccharide seems responsible for the glycoprotein attachments to the cell-surface baseplate ligatin. Using beta-/sup 32/P-UDP-/sup 3/H-glucose, we demonstrate in retinal homogenates the existence of a UDP-glucose:glycoprotein glucose-1-phosphotransferase (GlcPTase) that catalyzes the synthesis of such a linkage. Characterization of the doubly labeled product resulting from activity of the transferase reveals a family of endoglycosidase H-sensitive oligosaccharides displaying a cation-exchange profile similar to that of oligosaccharides derived from ligatin-associated proteins synthesized in vivo. Further analysis confirms that the incorporation of label is due to a terminal /sup 3/H-glucose joined via a /sup 32/P-phosphodiester linkage to carbon 6 of a penultimate mannose. We propose that GlcPTase may be a controlling enzyme for the targeting of certain newly synthesized proteins to the cell surface.

  5. Effects of bleomycin and methylprednisolone on the biosynthesis of oligosaccharide-lipids and glycoproteins in lung.

    PubMed

    Mohapatra, N; Lynn, W S; Bhattacharyya, S N

    1983-09-01

    Tissues from rabbit lung were found to incorporate radioactivity from [3H]mannose into the oligosaccharide of a polar lipid soluble in chloroform/methanol/water (10:10:3, by vol.). Only one oligosaccharide-lipid was formed and the composition of the radiolabelled carbohydrate moiety was Glc3Man9(GlcNAc)2. An antitumour antibiotic, bleomycin, and an anti-inflammatory steroid, methylprednisolone, partially inhibited the incorporation of [3H]mannose into oligosaccharide-lipids and in addition resulted in the production of two new components, Man5(GlcNAc)2 and Man2(GlcNAc)2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the glycoproteins synthesized from the oligosaccharide-lipid intermediates showed labelled components with Mr greater than 200000, 130000, 80000 and 62000. The addition of the drugs resulted in the same pattern with the addition of a new component of Mr 36000. Endoglucosaminidase H-treatment of the glycopeptides isolated from the Pronase-digested glycoproteins indicated that the oligosaccharide units in the glycoproteins were of the high-mannose-type. PMID:6193779

  6. Effects of bleomycin and methylprednisolone on the biosynthesis of oligosaccharide-lipids and glycoproteins in lung.

    PubMed Central

    Mohapatra, N; Lynn, W S; Bhattacharyya, S N

    1983-01-01

    Tissues from rabbit lung were found to incorporate radioactivity from [3H]mannose into the oligosaccharide of a polar lipid soluble in chloroform/methanol/water (10:10:3, by vol.). Only one oligosaccharide-lipid was formed and the composition of the radiolabelled carbohydrate moiety was Glc3Man9(GlcNAc)2. An antitumour antibiotic, bleomycin, and an anti-inflammatory steroid, methylprednisolone, partially inhibited the incorporation of [3H]mannose into oligosaccharide-lipids and in addition resulted in the production of two new components, Man5(GlcNAc)2 and Man2(GlcNAc)2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the glycoproteins synthesized from the oligosaccharide-lipid intermediates showed labelled components with Mr greater than 200000, 130000, 80000 and 62000. The addition of the drugs resulted in the same pattern with the addition of a new component of Mr 36000. Endoglucosaminidase H-treatment of the glycopeptides isolated from the Pronase-digested glycoproteins indicated that the oligosaccharide units in the glycoproteins were of the high-mannose-type. Images Fig. 6. PMID:6193779

  7. Identification of pregnancy-associated glycoproteins by peptide mass fingerprinting in water buffalo (Bubalus bubalis).

    PubMed

    Kumar, Pradeep; Saxena, Abhishake; Singh, S K; Sharma, R K; Singh, I; Agarwal, S K

    2014-08-01

    Ruminant placentas synthesize pregnancy-associated glycoproteins (PAGs) during pregnancy, which serve as biomarkers of pregnancy. The present study was conducted to verify, whether PAGs are expressed in buffalo placenta by using lectin-based affinity chromatography and peptide mass finger printing (PMF). Fetal cotyledonary tissues were collected from gravid uteri procured from slaughtered house. Proteins were extracted and subjected to wheat germ agglutinin (WGA) lectin affinity chromatography to isolate the PAGs. The isolated glycoproteins were separated by one-dimensional SDS-PAGE. PMF results of the 75 kDa protein revealed presence of two PAGs (PAG-7 and -11). The PAG-7 consisted of about 170 mass signals, of which 16 were assigned to corresponding/translated cDNA sequences of buffalo PAG-7, leading to sequence coverage of 40%. PMF result of PAG-11 showed 170 mass signals, of which 15 were assigned to buffalo PAG-11, leading to sequence coverage of 34%. In conclusion, the glycoprotein isolated from placental extract corresponding to 75 kDa band on SDS PAGE gel was a mixture of PAG-7 and -11, which may help in development of suitable diagnostics for pregnancy in buffalo.

