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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. Multilocus patterns of genetic variation across Cryptosporidium species suggest balancing selection at the gp60 locus.

    PubMed

    Abal-Fabeiro, J L; Maside, X; Bello, X; Llovo, J; Bartolomé, C

    2013-09-01

    Cryptosporidium is an apicomplexan protozoan that lives in most vertebrates, including humans. Its gp60 gene is functionally involved in its attachment to host cells, and its high level of genetic variation has made it the reference marker for sample typing in epidemiological studies. To understand the origin of such high diversity and to determine the extent to which this classification applies to the rest of the genome, we analysed the patterns of variation at gp60 and nine other nuclear loci in isolates of three Cryptosporidium species. Most loci showed low genetic polymorphism (πS <1%) and similar levels of between-species divergence. Contrastingly, gp60 exhibited very different characteristics: (i) it was nearly ten times more variable than the other loci; (ii) it displayed a significant excess of polymorphisms relative to between-species differences in a maximum-likelihood Hudson-Kreitman-Aguadé test; (iii) gp60 subtypes turned out to be much older than the species they were found in; and (iv) showed a significant excess of polymorphic variants shared across species from random expectations. These observations suggest that this locus evolves under balancing selection and specifically under negative frequency-dependent selection (FDS). Interestingly, genetic variation at the other loci clusters very well within the groups of isolates defined by gp60 subtypes, which may provide new tools to understand the genome-wide patterns of genetic variation of the parasite in the wild. These results suggest that gp60 plays an active and essential role in the life cycle of the parasite and that genetic variation at this locus might be essential for the parasite's long-term success. PMID:23915002

  3. 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...

  4. 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

  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. Glycoprotein Analysis

    NASA Astrophysics Data System (ADS)

    Fernandes, Daryl; Spencer, Daniel

    This chapter provides an overview of practical methods for glycosylation analysis of glycoprotein therapeutics . The topics include glycoprofiling methods for glycoforms, monosaccharides (neutral and N-acetylated species as well as sialic acids), oligosaccharides (chemical and enzymatic methods for glycan release, post-release purification, labeling and derivatization, different types of glycan HPLC and MS), and glycosylation site profiling.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Glycosylation Engineering of Glycoproteins

    NASA Astrophysics Data System (ADS)

    Sadamoto, Reiko; Nishimura, Shin-Ichiro

    Naturally occurring glycosylation of glycoproteins varies in glycosylation site and in the number and structure of glycans. The engineering of well-defined glycoproteins is an important technology for the preparation of pharmaceutically relevant glycoproteins and in the study of the relationship between glycans and proteins on a structure-function level. In pharmaceutical applications of glycoproteins, the presence of terminal sialic acids on glycans is particularly important for the in vivo circulatory half life, since sialic acid-terminated glycans are not recognized by asialoglycoprotein receptors. Therefore, there have been a number of attempts to control or modify cellular metabolism toward the expression of glycoproteins with glycosylation profiles similar to that of human glycoproteins. In this chapter, recent methods for glycoprotein engineering in various cell culture systems (mammalian cells, plant, yeast, and E. coli) and advances in the chemical approach to glycoprotein formation are described.

  18. 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.

  19. Pulsatile glycoprotein hormone secretion in glycoprotein-producing pituitary tumors.

    PubMed

    Samuels, M H; Henry, P; Kleinschmidt-Demasters, B K; Lillehei, K; Ridgway, E C

    1991-12-01

    To study patterns of hormone production and secretion in glycoprotein-producing pituitary tumors, 12 patients with such tumors underwent the following studies. Preoperatively, all patients had serum TSH, LH, FSH, and alpha-subunit levels measured every 15 min for 24 h. Hormone pulses were located by cluster analysis, and pulse parameters were compared to those in healthy young men, healthy young women, healthy postmenopausal women, and subjects with primary hypothyroidism. After surgery, immunocytochemistry for the four glycoproteins was performed on all tumors, and Northern blot analysis was performed in six tumors with probes for the four subunits. By immunocytochemistry, 42% of the tumors were positive for TSH beta, 83% for LH beta, 75% for FSH beta, and 92% for alpha-subunit. Preoperative serum hormone levels varied widely between patients and were not well correlated with the intensity of immunocytochemical staining. Northern blot analysis did not appear to be as sensitive as immunocytochemistry for detection of the glycoproteins. All patients had pulsatile glycoprotein secretion, with pulses of normal frequency but varied amplitude. These results suggest that in patients with glycoprotein tumors, hormone pulses may be an integral part of autonomous secretion, or that hypothalamic control is involved in glycoprotein secretion and, perhaps, in the pathogenesis of these tumors. PMID:1955510

  20. 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.

  1. 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

  2. Glycan analysis of therapeutic glycoproteins

    PubMed Central

    Zhang, Lei; Luo, Shen; Zhang, Baolin

    2016-01-01

    ABSTRACT Therapeutic monoclonal antibodies (mAbs) are glycoproteins produced by living cell systems. The glycan moieties attached to the proteins can directly affect protein stability, bioactivity, and immunogenicity. Therefore, glycan variants of a glycoprotein product must be adequately analyzed and controlled to ensure product quality. However, the inherent complexity of protein glycosylation poses a daunting analytical challenge. This review provides an update of recent advances in glycan analysis, including the potential utility of lectin-based microarray for high throughput glycan profiling. Emphasis is placed on comparison of the major types of analytics for use in determining unique glycan features such as glycosylation site, glycan structure, and content. PMID:26599345

  3. 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.

  4. Phosphorylation of the multidrug resistance associated glycoprotein.

    PubMed

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance 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 MgCl2 was enhanced a minimum of 2-fold by 10 microM 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-32P]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. PMID:3427052

  5. 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.

  6. 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

  7. Osmotic water permeability in glycoprotein containing liposomes.

    PubMed

    Neitchev, V Z; Kostadinov, A P

    1987-01-01

    The kinetics of osmotic water permeability in proteoliposomes containing alpha 1-acid glycoprotein was investigated by means of stopped-flow spectrophotometry. A biphasic time-course of scattered light with time was registered. The rate constants calculated from fits to an exponential function in the first phase were proportional to the final medium osmolarity. The apparent second order rate constants Kapp (Osm-1 sec-1) were determined at different glycoprotein concentrations in the original mixture for preparation of proteoliposomes. The value of Kapp at lipid:glycoprotein weight ratio = 1 was plotted in Arrhenius coordinates. The calculated activation energy for water permeation through the lipid bilayer suggests that eventual channel mechanism may be involved due to the presence of glycoprotein molecule in the liposomes. PMID:3431542

  8. 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

  9. 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

  10. 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

  11. Glycoprotein Structural Genomics: Solving the Glycosylation Problem

    PubMed Central

    Chang, Veronica T.; Crispin, Max; Aricescu, A. Radu; Harvey, David J.; Nettleship, Joanne E.; Fennelly, Janet A.; Yu, Chao; Boles, Kent S.; Evans, Edward J.; Stuart, David I.; Dwek, Raymond A.; Jones, E. Yvonne; Owens, Raymond J.; Davis, Simon J.

    2007-01-01

    Summary Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the “glycosylation problem” can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis. PMID:17355862

  12. Phosphorylation of the multidrug resistant associated glycoprotein (p-glycoprotein): Preparation and characterization of 7-acetyltaxol

    SciTech Connect

    Mellado, W.

    1988-01-01

    To assess the role of phosphorylation in P-glycoprotein function, phosphorylation of P-glycoprotein in intact cells and in cell-free membrane fractions has been studied. Results obtained with cell-free membrane fractions indicate that P-glycoprotein is a substrate for a membrane-associated protein kinase A (PK-A). To assess whether P-glycoprotein was phosphorylated in vivo by PK-A, MDR cells were incubated with ({sup 32}P)Pi in the presence or absence of 100 uM 8Br-cAMP. The tryptic phosphopeptides of six P-glycoproteins from five independently derived MDR cell lines were analyzed by HPLC. A similar analysis carried out with two other P-glycoproteins (from J7.V3-1 and the lower band of J7.T1-50) demonstrated a major phosphopeptide with a retention time of 26 min. Fraction 26 was resolved as a single phosphopeptide by 2-D mapping. The phosphorylation of fraction 26 which was derived from P-glycoprotein in J7.V3-1 or the J7.T1-50 lower band was enhanced when the cells were treated with 8BrcAMP.

  13. Adherence of oral streptococci to salivary glycoproteins.

    PubMed Central

    Murray, P A; Prakobphol, A; Lee, T; Hoover, C I; Fisher, S J

    1992-01-01

    We used an overlay method to study the ability of human salivary glycoproteins to serve as receptors for several strains of streptococci that colonize the oral cavity. Parotid and submandibular-sublingual salivas were collected as ductal secretions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The resulting blots were overlaid with [35S]methionine-labeled bacteria, and salivary components to which the bacteria bound were detected by autoradiography. Potential glycoprotein receptors were identified for 8 of the 16 strains tested. In three cases (Streptococcus sanguis 72-40 and 804 and Streptococcus sobrinus OMZ176), highly specific interactions with a single salivary component were detected. Removal of sialic acid residues from the low-molecular-weight salivary mucin prevented adherence of one of these strains (S. sanguis 72-40), suggesting that this saccharide either mediates binding or is a critical component of the receptor site. In the remaining five strains (Streptococcus gordonii G9B and 10558, S. sanguis 10556, and Streptococcus oralis 10557 and 72-41), interactions with multiple salivary components, including the low-molecular-weight salivary mucin, highly glycosylated proline-rich glycoproteins, and alpha-amylase, were detected. These results suggest that some oral streptococci can bind specifically to certain of the salivary glycoproteins. The interactions identified may play an important role in governing bacterial adherence and clearance within the oral cavity. Images PMID:1729194

  14. Interaction of Sindbis virus glycoproteins during morphogenesis.

    PubMed Central

    Jones, K J; Scupham, R K; Pfeil, J A; Wan, K; Sagik, B P; Bose, H R

    1977-01-01

    In cells infected with the Sindbis temperature-sensitive mutants ts-23 and ts-10 (complementation group D), which contain a defect in the envelope glycoprotein E1, the precursor polypeptide PE2 is not cleaved to the envelope glycoprotein E2 at the nonpermissive temperature. This defect is phenotypically identical to the defect observed in the complementation group E mutant, ts-20. The lesion in ts-23 is reversible upon shift to permissive temperature, whereas that of ts-10 is not. Antiserum against whole virus, E1, or E2 also prevents the cleavage of PE2 in cells infected with wild-type Sindbis virus. Because the cleavage of PE2 is inhibited by the lesion in mutants that are genotypically distinct and by anti-E1 or -E2 serum, it appears that PE2 and E1 exist as a complex in the membrane of the infected cell. Images PMID:833949

  15. 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

  16. 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

  17. Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

    PubMed Central

    Wang, Lai-Xi; Amin, Mohammed N.

    2014-01-01

    Summary Glycoproteins are an important class of biomolecules involved in a number of biological recognition processes. However, natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ in the structures of the pendent glycans, which are difficult to separate in pure glycoforms. As a result, synthetic homogeneous glycopeptides and glycoproteins have become indispensable probes for detailed structural and functional studies. A number of elegant chemical and biological strategies have been developed for synthetic construction of tailor-made, full-size glycoproteins to address specific biological problems. In this review, we highlight recent advances in chemical and chemoenzymatic synthesis of homogeneous glycoproteins. Selected examples are given to demonstrate the applications of tailor-made, glycan-defined glycoproteins for deciphering glycosylation functions. PMID:24439206

  18. 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

  19. [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

  20. Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

    PubMed Central

    Klupp, Barbara G.; Nixdorf, Ralf; Mettenleiter, Thomas C.

    2000-01-01

    A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine

  1. 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)

  2. Properties of a glycopeptide isolated from human Tamm-Horsfall glycoprotein. Interaction with leucoagglutinin and anti-(human Tamm-Horsfall glycoprotein) antibodies.

    PubMed Central

    Abbondanza, A; Franceschi, C; Licastro, F; Serafini-Cessi, F

    1980-01-01

    A sialylated glycopeptide isolated after Pronase digestion of human Tamm-Horsfall glycoprotein behaves as a powerful monovalent hapten in the precipitin reaction between human Tamm-Horsfall glycoprotein and leucoagglutinin, but fails to inhibit the interaction of the glycoprotein with rabbit anti-(human Tamm-Horsfall glycoprotein) antibodies. The glycopeptide is much less active than the intact glycoprotein as an inhibitor of lymphocyte transformation induced by leucoagglutinin. PMID:6967312

  3. 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

  4. 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

  5. Annotating Human P-Glycoprotein Bioassay Data

    PubMed Central

    Zdrazil, Barbara; Pinto, Marta; Vasanthanathan, Poongavanam; Williams, Antony J; Balderud, Linda Zander; Engkvist, Ola; Chichester, Christine; Hersey, Anne; Overington, John P; Ecker, Gerhard F

    2012-01-01

    Abstract Huge amounts of small compound bioactivity data have been entering the public domain as a consequence of open innovation initiatives. It is now the time to carefully analyse existing bioassay data and give it a systematic structure. Our study aims to annotate prominent in vitro assays used for the determination of bioactivities of human P-glycoprotein inhibitors and substrates as they are represented in the ChEMBL and TP-search open source databases. Furthermore, the ability of data, determined in different assays, to be combined with each other is explored. As a result of this study, it is suggested that for inhibitors of human P-glycoprotein it is possible to combine data coming from the same assay type, if the cell lines used are also identical and the fluorescent or radiolabeled substrate have overlapping binding sites. In addition, it demonstrates that there is a need for larger chemical diverse datasets that have been measured in a panel of different assays. This would certainly alleviate the search for other inter-correlations between bioactivity data yielded by different assay setups. PMID:23293680

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. Properties of odour-binding glycoproteins from rat olfactory epithelium.

    PubMed

    Fesenko, E E; Novoselov, V I; Bystrova, M F

    1988-01-22

    The specific membrane glycoproteins with high affinity for camphor and decanal were isolated from rat olfactory epithelium. Antibodies to these glycoproteins inhibited both the electroolfactogram and the binding of odorants. The enzyme immunoassay has shown these glycoproteins to be present in the olfactory epithelium of rat, mouse, guinea-pig and hamster but not in that of frog and carp. The molecular mass of the odour-binding glycoproteins from rat olfactory epithelium solubilized by Triton X-100 was approx. 140 kDa. They consisted of two subunits (88 and 55 kDa). The 88 kDa subunit was capable of binding odorants. The data obtained suggest that the glycoproteins isolated have some properties that make them plausible candidates for olfactory receptor molecules. PMID:3337807

  12. 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. PMID:18096391

  13. 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.

  14. P-glycoprotein in autoimmune rheumatic diseases.

    PubMed

    García-Carrasco, M; Mendoza-Pinto, C; Macias Díaz, S; Vera-Recabarren, M; Vázquez de Lara, L; Méndez Martínez, S; Soto-Santillán, P; González-Ramírez, R; Ruiz-Arguelles, A

    2015-07-01

    P-glycoprotein (Pgp) is a transmembrane protein of 170 kD encoded by the multidrug resistance 1 (MDR-1) gene, localized on chromosome 7. More than 50 polymorphisms of the MDR-1 gene have been described; a subset of these has been shown to play a pathophysiological role in the development of inflammatory bowel disease, femoral head osteonecrosis induced by steroids, lung cancer and renal epithelial tumors. Polymorphisms that have a protective effect on the development of conditions such as Parkinson disease have also been identified. P-glycoprotein belongs to the adenosine triphosphate binding cassette transporter superfamily and its structure comprises a chain of approximately 1280 aminoacid residues with an N-C terminal structure, arranged as 2 homologous halves, each of which has 6 transmembrane segments, with a total of 12 segments with 2 cytoplasmic nucleotide binding domains. Many cytokines like interleukin 2 and tumor necrosis factor alpha increase Pgp expression and activity. Pgp functions as an efflux pump for a variety of toxins in order to protect particular organs and tissues as the central nervous system. Pgp transports a variety of substrates including glucocorticoids while other drugs such as tacrolimus and cyclosporine A act as modulators of this protein. The most widely used method to measure Pgp activity is flow cytometry using naturally fluorescent substrates such as anthracyclines or rhodamine 123. The study of drug resistance and its association to Pgp began with the study of resistance to chemotherapy in the treatment of cancer and antiretroviral therapy for human immunodeficiency virus; however, the role of Pgp in the treatment of systemic lupus erythematosus, rheumatoid arthritis and psoriatic arthritis has been a focus of study lately and has emerged as an important mechanism by which treatment failure occurs. The present review analyzes the role of Pgp in these autoimmune diseases. PMID:25712147

  15. N-Glycoproteomics of Human Seminal Plasma Glycoproteins.

    PubMed

    Saraswat, Mayank; Joenväärä, Sakari; Tomar, Anil Kumar; Singh, Sarman; Yadav, Savita; Renkonen, Risto

    2016-03-01

    Seminal plasma aids sperm by inhibiting premature capacitation, helping in the intracervical transport and formation of an oviductal sperm reservoir, all of which appear to be important in the fertilization process. Epitopes such as Lewis x and y are known to be present on seminal plasma glycoproteins, which can modulate the maternal immune response. It is suggested by multiple studies that seminal plasma glycoproteins play, largely undiscovered, important roles in the process of fertilization. We have devised a strategy to analyze glycopeptides from a complex, unknown mixture of protease-digested proteins. This analysis provides identification of the glycoproteins, glycosylation sites, glycan compositions, and proposed structures from the original sample. This strategy has been applied to human seminal plasma total glycoproteins. We have elucidated glycan compositions and proposed structures for 243 glycopeptides belonging to 73 N-glycosylation sites on 50 glycoproteins. The majority of the proposed glycan structures were complex type (83%) followed by high-mannose (10%) and then hybrid (7%). Most of the glycoproteins were either sialylated, fucosylated, or both. Many Lewis x/a and y/b epitopes bearing glycans were found, suggesting immune-modulating epitopes on multiple seminal plasma glycoproteins. The study also shows that large scale N-glycosylation mapping is achievable with current techniques and the depth of the analysis is roughly proportional to the prefractionation and complexity of the sample. PMID:26791533

  16. 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. PMID:24783808

  17. Immunomodulatory Effects of Nontoxic Glycoprotein Fraction Isolated from Rice Bran.

    PubMed

    Park, Ho-Young; Yu, A-Reum; Hong, Hee-Do; Kim, Ha Hyung; Lee, Kwang-Won; Choi, Hee-Don

    2016-05-01

    Rice bran, a by-product of brown rice milling, is a rich source of dietary fiber and protein, and its usage as a functional food is expected to increase. In this study, immunomodulatory effects of glycoprotein obtained from rice bran were studied in normal mice and mouse models of cyclophosphamide-induced immunosuppression. We prepared glycoprotein from rice bran by using ammonium precipitation and anion chromatography techniques. Different doses of glycoprotein from rice bran (10, 25, and 50 mg/kg) were administered orally for 28 days. On day 21, cyclophosphamide at a dose of 100 mg/kg was administered intraperitoneally. Glycoprotein from rice bran showed a significant dose-dependent restoration of the spleen index and white blood cell count in the immunocompromised mice. Glycoprotein from rice bran affected the immunomodulatory function by inducing the proliferation of splenic lymphocytes, which produce potential T and B cells. Moreover, it prevented cyclophosphamide-induced damage of Th1-type immunomodulatory function through enhanced secretion of Th1-type cytokines (interferon-γ and interleukin-12). These results indicate that glycoprotein from rice bran significantly recovered cyclophosphamide-induced immunosuppression. Based on these data, it was concluded that glycoprotein from rice bran is a potent immunomodulator and can be developed to recover the immunity of immunocompromised individuals. PMID:26891000

  18. Identification of members of the P-glycoprotein multigene family

    SciTech Connect

    Ng, W.F.; Sarangi, F.; Zastawny, R.L.; Veinot-Drebot, L.; Ling, V. )

    1989-03-01

    Overproduction of P-glycoprotein is intimately associated with multidrug resistance. This protein appears to be encoded by a multigene family. Thus, differential expression of different members of this family may contribute to the complexity of the multidrug resistance phenotype. Three lambda genomic clones isolated from a hamster genomic library represent different members of the hamster P-glycoprotein gene family. Using a highly conserved exon probe, the authors found that the hamster P-glycoprotein gene family consists of three genes. They also found that the P-glycoprotein gene family consists of three genes in mice but has only two genes in humans and rhesus monkeys. The hamster P-glycoprotein genes have similar exon-intron organizations within the 3' region encoding the cytoplasmic domains. The propose that the hamster P-glycoprotein gene family arose from gene duplication. The hamster pgpl and pgp2 genes appear to be more closely related to each other than either gene is to the pgp3 gene. They speculate that the hamster pgpl and pgp2 genes arose from a recent gene duplication event and that primates did not undergo this duplication and therefore contain only two P-glycoprotein genes.

  19. Labelling of membrane glycoprotein in erythrocytes infected with Plasmodium knowlesi*

    PubMed Central

    Trigg, P. I.; Hirst, S. I.; Shakespeare, P. G.; Tappenden, L.

    1977-01-01

    Normal rhesus monkey erythrocytes and erythrocytes infected by P. knowlesi were labelled with galactose oxidase (EC 1.1.3.9) and tritiated sodium borohydride. The glycoproteins of normal erythrocytes were not labelled unless the cells were pretreated with neuraminidase, when peaks of activity with apparent molecular weights of 170 000, 126 000, 90 000, 50 000, and 35 000 were observed. Schizont-infected erythrocytes showed an absence of glycoprotein labelling even after neuraminidase treatment. The results indicate that there is an alteration in the glycoproteins of schizont-infected erythrocytes, which may contribute to the increased permeability and the immunological alterations on the surface of these cells. PMID:412601

  20. 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

  1. 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....5420 Alpha-1-glycoproteins immunological test system. (a) Identification. An alpha-1-glycoproteins... alpha-1-glycoproteins (a group of plasma proteins found in the alpha-1 group when subjected...

  2. 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...

  3. 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.

  4. Increase of α1-acid glycoprotein after treatment with amitriptyline

    PubMed Central

    Baumann, P.; Tinguely, D.; Schöpf, J.

    1982-01-01

    Sixteen primary depressive patients were treated for 3 weeks with amitriptyline 150 mg daily. In thirteen patients the plasma level of α1-acid glycoprotein (AAG) significantly increased after the treatment but the albumin levels did not change. PMID:7104160

  5. 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

  6. 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

  7. Enzymatic sulfation of mucus glycoprotein in gastric mucosa

    SciTech Connect

    Liau, Y.H.; Carter, S.R.; Gwozdzinski, K.; Nadziejko, C.; Slomiany, A.; Slomiany, B.L.

    1986-05-01

    Among the posttranslational modifications that mucus glycoprotein undergo prior to secretion into the gastric lumen is the process of sulfation of the carbohydrate chains. These sulfate groups impart strongly negative charge to nucus glycoprotein and are thought to play a major role in the maintenance of gastric mucosal integrity. The authors report here the presence and some properties of an enzyme involved in the sulfation of gastric mucus glycoprotein. The sulfotransferase activity which catalyzes the transfer of sulfate ester group from PAPS to mucus glycoprotein was located in the detergent extracts of the microsomal fraction of rat gastric mucosa. Optimum enzymatic activity for sulfation of gastric mucin was obtained using 0.5% Triton X-100 and 25mM NaF at a pH of 6.8. ATP, ADP, MgCl/sub 2/ and MnCl/sub 2/ at concentrations examined were inhibitory. Under optimal conditions, the rate of sulfate incorporation was proportional to the microsomal enzyme protein concentration up to 50..mu..g and remained constant with time of incubation for at least 1h. The apparent Km value of the enzyme for gastric mucus glycoprotein was 8.3 x 10/sup -6/M. The /sup 35/S-labeled product of the enzyme reaction cochromatographed on Bio-Gel A-50 with gastric mucin, and gave on CsCl equilibrium density gradient centrifugation a band at the density of 1.48 in which the /sup 35/S label coincided with the glycoprotein.

  8. 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. PMID:26517909

  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. 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. PMID:25247386

  11. Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

    PubMed Central

    Jallet, Corinne; Jacob, Yves; Bahloul, Chokri; Drings, Astrid; Desmezieres, Emmanuel; Tordo, Noël; Perrin, Pierre

    1999-01-01

    The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6). PMID:9847325

  12. 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.

  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. Convallatoxin: a new P-glycoprotein substrate.

    PubMed

    Gozalpour, Elnaz; Greupink, Rick; Bilos, Albert; Verweij, Vivienne; van den Heuvel, Jeroen J M W; Masereeuw, Rosalinde; Russel, Frans G M; Koenderink, Jan B

    2014-12-01

    Digitalis-like compounds (DLCs), such as digoxin and digitoxin that are derived from digitalis species, are currently used to treat heart failure and atrial fibrillation, but have a narrow therapeutic index. Drug-drug interactions at the transporter level are frequent causes of DLCs toxicity. P-glycoprotein (P-gp, ABCB1) is the primary transporter of digoxin and its inhibitors influence pharmacokinetics and disposition of digoxin in the human body; however, the involvement of P-gp in the disposition of other DLCs is currently unknown. In present study, the transport of fourteen DLCs by human P-gp was studied using membrane vesicles originating from human embryonic kidney (HEK293) cells overexpressing P-gp. DLCs were quantified by liquid chromatography-mass spectrometry (LC-MS). The Lily of the Valley toxin, convallatoxin, was identified as a P-gp substrate (Km: 1.1±0.2 mM) in the vesicular assay. Transport of convallatoxin by P-gp was confirmed in rat in vivo, in which co-administration with the P-gp inhibitor elacridar, resulted in increased concentrations in brain and kidney cortex. To address the interaction of convallatoxin with P-gp on a molecular level, the effect of nine alanine mutations was compared with the substrate N-methyl quinidine (NMQ). Phe343 appeared to be more important for transport of NMQ than convallatoxin, while Val982 was particularly relevant for convallatoxin transport. We identified convallatoxin as a new P-gp substrate and recognized Val982 as an important amino acid involved in its transport. These results contribute to a better understanding of the interaction of DLCs with P-gp. PMID:25264938

  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. 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.

  17. Biosynthesis of heterogeneous forms of multidrug resistance-associated glycoproteins.

    PubMed

    Greenberger, L M; Williams, S S; Horwitz, S B

    1987-10-01

    Multidrug-resistant J774.2 mouse macrophage-like cells, selected for resistance to colchicine, vinblastine, or taxol, overexpress antigenically related glycoproteins with distinct electrophoretic mobilities. These plasma membrane glycoproteins are likely to play a pivotal role in the expression of the multidrug resistance phenotype. To determine how these multidrug resistance-associated glycoproteins differ, the biosynthesis and N-linked carbohydrate composition of these proteins were examined and compared. Vinblastineor colchicine-selected cells made a 125-kDa precursor that was rapidly processed (t1/2 approximately equal to 20 min) to mature forms of 135 and 140 kDa, respectively. Heterogeneity between the 135- and 140-kDa forms of the molecule can be attributed to N-linked carbohydrate. In contrast, taxol-selected cells made two precursors, 125 and 120 kDa, which appeared within 5 and 15 min after the onset of pulse labeling, respectively. They were processed to mature forms of 140 and 130 kDa. Since a single deglycosylated precursor or mature form was not observed after enzymatic removal of N-linked oligosaccharides, other differences, besides N-linked glycosylation, which occur in early processing compartments, are likely to account for the two multidrug resistance-associated glycoproteins in taxol-selected cells. These results demonstrate that a family of multidrug resistance-associated glycoproteins can be differentially expressed. PMID:2888763

  18. Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters.

    PubMed

    Wang, Fun-In; Deng, Ming-Chung; Huang, Yu-Liang; Chang, Chia-Yi

    2015-07-01

    Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field. PMID:26131960

  19. The HTLV-I envelope glycoproteins: structure and functions.

    PubMed

    Delamarre, L; Rosenberg, A R; Pique, C; Pham, D; Callebaut, I; Dokhélar, M C

    1996-01-01

    The human T-cell lymphotropic virus type I (HTLV-I) envelope has a structural organization shared by all retroviral envelopes, which contain two mature viral glycoproteins deriving from a common precursor: an external surface protein (SU), associated with a transmembrane protein (TM) responsible for anchoring the SU-TM complex at the cell surface or in the viral envelope. Our understanding of the tertiary structure of these proteins is extremely poor. The intracellular maturation follows the normal cellular secretory pathway, resulting in expression of the mature glycoproteins at the cell surface. The five potential N-glycosylation sites are glycosylated. Most mutations artificially introduced into the glycoproteins result in loss of function, mostly due to abnormal intracellular maturation. This probably indicates a very compact structure of these proteins, where the entire structure is involved in correct conformation. Studies using neutralizing antibodies or mutagenesis have defined functional domains in the SU protein, which is responsible for receptor binding. These domains occur throughout the SU glycoprotein. Sequence analysis of the HTLV-I TM predicts a structure, and probably functions, similar to other retrovirus TMs: involvement of this glycoprotein in the different oligomerization steps leading to a fusogenic SU-TM complex and in the fusion process itself. These features remain to be proven, and it is not yet understood why the free HTLV-I viral particle is not infectious. PMID:8797709

  20. Glycoprotein 2 antibodies in Crohn's disease.

    PubMed

    Roggenbuck, Dirk; Reinhold, Dirk; Werner, Lael; Schierack, Peter; Bogdanos, Dimitrios P; Conrad, Karsten

    2013-01-01

    The pathogenesis of Crohn's disease (CrD) and ulcerative colitis (UC), the two major inflammatory bowel diseases (IBD), remains poorly understood. Autoimmunity is considered to be involved in the triggering and perpetuation of inflammatory processes leading to overt disease. Approximately 30% of CrD patients and less than 8% of UC patients show evidence of humoral autoimmunity to exocrine pancreas, detected by indirect immunofluorescence. Pancreatic autoantibodies (PAB) were described for the first time in 1984, but the autoantigenic target(s) of PABs were identified only in 2009. Utilizing immunoblotting and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, the major zymogen granule membrane glycoprotein 2 (GP2) has been discovered as the main PAB autoantigen. The expression of GP2 has been demonstrated at the site of intestinal inflammation, explaining the previously unaddressed contradiction of pancreatic autoimmunity and intestinal inflammation. Recent data demonstrate GP2 to be a specific receptor on microfold (M) cells of intestinal Peyer's patches, which are considered to be the original site of inflammation in CrD. Novel ELISAs, employing recombinant GP2 as the solid phase antigen, have confirmed the presence of IgA and IgG anti-GP2 PABs in CrD patients and revealed an association of anti-GP2 IgA as well as IgG levels with a specific clinical phenotype in CrD. Also, GP2 plays an important role in modulating innate and acquired intestinal immunity. Its urinary homologue, Tamm-Horsfall protein or uromodulin, has a similar effect in the urinary tract, further indicating that GP2 is not just an epiphenomenon of intestinal destruction. This review discusses the role of anti-GP2 autoantibodies as novel CrD-specific markers, the quantification of which provides the basis for further stratification of IBD patients. Given the association with a disease phenotype and the immunomodulating properties of GP2 itself, an important role for GP2

  1. Histidine-rich glycoprotein inhibits contact activation of blood coagulation.

    PubMed

    Vestergaard, A B; Andersen, H F; Magnusson, S; Halkier, T

    1990-12-01

    Histidine-rich glycoprotein has been purified from bovine plasma employing two different purification procedures. The first procedure was one-step ion-exchange chromatography using phosphocellulose, while the second procedure involved fractionation using polyethyleneglycol 6000 followed by column chromatography employing CM-Sepharose and heparin-Sepharose. The effect of purified bovine histidine-rich glycoprotein on the contact activation of blood coagulation was studied in human plasma by using as activating surface either an ellagic acid-phospholipid suspension (Cephotest) or sulfatide. Contact activation was monitored by the generation of amidolytic activity towards a synthetic chromogenic substrate (S-2302) for factor XIIa and plasma kallikrein. Bovine histidine-rich glycoprotein inhibits the contact activation induced by both of these activating surfaces. PMID:2084959

  2. Viral glycoproteins: biological role and application in diagnosis.

    PubMed

    Banerjee, Nilotpal; Mukhopadhyay, Sumi

    2016-03-01

    The viruses that infect humans cause a huge global disease burden and produce immense challenge towards healthcare system. Glycoproteins are one of the major components of human pathogenic viruses. They have been demonstrated to have important role(s) in infection and immunity. Concomitantly high titres of antibodies against these antigenic viral glycoproteins have paved the way for development of novel diagnostics. Availability of appropriate biomarkers is necessary for advance diagnosis of infectious diseases especially in case of outbreaks. As human mobilization has increased manifold nowadays, dissemination of infectious agents became quicker that paves the need of rapid diagnostic system. In case of viral infection it is an emergency as virus spreads and mutates very fast. This review encircles the vast arena of viral glycoproteins, their importance in health and disease and their diagnostic applications. PMID:26925438

  3. The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

    PubMed

    Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

    2015-01-01

    Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. PMID:25456403

  4. 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.

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

    PubMed

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

    2014-03-15

    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

  6. 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

  7. 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...

  8. Reversible conformational changes and fusion activity of rabies virus glycoprotein.

    PubMed Central

    Gaudin, Y; Tuffereau, C; Segretain, D; Knossow, M; Flamand, A

    1991-01-01

    In an attempt to understand the implication of the rabies virus glycoprotein (G) in the first steps of the viral cycle, we studied the pH dependence of virus-induced fusion and hemagglutination, as well as modifications of the structure and properties of the viral glycoprotein following pH acidification. Our results suggest that the G protein adopts at least three distinct configurations, each associated with different properties. At neutral pH, G did not fuse membranes or hemagglutinate erythrocytes. It was insensitive to digestion with bromelain and trypsin. At pH 6.4, the glycoprotein became sensitive to proteases. Hemagglutination was at its maximum and then sharply decreased with the pH. No fusion was detected. Aggregation of virus was also observed. The third configuration, at below pH 6.1, was associated with the appearance of fusion. Some neutralizing monoclonal antibodies were able to differentiate these three configurations. Preincubation of the virus at below pH 6 inhibited fusion, but this inhibition, like the structural modifications of the glycoprotein, was reversible when G was reincubated at neutral pH. Images PMID:1870204

  9. 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...

  10. Synthesis of cell envelope glycoproteins of Cryptococcus laurentii.

    PubMed

    Schutzbach, John; Ankel, Helmut; Brockhausen, Inka

    2007-05-21

    Fungi of the genus Cryptococcus are encapsulated basidiomycetes that are ubiquitously found in the environment. These organisms infect both lower and higher animals. Human infections that are common in immune-compromised individuals have proven difficult to cure or even control with currently available antimycotics that are quite often toxic to the host. The virulence of Cryptococcus has been linked primarily to its polysaccharide capsule, but also to cell-bound glycoproteins. In this review, we show that Cryptococcus laurentii is an excellent model for studies of polysaccharide and glycoprotein synthesis in the more pathogenic relative C. neoformans. In particular, we will discuss the structure and biosynthesis of O-linked carbohydrates on cell envelope glycoproteins of C. laurentii. These O-linked structures are synthesized by at least four mannosyltransferases, two galactosyltransferases, and at least one xylosyltransferase that have been characterized. These glycosyltransferases have no known homologues in human tissues. Therefore, enzymes involved in the synthesis of cryptococcal glycoproteins, as well as related enzymes involved in capsule synthesis, are potential targets for the development of specific inhibitors for treatment of cryptococcal disease. PMID:17316583

  11. THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN

    PubMed Central

    Redman, Colvin M.; Cherian, M. George

    1972-01-01

    These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the 14C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae. PMID:5057975

  12. Modulation of glycan detection on specific glycoproteins by lectin multimerization

    PubMed Central

    Cao, Zheng; Partyka, Katie; McDonald, Mitchell; Brouhard, Elizabeth; Hincapie, Marina; Brand, Randall E.; Hancock, William S.; Haab, Brian B.

    2013-01-01

    Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents such as lectins and glycan-binding antibodies is a valuable complement to methods involving mass spectrometry and chromatography. Many lectins, however, are not useful as analytic tools due to low affinity in vitro. As an approach to increasing lectin avidity to targeted glycans, we tested the use of lectin multimerization. Several biotinylated lectins were linked together through streptavidin interactions. The binding of certain lectins for purified glycoproteins and glycoproteins captured directly out of biological solutions was increased using multimerization, resulting in the detection of lower concentrations of glycoprotein than possible using monomeric detection. The analysis of glycoproteins in plasma samples showed that the level of binding enhancement through multimerization was not equivalent across patient samples. Wheat germ agglutinin (WGA) reactive glycans on fibronectin and thrombospondin-5 were preferentially bound by multimers in pancreatic cancer patient samples relative to control samples, suggesting a cancer-associated change in glycan density that could be detected only through lectin multimerization. This strategy could lead to the more sensitive and informative detection of glycans in biological samples and a broader spectrum of lectins that are useful as analytical reagents. PMID:23286506

  13. 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

  14. Modulation of glycan detection on specific glycoproteins by lectin multimerization.

    PubMed

    Cao, Zheng; Partyka, Katie; McDonald, Mitchell; Brouhard, Elizabeth; Hincapie, Marina; Brand, Randall E; Hancock, William S; Haab, Brian B

    2013-02-01

    Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents such as lectins and glycan-binding antibodies is a valuable complement to methods involving mass spectrometry and chromatography. Many lectins, however, are not useful as analytic tools due to low affinity in vitro. As an approach to increasing lectin avidity to targeted glycans, we tested the use of lectin multimerization. Several biotinylated lectins were linked together through streptavidin interactions. The binding of certain lectins for purified glycoproteins and glycoproteins captured directly out of biological solutions was increased using multimerization, resulting in the detection of lower concentrations of glycoprotein than possible using monomeric detection. The analysis of glycoproteins in plasma samples showed that the level of binding enhancement through multimerization was not equivalent across patient samples. Wheat germ agglutinin (WGA) reactive glycans on fibronectin and thrombospondin-5 were preferentially bound by multimers in pancreatic cancer patient samples relative to control samples, suggesting a cancer-associated change in glycan density that could be detected only through lectin multimerization. This strategy could lead to the more sensitive and informative detection of glycans in biological samples and a broader spectrum of lectins that are useful as analytical reagents. PMID:23286506

  15. 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.

  16. 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.