  8. Roles of bovine viral diarrhea virus envelope glycoproteins in inducing autophagy in MDBK cells.

    PubMed

    Fu, Qiang; Shi, Huijun; Shi, Mengting; Meng, Luping; Bao, Haiyang; Zhang, Guoqi; Ren, Yan; Zhang, Hui; Guo, Fei; Qiao, Jun; Jia, Bin; Wang, Pengyan; Ni, Wei; Sheng, Jinliang; Chen, Chuangfu

    2014-11-01

    Macroautophagy (autophagy) is an evolutionarily conserved control process that maintains cellular homeostasis in eukaryotic cells. Autophagy principally serves an adaptive role to degrade dysfunctional proteins and to clean damaged organelles in response to pathogenic, viral, or microbial infection, nutrient deprivation and endoplasmic reticulum (ER) stress. In previous study, we showed bovine viral diarrhea virus (BVDV) NADL infection induced autophagy and significantly elevated the expression levels of autophagy-related genes, Beclin1 and ATG14, at 12 h post-infection in MDBK cells. However, the specific mechanisms involved in controlling autophagic activity remain unclear. Here, we investigate the effects of BVDV NADL envelope glycoproteins overexpression on inducing autophagy. The results show that viral envelope glycoproteins E(rns) and E2 overexpression mediated by lentivirus increase the formation of autophagosome, the percentage of GFP-LC3 puncta-positive cells and the expression levels of Beclin1 and ATG14. Whereas E1 overexpression doesn't affect autophagic activity. Collectively, these findings suggest that the viral envelope glycoproteins E(rns) and E2 are involved in inducing autophagy, and provide a mechanistic insight into the regulation of autophagy in viral infected cells.

  9. Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients

    PubMed Central

    Douam, Florian; Bobay, Louis-Marie; Maurin, Guillemette; Fresquet, Judith; Calland, Noémie; Maisse, Carine; Durand, Tony; Cosset, François-Loïc; Féray, Cyrille

    2015-01-01

    ABSTRACT Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. IMPORTANCE Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting

  10. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  11. Glycoprotein gp50-negative pseudorabies virus: a novel approach toward a nonspreading live herpesvirus vaccine.

    PubMed Central

    Heffner, S; Kovács, F; Klupp, B G; Mettenleiter, T C

    1993-01-01

    Essential herpesvirus glycoproteins are involved in membrane fusion processes during infection, e.g., viral penetration and direct cell-to-cell transmission. We previously showed that the gD-homologous glycoprotein gp50 of pseudorabies virus (PrV) is essential for virus entry into target cells but proved to be dispensable for direct viral cell-to-cell spread in cell culture (I. Rauh and T. C. Mettenleiter, J. Virol. 65:5348-5456, 1991). For gp50-negative (gp50-) viruses, after phenotypic complementation necessary for primary infection, the only means of viral spread is by way of direct cell-to-cell transmission. In contrast, virus mutants lacking the essential gB-homologous glycoprotein gII after phenotypic complementation are only able to infect primary target cells and are blocked in further viral spread. To analyze how these in vitro phenotypes translate into virus replication in the animal, mice were infected intranasally with gp50- or gII- PrV mutants after prior phenotypic complementation by propagation on cell lines providing the essential glycoprotein in trans. Our results show that whereas the gII- mutants did not cause disease or any symptoms, gp50- mutants derived from two different PrV strains were fully virulent, with animals exhibiting severe symptoms ultimately leading to death. However, free infectious virus could not be recovered from either gp50- or gII- PrV-infected animals. We conclude that direct cell-to-cell transmission as the only means of viral spread of the gp50- mutants is sufficient for a full virulent phenotype in mice. After infection of pigs with phenotypically complemented gp50- PrV, only mild symptoms were observed, whereas the gII- mutant was totally avirulent. In both cases, shedding of infectious virus did not occur, in contrast to results with animals infected by gX- PrV that showed severe signs of disease and extensive virus shedding. After challenge infection with the highly virulent NIA-3 strain, the previously gII- Pr

  12. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  13. Dissection of the Antibody Response against Herpes Simplex Virus Glycoproteins in Naturally Infected Humans

    PubMed Central

    Huang, Zhen-Yu; Whitbeck, J. Charles; Ponce de Leon, Manuel; Lou, Huan; Wald, Anna; Krummenacher, Claude; Eisenberg, Roselyn J.; Cohen, Gary H.