  17. Bio-vaterite formation by glycoproteins from freshwater pearls.

    PubMed

    Natoli, Antonino; Wiens, Matthias; Schröder, Heinz-Christoph; Stifanic, Mauro; Batel, Renato; Soldati, Analia L; Jacob, Dorrit E; Müller, Werner E G

    2010-06-01

    A 48 kDa acidic and putative calcium-binding glycoprotein was isolated from pearls of the freshwater mussel Hyriopsis cumingii. This protein was compared with a related 46 kDa polypeptide, obtained from the nacreous shell of the same species. Separation by two-dimensional gel electrophoresis revealed that the difference in molecular weight is due to the higher extent of glycosylation of the 48 kDa protein existing in pearls. Evidence is presented that the sugar moieties of the protein contribute to crystal growth, starting with the nucleation step. In in vitro precipitation experiments, the 48 kDa glycoprotein of pearls directed the formation of round-shaped vaterite crystals while the 46 kDa glycoprotein of shells promoted formation of small irregular calcite particles. Furthermore, both proteins, 48 kDa/46 kDa, comprised carbonic anhydrase activity that has been implicated in CaCO(3) formation. Thus, a function of the isolated glycoproteins in biomineralization is proposed together with the mechanism by which they can stabilize different calcium carbonate polymorphs. PMID:20171896

  18. 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.

  19. Inflammatory glycoproteins in cardiometabolic disorders, autoimmune diseases and cancer.

    PubMed

    Connelly, Margery A; Gruppen, Eke G; Otvos, James D; Dullaart, Robin P F

    2016-08-01

    The physiological function initially attributed to the oligosaccharide moieties or glycans on inflammatory glycoproteins was to improve protein stability. However, it is now clear that glycans play a prominent role in glycoprotein structure and function and in some cases contribute to disease states. In fact, glycan processing contributes to pathogenicity not only in autoimmune disorders but also in atherosclerotic cardiovascular disease, diabetes and malignancy. While most clinical laboratory tests measure circulating levels of inflammatory proteins, newly developed diagnostic and prognostic tests are harvesting the information that can be gleaned by measuring the amount or structure of the attached glycans, which may be unique to individuals as well as various diseases. As such, these newer glycan-based tests may provide future means for more personalized approaches to patient stratification and improved patient care. Here we will discuss recent progress in high-throughput laboratory methods for glycomics (i.e. the study of glycan structures) and glycoprotein quantification by methods such as mass spectrometry and nuclear magnetic resonance spectroscopy. We will also review the clinical utility of glycoprotein and glycan measurements in the prediction of common low-grade inflammatory disorders including cardiovascular disease, diabetes and cancer, as well as for monitoring autoimmune disease activity. PMID:27312321

  20. 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

  1. Envelope Glycoprotein Trimers as HIV-1 Vaccine Immunogens

    PubMed Central

    Sattentau, Quentin J.

    2013-01-01

    The HIV-1 envelope glycoprotein spike is the target of neutralizing antibody attack, and hence represents the only relevant viral antigen for antibody-based vaccine design. Various approaches have been attempted to recapitulate Env in membrane-anchored and soluble forms, and these will be discussed here in the context of recent successes and challenges still to be overcome. PMID:26344344

  2. Distinct structural rearrangements of the VSV glycoprotein drive membrane fusion

    PubMed Central

    Libersou, Sonia; Albertini, Aurélie A.V.; Ouldali, Malika; Maury, Virginie; Maheu, Christine; Raux, Hélène; de Haas, Felix; Roche, Stéphane

    2010-01-01

    The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes, driven by conformational changes in viral glycoproteins. Many studies have shown that fusion involves the cooperative action of a large number of these glycoproteins, but the underlying mechanisms are unknown. We used electron microscopy and tomography to study the low pH–induced fusion reaction catalyzed by vesicular stomatitis virus glycoprotein (G). Pre- and post-fusion crystal structures were observed on virions at high and low pH, respectively. Individual fusion events with liposomes were also visualized. Fusion appears to be driven by two successive structural rearrangements of G at different sites on the virion. Fusion is initiated at the flat base of the particle. Glycoproteins located outside the contact zone between virions and liposomes then reorganize into regular arrays. We suggest that the formation of these arrays, which have been shown to be an intrinsic property of the G ectodomain, induces membrane constraints, achieving the fusion reaction. PMID:20921141

  3. 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.

  4. 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.

  5. Microfluidic digital isoelectric fractionation for rapid multidimensional glycoprotein analysis.

    PubMed

    Mai, Junyu; Sommer, Gregory J; Hatch, Anson V

    2012-04-17

    Here we present an integrated microfluidic device for rapid and automated isolation and quantification of glycoprotein biomarkers directly from biological samples on a multidimensional analysis platform. In the first dimension, digital isoelectric fractionation (dIEF) uses discrete pH-specific membranes to separate proteins and their isoforms into precise bins in a highly flexible spatial arrangement on-chip. dIEF provides high sample preconcentration factors followed by immediate high-fidelity transfer of fractions for downstream analysis. We successfully fractionate isoforms of two potential glycoprotein cancer markers, fetuin and prostate-specific antigen (PSA), with 10 min run time, and results are compared qualitatively and quantitatively to conventional slab gel IEF. In the second dimension, functionalized monolithic columns are used to capture and detect targeted analytes from each fraction. We demonstrate rapid two-dimensional fractionation, immunocapture, and detection of C-reactive protein (CRP) spiked in human serum. This rapid, flexible, and automated approach is well-suited for glycoprotein biomarker research and verification studies and represents a practical avenue for glycoprotein isoform-based diagnostic testing. PMID:22409593

  6. Novel bifidobacterial glycosidases acting on sugar chains of mucin glycoproteins.

    PubMed

    Katayama, Takane; Fujita, Kiyotaka; Yamamoto, Kenji

    2005-05-01

    Bifidobacterium bifidum was found to produce a specific 1,2-alpha-L-fucosidase. Its gene (afc A) has been cloned and the DNA sequence was determined. The Afc A protein consisting of 1959 amino acid residues with a predicted molecular mass of 205 kDa can be divided into three domains; the N-terminal function-unknown domain (576 aa), the catalytic domain (898 aa), and the C-terminal bacterial Ig-like domain (485 aa). The recombinant catalytic domain specifically hydrolyzed the terminal alpha-(1-->2)-fucosidic linkages of various oligosaccharides and sugar chains of glycoproteins. The primary structure of the catalytic domain exhibited no similarity to those of any glycoside hydrolases but showed similarity to those of several hypothetical proteins in a database, which resulted in establishment of a novel glycoside hydrolase family (GH family 95). Several bifidobacteria were found to produce a specific endo-alpha-N-acetylgalactosaminidase, which is the endoglycosidase liberating the O-glycosidically linked galactosyl beta1-->3 N-acetylgalactosamine disaccharide from mucin glycoprotein. The molecular cloning of endo-alpha-N-acetylgalactosaminidase was carried out on Bifidobacterium longum based on the information in the database. The gene was found to comprise 1966 amino acid residues with a predicted molecular mass of 210 kDa. The recombinant protein released galactosyl beta1-->3 N-acetylgalactosamine disaccharide from natural glycoproteins. This enzyme of B. longum is believed to be involved in the catabolism of oligosaccharide of intestinal mucin glycoproteins. Both 1,2-alpha-L-fucosidase and endo-alpha-N-acetylgalactosaminidase are novel and specific enzymes acting on oligosaccharides that exist mainly in mucin glycoproteins. Thus, it is reasonable to conclude that bifidobacteria produce these enzymes to preferentially utilize the oligosaccharides present in the intestinal ecosystem. PMID:16233817

  7. Structural and functional attributes of zona pellucida glycoproteins.

    PubMed

    Gupta, Satish K; Chakravarty, Sanchita; Suraj, K; Bansal, Pankaj; Ganguly, Anasua; Jain, Manish K; Bhandari, Beena

    2007-01-01

    A translucent matrix termed the zona pellucida (ZP) surrounds the mammalian oocyte. It plays a critical role in fertilization by acting as a "docking site" for binding of spermatozoa followed by induction of the acrosome reaction in the zona bound sperm. Recent analyses of the genes of the human oocyte revealed that the ZP matrix is composed of four glycoproteins, designated as ZP1, ZP2, ZP3 and ZP4, instead of 3 found in the mouse ZP. Comparison of the deduced amino acid (aa) sequences of the human ZP glycoproteins with those from various species, revealed that these are evolutionarily conserved. Phylogenetic analysis revealed that ZP1 and ZP4 may be related as these have the highest sequence identity at the aa level within a given species. Each zona protein has a signal sequence driving these proteins to the endoplasmic reticulum, a aproximately 260 aa long 'ZP domain' comprising of 8-10 conserved cysteine residues, a C-terminal, hydrophobic transmembrane-like region and a short cytoplasmic tail. In order to understand the structure-function relationship of human ZP glycoproteins, our lab has cloned and expressed ZP2, ZP3 and ZP4 proteins both in E. coli as well as baculovirus expression systems. Simultaneously, our group has been able to amplify the cDNA encoding human ZP1. Employing baculovirus-expressed recombinant ZP glycoproteins; our group has provided evidence for the first time that in human, in addition to ZP3, ZP4 is also able to induce acrosomal exocytosis in the capacitated spermatozoa. ZP3 mediated induction of the acrosome reaction can be inhibited by pertussis toxin suggesting the involvement of G, protein in downstream signaling in contrast to ZP4, which follows a G, protein independent pathway. Hence, elucidation of the role of individual ZP glycoproteins in humans will provide a better insight into the gamete interaction culminating in fertilization. PMID:17566274

  8. 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

  9. Human blood platelet membrane glycoproteins. Resolution in different polyacrylamide gel electrophoretic systems.

    PubMed

    Jenkins, C S; Ali-Briggs, E F; Zonneveld, G T; Sturk, A; Clemetson, K J

    1980-02-29

    The separation of the major platelet membrane glycoproteins of normal subjects and subjects with well defined platelet membrane glycoprotein abnormalities have been examined using four different polyacrylamide gel electrophoretic techniques (continuous and discontinuous). The mobilities of the resolved glycoprotein bands have been correlated with the glycoprotein nomenclature proposed for the conventional sodium dodecyl sulphate-phosphate buffer system. Since the glycoprotein distribution varies depending on the system used, the merits of each method should be considered before application to a specific problem. PMID:6768152

  10. Antifreeze glycoproteins from an Antarctic fish. Quasi-elastic light scattering studies of the hydrodynamic conformations of antifreeze glycoproteins.

    PubMed

    Ahmed, A I; Feeney, R E; Osuga, D T; Yeh, Y

    1975-05-10

    A quasi-elastic light-scattering technique was used to study the hydrodynamic conformations of antifreeze glycoproteins from an Antarctic fish. Antifreeze glycoprotein is composed of repeating units of Ala-Ala-Thr, with each threonine O-linked to a disaccharide, and it exists as several polymers of different numbers of this repeating unit. Molecular weights of the two major active polymers are 10,500 and 17,500 by such methods as centrifugation and osmotic pressure, but smaller than 20 by freezing-point depression. Translational diffusion coefficients at 20 degrees were 8.35 times 10-7 cm2 s-1 and 6.15 times 10-7 cm2 s-1 for the M-r-10,500 and 17,500 polymers, respectively. Measurements at -0.2 degrees in the presence of ice crystals did not indicate any conformational changes that might be related to the lowering of the freezing temperature. Lowering the temperature of these glycoprotein solutions close to temperatures of freezing caused a decrease in the effective hydrodynamic radius of both active and inactive glycoprotein components. PMID:1168194

  11. 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

  12. Expression of the CD68 glycoprotein in the rat epididymis.

    PubMed

    Liguori, Giovanna; De Pasquale, Valeria; Della Morte, Rossella; Avallone, Luigi; Costagliola, Anna; Vittoria, Alfredo; Tafuri, Simona

    2015-11-01

    The 110 kDa trans-membrane glycoprotein CD68 is highly expressed by human monocytes and tissue macrophages. However, in addition to the monocyte/macrophage system, CD68 has been also found in normal and tumor cells with no macrophagic activity such as lymphocytes, fibroblasts, endothelial cells, small intestinal epithelial cells, and neoplastic cells of different origins. Here, for the first time we demonstrate the immunohistochemical localization of CD68 in the principal cells of the cranial and caudal segments of rat epididymis. These results were confirmed by biochemical analyses showing the expression of CD68 mRNA transcripts and the protein in the epididymis tissues. Our findings, while providing further evidence that CD68 expression is not restricted to the monocyte/macrophage system, suggest that the glycoprotein might be involved in the functions of epididymal principal cells that contribute to spermatozoa maturation process. PMID:26433032

  13. 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

  14. 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

  15. Some factors affecting the production, by cultured baby-hamster kidney cells, of BHK glycoprotein I which cross-reacts immunologically with Tamm-Horsfall glycoprotein.

    PubMed Central

    Bloomfield, F J; Dunstan, D R; Foster, C L; Serafini-Cessi, F; Marshall, R D

    1977-01-01

    Cultured baby-hamster kidney cells (BHK-21/C13), which are adapted to grow in suspension (strain 2P), roduce a glycoprotein, termed BHK glycoprotein I, which cross-reacts immunologically with hamster urinary (Tamm-Horsfall glycoprotein. BHK glycoprotein I was isolated in an electrophoretically (sodium dodecyl sulphate/polyacrylamide gel) homogeneous form by application of affinity chromatography to the medium in which cells had been cultured. Insolubilized anti-(Tamm-Horsfall glycoprotein immunoglobulin G) was used as the adsorbent. The amount of BHK glycoprotein I associated with the cultured cells was found by both radioimmunoassay and immunofluorescence to be related to the amount of Ca2+ in the medium and to the particular stage of the cell cycle. 5'-Nucleotidase was also shed by the cells into the culture medium in amounts related to the stage of the cell cycle. The turnover of hamster Tamm-Horsfall glycoprotein in vivo appeared to be considerably more rapid than can be accounted for by cell turnover. Hamster Tamm-Horsfall glycoprotein was shown to be ineffective in inhibiting agglutination of chicken erythrocytes caused by influenza virus. Images PLATE 1 PLATE 2 PLATE 3 PLATE 4 PMID:328011

  16. High efficiency labeling of glycoproteins on living cells

    PubMed Central

    Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

    2010-01-01

    We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

  17. 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.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L

    PubMed Central

    Beevers, Leonard; Mense, Rose M.

    1977-01-01

    Particulate preparations from developing cotyledons of Pisum sativum L. cv. Burpeeana catalyze glycosyl transfer from UDP-[14C]N-acetylglucosamine and GDP-[14C]mannose. Radioactivity is transferred to lipid components soluble in chloroform-methanol (2:1) and chloroform-methanol-water (1:1:0.3) and into a water-insoluble and lipid-free residue. The chloroform-methanol-soluble component formed from GDP-[14C]mannose appears to be a mannosyl lipid, whereas the chloroform-methanol-water-soluble fraction is probably a mixed oligosaccharide-lipid containing N-acetylglucosamine and mannose residues. The chloroform-methanol-soluble component formed from UDP-[14C]N-acetylglucosamine appears to be N,N′-diacetylchitibiosyl lipid, which may be incorporated with mannose to form the chloroform-methanol-water-soluble mixed oligosaccharide lipid. The oligosaccharide lipid appears to function as a precursor for the transfer of the oligosaccharide to the peptide moiety in the formation of the glycoproteins. The bulk of the radioactivity, arising from UDP-[14C]N-acetylglucosamine, incorporated into the insoluble residue, is associated with glycoprotein. In contrast only a small percentage of radioactivity in the insoluble residue, arising from GDP-[14C]mannose incorporation, appears to be associated with glycoprotein. The majority of the radioactivity found in the residue fraction labeled from GDP-[14C]mannose appears to be associated with oligomannosyl residues. PMID:16660168

  3. Ultrasensitive impedimetric lectin based biosensor for glycoproteins containing sialic acid

    PubMed Central

    Bertok, Tomas; Gemeiner, Pavol; Mikula, Milan; Gemeiner, Peter; Tkac, Jan

    2016-01-01

    We report on an ultrasensitive label-free lectin-based impedimetric biosensor for the determination of the sialylated glycoproteins fetuin and asialofetuin. A sialic acid binding agglutinin from Sambucus nigra I was covalently immobilised on a mixed self-assembled monolayer (SAM) consisting of 11-mercaptoundecanoic acid and 6-mercaptohexanol. Poly(vinyl alcohol) was used as a blocking agent. The sensor layer was characterised by atomic force microscopy, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy. The biosensor exhibits a linear range that spans 7 orders of magnitude for both glycoproteins, with a detection limit as low as 0.33 fM for fetuin and 0.54 fM for asialofetuin. We also show, by making control experiments with oxidised asialofetuin, that the biosensor is capable of quantitatively detecting changes in the fraction of sialic acid on glycoproteins. We conclude that this work lays a solid foundation for future applications of such a biosensor in terms of the diagnosis of diseases such as chronic inflammatory rheumatoid arthritis, genetic disorders and cancer, all of which are associated with aberrant glycosylation of protein biomarkers. PMID:27231402

  4. Australine, a pyrrolizidine alkaloid that inhibits amyloglucosidase and glycoprotein processing

    SciTech Connect

    Tropea, J.E.; Molyneux, R.J.; Kaushal, G.P.; Pan, Y.T.; Mitchell, M.; Elbein, A.D. )

    1989-03-07

    Australine is a polyhydroxylated pyrrolizidine alkaloid that was isolated from the seeds of the Australian tree Castanospermum australe and characterized by NMR and X-ray diffraction analysis. Since swainsonine and catanospermine are polyhydroxylated indolizidine alkaloids that inhibit specific glycosidases, the authors tested australine against a variety of exoglycosidases to determine whether it would inhibit any of these enzymes. This alkaloid proved to be a good inhibitor of the {alpha}-glucosidase amyloglucosidase (50% inhibition at 5.8 {mu}M), but it did not inhibit {beta}-glucosidase, {alpha}- or {beta}-mannosidase, or {alpha}- or {beta}-galactosidase. The inhibition of amyloglucosidase was of a competitive nature. Australine also inhibited the glycoprotein processing enzyme glucosidase I, but had only slight activity toward glucosidase II. When incubated with cultured cells, this alkaloid inhibited glycoprotein processing at the glucosidase I step and caused the accumulation of glycoproteins with Glc{sub 3}Man{sub 7-9}(GlcNAc){sub 2}-oligosaccharides.

  5. 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

  6. 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

  7. 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

  8. The immunomodulating roles of glycoproteins in epithelial ovarian cancer

    PubMed Central

    Patankar, Manish S.; Gubbels, Jennifer A.A.; Felder, Mildred; Connor, Joseph P.

    2015-01-01

    The complexity of the immune system demands an intricate defense mechanism by tumors. Ovarian and other tumors employ specific glycoproteins and the associated glycan sequences to modulate immune responses. Glycoproteins enable tumor cells that express or secrete these molecules to evade immune cell attack and induce the immune system to promote tumor growth. This review focuses first on the immune environment in ovarian cancer, and the mechanisms of activation and inhibition that immune cells undergo in order to either attack or ignore a target cell. Next we illustrate the immunomodulatory roles of ovarian cancer-associated glycans and glycoproteins in 1. preventing immune synapse formation, 2. serving as ligands of immune cell receptors, 3. scavenging cytokines and chemokines, and 4. participating in the formation of autoantibodies against the tumor. The importance of these immunomodulating strategies from the view points of understanding the tumor immunology of ovarian tumors, potential origin of such mechanisms, and specific strategies to circumvent the glycoconjugate-mediated suppression of immune responses is discussed in this review. PMID:22201900

  9. 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

  10. 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

  11. Characterization of the O- and N-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni

    SciTech Connect

    Nyame, A.K.

    1987-01-01

    The structures of the O- and N-linked oligosaccharides in glycoproteins synthesized by larval and adult schistosomes of Schistosoma mansoni have been investigated. Mechanically transformed schistosomula or adult schistosomes were incubated in media containing either (/sup 3/H)mannose, (/sup 3/H)glucosamine or (/sup 3/H)galactose for 48 and 24 hr, respectively, to radiolabel metabolically the oligosaccharide moieties of newly synthesized glycoproteins. Analyses of the radiolabeled glycoproteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and fluorography demonstrated that numerous glycoproteins from 48-hr old schistosomula and adult schistosomes were labeled by both the (/sup 3/H)mannose and (/sup 3/H)glucosamine precursors. The (/sup 3/H)galactose precursor was incorporated into numerous glycoproteins in adult schistosomes; however, few, if any, glycoproteins in schistosomula were labeled by this radioactive sugar precursor.

  12. Identification and antigenicity of the major envelope glycoprotein of lymphadenopathy-associated virus

    SciTech Connect

    Montagnier, L.; Clavel, F.; Krust, B.; Chamaret, S.; Rey, F.; Barre-Sinoussi, F.; Chermann, J.C.

    1985-07-15

    The major envelope glycoprotein of the causative agent of Acquired Immune Deficiency Syndrome (AIDS) lymphadenopathy-associated virus (LAV) has been identified and characterized. The glycoprotein has an apparent molecular weight of 110,000-120,000 under denaturing conditions in polyacrylamide gel electrophoresis. Upon deglycosylation by a specific endoglycosydase, its size is reduced to 80,000. Cellular precursors of this glycoprotein have been detected with apparent molecular weight of 150,000 and 135,000. Nearly all AIDS and pre-AIDS patients have detectable antibodies against this viral glycoprotein.

  13. The role of interfacial structured water on the glycoprotein arrangement in liposomes.

    PubMed

    Neitchev, V Z; Kostadinov, A P

    1986-01-01

    The effect of perturbing the interfacial water structure in liposomes on the glycoprotein arrangement in the bilayer was investigated. This perturbation was achieved by a series of reagents called structure makers and breakers. The glycoprotein arrangement in the liposomes was determined by fluorescence measurement with 1-anilino-2-naphthalene sulphonate (ANS). A dependence of (n) (number of binding sites for ANS on the glycoprotein molecule) with concentration of structure maker and breaker reagents was observed. The results have been interpreted as a possible new arrangement of membrane-bound glycoprotein, due to the effect of perturbing the interfacial water structure in the liposomes. PMID:3807904

  14. RELATIONSHIPS BETWEEN ACCUMULATION OF A 60KDA STRESS PROTEIN AND SCOPE-FOR-GROWTH IN MYTILIS EDULIS EXPOSED TO A RANGE OF COPPER CONCENTRATIONS

    EPA Science Inventory

    Blue mussels, Mytilus edulis, were exposed in the laboratory for 7 days to a range of Cu concentrations (O to 100 ug/liter) to test the feasibility of using the accumulation of a 60 kDa stress protein, also referred to as heat shock protein 60 (hsp60) as a molecular indicator of ...

  15. Analogies and homologies in lipopolysaccharide and glycoprotein biosynthesis in bacteria.

    PubMed

    Hug, Isabelle; Feldman, Mario F

    2011-02-01

    Bacteria generate and attach countless glycan structures to diverse macromolecules. Despite this diversity, the mechanisms of glycoconjugate biosynthesis are often surprisingly similar. The focus of this review is on the commonalities between lipopolysaccharide (LPS) and glycoprotein assembly pathways and their evolutionary relationship. Three steps that are essential for both pathways are completed by membrane proteins. These include the initiation of glycan assembly through the attachment of a first sugar residue onto the lipid carrier undecaprenyl pyrophosphate, the translocation across the plasma membrane and the final transfer onto proteins or lipid A-core. Two families of initiating enzymes have been described: the polyprenyl-P N-acetylhexosamine-1-P transferases and the polyprenyl-P hexosamine-1-P transferases, represented by Escherichia coli WecA and Salmonella enterica WbaP, respectively. Translocases are either Wzx-like flippases or adenosine triphosphate (ATP)-binding cassette transporters (ABC transporters). The latter can consist either of two polypeptides, Wzt and Wzm, or of a single polypeptide homolog to the Campylobacter jejuni PglK. Finally, there are two families of conjugating enzymes, the N-oligosaccharyltransferases (N-OTase), best represented by C. jejuni PglB, and the O-OTases, including Neisseria meningitidis PglL and the O antigen ligases involved in LPS biosynthesis. With the exception of the N-OTases, probably restricted to glycoprotein synthesis, members of all these transmembrane protein families can be involved in the synthesis of both glycoproteins and LPS. Because many translocation and conjugation enzymes display relaxed substrate specificity, these bacterial enzymes could be exploited in engineered living bacteria for customized glycoconjugate production, generating potential vaccines and therapeutics. PMID:20871101

  16. Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma.

    PubMed

    Yu, Han-Ling; Chertkow, Howard M; Bergman, Howard; Schipper, Hyman M

    2003-11-01

    A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition. PMID:14595822

  17. Alcelaphine herpesvirus 1 glycoprotein B: recombinant expression and antibody recognition.

    PubMed

    Dry, Inga; Todd, Helen; Deane, David; Percival, Ann; Mclean, Kevin; Inglis, Neil F; Manson, Erin D T; Haig, David M; Nayuni, Shilpa; Hutt-Fletcher, Lindsey M; Grant, Dawn M; Bartley, Kathryn; Stewart, James P; Russell, George C

    2016-03-01

    The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes fatal malignant catarrhal fever (MCF) in susceptible species including cattle, but infects its reservoir host, wildebeest, without causing disease. Pathology in cattle may be influenced by virus-host cell interactions mediated by the virus glycoproteins. Cloning and expression of a haemagglutinin-tagged version of the AlHV-1 glycoprotein B (gB) was used to demonstrate that the AlHV-1-specific monoclonal antibody 12B5 recognised gB and that gB was the main component of the gp115 complex of AlHV-1, a glycoprotein complex of five components identified on the surface of AlHV-1 by immunoprecipitation and radiolabelling. Analysis of AlHV-1 virus particles showed that the native form of gB was detected by mAb 12B5 as a band of about 70 kDa, whilst recombinant gB expressed by transfected HEK293T cells appeared to be subject to additional cleavage and incomplete post-translational processing. Antibody 12B5 recognised an epitope on the N-terminal furin-cleaved fragment of gB on AlHV-1 virus particles. It could be used to detect recombinant and virus-expressed gB on western blots and on the surface of infected cells by flow cytometry, whilst recombinant gB was detected on the surface of transfected cells by immunofluorescence. Recombinant gB has potential as an antigen for ELISA detection of MCF virus infection and as a candidate vaccine antigen. PMID:26650040

  18. Expression of alpha-GalNAc glycoproteins by breast cancers.

    PubMed Central

    Brooks, S. A.; Leathem, A. J.

    1995-01-01

    The expression of complex carbohydrates recognised by Helix pomatia lectin (HPA, nominal monosaccharide binding specificity alpha-GalNAc) has been shown to predict unfavourable prognosis in breast and other cancers. It has been suggested that the prognostic significance of HPA binding may be through recognition of either Tn epitope (alpha-GalNAc-O-serine/threonine) or blood group A antigen (terminal alpha-1-->3GalNAc attached to the basic H-antigen, Fuc-alpha-1-->2-Gal-beta-1-->4(or 3) GlcNAc-->R). In this study, the expression of glycoproteins terminating in alpha-GalNAc residues was investigated immunohistochemically using HPA and two monoclonal antibodies--BRIC 66 (anti-alpha-GalNAc) and BRIC 111 (anti-Tn). In paraffin sections, 74/87 (85%) of breast cancers expressed HPA-binding ligands, while 28/87 (32%) were positive for BRIC 66 binding and 25/87 (29%) expressed Tn. Distribution of staining patterns were distinctive and different with the three markers. BRIC 66, BRIC 111 and HPA binding to glycoproteins derived from breast cancer homogenates and to blood group A and Tn positive glycoproteins in Western blots confirmed the immunohistochemistry data. The results suggest that the prognostic significance of HPA binding in breast cancer is unlikely to be simply through recognition of blood group A antigen or Tn epitope on cancer cells. Breast cancers may express a complex profile of related but distinct glycans sharing similar terminal immunodominant sugar GalNAc, which may be implicated in aggressive biological behaviour. Images Figure 1 Figure 2 PMID:7537516

  19. 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

  20. Antigiardial activity of glycoproteins and glycopeptides from Ziziphus honey.

    PubMed

    Mohammed, Seif Eldin A; Kabashi, Ahmed S; Koko, Waleed S; Azim, M Kamran

    2015-01-01

    Natural honey contains an array of glycoproteins, proteoglycans and glycopeptides. Size-exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular masses in the range from 10 to >200 kDa. The fractionated proteins exhibited in vitro activities against Giardia lamblia with IC50 values ≤ 25 μg/mL. Results indicated that honey proteins were more active as antiprotozoal agents than metronidazole. This study indicated the potential of honey proteins and peptides as novel antigiardial agents. PMID:25587739

  1. 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

  2. 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

  3. 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

  4. Identification of a mouse synaptic glycoprotein gene in cultured neurons.

    PubMed

    Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

    2005-10-01

    Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation. PMID:16341590

  5. Entrapment in anti myelin-associated glycoprotein neuropathy.

    PubMed

    Faber, Catharina G; Notermans, Nicolette C; Wokke, John H J; Franssen, Hessel

    2009-04-01

    Anti-myelin associated glycoprotein (MAG) neuropathy is a chronic disorder in which IgM antibodies react with Schwann cell glycoproteins, including MAG and peripheral myelin protein 22 (PMP22). Nerve conduction studies show features of axon loss and predominantly distal slowing consistent with demyelination. Because a genetic loss of PMP22 function yields hereditary neuropathy with liability to pressure palsies (HNPP), loss of PMP22 function due to anti- MAG antibodies may result in increased sensitivity to entrapment. We investigated this by performing standardized electrophysiological studies in 16 patients with anti-MAG neuropathy and 16 disease controls with genetically confirmed HNPP. Disproportionate slowing relative to adjacent segments occurred in similar proportions of patients with anti-MAG neuropathy and HNPP, and was of the same magnitude in each group. Affected were the elbow, carpal tunnel and the wrist-hand segments of the median and ulnar nerves. However, in anti-MAG neuropathy as compared to HNPP, absolute values of distal motor latencies and conduction velocities outside entrapment sites were slower and amplitudes were lower. In conclusion, increased sensitivity for entrapment may occur in anti-MAG neuropathy and contribute to part of the nerve damage. PMID:19306083

  6. 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. PMID:25496517

  7. Glycosylation of closely spaced acceptor sites in human glycoproteins

    PubMed Central

    Shrimal, Shiteshu; Gilmore, Reid

    2013-01-01

    Summary Asparagine-linked glycosylation of proteins by the oligosaccharyltransferase (OST) occurs when acceptor sites or sequons (N-x≠P-T/S) on nascent polypeptides enter the lumen of the rough endoplasmic reticulum. Metazoan organisms assemble two isoforms of the OST that have different catalytic subunits (STT3A or STT3B) and partially non-overlapping cellular roles. Potential glycosylation sites move past the STT3A complex, which is associated with the translocation channel, at the protein synthesis elongation rate. Here, we investigated whether close spacing between acceptor sites in a nascent protein promotes site skipping by the STT3A complex. Biosynthetic analysis of four human glycoproteins revealed that closely spaced sites are efficiently glycosylated by an STT3B-independent process unless the sequons contain non-optimal sequence features, including extreme close spacing between sequons (e.g. NxTNxT) or the presence of paired NxS sequons (e.g. NxSANxS). Many, but not all, glycosylation sites that are skipped by the STT3A complex can be glycosylated by the STT3B complex. Analysis of a murine glycoprotein database revealed that closely spaced sequons are surprisingly common, and are enriched for paired NxT sites when the gap between sequons is less than three residues. PMID:24105266

  8. 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

  9. Characterization of pseudorabies virus glycoprotein B expressed by canine herpesvirus.

    PubMed

    Nishikawa, Y; Xuan, X; Kimura, M; Otsuka, H

    1999-10-01

    A recombinant canine herpesvirus (CHV) which expressed glycoprotein B (gB) of pseudorabies virus (PrV) was constructed. The antigenicity of the PrV gB expressed by the recombinant CHV is similar to that of the native PrV. The expressed PrV gB was shown to be transported to the surface of infected cells as judged by an indirected immunofluorescence test. Antibodies raised in mice immunized with the recombinant CHV neutralized the infectivity of PrV in vitro. It is known that the authentic PrV gB exists as a glycoprotein complex, which consists of gBa, gBb and gBc. In MDCK cells, PrV gB expressed by the recombinant CHV was processed like authentic PrV gB, suggesting that the cleavage mechanism of PrV gB depends on a functional cleavage domain from PrV gB gene and protease from infected cells. PMID:10563288

  10. Differential expression of salivary glycoproteins in aggressive and chronic periodontitis

    PubMed Central

    ROCHA, Daniela de Morais; ZENÓBIO, Elton Gonçalves; VAN DYKE, Thomas; SILVA, Karine Simões; COSTA, Fernando Oliveira; SOARES, Rodrigo Villamarim

    2012-01-01

    Objectives The aim of this study was to compare the pattern of secretion and the expression of mucin glycoprotein-2 (MG2) and lactoferrin in individuals with or without periodontitis. Material and Methods Five individuals with aggressive periodontitis (APG), 5 with generalized chronic periodontitis (CPG) and 5 without periodontitis (CG) were enrolled after informed consent. Non-stimulated and stimulated submandibular and sublingual saliva was collected and samples analyzed by Western blot probed with specific antibodies. Results Stimulated and non-stimulated salivary flow rates did not differ among groups. Western blot analysis revealed that stimulation led to: an increase in MG2 expression in all groups, and to lactoferrin expression in APG and CPG. In non-stimulated saliva, CG exhibited the highest expression of both glycoproteins. In stimulated saliva, CG exhibited the highest expression of MG2, whereas APG the highest of lactoferrin. Conclusions The pattern of secretion of MG2 and lactoferrin in health and disease is complex. Although the present study analyzed samples from a limited number of participants, the reduced expression of MG2 and lactoferrin in APG and CPG under non-stimulated condition, the predominant circumstance of salivary secretion during the day, suggests that these salivary constituents may play a role in the etiopathogenesis of these diseases. PMID:22666834

  11. Sequence polymorphism of the predicted human metapneumovirus G glycoprotein.

    PubMed

    Peret, Teresa C T; Abed, Yacine; Anderson, Larry J; Erdman, Dean D; Boivin, Guy

    2004-03-01

    The putative G glycoprotein genes of 25 human metapneumovirus (hMPV) field isolates obtained during five consecutive epidemic seasons (1997 to 2002) were sequenced. Sequence alignments identified two major genetic groups, designated groups 1 and 2, and two minor genetic clusters within each major group, designated subgroups A and B. Extensive nucleotide and deduced amino acid sequence variability was observed, consisting of high rates of nucleotide substitutions, use of alternative transcription-termination codons and insertions that retained the reading frame. Deduced amino acid sequences showed the greatest variability, with most differences located in the extracellular domain of the protein: nucleotide and amino acid sequence identities for the entire open reading frame ranged from 52 to 58 % and 31 to 35 %, respectively, between the two major groups. Like the closely related avian pneumovirus and human and bovine respiratory syncytial viruses, the predicted G protein of hMPV shared the basic features of a type II mucin-like glycosylated protein. However, differences from these related viruses were also observed, e.g. lack of conserved cysteine clusters as seen in human respiratory syncytial virus and avian pneumovirus. The displacement of genetic groups of hMPV observed during the study period suggests that potential antigenic differences in the G glycoprotein, which have evolved in response to immune-mediated pressure, may influence the circulation patterns of hMPV strains. PMID:14993653

  12. Inhibition of bacterial ice nucleators by fish antifreeze glycoproteins.

    PubMed

    Parody-Morreale, A; Murphy, K P; Di Cera, E; Fall, R; DeVries, A L; Gill, S J

    1988-06-23

    Certain bacteria promote the formation of ice in super-cooled water by means of ice nucleators which contain a unique protein associated with the cell membrane. Ice nucleators in general are believed to act by mimicking the structure of an ice crystal surface, thus imposing an ice-like arrangement on the water molecules in contact with the nucleating surface and lowering the energy necessary for the initiation of ice formation. Quantitative investigation of the bacterial ice-nucleating process has recently been made possible by the discovery of certain bacteria that shed stable membrane vesicles with ice nucleating activity. The opposite effect, inhibition of ice formation, has been described for a group of glycoproteins found in different fish and insect species. This group of substances, termed antifreeze glycoproteins (AFGPs), promotes the supercooling of water with no appreciable effect on the equilibrium freezing point or melting temperature. Substantial evidence now indicates that AFGPs act by binding to a growing ice crystal and slowing crystal growth. As the ice-nucleating protein surface is believed to have a structure similar to an embryonic ice crystal, AFGPs might be predicted to interact directly with a bacterial ice-nucleating site. We report here that AFGPs from the antarctic fish Dissostichus mawsoni inhibit the ice-nucleating activity of membrane vesicles from the bacterium Erwinia herbicola. The inhibition effect shows saturation at high concentration of AFGP and conforms to a simple binding reaction between the AFGP and the nucleation centre. PMID:3386720

  13. Cell wall proteome of Clostridium thermocellum and detection of glycoproteins.

    PubMed

    Yu, Tingting; Xu, Xinping; Peng, Yanfeng; Luo, Yuanming; Yang, Keqian

    2012-06-20

    Clostridium thermocellum, a thermophilic anaerobe, has the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. In this work, the cell wall proteome of C. thermocellum was investigated. The proteins in the cell wall fraction of C. thermocellum prepared by the boiling SDS method were released by mutanolysin digestion and resolved on two-dimensional (2D) gel. One hundred and thirty-two proteins were identified by mass spectrometry, among which the extracellular solute-binding protein (CbpB/cthe_1020), enolase, glyceraldehyde-3-phosphate dehydrogenase and translation elongation factor EF-Tu were detected as highly abundant proteins. Besides the known surface localized proteins, including FtsZ, MinD, GroEL, DnaK, many enzymes involved in bioenergetics, such as alcohol dehydrogenases and hydrogenases were also detected. By glycan stain and MS analysis of glycopeptides, we identified CbpB as a glycoprotein, which is the second glycoprotein from C. thermocellum characterized. The fact that CbpB was highly abundant in the cell wall region and glycosylated, reflects its importance in substrate assimilation. Our results indicate cell wall proteins constitute a significant portion of cellular proteins and may play important physiological roles (i.e. bioenergetics) in this bacterium. The insights described are relevant for the development of C. thermocellum as a biofuel producer. PMID:22494898

  14. Immunomodulatory roles of the carcinoembryonic antigen family of glycoproteins.

    PubMed

    Shao, Ling; Allez, Matthieu; Park, Mee-Sook; Mayer, Lloyd

    2006-08-01

    One of the most remarkable aspects of the immune system is its ability to fashion an immune response most appropriate to the activating stimulus. Although the immune system possesses a number of adaptations to accomplish this, an important theme is local immune regulation by site-specific expression of receptors and ligands. One family of molecules that is gaining attention as modulators of the immune system is the carcinoembryonic antigen cell-adhesion molecule family (CEACAM). Functionally, the carcinoembryonic antigen family can mediate cell-cell contact, host-pathogen interactions, and immune regulation. For example, biliary glycoprotein (CEACAM1) can have direct activity on T cells, leading to the inhibition of helper or cytotoxic T cell function. The expression of carcinoembryonic antigen (CEACAM5) on intestinal epithelial cells is involved in the activation of populations of regulatory CD8(+) T cells, while a distinct subset of regulatory CD8+ T cells is activated by nonspecific cross-reacting antigen (CEACAM6) on placental trophoblasts. Interestingly, the function and phenotype of these cells depend upon the specific member of the carcinoembryonic antigen family expressed, as well as the antigen-presenting molecule with which it associates. Thus, these glycoproteins comprise a family of molecules whose functions can depend on their nature and context. PMID:17057200

  15. 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

  16. [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. PMID:23233923

  17. A facile and general approach for preparation of glycoprotein-imprinted magnetic nanoparticles with synergistic selectivity.