    2014-01-01

    ABSTRACT Relatively little is known about the extent of the polyclonal antibody (PAb) repertoire elicited by herpes simplex virus (HSV) glycoproteins during natural infection and how these antibodies affect virus neutralization. Here, we examined IgGs from 10 HSV-seropositive individuals originally classified as high or low virus shedders. All PAbs neutralized virus to various extents. We determined which HSV entry glycoproteins these PAbs were directed against: glycoproteins gB, gD, and gC were recognized by all sera, but fewer sera reacted against gH/gL. We previously characterized multiple mouse monoclonal antibodies (MAbs) and mapped those with high neutralizing activity to the crystal structures of gD, gB, and gH/gL. We used a biosensor competition assay to determine whether there were corresponding human antibodies to those epitopes. All 10 samples had neutralizing IgGs to gD epitopes, but there were variations in which epitopes were seen in individual samples. Surprisingly, only three samples contained neutralizing IgGs to gB epitopes. To further dissect the nature of these IgGs, we developed a method to select out gD- and gB-specific IgGs from four representative sera via affinity chromatography, allowing us to determine the contribution of antibodies against each glycoprotein to the overall neutralization capacity of the serum. In two cases, gD and gB accounted for all of the neutralizing activity against HSV-2, with a modest amount of HSV-1 neutralization directed against gC. In the other two samples, the dominant response was to gD. IMPORTANCE Antibodies targeting functional epitopes on HSV entry glycoproteins mediate HSV neutralization. Virus-neutralizing epitopes have been defined and characterized using murine monoclonal antibodies. However, it is largely unknown whether these same epitopes are targeted by the humoral response to HSV infection in humans. We have shown that during natural infection, virus-neutralizing antibodies are principally

  14. Prestaining of glycoproteins in SDS-PAGE via 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide with weak influence on protein mobility.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Yu, Qing; Zhu, Xinliang; Niu, Chao; Cong, Weitao; Jin, Litai

    2014-12-01

    A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH). The prestained gels were readily imaged after electrophoresis without any time-consuming steps needed for poststain. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to the most commonly used Pro-Q Emerald 488 glycoprotein stain. In addition, subsequent study of deglycosylation, glycoprotein affinity chromatography, and LC-MS/MS analysis were performed to confirm the specificity of the newly developed method. As a result, BH prestain provides a new choice for quick, sensitive, specific, economical, and MS compatible visualization of gel-separated glycoproteins. PMID:25229714

  15. Prestaining of glycoproteins in SDS-PAGE via 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide with weak influence on protein mobility.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Yu, Qing; Zhu, Xinliang; Niu, Chao; Cong, Weitao; Jin, Litai

    2014-12-01

    A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH). The prestained gels were readily imaged after electrophoresis without any time-consuming steps needed for poststain. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to the most commonly used Pro-Q Emerald 488 glycoprotein stain. In addition, subsequent study of deglycosylation, glycoprotein affinity chromatography, and LC-MS/MS analysis were performed to confirm the specificity of the newly developed method. As a result, BH prestain provides a new choice for quick, sensitive, specific, economical, and MS compatible visualization of gel-separated glycoproteins.

  16. Kinetic Validation of the Models for P-Glycoprotein ATP Hydrolysis and Vanadate-Induced Trapping. Proposal for Additional Steps

    PubMed Central

    Lugo, Miguel Ramón; Sharom, Frances Jane

    2014-01-01

    P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s) include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the nature of the

  17. The evolution of N-glycan-dependent endoplasmic reticulum quality control factors for glycoprotein folding and degradation.

    PubMed

    Banerjee, Sulagna; Vishwanath, Prashanth; Cui, Jike; Kelleher, Daniel J; Gilmore, Reid; Robbins, Phillips W; Samuelson, John

    2007-07-10

    Asn-linked glycans (N-glycans) play important roles in the quality control (QC) of glycoprotein folding in the endoplasmic reticulum (ER) lumen and in ER-associated degradation (ERAD) of proteins by cytosolic proteasomes. A UDP-Glc:glycoprotein glucosyltransferase glucosylates N-glycans of misfolded proteins, which are then bound and refolded by calreticulin and/or calnexin in association with a protein disulfide isomerase. Alternatively, an alpha-1,2-mannosidase (Mns1) and mannosidase-like proteins (ER degradation-enhancing alpha-mannosidase-like proteins 1, 2, and 3) are part of a process that results in the dislocation of misfolded glycoproteins into the cytosol, where proteins are degraded in the proteasome. Recently we found that numerous protists and fungi contain 0-11 sugars in their N-glycan precursors versus 14 sugars in those of animals, plants, fungi, and Dictyostelium. Our goal here was to determine what effect N-glycan precursor diversity has on N-glycan-dependent QC systems of glycoprotein folding and ERAD. N-glycan-dependent QC of folding (UDP-Glc:glycoprotein glucosyltransferase, calreticulin, and/or calnexin) was present and active in some but not all protists containing at least five mannose residues in their N-glycans and was absent in protists lacking Man. In contrast, N-glycan-dependent ERAD appeared to be absent from the majority of protists. However, Trypanosoma and Trichomonas genomes predicted ER degradation-enhancing alpha-mannosidase-like protein and Mns1 orthologs, respectively, each of which had alpha-mannosidase activity in vitro. Phylogenetic analyses suggested that the diversity of N-glycan-dependent QC of glycoprotein folding (and possibly that of ERAD) was best explained by secondary loss. We conclude that N-glycan precursor length has profound effects on N-glycan-dependent QC of glycoprotein folding and ERAD.