    PubMed

    Hao, Yi; Gao, Ruixia; Liu, Dechun; He, Gaiyan; Tang, Yuhai; Guo, Zengjun

    2016-06-01

    In light of the significance of glycoprotein biomarkers for early clinical diagnostics and treatments of diseases, it is essential to develop efficient and selective enrichment platforms for glycoproteins. In this study, we present a facile and general strategy to prepare the boronate affinity-based magnetic imprinted nanoparticles. Boronic acid ligands were first grafted on the directly aldehyde-functionalized magnetic nanoparticles through amidation reaction. Then, template glycoproteins were immobilized on the boronic acid-modified magnetic nanoparticles via boronate affinity binding. Subsequently, a thin layer of dopamine was formed to coat the surface of magnetic nanoparticles through self-polymerization. After the template glycoproteins were removed, the cavities that can specific bind the template glycoproteins were fabricated. Adopting horseradish peroxidase as model template, the effects of imprinting conditions as well as the properties and performance of the obtained products were investigated. The resultant imprinted materials exhibit highly favorable features, including uniform surface morphology with thin imprinted shell of about 8nm, super-paramagnetic property, fast kinetics of 40min, high adsorption capacity of 60.3mgg(-1), and satisfactory reusability for at least five cycles of adsorption-desorption without obvious deterioration. Meanwhile, the obtained magnetic imprinted nanoparticles could capture target glycoprotein from nonglycoproteins, but also from other glycoproteins because the synergistic selectivity of boronate affinity and imprinting effect. In addition, the facile preparation method shows feasibility in the imprinting of different glycoproteins. PMID:27130111

  18. 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.

  19. 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…

  20. Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins.

    PubMed Central

    Salzwedel, K; Johnston, P B; Roberts, S J; Dubay, J W; Hunter, E

    1993-01-01

    Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome. Images PMID:8102410

  1. [Electrophoretic analysis of urinary glycoproteins in diabetic nephropathy using peroxidase-lectins].

    PubMed

    Nakashima, Y; Dohi, K; Dohi, Y; Nishiura, K; Kanauchi, M; Ishii, K; Kawano, T; Takenaka, M; Sugimoto, K; Moriyama, T

    1989-11-01

    We examined the clinical usefulness determined by polyacrylamide gel electrophoresis, followed by reaction with peroxidase-coupled lectins using urinary glycoproteins for diabetic nephropathy in 20 patients with diabetes mellitus. Lectins used were Triticum vulgaris (WGA), Phaseolus vulgaris (PHA-E4), Dolichos biflorus (DBA), and Lens culinaris (LCA), which have high affinity for beta 1----4N-acetyl-D-glucosamine (GlcNAc beta 1----4GlcNAc), N-acetyl-D-galactosamine (GalNAc), alpha-galactosamine (alpha-GalNAc), and alpha-mannose (alpha-Man) residues, respectively. Electrophoretic patterns of urinary glycoproteins clearly showed the presence of lectin-reactive glycoproteins with molecular weights lower than that of albumin. The molecular weight of the main bands reacted with WGA, PHA-E4 or LCA were 50,000 and 38,000, and increased with the progress of diabetic nephropathy. WGA reacted strongly with many glycoproteins having a wide range of molecular weights. LCA and PHA-E4 reacted preferentially with glycoproteins of molecular weights glycoproteins of molecular weights lower than 50,000, but no reaction was observed by DBA. These results suggest that low molecular urinary glycoproteins have abundant carbohydrate residues such as GlcNAc beta 1----4GlcNAc, GalNAc, and alpha-Man. The excretion of low molecular weight glycoproteins with high affinities for some lectins suggests functional impairment in diabetic nephropathy. PMID:2483179

  2. 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... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of...

  3. 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... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in...

  4. 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. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of...

  5. 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. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of...

  6. 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... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of...

  7. 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... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in...

  8. 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. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of...

  9. 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... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in...

  10. 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... electrophoresis) in serum and other body fluids. Measurement of specific alpha-1-glycoproteins may aid in...

  11. 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... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of...

  12. 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. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the beta-2-glycoprotein I (a serum protein) in serum and other body fluids. Measurement of...

  13. 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... the beta-2-glycoprotein III (a serum protein) in serum and other body fluids. Measurement of...

  14. 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

  15. 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.

  16. 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

  17. Mucus glycoprotein secretion by duodenal mucosa in response to luminal arachidonic acid.

    PubMed

    Kosmala, M; Carter, S R; Konturek, S J; Slomiany, A; Slomiany, B L

    1986-12-10

    The effect of luminal application of arachidonic acid on the alkaline secretion, prostaglandin generation, and mucus glycoprotein output and composition was studied in proximal and distal duodenum of conscious dogs. Surgically prepared duodenal loops were instilled in vivo for up to 2 h with saline (control) followed by various concentrations (12.5-100 micrograms/ml) of arachidonic acid. The experiments were conducted with and without intravenous pretreatment with indomethacin. The recovered instillates were assayed for the content of prostaglandin and HCO3-, and used for the isolation of mucus glycoprotein. Exposure of duodenal mucosa to arachidonic acid led to concentration-dependent increase in the output of HCO3- and prostaglandin generation. In both cases this response was greater in the proximal duodenum. Pretreatment with indomethacin caused reduction in the basal HCO3- and prostaglandin output, and prevented the increments evoked by arachidonic acid. The proximal and distal duodenum displayed similar basal output and composition of mucus glycoprotein. Comparable increases in these glycoproteins were also obtained with arachidonic acid, the effect of which was abolished by indomethacin. Compared to basal conditions, mucus glycoproteins elaborated in response to arachidonic acid exhibited higher contents of associated lipids and covalently bound fatty acids, and contained less protein. The associated lipids of mucus glycoproteins elaborated in the presence of arachidonic acid showed enrichment in phospholipids and decrease in neutral lipids. The carbohydrate components in these glycoproteins also exhibited higher proportions of sialic acid and sulfate. The changes brought about by arachidonic acid were prevented by indomethacin pretreatment, and in both cases the glycoprotein composition returned to that obtained under basal conditions. The enrichment of mucus glycoprotein in lipids, sialic acid and sulfate in response to endogenous prostaglandin may be of

  18. 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

  19. The future of glycoprotein VI as an antithrombotic target.

    PubMed

    Zahid, M; Mangin, P; Loyau, S; Hechler, B; Billiald, P; Gachet, C; Jandrot-Perrus, M

    2012-12-01

    The treatment of acute coronary syndromes has been considerably improved in recent years with the introduction of highly efficient antiplatelet drugs. However, there are still significant limitations: the recurrence of adverse vascular events remains a problem, and the improvement in efficacy is counterbalanced by an increased risk of bleeding, which is of particular importance in patients at risk of stroke. One of the most attractive targets for the development of new molecules with potential antithrombotic activity is platelet glycoprotein (GP)VI, because its blockade appears to ideally combine efficacy and safety. This review summarizes current knowledge on GPVI regarding its structure, its function, and its role in physiologic hemostasis and thrombosis. Strategies for inhibiting GPVI are presented, and evidence of the antithrombotic efficacy and safety of GPVI antagonists is provided. PMID:23020554

  20. 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.

  1. Raccoon poxvirus rabies virus glycoprotein recombinant vaccine in sheep.

    PubMed

    DeMartini, J C; Bickle, H M; Brodie, S J; He, B X; Esposito, J J

    1993-01-01

    Twenty sheep were divided into groups and inoculated by various routes with recombinant raccoon poxvirus expressing the CVS rabies virus glycoprotein (rRCNV-G) or with raccoon poxvirus (RCNV). The apparent innocuous pathologic responses to each virus coupled with development of high levels of rabies virus neutralizing antibodies in animals vaccinated with rRCNV-G intradermally or intramuscularly suggested that the recombinant is effective and that RCNV would be a suitable substrate for further development of sheep vaccines. Poor antibody response to rRCNV-G given orally implied that it would be relatively harmless if inadvertently ingested by sheep. Virus transmission between vaccinated and sentinel sheep was not observed or detected serologically. PMID:8240013

  2. 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

  3. Modulation of P-glycoprotein ATPase activity by some phytoconstituents.

    PubMed

    Najar, I A; Sachin, B S; Sharma, S C; Satti, N K; Suri, K A; Johri, R K

    2010-03-01

    In the present investigation 16 phytoconstituents, which are active moieties found in several medicinal herbs, have been evaluated for their P-glycoprotein (P-gp) stimulation/inhibition profiles using a P-gp-dependent ATPase assay in rat jejunal membrane (in vitro). Acteoside, agnuside, catechin, chlorogenic acid, picroside -II and santonin showed an inhibitory effect. Negundoside, picroside -I and oleanolic acid caused a stimulatory effect. Andrographolide, apocyanin, berberine, glycyrrhizin, magniferin and piperine produced a biphasic response (stimulation at low concentration and inhibition at high concentration). The results suggested that a possible interaction of these phytoconstituents at the level of P-gp, could be an important parameter in determining their role in several key pharmacodynamic events. PMID:19653312

  4. Glycoprotein IIb/IIIa inhibitors: The resurgence of tirofiban.

    PubMed

    King, Shawn; Short, Marintha; Harmon, Cassidy

    2016-03-01

    Glycoprotein (GP) IIb/IIIa inhibitors block platelet aggregation, reducing thrombotic events in acute coronary syndrome. They are most often utilized in patients who likely have an intracoronary thrombus. Tirofiban, eptifibatide, and abciximab are the three GP IIb/IIIa inhibitors approved for use in the United States. Each agent has unique pharmacological properties. They all have a rapid onset and are most often utilized in conjunction with heparin. Tirofiban, in particular, fell out of favor due to inferior dosing with its original Food and Drug Administration (FDA) approved indication, but has re-emerged in the market with a high-dose bolus regimen that is considered equally as effective as the FDA approved dosing regimens of other GP IIb/IIIa inhibitors. This review looks at pharmacological properties of all three agents, significant clinical trials associated with their use, and their place in current guidelines. PMID:26187354

  5. 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.

  6. Specificity analysis of lectins and antibodies using remodeled glycoproteins.

    PubMed

    Iskratsch, Thomas; Braun, Andreas; Paschinger, Katharina; Wilson, Iain B H

    2009-03-15

    Due to their ability to bind specifically to certain carbohydrate sequences, lectins are a frequently used tool in cytology, histology, and glycan analysis but also offer new options for drug targeting and drug delivery systems. For these and other potential applications, it is necessary to be certain as to the carbohydrate structures interacting with the lectin. Therefore, we used glycoproteins remodeled with glycosyltransferases and glycosidases for testing specificities of lectins from Aleuria aurantia (AAL), Erythrina cristagalli (ECL), Griffonia simplicifolia (GSL I-B(4)), Helix pomatia agglutinin (HPA), Lens culinaris (LCA), Lotus tetragonolobus (LTA), peanut (Arachis hypogaeae) (PNA), Ricinus communis (RCA I), Sambucus nigra (SNA), Vicia villosa (VVA), and wheat germ (Triticum vulgaris) (WGA) as well as reactivities of anti-carbohydrate antibodies (anti-bee venom, anti-horseradish peroxidase [anti-HRP], and anti-Lewis(x)). After enzymatic remodeling, the resulting neoglycoforms display defined carbohydrate sequences and can be used, when spotted on nitrocellulose or in enzyme-linked lectinosorbent assays, to identify the sugar moieties bound by the lectins. Transferrin with its two biantennary complex N-glycans was used as scaffold for gaining diverse N-glycosidic structures, whereas fetuin was modified using glycosidases to test the specificities of lectins toward both N- and O-glycans. In addition, alpha(1)-acid glycoprotein and Schistosoma mansoni egg extract were chosen as controls for lectin interactions with fucosylated glycans (Lewis(x) and core alpha1,3-fucose). Our data complement and expand the existing knowledge about the binding specificity of a range of commercially available lectins. PMID:19123999

  7. 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

  8. A comparative study of the major glycoprotein isolated from normal and neoplastic gastric mucosa.

    PubMed

    Schrager, J; Oates, M D

    1973-04-01

    The isolation and composition of glycoproteins from mucosae of normal stomachs, of stomachs with gastric ulcer, and of stomachs with carcinoma is described. The glycoproteins from the mucosae of normal stomachs and with gastric ulcer showed virtually the same carbohydrate and amino acid content as the principal gastric glycoprotein isolated from gastric aspirates. They all revealed a common basic carbohydrate composition: galactose, fucose, glucosamine, and galactosamine were present in approximate molar ratios of 4:3:3:1. THE RESULTS SUGGEST THAT THE GLYCOPROTEINS ISOLATED FROM GASTRIC ASPIRATES FROM NORMAL AND NEOPLASTIC GASTRIC MUCOSAE SHARE A NUMBER OF STRUCTURAL FEATURES: (1) a protein core with a characteristic amino acid composition; (2) the range of sugars forming the carbohydrate side chains; (3) galactosamine approximately equimolar with the sum of threonine and serine; (4) galactose approximately equimolar with the sum of glucosamine and galactosamine; (5) absence of mannose; (6) a high carbohydrate content (80-85%); and (7) blood group activity. The neoplastic glycoproteins differed from the normal glycoproteins in that the quantitative relationships of the carbohydrate components of the neoplastic glycoproteins showed variations dividing the extracts investigated into groups, each group with a distinctive and constant carbohydrate composition. The blood group specificity of 15 out of 24 cases investigated differed from that of the hosts' red cells. PMID:4706916

  9. The lectin riddle: glycoproteins fractionated from complex mixtures have similar glycomic profiles.

    PubMed

    Lee, Albert; Nakano, Miyako; Hincapie, Marina; Kolarich, Daniel; Baker, Mark S; Hancock, William S; Packer, Nicolle H

    2010-08-01

    One common method used for analyzing the glycoproteome is chromatography using multiple lectins that display different affinities toward oligosaccharide structures. Much has been done to determine lectin affinity using standard glycoproteins with known glycosylation; however, a knowledge of the selectivity and specificity of lectins exposed to complex mixtures of proteins is required if they are to be used as a means of studying the glycoproteome. In the present study, three lectins (Concanavalin A, Jacalin, and Wheat Germ Agglutinin) were used to fractionate glycoproteins from two different complex environments: (1) cell membranes and (2) plasma. Reproducible enrichment of glycoproteins from these samples has been shown to result from the combined use of these lectins. However, the global glycan profiles of the released N- and O-linked oligosaccharides from the glycoproteins retained by the lectins, and from those glycoproteins that did not bind, using both these complex samples, were found to be very similar. That is, although the lectins selectively and reproducibly retained some glycoproteins, other proteins with the same attached oligosaccharide structures did not bind. Some small N- and O-glycan differences were observed in the bound fractions but there was little absolute specificity toward individual oligosaccharide structures known to have high affinity to these lectins. These data indicate that lectins are useful for fractionating glycoproteins from complex mixtures, but that the overall glycoproteome is not isolated by this approach. PMID:20726804

  10. 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. PMID:26743719

  11. 1-Cinnamoyl-3,11-dihydroxymeliacarpin delays glycoprotein transport restraining virus multiplication without cytotoxicity.

    PubMed

    Bueno, Carlos A; Alché, Laura E; Barquero, Andrea A

    2010-02-26

    The 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), isolated from extracts of Melia azedarach L., displays antiviral and immunomodulating properties. CDM is the first reported tetranortriterpenoid responsible for the alkalinization of intracellular compartments affecting both, viral endocytic and exocytic pathways. Considering that viral glycoprotein synthesis is completely dependent upon cellular membrane trafficking, we questioned whether CDM might also interfere with the normal transport of cellular glycoproteins. This study demonstrates that CDM promoted a transient block in the transport of two cellular glycoproteins, the transferrin receptor (TfR) and TNF-alpha. Nevertheless, CDM did not affect the transferrin binding ability of TfR and did not impede the TNF-alpha secretion. On the other hand, CDM disturbed the intracellular localization of capsid, glycoprotein and tegument proteins simultaneously in the same HSV-1 infected cells. Besides, we show that concanamycin A and monensin provoke a permanent blockage of viral and cellular glycoproteins, in contrast to the delay observed after CDM treatment. Thus, the delay on glycoprotein transport caused by CDM would account for the strong inhibition on virus multiplication without interfering with the bioactivity of cellular glycoproteins. PMID:20097166

  12. 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. PMID:26066908

  13. Purification of a Trypanosoma cruzi trypomastigote 60-kilodalton surface glycoprotein that primes and activates murine lymphocytes.

    PubMed Central

    Villalta, F; Lima, M F; Howard, S A; Zhou, L; Ruiz-Ruano, A

    1992-01-01

    We have purified a glycoprotein with a relative molecular mass of 60 kDa and present on the surface of Trypanosoma cruzi trypomastigotes and studied its ability to prime and stimulate the proliferation of murine spleen cells. T. cruzi trypomastigote membrane proteins were separated by preparative isoelectrofocusing. A trypomastigote 60-kDa surface protein with an isoelectric point of 4.2 was enriched by chromatofocusing and was readily purified in native form to homogeneity by gel filtration on a Superose column by use of a fast protein liquid chromatography system. Biotinylated wheat germ agglutinin, Ricinus communis agglutinin, and Datura stramonium agglutinin bound to blots containing the purified trypomastigote 60-kDa surface protein, indicating that this protein was glycosylated. The purified trypomastigote 60-kDa glycoprotein was recognized by antibodies produced during human infection, and immunoglobulin G against the purified glycoprotein immunoprecipitated a biotinylated 60-kDa molecule from the surface of trypomastigotes but not epimastigotes. Specific immunoglobulin G against the 60-kDa glycoprotein also increased the uptake of trypomastigotes and promoted parasite killing by macrophages. The purified 60-kDa glycoprotein was able to specifically activate primed lymphocytes, since there was a significant increase in [3H]thymidine incorporation by spleen cells obtained from CBA mice primed with this glycoprotein, with respect to control values. Furthermore, the 60-kDa glycoprotein did not stimulate unprimed spleen cells, indicating that the lymphoproliferation induced by this glycoprotein was specific and was not due to polyclonal activation. Our findings indicate that this T. cruzi trypomastigote 60-kDa surface glycoprotein primes and activates lymphocytes, which could lead to a beneficial immune response in the host. Images PMID:1639469

  14. Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain.

    PubMed Central

    Quarles, R H

    1976-01-01

    Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content. PMID:942396

  15. 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

  16. 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.

  17. 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....

  18. 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....

  19. 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....

  20. 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....

  1. 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

  2. Glucocorticoid-Dependent Complementation of a Hepatoma Cell Variant Defective in Viral Glycoprotein Sorting

    NASA Astrophysics Data System (ADS)

    John, Nancy J.; Bravo, Deborah A.; Haffar, Omar K.; Firestone, Gary L.

    1988-02-01

    We have utilized the rat hepatoma (HTC) cell sorting variant CR4 to examine the glucocorticoid-regulated pathways that localize mouse mammary tumor virus glycoproteins to the cell surface. The defective sorting of cell surface mouse mammary tumor virus glycoproteins in CR4 cells was complemented after fusion with either normal rat hepatocytes or uninfected HTC cells. Indirect immunofluorescence of transient heterokaryons revealed that the regulated localization of mouse mammary tumor virus glycoproteins was dependent upon glucocorticoid treatment and required de novo RNA and protein synthesis. Thus, a glucocorticoid-regulated trafficking activity, unrelated to mouse mammary tumor virus sequences, which is induced in both adult rat liver and cultured hepatoma cells, can act in trans to mediate an intracellular sorting pathway for membrane glycoproteins.

  3. 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

  4. 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....

  5. The three-dimensional structure of the cell wall glycoprotein of Chlorogonium elongatum.

    PubMed

    Shaw, P J; Hills, G J

    1984-06-01

    The green alga Chlorogonium elongatum, a member of the Volvocales, possesses a crystalline cell wall composed of hydroxyproline-rich glycoprotein similar to the primary cell wall glycoproteins of higher plants. Electron microscopy and computer image processing have been used to determine the crystal structure of the Chlorogonium cell wall in three dimensions to a resolution of 2.0 nm. The structure is composed of heterologous dimers. Each subunit of the dimer comprises a long, thin spacer domain and a large globular domain, which is the site of the intra- and inter-dimer interactions. There are also sites of intersubunit interactions at the opposite ends of the rod domains. We suggest that the rods are composed predominantly of glycosylated polyproline helix, as has been suggested for higher plant cell wall glycoproteins and has been shown for the cell wall glycoprotein of Chlamydomonas reinhardtii, which is closely related to Chlorogonium. PMID:6490737

  6. Resolution of two surface glycoproteins from human parainfluenza-3 virus by crossed immunoelectrophoresis.

    PubMed

    Holling, R A; Guskey, L E

    1984-07-01

    The technique of two-dimensional crossed immunoelectrophoresis (CIE) was used to resolve two glycoproteins from purified human parainfluenza type 3 virus. Virus preparations were extracted with Triton X-100 and fractionated by centrifugation in a Beckman airfuge. Two immunoprecipitates were detected by CIE in the supernatant fractions, but were not found in the pellets from extracted virus. Viral glycoproteins labeled with [35S]methionine were isolated by affinity chromatography on concanavalin A (Con A) agarose columns, resolved by CIE and detected by autoradiography. Resolution of two glycoprotein peaks from as little as 4.5 micrograms of protein from extracted virus is consistent with results from polyacrylamide gel patterns showing two unique glycoproteins with molecular weights of 48 kd and 65 kd. PMID:6088566

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Rabies virus (RV) glycoprotein expression levels are not critical for pathogenicity of RV.

    PubMed

    Wirblich, Christoph; Schnell, Matthias J

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain. PMID:21068252

  12. Rabies Virus (RV) Glycoprotein Expression Levels Are Not Critical for Pathogenicity of RV▿

    PubMed Central

    Wirblich, Christoph; Schnell, Matthias J.

    2011-01-01

    Previous comparisons of different rabies virus (RV) strains suggested an inverse relationship between pathogenicity and the amount of glycoprotein produced in infected cells. In order to provide more insight into this relationship, we pursued an experimental approach that allowed us to alter the glycoprotein expression level without altering the glycoprotein sequence, thereby eliminating the contribution of amino acid changes to differences in viral virulence. To this end, we constructed an infectious clone of the highly pathogenic rabies virus strain CVS-N2c and replaced its cognate glycoprotein gene with synthetic versions in which silent mutations were introduced to replace wild-type codons with the most or least frequently used synonymous codons. A recombinant N2c variant containing the fully codon-optimized G gene and three variants carrying a partially codon-deoptimized G gene were recovered on mouse neuroblastoma cells and shown to express 2- to 3-fold more and less glycoprotein, respectively, than wild-type N2c. Pathogenicity studies in mice revealed the WT-N2c virus to be the most pathogenic strain. Variants containing partially codon-deoptimized glycoprotein genes or the codon-optimized gene were less pathogenic than WT-N2c but still caused significant mortality. We conclude that the expression level of the glycoprotein gene does have an impact on pathogenicity but is not a dominant factor that determines pathogenicity. Thus, strategies such as changes in codon usage that aim solely at altering the expression level of the glycoprotein gene do not suffice to render a pathogenic rabies virus apathogenic and are not a viable and safe approach for attenuation of a pathogenic strain. PMID:21068252

  13. Modulation of heparin cofactor II activity by histidine-rich glycoprotein and platelet factor 4.

    PubMed Central

    Tollefsen, D M; Pestka, C A

    1985-01-01

    Heparin cofactor II is a plasma protein that inhibits thrombin rapidly in the presence of either heparin or dermatan sulfate. We have determined the effects of two glycosaminoglycan-binding proteins, i.e., histidine-rich glycoprotein and platelet factor 4, on these reactions. Inhibition of thrombin by heparin cofactor II and heparin was completely prevented by purified histidine-rich glycoprotein at the ratio of 13 micrograms histidine-rich glycoprotein/microgram heparin. In contrast, histidine-rich glycoprotein had no effect on inhibition of thrombin by heparin cofactor II and dermatan sulfate at ratios of less than or equal to 128 micrograms histidine-rich glycoprotein/microgram dermatan sulfate. Removal of 85-90% of the histidine-rich glycoprotein from plasma resulted in a fourfold reduction in the amount of heparin required to prolong the thrombin clotting time from 14 s to greater than 180 s but had no effect on the amount of dermatan sulfate required for similar anti-coagulant activity. In contrast to histidine-rich glycoprotein, purified platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of either heparin or dermatan sulfate at the ratio of 2 micrograms platelet factor 4/micrograms glycosaminoglycan. Furthermore, the supernatant medium from platelets treated with arachidonic acid to cause secretion of platelet factor 4 prevented inhibition of thrombin by heparin cofactor II in the presence of heparin or dermatan sulfate. We conclude that histidine-rich glycoprotein and platelet factor 4 can regulate the antithrombin activity of heparin cofactor II. Images PMID:3838317

  14. 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

  15. 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

  16. A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation

    PubMed Central

    Schwarz, Flavio; Huang, Wei; Li, Cishan; Schulz, Benjamin L.; Lizak, Christian; Palumbo, Alessandro; Numao, Shin; Neri, Dario; Aebi, Markus; Wang, Lai-Xi

    2010-01-01

    We describe a novel method for producing homogeneous eukaryotic N-glycoproteins. The method involves the engineering and functional transfer of the C. jejuni glycosylation machinery in E. coli to express glycosylated proteins with the key GlcNAc-Asn linkage. The bacterial glycans were then trimmed and remodeled in vitro by enzymatic transglycosylation to fulfill a eukaryotic N-glycosylation. It provides a potentially general platform for producing eukaryotic N-glycoproteins. PMID:20190762

  17. 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

  18. Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

    PubMed Central

    Melchior, Donald L.; Sharom, Frances J.; Evers, Raymond; Wright, George E.; Chu, Joseph W.K.; Wright, Stephen E.; Chu, Xiaoyan; Yabut, Jocelyn

    2012-01-01

    Introduction P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Methods Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Results Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2 = 0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans. Discussion This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 minutes, and requires minimal quantities of test

  19. 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.

    1986-01-01

    The humoral immune responses 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. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with (/sup 35/S) methinonine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by sera of infected mice. In contrast, sera of vaccinated mice uniquely precipitated a 38,000 m.w. glycoprotein of schistosomula cultured for 5 days and a 94,000 m.w. glycoprotein of adult male worms. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. In particular, an egg glycoprotein of 85,000 to 70,000 and isoelectric point of 4.8 showed an enhanced reactivity with sera of vaccinated mice in comparison with infected mice. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.

  20. Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

    PubMed Central

    Marr, Anne M. S.; Neuberger, A.; Ratcliffe, Wendy A.

    1971-01-01

    1. Tamm–Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm–Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000±6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm–Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm–Horsfall antiserum. ImagesFig. 2.Fig. 4.Fig. 5. PMID:5129252

  1. 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. PMID:22975980

  2. 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

  3. Exploiting bacterial glycosylation machineries for the synthesis of a Lewis antigen-containing glycoprotein.

    PubMed

    Hug, Isabelle; Zheng, Blake; Reiz, Bela; Whittal, Randy M; Fentabil, Messele A; Klassen, John S; Feldman, Mario F

    2011-10-28

    Glycoproteins constitute a class of compounds of increasing importance for pharmaceutical applications. The manipulation of bacterial protein glycosylation systems from Gram-negative bacteria for the synthesis of recombinant glycoproteins is a promising alternative to the current production methods. Proteins carrying Lewis antigens have been shown to have potential applications for the treatment of diverse autoimmune diseases. In this work, we developed a mixed approach consisting of in vivo and in vitro steps for the synthesis of glycoproteins containing the Lewis x antigen. Using glycosyltransferases from Haemophilus influenzae, we engineered Escherichia coli to assemble a tetrasaccharide on the lipid carrier undecaprenylphosphate. This glycan was transferred in vivo from the lipid to a carrier protein by the Campylobacter jejuni oligosaccharyltransferase PglB. The glycoprotein was then fucosylated in vitro by a truncated fucosyltransferase from Helicobacter pylori. Diverse mass spectrometry techniques were used to confirm the structure of the glycan. The strategy presented here could be adapted in the future for the synthesis of diverse glycoproteins. Our experiments demonstrate that bacterial enzymes can be exploited for the production of glycoproteins carrying glycans present in human cells for potential therapeutic applications. PMID:21878645

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. P-glycoprotein ABCB1: a major player in drug handling by mammals.

    PubMed

    Borst, Piet; Schinkel, Alfred H

    2013-10-01

    Mammalian P-glycoproteins are active drug efflux transporters located in the plasma membrane. In the early nineties, we generated knockouts of the three P-glycoprotein genes of mice, the Mdr1a, Mdr1b, and Mdr2 P-glycoproteins, now known as Abcb1a, Abcb1b, and Abcb4, respectively. In the JCI papers that are the subject of this Hindsight, we showed that loss of Mdr1a (Abcb1a) had a profound effect on the tissue distribution and especially the brain accumulation of a range of drugs frequently used in humans, including dexamethasone, digoxin, cyclosporin A, ondansetron, domperidone, and loperamide. All drugs were shown to be excellent substrates of the murine ABCB1A P-glycoprotein and its human counterpart, the MDR1 P-glycoprotein, ABCB1. We found that the ability of ABCB1 to prevent accumulation of some drugs in the brain is a prerequisite for their clinical use, as absence of the transporter led to severe toxicity or undesired CNS pharmacodynamic effects. Subsequent work has fully confirmed the profound effect of the drug-transporting ABCB1 P-glycoprotein on the pharmacokinetics of drugs in humans. In fact, every new drug is now screened for transport by ABCB1, as this limits oral availability and penetration into sanctuaries protected by ABCB1, such as the brain. PMID:24084745

  9. 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

  10. Distinct P-glycoprotein precursors are overproduced in independently isolated drug-resistant cell lines.

    PubMed

    Greenberger, L M; Lothstein, L; Williams, S S; Horwitz, S B

    1988-06-01

    A family of P-glycoproteins are overproduced in multidrug-resistant cells derived from the murine macrophage-like line J774.2. To determine whether individual family members are overproduced in response to different drugs, the P-glycoprotein precursors in several independently isolated cell lines, which were selected for resistance to vinblastine or taxol, were compared. Individual cell lines selected with vinblastine overproduced P-glycoprotein precursors of either 120 or 125 kDa. Taxol-selected cell lines overproduced either the 125-kDa precursor or both precursors simultaneously. Two similar but distinct peptide maps for the mature P-glycoproteins were observed. These maps corresponded to each precursor regardless of the drug used for selection. One vinblastine-resistant cell line switched from the 125- to the 120-kDa precursor when grown in increasing concentrations of drug. This change coincided with the overexpression of a distinct subset of mRNA species that code for P-glycoprotein. It is concluded that precursor expression is not drug-specific. These data suggest that individual overproduced P-glycoprotein family members are translated as distinct polypeptides. The results may help to explain the diversity in the multidrug-resistant phenotype. PMID:2897689

  11. Reaction Dynamics of ATP Hydrolysis Catalyzed by P-Glycoprotein

    PubMed Central

    2015-01-01

    P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug–drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ18O4-labeled ATP, no positional isotope exchange is detectable at the bridging β-phosphorus–O−γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three 18O/two 18O/one 18O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO42– (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states. PMID:24506763

  12. Predicting Binding to P-Glycoprotein by Flexible Receptor Docking

    PubMed Central

    Dolghih, Elena; Bryant, Clifford; Renslo, Adam R.; Jacobson, Matthew P.

    2011-01-01

    P-glycoprotein (P-gp) is an ATP-dependent transport protein that is selectively expressed at entry points of xenobiotics where, acting as an efflux pump, it prevents their entering sensitive organs. The protein also plays a key role in the absorption and blood-brain barrier penetration of many drugs, while its overexpression in cancer cells has been linked to multidrug resistance in tumors. The recent publication of the mouse P-gp crystal structure revealed a large and hydrophobic binding cavity with no clearly defined sub-sites that supports an “induced-fit” ligand binding model. We employed flexible receptor docking to develop a new prediction algorithm for P-gp binding specificity. We tested the ability of this method to differentiate between binders and nonbinders of P-gp using consistently measured experimental data from P-gp efflux and calcein-inhibition assays. We also subjected the model to a blind test on a series of peptidic cysteine protease inhibitors, confirming the ability to predict compounds more likely to be P-gp substrates. Finally, we used the method to predict cellular metabolites that may be P-gp substrates. Overall, our results suggest that many P-gp substrates bind deeper in the cavity than the cyclic peptide in the crystal structure and that specificity in P-gp is better understood in terms of physicochemical properties of the ligands (and the binding site), rather than being defined by specific sub-sites. PMID:21731480

  13. Organization of the gene for platelet glycoprotein IIb

    SciTech Connect

    Heidenreich, R. ); Eisman, R.; Surrey, S.; Delgrosso, K.; Schwartz, E.; Poncz, M. Univ. of Pennsylvania, Philadelphia ); Bennett, J.S. Univ. of Pennsylvania, Philadelphia )

    1990-02-06

    The glycoprotein (GP) IIb/IIIa heterodimer functions as a receptor for fibrinogen, von Willebrand factor, and fibronectin on activated platelets; it is dysfunctional in the bleeding diathesis Glanzmann's thrombasthenia. This receptor is a member of the integrin family, which includes homologous membrane receptors involved in a number of different cell-cell and cell-matrix adhesive interactions. Knowledge of the sequence and organization of the GPIIb and GPIIIa genes will help in understanding evolutionary relationships and functional homologies of this family of adhesion protein receptors and will facilitate analysis of molecular defects responsible for thrombasthenia. Using the GPIIb cDNA as a probe, the authors have isolated overlapping genomic clones encompassing the entire coding region, the 5{prime}- and 3{prime}-untranslated sequences, and the immediate flanking regions for the GPIIb gene. The gene spans approximately 17.2 kilobases (kb); all but approximately 2.6 kb of intronic DNA sequence has been determined. The GPIIb gene contains 30 exons whose demarcations do not correlate with previously suggested functional domains. Two intron/exon borders have the rare GC splice donor sequence instead of the consensus GT sequence. There are at least seven complete and three partial AluI sequence repeats within the intron sequences. The immediate 5{prime}-flanking sequence of rodent GPIIb demonstrates complete identity near the proposed cap site with its human counterpart, but again, no TATA or CAAT boxes are apparent.

  14. Hyposialylated α1-acid glycoprotein inhibits phagocytosis of feline neutrophils.

    PubMed

    Rossi, G; Capitani, L; Ceciliani, F; Restelli, L; Paltrinieri, S

    2013-10-01

    Feline α1-acid glycoprotein (fAGP) modifies both its serum concentration and its glycan moiety during diseases. fAGP is hyposialylated in cats with feline infectious peritonitis (FIP), but not in clinically healthy cats or in cats with other diseases. This study was aimed to determine whether hyposialylated fAGP influences phagocytosis. A flow cytometric method based on ingestion of fluoresceinated bacteria and adapted to feline blood was used to assess phagocytosis of leukocytes incubated with 'non-pathological' fAGP (purified from sera with normal concentrations of AGP) and 'pathological' fAGP (purified from sera with >1.5mg/mL hyposialylated AGP). The flow cytometric method provided repeatable results for neutrophils (coefficients of variations, CVs <15%) but not for monocytes (CVs>20%) which had also a high individual variability. Compared with saline solution and with non-pathological fAGP, pathological fAGP significantly decreased phagocytosis in neutrophils and monocytes. This study demonstrated that hyposialylated fAGP down-regulates the phagocytic activity of feline neutrophils. PMID:23726663

  15. 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

  16. P-Glycoprotein and Drug Resistance in Systemic Autoimmune Diseases

    PubMed Central

    Picchianti-Diamanti, Andrea; Rosado, Maria Manuela; Scarsella, Marco; Laganà, Bruno; D’Amelio, Raffaele

    2014-01-01

    Autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are chronic inflammatory disorders of unknown etiology characterized by a wide range of abnormalities of the immune system that may compromise the function of several organs, such as kidney, heart, joints, brain and skin. Corticosteroids (CCS), synthetic and biologic immunosuppressive agents have demonstrated the capacity to improve the course of autoimmune diseases. However, a significant number of patients do not respond or develop resistance to these therapies over time. P-glycoprotein (P-gp) is a transmembrane protein that pumps several drugs out of the cell, including CCS and immunosuppressants; thus, its over-expression or hyper-function has been proposed as a possible mechanism of drug resistance in patients with autoimmune disorders. Recently, different authors have demonstrated that P-gp inhibitors, such as cyclosporine A (CsA) and its analogue Tacrolimus, are able to reduce P-gp expression and or function in SLE, RA and PsA patients. These observations suggest that P-gp antagonists could be adopted to revert drug resistance and improve disease outcome. The complex inter-relationship among drug resistance, P-gp expression and autoimmunity still remains elusive. PMID:24658440

  17. 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

  18. Platelet glycoprotein Ibα supports experimental lung metastasis

    PubMed Central

    Jain, Shashank; Zuka, Masahiko; Liu, Jungling; Russell, Susan; Dent, Judith; Guerrero, José A.; Forsyth, Jane; Maruszak, Brigid; Gartner, T. Kent; Felding-Habermann, Brunhilde; Ware, Jerry