  18. Interaction of a 60-kilodalton D-mannose-containing salivary glycoprotein with type 1 fimbriae of Escherichia coli.

    PubMed

    Babu, J P; Abraham, S N; Dabbous, M K; Beachey, E H

    1986-10-01

    A 60-kilodalton glycoprotein previously isolated and purified from human saliva (J. B. Babu, E. H. Beachey, D. L. Hasty, and W. A. Simpson, Infect. Immun. 51: 405-413, 1986) was found to interact with type 1 fimbriae and prevent adhesion of type 1 fimbriated Escherichia coli to animal cells in a D-mannose-sensitive manner. Purified salivary glycoprotein agglutinated type 1 fimbriated E. coli and, at subagglutinating concentrations, blocked the ability of type 1 fimbriated E. coli to attach to human buccal epithelial cells or agglutinate guinea pig erythrocytes. Both interactions were inhibited by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Complexing of the glycoprotein to type 1 fimbriae was demonstrated by molecular sieve chromatography and modified Western blots. When mixed with type 1 fimbriae, the radiolabeled salivary glycoprotein coeluted with type 1 fimbriae from a column of Sepharose 4B. When blotted from a sodium dodecyl sulfate gel to nitrocellulose sheets, the glycoprotein interacted directly with type 1 fimbriae applied to the blots. Both of the latter interactions also were blocked by alpha-methyl-D-mannoside but not by alpha-methyl-D-glucoside. Chemical modification of the glycoprotein with sodium metaperiodate abolished its ability to interact with isolated type 1 fimbriae or type 1 fimbriated E. coli. These results suggest that the carbohydrate moiety of the 60-kilodalton glycoprotein serves as a receptor for type 1 fimbriae in the oral cavity, and we postulate that the interaction may cause agglutination and early removal of E. coli, thereby preventing colonization by these organisms of oropharyngeal mucosae and dental tissues. PMID:2875948

  19. Effect of collecting duct-specific deletion of both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on renal response to metabolic acidosis.

    PubMed

    Lee, Hyun-Wook; Verlander, Jill W; Handlogten, Mary E; Han, Ki-Hwan; Weiner, I David

    2014-02-15

    The Rhesus (Rh) glycoproteins, Rh B and Rh C Glycoprotein (Rhbg and Rhcg, respectively), are ammonia-specific transporters expressed in renal distal nephron and collecting duct sites that are necessary for normal rates of ammonia excretion. The purpose of the current studies was to determine the effect of their combined deletion from the renal collecting duct (CD-Rhbg/Rhcg-KO) on basal and acidosis-stimulated acid-base homeostasis. Under basal conditions, urine pH and ammonia excretion and serum HCO3(-) were similar in control (C) and CD-Rhbg/Rhcg-KO mice. After acid-loading for 7 days, CD-Rhbg/Rhcg-KO mice developed significantly more severe metabolic acidosis than did C mice. Acid loading increased ammonia excretion, but ammonia excretion increased more slowly in CD-Rhbg/Rhcg-KO and it was significantly less than in C mice on days 1-5. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice on days 1, 3, and 5 of acid loading. Metabolic acidosis increased phosphenolpyruvate carboxykinase (PEPCK) and Na(+)/H(+) exchanger NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that 1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis; 2) the significantly greater changes in PEPCK, NHE-3, and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared with acid-loaded C mice cause the role of Rhbg and Rhcg to be underestimated quantitatively; and 3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3, and GS expression despite the presence of persistent metabolic acidosis.

  20. Effect of the carbohydrate moiety on the secondary structure of beta 2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins.

    PubMed

    Walsh, M T; Watzlawick, H; Putnam, F W; Schmid, K; Brossmer, R

    1990-07-01

    By use of six highly purified exoglycosidases with well-defined specificity, the oligosaccharide units of human plasma beta 2-glycoprotein I (beta 2I) were modified by sequential enzymatic degradation. The released monosaccharides (NeuAc, Gal, GlcNAc, and Man) were quantified, and the carbohydrate compositions of the resulting glycoprotein (gp) derivatives were determined. The gp was found to be both partially sialylated and galactosylated. These findings which are in agreement with earlier reports suggest that the carbohydrate moiety of beta 2I possesses more bi- than tri-antennas, probably three of the former and two of the latter carbohydrate units. Circular dichroic (CD) spectra of native beta 2I and its derivatives were measured in aqueous buffer and 2-chloroethanol (2-CE). Analysis of these spectra for elements of secondary structure showed beta 2I and most of the derivatives to contain predominantly beta-sheet and beta-turn structures. The lack of alpha-helical structures in aqueous buffer was noted. Removal of a large portion of the carbohydrate moiety did not alter the CD spectra or secondary structure of beta 2I in either aqueous buffer or in 2-CE. However, after enzymatic removal of approximately 96% of the carbohydrate moiety, large significant changes in the spectra and secondary structures were observed. In aqueous buffer a shift in the wavelength minimum occurred, accompanied by an increase in the magnitude of the molar ellipticity and the amount of beta-turn, with a reduction in random coil. One-third of the amino acids which were originally in random coil conformation assumed beta-turns after removal of 96% of the carbohydrate moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells.

    PubMed

    Lee, Sei-Jung; Lim, Kye-Taek

    2006-04-01

    This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays, respectively. SNL glycoprotein has an apparent cytotoxic and apoptotic effect at a concentration of 40 microg/ml after 4 h. To further verify the apoptotic effect, we investigated the changes in activity of the apoptotic-related proteins [Bid, cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)] triggered by SNL glycoprotein, using a western blot analysis. The results in this study indicated that SNL glycoprotein has a stimulatory effect on Bid activation, resulting in the release of cytochrome c, the stimulation of caspase-8, -9 and -3 activities, and the cleavage of PARP in HCT-116 cells. However, SNL glycoprotein did not significantly stimulate an increase in levels of intracellular reactive oxygen species (ROS). From the results in this experiment, it is suggested that SNL glycoprotein induces apoptosis through the mitochondrial apoptotic signal pathway in HCT-116 cells, rather than through intracellular ROS. PMID:16208518

  2. Amino acid sequence and location of the disulfide bonds in bovine beta 2 glycoprotein I: the presence of five Sushi domains.

    PubMed

    Kato, H; Enjyoji, K

    1991-12-17

    beta 2 glycoprotein I is a plasma protein with the ability to bind with various kinds of negatively charged substances. The complete amino acid sequence and the location of all the disulfide bonds of bovine beta 2 glycoprotein I were determined. Bovine beta 2 glycoprotein I consists of 326 amino acid residues with five asparagine-linked carbohydrate chains. Homology with the human protein was calculated to be 83%. Eleven disulfide bonds in bovine beta 2 glycoprotein I constitute four characteristic domains, Sushi domains, and one modified form of a Sushi domain.

  3. Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus.

    PubMed Central

    Smith, G W; Hightower, L E

    1982-01-01

    Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells. PMID:6896347

  4. Measurement of glycoprotein IIb/IIIa blockade by flow cytometry with fluorescein isothiocyanate-conjugated crotavirin, a member of disintegrins.

    PubMed

    Liu, C Z; Hur, B T; Huang, T F

    1996-10-01

    The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

  5. Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog

    SciTech Connect

    Akiyama, S.; Cornwell, M.M.; Kuwano, M.; Pastan, I.; Gottesman, M.M.

    1988-02-01

    Multidrug-resistant human KB carcinoma cells express a 170,000-dalton membrane glycoprotein (P-glycoprotein) that can be photoaffinity labeled with the vinblastine analog N-(p-azido-(3-/sup 125/I)salicyl)-N'-(beta-aminoethyl)vindesine. Several agents that suppress the multidrug-resistant phenotype, including N-solanesyl-N,N'-bis(3,4-dimethylbenzyl)ethylenediamine, cepharanthine, quinidine, and reserpine, were found to inhibit photolabeling of P-glycoprotein at doses comparable to those that reverse multidrug resistance. However, the phenothiazines chlorpromazine and trifluoperazine, which also effectively reverse multidrug resistance, were poor inhibitors of the photoaffinity labeling of P-glycoprotein. Chloroquine, propranolol, or atropine, which only partially reversed the drug resistance, also did not inhibit photolabeling. Naphthalene sulfonamide calmodulin inhibitors, W7 and W5, as well as many other drugs that did not circumvent multidrug resistance, did not inhibit photolabeling. These studies suggest that most, but not all, agents that phenotypically suppress multidrug resistance also inhibit drug binding to a site on P-glycoprotein with which a photoaffinity analog of vinblastine interacts.

  6. Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

    PubMed Central

    Dutta, Somnath; Yan, Lianying; Feng, YanRu; Wang, Lin-Fa; Skiniotis, Georgios; Lee, Benhur; Zhou, Z. Hong; Broder, Christopher C.; Aguilar, Hector C.; Nikolov, Dimitar B.

    2015-01-01

    Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein. PMID:26646856

  7. Modulation of Mannose and Asialoglycoprotein Receptor Expression Determines Glycoprotein Hormone Half-life at Critical Points in the Reproductive Cycle*

    PubMed Central

    Mi, Yiling; Lin, Angela; Fiete, Dorothy; Steirer, Lindsay; Baenziger, Jacques U.