    2007-01-01

    The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy. PMID:17494758

  19. 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

  20. 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

  1. 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

  2. Antibodies to myelin-associated glycoprotein accelerate preferential motor reinnervation.

    PubMed

    Mears, Simon; Schachner, Melitta; Brushart, Thomas M

    2003-06-01

    Predegeneration of nerve enhances its ability to support axon regeneration. Trophic factors are upregulated by reactive Schwann cells while potentially inhibitory molecules are removed. These experiments isolate the effects of one such inhibitory molecule, the myelin-associated glycoprotein (MAG), to determine its role in modifying regeneration after nerve repair. Suture of the mouse femoral nerve was followed by daily intraperitoneal injection of antibodies to MAG, antibodies to HNK-1, a specific muscle pathway marker, or no further treatment. Regeneration was assayed by double-labeling the femoral cutaneous and muscle branches with horseradish peroxidase and fluoro-gold after 4 weeks or 6 weeks of regeneration. Four weeks after nerve repair, selective reinnervation of the muscle branch by motoneurons, or preferential motor reinnervation (PMR), was not seen in either controls or L2-antibody-treated animals. In contrast, treatment with MAG antibodies resulted in dramatic PMR. By 6 weeks, the controls had achieved borderline specificity, substantial PMR developed in the L2 antibody group and the MAG group changed little. Blocking access to MAG in the distal nerve stump thus accelerated and enhanced PMR. Sensory regeneration was depressed by both antibody treatments at 4 weeks but recovered by 6 weeks. Antibody administration has a generalized effect on sensory regeneration that is unrelated to the behavior of motoneurons in the same nerve. PMID:12795713

  3. Translational dynamics of antifreeze glycoprotein in supercooled water.

    PubMed

    Krishnan, V V; Fink, William H; Feeney, Robert E; Yeh, Yin

    2004-08-01

    Structure and dynamics of biomolecules in supercooled water assume a particular and distinct importance in the case of antifreeze glycoproteins (AFGPs), which function at sub-zero temperatures. To investigate whether any large-scale structural digressions in the supercooled state are correlated to the function of AFGPs, self-diffusion behavior of the AFGP8, the smallest AFGP is monitored as a function of temperature from 243 to 303 K using nuclear magnetic resonance (NMR) spectroscopy. The experimental results are compared with the hydrodynamic calculations using the viscosity of water at the same temperature range. In order to evaluate results on AFGP8, the smallest AFGP, constituting approximately two-thirds of the total AFGP fraction in fish blood serum, similar experimental and computational calculations were also performed on a set of globular proteins. These results show that even though the general trend of translational dynamics of AFGP is similar to that of the other globular proteins, AFGP8 appears to be more hydrated (approximately 30% increase in the bead radius) than the others over the temperature range studied. These results also suggest that local conformational changes such as segmental librations or hydrogen bond dynamics that are closer to the protein surface are more likely the determining dynamic factors for the function of AFGPs rather than any large-scale structural rearrangements. PMID:15228958

  4. 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

  5. Characterization of the glycoproteins of bat-derived influenza viruses.

    PubMed

    Maruyama, Junki; Nao, Naganori; Miyamoto, Hiroko; Maeda, Ken; Ogawa, Hirohito; Yoshida, Reiko; Igarashi, Manabu; Takada, Ayato

    2016-01-15

    Recently found bat-derived influenza viruses (BatIVs) have hemagglutinin (HA) and neuraminidase (NA) gene segments distinct from those of previously known influenza A viruses. However, pathogenicities of these BatIVs remain unknown since infectious virus strains have not been isolated yet. To gain insight into the biological properties of BatIVs, we generated vesicular stomatitis viruses (VSVs) pseudotyped with the BatIV HA and NA. We found that VSVs pseudotyped with BatIV HAs and NAs efficiently infected particular bat cell lines but not those derived from primates, and that proteolytic cleavage with a trypsin-like protease was necessary for HA-mediated virus entry. Treatment of the susceptible bat cells with some enzymes and inhibitors revealed that BatIV HAs might recognize some cellular glycoproteins as receptors rather than the sialic acids used for the other known influenza viruses. These data provide fundamental information on the mechanisms underlying the cellular entry and host restriction of BatIVs. PMID:26605499

  6. Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

    PubMed

    Ben Azoun, Safa; Belhaj, Aicha Eya; Göngrich, Rebecca; Gasser, Brigitte; Kallel, Héla

    2016-05-01

    In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV-G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV-G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV-G gene was not tremendously increased as compared with the non-optimized one. In addition, similar levels of RABV-G were obtained when α-factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high-level expression of RABV-G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV-G expression cassette indicating that folding was the limiting step for RABV-G secretion. To circumvent this limitation, co-overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV-G reached 1230 ng ml(-1) . Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host. PMID:26880068

  7. 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.

  8. Seroreactive recombinant herpes simplex virus type 2-specific glycoprotein G.

    PubMed Central

    Parkes, D L; Smith, C M; Rose, J M; Brandis, J; Coates, S R

    1991-01-01

    The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes. A portion of this gG gene has been expressed as a fusion protein in Escherichia coli. Expression was regulated by a lambda phage pL promoter. The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography. Amino acid sequence analysis confirmed the N terminus of the purified protein. Mice immunized with recombinant gG developed antibodies reactive with native HSV-2 protein, but not with HSV-1 protein, in an indirect immunofluorescence assay. The serological activity of this purified recombinant gG protein was evaluated by immunoblot assay. This protein was reactive with an HSV-2 gG monoclonal antibody. It was also reactive with HSV-2 rabbit antiserum but not with HSV-1 rabbit antiserum. Of 15 patient serum samples known to have antibody to HSV-2, 14 were reactive with this recombinant type 2-specific gG protein, and none of 15 HSV antibody-negative patient serum samples showed reactivity. In agreement with the expected prevalence of HSV-2 infection, 27.6% of 134 serum samples from random normal individuals had antibodies reactive with recombinant gG. This recombinant gG protein may be of value in detecting HSV-2-specific antibody responses in patients infected with HSV-2. Images PMID:1653787

  9. 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

  10. Understanding and exploiting 5T4 oncofoetal glycoprotein expression.

    PubMed

    Stern, Peter L; Brazzatti, Julie; Sawan, Saladin; McGinn, Owen J

    2014-12-01

    Oncofoetal antigens are present during foetal development with generally limited expression in the adult but are upregulated in cancer. These molecules can sometimes be used to diagnose or follow treatment of tumours or as a target for different immunotherapies. The 5T4 oncofoetal glycoprotein was identified by searching for shared surface molecules of human trophoblast and cancer cells with the rationale that they may function to allow survival of the foetus as a semi-allograft in the mother or a tumour in its host, potentially influencing growth, invasion or altered immune surveillance of the host. 5T4 tumour selective expression has stimulated the development of 5T4 vaccine, 5T4 antibody targeted-superantigen and 5T4 antibody-drug therapies through preclinical and into clinical studies. It is now apparent that 5T4 expression is a marker of the use (or not) of several cellular pathways relevant to tumour growth and spread. Thus 5T4 expression is mechanistically associated with the directional movement of cells through epithelial mesenchymal transition, facilitation of CXCL12/CXCR4 chemotaxis, blocking of canonical Wnt/beta-catenin while favouring non-canonical pathway signalling. These processes are highly regulated in development and in normal adult tissues but can contribute to the spread of cancer cells. Understanding the differential impact of these pathways marked by 5T4 can potentially improve existing, or aid development of novel cancer treatment strategies. PMID:25066861

  11. 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

  12. [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

  13. Interactions of attention-deficit/hyperactivity disorder therapeutic agents with the efflux transporter P-glycoprotein

    PubMed Central

    Zhu, Hao-Jie; Wang, Jun-Sheng; Donovan, Jennifer L.; Jiang, Yan; Gibson, Bryan B.; DeVane, C. Lindsay; Markowitz, John S.

    2009-01-01

    The objective of this study was to assess the potential interactions of the drug transporter P-glycoprotein with attention-deficit/hyperactivity disorder (ADHD) therapeutic agents atomoxetine — and the individual isomers of methylphenidate, amphetamine, and modafinil utilizing established in vitro assay. An initial ATPase assay indicated that both d- and l-methylphenidate have weak affinity for P-glycoprotein. The intracellular accumulation of P-glycoprotein substrates doxorubicin and rhodamine123 in the P-glycoprotein overexpressing cell line LLC-PK1/MDR1 was determined to evaluate potential inhibitory effects on P-glycoprotein. The results demonstrated that all compounds, except both modafinil isomers, significantly increased doxorubicin and rhodamine123 accumulation in LLC-PK1/MDR1 cells at higher concentrations. To investigate the P-glycoprotein substrate properties, the intracellular concentrations of the tested compounds in LLC-PK1/MDR1 and P-glycoprotein negative LLC-PK1 cells were measured in the presence and absence of the P-glycoprotein inhibitor PSC833. The results indicate that the accumulation of d-methylphenidate in LLC-PK1 cells was 32.0% higher than in LLC-PK1/MDR1 cells. Additionally, coadministration of PSC833 leads to 52.9% and 45.6% increases in d-modafinil and l-modafinil accumulation, respectively, in LLC-PK1/MDR1 cells. Further studies demonstrated that l-modafinil transport across LLC-PK1/MDR1 cell monolayers in the basolateral-to-apical (B–A) direction was significantly higher than in the apical-to-basolateral (A–B) direction. PSC833 treatment significantly decreased the transport of l-modafinil in B–A direction. In conclusion, our results suggest that all tested agents with the exception of modafinil isomers are relatively weak P-glycoprotein inhibitors. Furthermore, P-glycoprotein may play a minor role in the transport of d-methylphenidate, d-modafinil, and l-modafinil. PMID:17963743

  14. 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

  15. 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

  16. 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.

  17. Effect of the ionophore monensin on herpes simplex virus type 1-induced cell fusion, glycoprotein synthesis, and virion infectivity.

    PubMed

    Kousoulas, K G; Bzik, D J; Person, S

    1983-01-01

    The ionophore monensin inhibited the formation of mature, fully glycosylated glycoproteins gB, gC, and gD during herpes simplex virus type 1 infection of human embryonic lung cells. Underglycosylated forms, including the apparent high-mannose precursor forms of the major glycoproteins, appeared. Monensin inhibited virus-induced cell fusion. Infectious virions produced in the presence of monensin appeared to contain predominantly underglycosylated glycoproteins. PMID:6307921

  18. Requirements for Cell Rounding and Surface Protein Down-Regulation by Ebola Virus Glycoprotein

    PubMed Central

    Francica, Joseph R.; Matukonis, Meghan K.; Bates, Paul

    2009-01-01

    Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of β1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects. PMID:19013626

  19. 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

  20. Lp(a) glycoprotein phenotypes. Inheritance and relation to Lp(a)-lipoprotein concentrations in plasma.

    PubMed Central

    Utermann, G; Menzel, H J; Kraft, H G; Duba, H C; Kemmler, H G; Seitz, C

    1987-01-01

    The Lp(a) lipoprotein represents a quantitative genetic trait. It contains two different polypeptide chains, the Lp(a) glycoprotein and apo B-100. We have demonstrated the Lp(a) glycoprotein directly in human sera by sodium dodecyl sulfate-gel electrophoresis under reducing conditions after immunoblotting using anti-Lp(a) serum and have observed inter- and intraindividual size heterogeneity of the glycoprotein with apparent molecular weights ranging from approximately 400,000-700,000 D. According to their relative mobilities compared with apo B-100 Lp(a) patterns were categorized into phenotypes F (faster than apo B-100), B (similar to apo B-100), S1, S2, S3, and S4 (all slower than apo B-100), and into the respective double-band phenotypes. Results from neuraminidase treatment of isolated Lp(a) glycoprotein indicate that the phenotypic differences do not reside in the sialic acid moiety of the glycoprotein. Family studies are compatible with the concept that Lp(a) glycoprotein phenotypes are controlled by a series of autosomal alleles (Lp[a]F, Lp[a]B, Lp[a]S1, Lp[a]S2, Lp[a]S3, Lp[a]S4, and Lp[a]0) at a single locus. Comparison of Lp(a) plasma concentrations in different phenotypes revealed a highly significant association of phenotype with concentration. Phenotypes B, S1, and S2 are associated with high and phenotypes S3 and S4 with low Lp(a) concentrations. This suggests that the same gene locus is involved in determining Lp(a) glycoprotein phenotypes and Lp(a) lipoprotein concentrations in plasma and is the first indication for structural differences underlying the quantitative genetic Lp(a)-trait. Images PMID:2956279

  1. 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.

  2. 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.

  3. 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.

  4. Growth and metabolism of fucosylated plasma-membrane glycoproteins in mouse neuroblastoma N2a cells

    PubMed Central

    Milenkovic, Ada G.; Rachmeler, Martin; Johnson, Terry C.

    1978-01-01

    The presence of 1.0mm-dibutyryl cyclic AMP (N6,O2′-dibutyryladenosine 3′:5′-cyclic monophosphate) and 1.5mm-theophylline completely inhibits the growth of mouse neuroblastoma N2a cells by 24–36h. When compared with N2a cultures without inhibitors (controls), the proportion of cells in S phase, measured by radioautography with [3H]-thymidine, was decreased from 55 to 12%. In addition, the presence of the inhibitors decreased apparent [3H]fucose incorporation into glycoproteins by 50%, and removing the inhibitors resulted in a rapid recovery of both DNA synthesis and glycoprotein metabolism. Measurement of intracellular acid-soluble radioactive fucose revealed that decreased fucose uptake could account for the apparent change in incorporation. Removing dibutyryl cyclic AMP and theophylline from the medium resulted in a rapid uptake of radioactive fucose to within control values, which illustrated that the inhibitors decreased transport of the carbohydrate, although the cells remained viable. Treatment with dibutyryl cyclic AMP and theophylline also reversibly inhibited glycoprotein degradation. Plasma membranes isolated from growing cells and from growth-inhibited cells labelled with [14C]fucose and [3H]fucose respectively were co-electrophoresed on sodium dodecyl sulphate/polyacrylamide gels. These displayed no apparent differences in synthesis of specific membrane glycoproteins. Electrophoresis of plasma membranes isolated from cultures pulse–chased with [14C]fucose and [3H]fucose was used to discern turnover patterns of specific plasma-membrane glycoproteins. High-molecular-weight glycoproteins exhibited rapid rates of turnover in membranes from growing cells, but moderate turnover rates in growth-inhibited cells and cells reversed from growth inhibition. These data indicate that growth arrest of N2a cells results in alterations in the metabolic turnover of plasma-membrane glycoproteins. PMID:218551

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

    PubMed

    Sorrell, M F; Nauss, J M; Donohue, T M; 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 [14C]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 [14C]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, [14C]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. PMID:6822326

  6. 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

  7. Multiple Drug Transport Pathways through human P-Glycoprotein(†)

    PubMed Central

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

    2015-01-01

    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 to 12 Å through the plane of the membrane as P-gp progressed through a catalytic cycle. Methyl-pyrophosphate, 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 is presented that involves stabilization of an outward open conformation with tariquidar bound in intracellular loops or at the drug binding domain of P-gp. PMID:26125482

  8. Generating inhibitors of P-glycoprotein: where to, now?

    PubMed

    Crowley, Emily; McDevitt, Christopher A; Callaghan, Richard

    2010-01-01

    The prominent role for the drug efflux pump ABCB1 (P-glycoprotein) in mediating resistance to chemotherapy was first suggested in 1976 and sparked an incredible drive to restore the efficacy of anticancer drugs. Achieving this goal seemed inevitable in 1982 when a series of calcium channel blockers were demonstrated to restore the efficacy of chemotherapy agents. A large number of other compounds have since been demonstrated to restore chemotherapeutic sensitivity in cancer cells or tissues. Where do we stand almost three decades since the first reports of ABCB1 inhibition? Unfortunately, in the aftermath of extensive fundamental and clinical research efforts the situation remains gloomy. Only a small handful of compounds have reached late stage clinical trials and none are in routine clinical usage to circumvent chemoresistance. Why has the translation process been so ineffective? One factor is the multifactorial nature of drug resistance inherent to cancer tissues; ABCB1 is not the sole factor. However, expression of ABCB1 remains a significant negative prognostic indicator and is closely associated with poor response to chemotherapy in many cancer types. The main difficulties with restoration of sensitivity to chemotherapy reside with poor properties of the ABCB1 inhibitors: (1) low selectivity to ABCB1, (2) poor potency to inhibit ABCB1, (3) inherent toxicity and/or (4) adverse pharmacokinetic interactions with anticancer drugs. Despite these difficulties, there is a clear requirement for effective inhibitors and to date the strategies for generating such compounds have involved serendipity or simple chemical syntheses. This chapter outlines more sophisticated approaches making use of bioinformatics, combinatorial chemistry and structure informed drug design. Generating a new arsenal of potent and selective ABCB1 inhibitors offers the promise of restoring the efficacy of a key weapon in cancer treatment--chemotherapy. PMID:19949934

  9. Interactome analysis of herpes simplex virus 1 envelope glycoprotein H.

    PubMed

    Hirohata, Yoshitaka; Kato, Akihisa; Oyama, Masaaki; Kozuka-Hata, Hiroko; Koyanagi, Naoto; Arii, Jun; Kawaguchi, Yasushi

    2015-06-01

    Herpes simplex virus 1 (HSV-1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV-1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry-based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive-1 (GCN1), a trans-acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV-1-infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV-1-infected cells and that the gH-null mutation increases eIF2α in HSV-1-infected cells, whereas gH overexpression in the absence of other HSV-1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV-1-infected cells and that the GCN1-binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV-1-infected cells. PMID:25808324

  10. Acetaminophen inhibits intestinal p-glycoprotein transport activity.

    PubMed

    Novak, Analia; Carpini, Griselda Delli; Ruiz, María Laura; Luquita, Marcelo G; Rubio, Modesto C; Mottino, Aldo D; Ghanem, Carolina I

    2013-10-01

    Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 μM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP. PMID:23897240

  11. 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

  12. Autoantibodies induced by chimeric cytokine - HIV envelope glycoprotein immunogens

    PubMed Central

    Isik, Gözde; van Montfort, Thijs; Chung, Nancy P.Y.; Moore, John P.; Sanders, Rogier W.