    2014-01-01

    The rate at which glycoproteins are cleared from the circulation has a critical impact on their biologic activity in vivo. We have shown that clearance rates for glycoproteins such as luteinizing hormone (LH) that undergo regulated release into the circulation determine their potency. Two highly abundant, carbohydrate-specific, endocytic receptors, the asialoglycoprotein receptor (ASGR) and the mannose receptor (ManR) are expressed in the liver by parenchymal and sinusoidal endothelial cells, respectively. We demonstrate that the ManR mediates the clearance of glycoproteins such as LH that bear N-linked glycans terminating with β1,4-linked GalNAc-4-SO4, as well as glycoproteins bearing glycans that terminate with Man. Steady state levels of mRNA encoding the ASGR and the ManR are regulated by progesterone in pregnant mice, reaching maximal levels on day 12.5 of pregnancy. Protein expression and glycan-specific binding activity also increase in the livers of pregnant mice. In contrast, ManR mRNA, but not ASGR mRNA, decreases in male mice at the time of sexual maturation. We show that levels of ManR and ASGR expression control the clearance rate for glycoproteins bearing recognized glycans. Thus, reduced expression of the ManR at the time of sexual maturation will increase the potency of LH in vivo, whereas increased expression during pregnancy will reduce LH potency until progesterone and receptor levels fall prior to parturition. PMID:24619407

  8. Fibrinogen-binding properties of the human platelet glycoprotein IIb-IIIa complex: a study using crossed-radioimmunoelectrophoresis

    SciTech Connect

    Gogstad, G.O.; Brosstad, F.; Krutnes, M.B.; Hagen, I.; Solum, N.O.

    1982-09-01

    Fibrinogen-binding platelet proteins have been examined by crossed-immunoelectrophoresis of solubilized, washed platelets followed by the incubation of the immunoplates with /sup 125/I-fibrinogen and exposure to x-ray films. Incubation with 0.1 mg/ml of /sup 125/I-fibrinogen revealed the binding of fibrinogen to the immunoprecipitates representing the glycoprotein IIb-IIIa complex, factor XIIIa chain, a granule membrane protein termed G4, fibrinogen, and albumin. Only the glycoprotein IIb-IIIa precipitate and the fibrinogen precipitate showed significant binding when the concentration of /sup 125/I-fibrinogen was lowered to 0.01 mg/ml. Thi indicates that the binding of fibrinogen is specific. The binding of /sup 125/I-fibrinogen to the precipitates representing the glycoprotein IIb-IIIa complex, the factor XIIIa chain, and G4, but not to the albumin precipitate, was significantly lowered in the presence of EDTA. This effect of EDTA increased with increasing pH, with no binding at pH 8.7. The results indicate that the glycoprotein IIb-IIIa complex, but not the separate glycoproteins IIb and IIIa, can act as Ca/sup 2 +/ or Mg/sup 2 +/-dependent fibrinogen receptor, under proper physiologic conditions.

  9. IgG and IgM antibodies to viral glycoproteins in respiratory syncytial virus infections of graded severity.

    PubMed Central

    Toms, G L; Webb, M S; Milner, P D; Milner, A D; Routledge, E G; Scott, R; Stokes, G M; Swarbrick, A; Taylor, C E

    1989-01-01

    Serum antibodies to the fusion (F) and large glycoprotein (G) of respiratory syncytial virus in the serum of 57 infected infants were measured by enzyme linked immunosorbent assay (ELISA). Most serum samples taken at the time of admission to hospital contained antibodies to both glycoproteins, and overall there was no significant evidence of a selective deficiency of antibody to either viral antigen. Less than a quarter of the infants showed rising IgG antibody titres to either glycoprotein after infection, whereas over threequarters produced an IgM response. There was a significant correlation between IgG response to viral glycoproteins and the age of the infant. The correlation of age with the IgM response was less pronounced, and there was no correlation between serum IgG antibody derived transplancentally in the acute phase of infection and IgM response to either glycoprotein. Neither IgG or IgM responses correlated with a clinical assessment of the severity of infection in the infants. IgM responses, however, were weakly correlated with reduced secretion of infectious virus in the upper respiratory tract. PMID:2624472

  10. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    PubMed

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  11. Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins.

    PubMed

    Patel, T; Bruce, J; Merry, A; Bigge, C; Wormald, M; Jaques, A; Parekh, R

    1993-01-19

    The use of hydrazine to release unreduced N- and O-linked oligosaccharides from glycoproteins has been investigated using several "standard" glycoproteins of previously defined glycosylation. It is shown that hydrazinolysis can be used to release intact N- and O-linked oligosaccharides in an unreduced form. The release of O-linked oligosaccharides occurs with a lower temperature dependence than the release of N-linked oligosaccharides, and the kinetic parameters governing release of oligosaccharides from these standard glycoproteins have been determined. These parameters allow a definition of reaction conditions under which anhydrous hydrazinolysis can be used to selectively release O-linked oligosaccharides (60 degrees C, 5 h) or release both N- and O-linked oligosaccharides (95 degrees C, 4 h) in high yield (> 85%) from all glycoproteins investigated (n = 11). Under these reaction conditions, the recovered N- and O-linked oligosaccharides are structurally intact (as judged by 600-MHz 1H-NMR, laser-desorption mass spectrometry, HPAEC-PAD, gel filtration, and glycosidase digestion), with the possible exception of certain N- and O-acyl substituents of sialic acid. This use of mild hydrazinolysis therefore allows both the simultaneous and sequential chemical release from glycoproteins of O- and N-linked oligosaccharides in their intact unreduced form.

  12. Selective extraction and enrichment of glycoproteins based on boronate affinity SPME and determination by CIEF-WCID.

    PubMed

    Li, Lixian; Xia, Zhining; Pawliszyn, Janusz

    2015-07-30

    In this study, a new thin-film boronic acid coating was developed for solid-phase microextraction (SPME) followed by capillary isoelectric focusing with whole-column imaging detection (CIEF-WCID). Boronate functionalized particles of phenylboronic acid (PBA) and 3-aminophenylboronic acid (3-aPBA) were utilized as boronate affinity solid phase coating on thin-film stainless steel blades for selective extraction and enrichment of glycoproteins. The process of extraction and elution could be easily controlled by adjusting pH. To test specificity, asialofetuin and lactoferrin were selected as glycoproteins test molecules, while BSA and myoglobin were used as control non-glycoproteins in this study. The boronate affinity coating was characterized. The effect of buffer, pH, extraction profiles and elution profiles were investigated. The developed method was successfully applied to extract glycoproteins from standard buffer, PBS, human plasma and 10-fold diluted human blood using two kinds of boronate affinity blades. Boronate affinity SPME could be a promising tool for selective extraction and enrichment of low-abundance glycoproteins in real biological samples.

  13. Emerging structural insights into glycoprotein quality control coupled with N-glycan processing in the endoplasmic reticulum.

    PubMed

    Satoh, Tadashi; Yamaguchi, Takumi; Kato, Koichi

    2015-01-30

    In the endoplasmic reticulum (ER), the sugar chain is initially introduced onto newly synthesized proteins as a triantennary tetradecasaccharide (Glc3Man9GlcNAc2). The attached oligosaccharide chain is subjected to stepwise trimming by the actions of specific glucosidases and mannosidases. In these processes, the transiently expressed N-glycans, as processing intermediates, function as signals for the determination of glycoprotein fates, i.e., folding, transport, or degradation through interactions of a series of intracellular lectins. The monoglucosylated glycoforms are hallmarks of incompletely folded states of glycoproteins in this system, whereas the outer mannose trimming leads to ER-associated glycoprotein degradation. This review outlines the recently emerging evidence regarding the molecular and structural basis of this glycoprotein quality control system, which is regulated through dynamic interplay among intracellular lectins, glycosidases, and glycosyltransferase. Structural snapshots of carbohydrate-lectin interactions have been provided at the atomic level using X-ray crystallographic analyses. Conformational ensembles of uncomplexed triantennary high-mannose-type oligosaccharides have been characterized in a quantitative manner using molecular dynamics simulation in conjunction with nuclear magnetic resonance spectroscopy. These complementary views provide new insights into glycoprotein recognition in quality control coupled with N-glycan processing.

  14. Modulation of mannose and asialoglycoprotein receptor expression determines glycoprotein hormone half-life at critical points in the reproductive cycle.

    PubMed

    Mi, Yiling; Lin, Angela; Fiete, Dorothy; Steirer, Lindsay; Baenziger, Jacques U

    2014-04-25

    The rate at which glycoproteins are cleared from the circulation has a critical impact on their biologic activity in vivo. We have shown that clearance rates for glycoproteins such as luteinizing hormone (LH) that undergo regulated release into the circulation determine their potency. Two highly abundant, carbohydrate-specific, endocytic receptors, the asialoglycoprotein receptor (ASGR) and the mannose receptor (ManR) are expressed in the liver by parenchymal and sinusoidal endothelial cells, respectively. We demonstrate that the ManR mediates the clearance of glycoproteins such as LH that bear N-linked glycans terminating with β1,4-linked GalNAc-4-SO4, as well as glycoproteins bearing glycans that terminate with Man. Steady state levels of mRNA encoding the ASGR and the ManR are regulated by progesterone in pregnant mice, reaching maximal levels on day 12.5 of pregnancy. Protein expression and glycan-specific binding activity also increase in the livers of pregnant mice. In contrast, ManR mRNA, but not ASGR mRNA, decreases in male mice at the time of sexual maturation. We show that levels of ManR and ASGR expression control the clearance rate for glycoproteins bearing recognized glycans. Thus, reduced expression of the ManR at the time of sexual maturation will increase the potency of LH in vivo, whereas increased expression during pregnancy will reduce LH potency until progesterone and receptor levels fall prior to parturition.

  15. Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

    PubMed

    Asfor, A S; Wakeley, P R; Drew, T W; Paton, D J

    2014-08-30

    Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells.

  16. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  17. Mediation of Cryptosporidium parvum Infection In Vitro by Mucin-Like Glycoproteins Defined by a Neutralizing Monoclonal Antibody

    PubMed Central

    Cevallos, Ana María; Bhat, Najma; Verdon, Renaud; Hamer, Davidson H.; Stein, Barry; Tzipori, Saul; Pereira, Miercio E. A.; Keusch, Gerald T.; Ward, Honorine D.

    2000-01-01

    The protozoan parasite Cryptosporidium parvum is a significant cause of diarrheal disease worldwide. Attachment to and invasion of host intestinal epithelial cells by C. parvum sporozoites are crucial steps in the pathogenesis of cryptosporidiosis. The molecular basis of these initial interactions is unknown. In order to identify putative C. parvum adhesion- and invasion-specific proteins, we raised monoclonal antibodies (MAbs) to sporozoites and evaluated them for inhibition of attachment and invasion in vitro. Using this approach, we identified two glycoproteins recognized by 4E9, a MAb which neutralized C. parvum infection and inhibited sporozoite attachment to intestinal epithelial cells in vitro. 4E9 recognized a 40-kDa glycoprotein named gp40 and a second, >220-kDa protein which was identified as GP900, a previously described mucin-like glycoprotein. Glycoproteins recognized by 4E9 are localized to the surface and apical region of invasive stages and are shed in trails from the parasite during gliding motility. The epitope recognized by 4E9 contains α-N-acetylgalactosamine residues, which are present in a mucin-type O-glycosidic linkage. Lectins specific for these glycans bind to the surface and apical region of sporozoites and block attachment to host cells. The surface and apical localization of these glycoproteins and the neutralizing effect of the MAb and α-N-acetylgalactosamine-specific lectins strongly implicate these proteins and their glycotopes as playing a role in C. parvum-host cell interactions. PMID:10948140

  18. Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus.

    PubMed

    Blackhall, W J; Liu, H Y; Xu, M; Prichard, R K; Beech, R N

    1998-09-15

    Resistance to anthelmintics that are used to control parasite populations in domestic animals has become a serious problem worldwide. The development of resistance is an evolutionary process that leads to genetic changes in parasite populations in response to drug exposure. The anthelmintic ivermectin is known to bind to the human membrane transport protein, P-glycoprotein, and P-glycoprotein-deficient mice treated with ivermectin have shown signs of neurotoxicity. P-glycoprotein is believed to be involved in the multidrug resistance phenotype seen in some human cancers and for drug resistance in some protists. We have examined the genetic variation of a P-glycoprotein homologue from the nematode Haemonchus contortus to see if an association exists between specific alleles of this gene and survival to exposure to ivermectin or moxidectin. Two parasite strains passaged without drug treatment and three strains, subjected to anthelmintic selection and derived from the unselected strains, were examined. Allelic variation in the unselected strains showed this locus to be highly polymorphic. chi 2 analyses of allele frequencies showed significant differences between the unselected and the drug-selected derived strains. In all three drug-selected strains, an apparent selection for the same allele was observed. These findings suggest that P-glycoprotein may be involved in resistance to both ivermectin and moxidectin in H. contortus.

  19. Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

    PubMed

    Martínez-Menárguez, J A; Ballesta, J; Avilés, M; Castells, M T; Madrid, J F

    1992-01-01

    The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane. PMID:1500300

  20. Transfusion of murine RBCs expressing the human KEL glycoprotein induces clinically significant alloantibodies

    PubMed Central

    Stowell, Sean R.; Girard-Pierce, Kathryn R.; Smith, Nicole H.; Henry, Kate L.; Arthur, C. Maridith; Zimring, James C.; Hendrickson, Jeanne E.

    2013-01-01

    Background Red blood cell (RBC) alloantibodies to non-self antigens may develop following transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans. Study Design and Methods We recently generated transgenic mice with RBC specific expression of the human KEL glycoprotein, with the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system. Results Transfusion of RBCs from KEL2 donors into wild type recipients (lacking the human KEL protein but expressing the murine KEL orthologue) resulted in dose dependent anti-KEL glycoprotein IgM and IgG antibody responses, enhanced by recipient inflammation with poly (I:C). Boostable responses were evident upon repeat transfusion, with morbid appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic following transfusion into wild type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti-KEL1 or anti-KEL2 responses. Conclusions This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long term translational goals of these studies include improving transfusion safety for at risk patients. PMID:23621760