    2014-01-01

    Cytokines are often used as adjuvants to increase the immunogenicity of vaccines as they can improve the immune response and/or direct it into a desired direction. As an alternative to co-delivering antigens and cytokines separately they can be fused into a composite protein, with the advantage that both moieties act on the same immune cells. The HIV-1 envelope glycoprotein (Env) spike, located on the outside of virus particles and the only relevant protein for the induction of neutralizing antibodies (NAbs), is poorly immunogenic. The induction of anti-Env Abs can be improved by coupling Env proteins to co-stimulatory molecules such as a proliferation inducing ligand (APRIL). Here, we evaluated the immunogenicity of chimeric molecules containing uncleaved Env gp140 fused to the species-matched cytokines IL-21 or GM-CSF in rabbits and mice. Each cytokine was either fused to the C-terminus of Env or embedded within Env at the position of the variable loops 1 and 2 (V1V2). The cytokine components of the chimeric Env-GM-CSF and Env-IL-21 molecules were functional in vitro, but none of the Env-cytokine fusion proteins resulted in improved Ab responses in vivo. Both the Env-GM-CSF and the Env-IL-21 molecules induced strong anti-cytokine Ab responses, in both test species. These autoimmune responses were independent of the location of the cytokine in the chimeric Env molecules; in that they were induced by cytokines inserted within the V1V2 of Env or fused to its Ct. The induction of undesired autoimmune responses should be considered when using cytokines as co-stimulatory molecules in fusion proteins. PMID:24729614

  13. Conformational changes of P-glycoprotein by nucleotide binding.

    PubMed Central

    Wang, G; Pincheira, R; Zhang, M; Zhang, J T

    1997-01-01

    P-glycoprotein (Pgp) is a membrane protein that transports chemotherapeutic drugs, causing multidrug resistance in human cancer cells. Pgp is a member of the ATP-binding cassette superfamily and functions as a transport ATPase. It has been suggested that the conformation of Pgp changes in the catalytic cycle. In this study, we tested this hypothesis by using limited proteolysis as a tool to detect different conformational states trapped by binding of nucleotide ligands and inhibitors. Pgp has high basal ATPase activity; that is, ATP hydrolysis by Pgp is not rigidly associated with drug transport. This activity provides a convenient method for studying the conformational change of Pgp induced by nucleotide ligands, in the absence of drug substrates which may generate complications due to their own binding. Inside-out membrane vesicles containing human Pgp were isolated from multidrug-resistant SKOV/VLB cells and treated with trypsin in the absence or presence of MgATP, Mg-adenosine 5'-[beta,gamma-imido]triphosphate (Mg-p[NH]ppA) and MgADP. Changes in the proteolysis profile of Pgp owing to binding of nucleotides were used to indicate the conformational changes in Pgp. We found that generation of tryptic fragments, including the loop linking transmembrane (TM) regions TM8 and TM9 of Pgp, were stimulated by the binding of Mg-p[NH]ppA, MgATP and MgADP, indicating that the Pgp conformation was changed by the binding of these nucleotides. The effects of nucleotides on Pgp conformation are directly associated with the binding and/or hydrolysis of these ligands. Four conformational states of Pgp were stabilized under different conditions with various ligands and inhibitors. We propose that cycling through these four states couples the Pgp-mediated MgATP hydrolysis to drug transport. PMID:9396736

  14. 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

  15. Pseudomonas aeruginosa outer membrane adhesins for human respiratory mucus glycoproteins.

    PubMed Central

    Carnoy, C; Scharfman, A; Van Brussel, E; Lamblin, G; Ramphal, R; Roussel, P

    1994-01-01

    The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesion assays have been developed and have suggested that nonpilus adhesins of P. aeruginosa are the most important ones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outer membrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoN mutants, 1244-N3 and PAK-N1, were prepared by a mild extraction with Zwittergent 3-14. Mucin-binding adhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulose replicas, using human bronchial mucins labeled with 125I. The binding properties of these OMP with lactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucins detected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, five bands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized by radiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeled respiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydrate sites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate that outer membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinity for human respiratory mucins and/or lactotransferrin. Images PMID:8168955

  16. Interaction of Common Azole Antifungals with P Glycoprotein

    PubMed Central

    Wang, Er-jia; Lew, Karen; Casciano, Christopher N.; Clement, Robert P.; Johnson, William W.

    2002-01-01

    Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and exposure to azole antifungal therapeutics and partially explain the clinical drug interactions observed with some antifungals. Using a whole-cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the abilities of the most widely prescribed orally administered azole antifungals to inhibit the function of this transporter. In a cell line presenting an overexpressed amount of the human P-gp transporter, itraconazole and ketoconazole inhibited P-gp function with 50% inhibitory concentrations (IC50s) of ∼2 and ∼6 μM, respectively. Cyclosporin A was inhibitory with an IC50 of 1.4 μM in this system. Uniquely, fluconazole had no effect in this assay, a result consistent with known clinical interactions. The effects of these azole antifungals on ATP consumption by P-gp (representing transport activity) were also assessed, and the Km values were congruent with the IC50s. Therefore, exposure of tissue to the azole antifungals may be modulated by human P-gp, and the clinical interactions of azole antifungals with other drugs may be due, in part, to inhibition of P-gp transport. PMID:11751127

  17. 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

  18. 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

  19. 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.

  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. 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. PMID:26484739

  2. 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

  3. 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.

  4. Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein

    PubMed Central

    Breithaupt, Constanze; Schubart, Anna; Zander, Hilke; Skerra, Arne; Huber, Robert; Linington, Christopher; Jacob, Uwe

    2003-01-01

    Multiple sclerosis is a chronic disease of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal loss. The immunopathogenesis of demyelination in multiple sclerosis involves an autoantibody response to myelin oligodendrocyte glycoprotein (MOG), a type I transmembrane protein located at the surface of CNS myelin. Here we present the crystal structures of the extracellular domain of MOG (MOGIgd) at 1.45-Å resolution and the complex of MOGIgd with the antigen-binding fragment (Fab) of the MOG-specific demyelinating monoclonal antibody 8-18C5 at 3.0-Å resolution. MOGIgd adopts an IgV like fold with the A′GFCC′C″ sheet harboring a cavity similar to the one used by the costimulatory molecule B7-2 to bind its ligand CTLA4. The antibody 8-18C5 binds to three loops located at the membrane-distal side of MOG with a surprisingly dominant contribution made by MOG residues 101–108 containing a strained loop that forms the upper edge of the putative ligand binding site. The sequence R101DHSYQEE108 is unique for MOG, whereas large parts of the remaining sequence are conserved in potentially tolerogenic MOG homologues expressed outside the immuno-privileged environment of the CNS. Strikingly, the only sequence identical to DHSYQEE was found in a Chlamydia trachomatis protein of unknown function, raising the possibility that Chlamydia infections may play a role in the MOG-specific autoimmune response in man. Our data provide the structural basis for the development of diagnostic and therapeutic strategies targeting the pathogenic autoantibody response to MOG. PMID:12874380

  5. Conformation of the antifreeze glycoprotein of polar fish.

    PubMed

    Bush, C A; Ralapati, S; Matson, G M; Yamasaki, R B; Osuga, D T; Yeh, Y; Feeney, R E

    1984-08-01

    High-field proton and 13C NMR spectroscopy has been used to test and refine the recent proposal, based on vacuum uv circular dichroism results, of a threefold left-handed helical conformation for antifreeze glycoprotein (AFGP). Partial assignment of the protons of the glycotripeptide repeating unit has been made by comparison with spectra of model compounds, by selective decoupling, and by measurements of nuclear Overhauser effect (nOe). At 40 degrees C, AFGP fraction 8 (Mr 2600) shows 2-Hz linewidths which broaden at lower temperature. Neither 1H nor 13C chemical shifts depend strongly on temperature, suggesting no abrupt conformational transition. The nOe between alanine alpha and beta protons vary with temperature and with field strength, from small positive enhancements at 50 degrees C and 80 MHz to large negative effects at 3 degrees C and 300 MHz, indicating a substantial change of rotational correlation time with temperature. The higher-molecular-weight fraction 1-4 shows negative nOe at all temperatures. The CD spectra of fraction 1-4 show bands characteristic of the polyproline II structure at both 3 and 50 degrees C, while those bands in fraction 8 are weaker at 50 than 3 degrees C. The 1H nOe, the 13C T1, and CD data are interpreted as indicating that AFGP fraction 8 is an extended "rod-like" conformation at low temperature which becomes a flexible coil at high temperature, while fraction 1-4 is a flexible rod with sufficient segmental mobility to eliminate any long-range order. PMID:6087734

  6. 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.

  7. 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

  8. Confident Assignment of Site-Specific Glycosylation in Complex Glycoproteins in a Single Step

    PubMed Central

    2015-01-01

    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. PMID:25153361

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. Detergent-Assisted Glycoprotein Capture: A Versatile Tool for In-Depth N-Glycoproteome Analysis.

    PubMed

    Chen, Rui; Zou, Hanfa; Figeys, Daniel

    2016-06-01

    Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazide groups to oxidized glycoproteins, thus increasing the coverage of the N-glycoproteome. Compared with the conventional glycopeptide capture method, the detergent-assisted glycoprotein capture approach nearly doubled the number of N-glycosylation sites identified from HEK 293T cells with improved specificity. Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293T cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Furthermore, the application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immunodepletion or fractionation. Overall, the detergent-assisted glycoprotein capture method simplified the capture process, and it increased the number of sites observed on both hydrophobic membrane proteins and hydrophilic secreted proteins. PMID:27147131

  14. 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. PMID:26459807

  15. 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.

  16. 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

  17. Human herpesvirus 8 glycoprotein B binds the entry receptor DC-SIGN

    PubMed Central

    Hensler, Heather R; Tomaszewski, Monica J; Rappocciolo, Giovanna; Rinaldo, Charles R; Jenkins, Frank J

    2014-01-01

    We have previously shown that human herpesvirus 8 (HHV-8) uses DC-SIGN as an entry receptor for dendritic cells, macrophages and B cells. The viral attachment protein for DC-SIGN is unknown. HHV-8 virions contain 5 conserved herpesvirus glycoproteins, a single unique glycoprotein, and 2 predicted glycoproteins. Previous studies have shown that DC-SIGN binds highly mannosylated glycoproteins. The HHV-8 glycoprotein B (gB) has been reported to be highly mannosylated, and therefore we hypothesized that gB will bind to DC-SIGN. In this report we confirm that gB has a high mannose carbohydrate structure and demonstrate for the first time that it binds DC-SIGN in a dose-dependent manner. We also identify key amino acids in the DC-SIGN carbohydrate recognition domain that are required for HHV-8 infection and compare these results with published binding regions for ICAM-2/3 and HIV-1 gp120. These results clarify some of the initial events in HHV-8 entry and can be used for the design of targeted preventive therapies. PMID:25018023

  18. 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

  19. Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles.

    PubMed Central

    Mebatsion, T; Schnell, M J; Conzelmann, K K

    1995-01-01

    A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells. PMID:7853476

  20. Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis

    PubMed Central

    d'Amaro, Rocca; Scheidegger, Rolf; Blumer, Susan; Pazera, Pawel; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf

  1. Putative functions of extracellular matrix glycoproteins in secondary palate morphogenesis.

    PubMed

    d'Amaro, Rocca; Scheidegger, Rolf; Blumer, Susan; Pazera, Pawel; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Cleft palate is a common birth defect in humans. Elevation and fusion of paired palatal shelves are coordinated by growth and transcription factors, and mutations in these can cause malformations. Among the effector genes for growth factor signaling are extracellular matrix (ECM) glycoproteins. These provide substrates for cell adhesion (e.g., fibronectin, tenascins), but also regulate growth factor availability (e.g., fibrillins). Cleft palate in Bmp7 null mouse embryos is caused by a delay in palatal shelf elevation. In contrast, palatal shelves of Tgf-β3 knockout mice elevate normally, but a cleft develops due to their failure to fuse. However, nothing is known about a possible functional interaction between specific ECM proteins and Tgf-β/Bmp family members in palatogenesis. To start addressing this question, we studied the mRNA and protein distribution of relevant ECM components during secondary palate development, and compared it to growth factor expression in wildtypewild type and mutant mice. We found that fibrillin-2 (but not fibrillin-1) mRNA appeared in the mesenchyme of elevated palatal shelves adjacent to the midline epithelial cells, which were positive for Tgf-β3 mRNA. Moreover, midline epithelial cells started expressing fibronectin upon contact of the two palatal shelves. These findings support the hypothesis that fibrillin-2 and fibronectin are involved in regulating the activity of Tgf-β3 at the fusing midline. In addition, we observed that tenascin-W (but not tenascin-C) was misexpressed in palatal shelves of Bmp7-deficient mouse embryos. In contrast to tenascin-C, tenascin-W secretion was strongly induced by Bmp7 in embryonic cranial fibroblasts in vitro. These results are consistent with a putative function for tenascin-W as a target of Bmp7 signaling during palate elevation. Our results indicate that distinct ECM proteins are important for morphogenesis of the secondary palate, both as downstream effectors and as regulators of Tgf

  2. 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

  3. 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

  4. 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

  5. Characterization of intact neo-glycoproteins by hydrophilic interaction liquid chromatography.

    PubMed

    Pedrali, Alice; Tengattini, Sara; Marrubini, Giorgio; Bavaro, Teodora; Hemström, Petrus; Massolini, Gabriella; Terreni, Marco; Temporini, Caterina

    2014-01-01

    In this study, an HPLC HILIC-UV method was developed for the analysis of intact neo-glycoproteins. During method development the experimental conditions evaluated involved different HILIC columns (TSKgel Amide-80 and ZIC-pHILIC), and water-acetonitrile mixtures containing various types of acids and salts. The final selected method was based on a TSKgel Amide-80 column and a mobile phase composed of acetonitrile and water both containing 10 mM HClO4. The influence of temperature and sample preparation on the chromatographic performances of the HILIC method was also investigated. The method was applied to the separation of neo-glycoproteins prepared starting from the model protein RNase A by chemical conjugation of different glycans. Using the method here reported it was possible to monitor by UV detection the glycosylation reaction and assess the distribution of neo-glycoprotein isoforms without laborious sample workup prior to analysis. PMID:24983858

  6. Sialylation sensitive bands in the Raman spectra of oligosaccharides and glycoproteins

    NASA Astrophysics Data System (ADS)

    Oleinikov, V.; Kryukov, E.; Kovner, M.; Ermishov, M.; Tuzikov, A.; Shiyan, S.; Bovin, N.; Nabiev, I.

    1999-05-01

    N-Acetylneuraminic (sialic) acid which plays a key role in process of cell recognition and interaction was studied by Raman spectroscopy. It was revealed that the strong band at 873 cm -1 arising from the glycerol fragment vibrations can be used as a Raman marker of sialic acid. In the Raman spectra of oligosaccharides and glycoproteins this band is observed at 880 cm -1. The strong dependence of the 880 cm -1 band intensity on the sialic acid ( Sia) content in α 1-acid glycoprotein was shown. The data demonstrate the possibility to use Raman spectroscopy approach as a simple and non-destructive assay for the rapid registration and quantification of Sia in the glycoproteins and on the membranes of the living cells.

  7. 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.

  8. A neurodystrophic syndrome resembling carbohydrate-deficient glycoprotein syndrome type III.

    PubMed

    Stibler, H; Gylje, H; Uller, A

    1999-04-01

    A 10-month old girl is described with a serum transferrin isoform abnormality of the same kind as in two previously reported girls with carbohydrate-deficient glycoprotein syndrome type III. This patient presented with joint abnormalities and rapidly developing hypsarrhythmia, hypotonia, psychomotor delay and growth retardation. Fingers, toes, nails and local skin were dysmorphic. She had pale optic discs, thoracic syringomyelia and frontal lobe atrophy at three months. The CDT value in serum was greatly elevated. Several carbohydrate-deficient isoforms were found in transferrin (four), alpha1-antitrypsin (three), antithrombin (two) and thyroxine-binding globulin (four). Mutations in the CDGS 1-gene were excluded. The CDGS III glycoprotein abnormality most probably represents a distinct disorder of glycoprotein metabolism, and needs to be considered in unclear hypsarrhythmia with developmental delay. Dysmorphic features may be added to this syndrome. PMID:10401691

  9. Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

    PubMed

    Sheta, Razan; Roux-Dalvai, Florence; Woo, Christina M; Fournier, Frédéric; Bourassa, Sylvie; Bertozzi, Carolyn R; Droit, Arnaud; Bachvarov, Dimcho

    2016-09-01

    This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets. PMID:27331112

  10. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  11. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection.

    PubMed

    Deng, Kai; Liu, Ruyu; Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1-6 was determined. We defined a domain comprising amino acids (aa) 192-221, 232-251, 262-281 and 292-331 of E1, and 421-543, 564-583, 594-618 and 634-673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444-463) and PUHI45 (aa 604-618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  12. Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns.

    PubMed

    Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A

    2009-08-01

    Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a

  13. Glycan structures contain information for the spatial arrangement of glycoproteins in the plasma membrane.

    PubMed

    Hall, M Kristen; Weidner, Douglas A; Chen, Jian ming; Bernetski, Christopher J; Schwalbe, Ruth A

    2013-01-01

    Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-)5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells. PMID:24040379

  14. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

    PubMed Central

    Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  15. Glycoprotein-based enzyme-linked immunosorbent assays for serodiagnosis of infectious laryngotracheitis.

    PubMed

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta; Samal, Siba K

    2015-05-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

  16. A Single gD Glycoprotein Can Mediate Infection by Herpes simplex Virus

    PubMed Central

    2013-01-01

    Herpes simplex viruses display hundreds of gD glycoproteins, and yet their neutralization requires tens of thousands of antibodies per virion, leading us to ask whether a wild-type virion with just a single free gD is still infective. By quantitative analysis of fluorescently labeled virus particles and virus neutralization assays, we show that entry of a wild-type HSV virion to a cell does indeed require just one or two of the approximately 300 gD glycoproteins to be left unbound by monoclonal antibody. This indicates that HSV entry is an extraordinarily efficient process, functioning at the level of single molecular complexes. PMID:23837576

  17. Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection.

    PubMed

    Mahmoud, Nora F; Jasirwan, Chyntia; Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki; Nagamata, Satoshi; Kawabata, Akiko; Tang, Huamin; Mori, Yasuko

    2016-03-01

    Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. PMID:26802210

  18. Glycoprotein-Based Enzyme-Linked Immunosorbent Assays for Serodiagnosis of Infectious Laryngotracheitis

    PubMed Central

    Kanabagatte Basavarajappa, Mallikarjuna; Song, Haichen; Lamichhane, Chinta

    2015-01-01

    For detection of infectious laryngotracheitis virus (ILTV) antibody, glycoprotein B-, C-, and D-based enzyme-linked immunosorbent assays (B-, C-, and D-ELISAs, respectively) were developed. The B- and D-ELISAs showed enhanced detection of anti-ILTV antibodies in infected chickens compared to that of the commercial ELISA. Furthermore, the D-ELISA was efficient in detecting seroconversion with vectored vaccine, using recombinant Newcastle disease virus (rNDV) expressing glycoprotein D (gD) as the vaccine vector. PMID:25694519

  19. 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

  20. 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

  1. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles

    PubMed Central

    Freitag, Tanja C.

    2015-01-01

    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

  2. ICP-MS-Based Multiplex Profiling of Glycoproteins Using Lectins Conjugated to Lanthanide-Chelating Polymers

    PubMed Central

    Leipold, Michael D.; Herrera, Isaac; Ornatsky, Olga; Baranov, Vladimir; Nitz, Mark

    2009-01-01

    Lectins have been increasingly important in the study of glycoproteins. Here we report a glycoprofiling method based on the covalent attachment of metal-chelating polymers to lectins for use in an ICP-MS-based assays. The labeled lectins are able to distinguish between glycoproteins covalently attached to a microtiter plate and their binding can be directly quantified by ICP-MS. Since each conjugate contains a different lanthanide, the assays can be conducted in a single or multiplex fashion, and may be readily elaborated to many different assay formats. PMID:19072657

  3. 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.

  4. 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.

  5. 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

  6. A haemagglutinin in the tissue fluid of the Pacific oyster, Crassostrea gigas, with specificity for sialic acid residues in glycoproteins.

    PubMed

    Hardy, S W; Grant, P T; Fletcher, T C

    1977-06-15

    An agglutinin for human red cells has a specificity for sialic acid and a high affinity for bovine salivary glycoprotein. Digestion of the glycoprotein with Pronase or neuraminidas indicated that binding of sialic acid to receptors in the agglutinin is the first step in the mechanism of formation of a stable complex between ligand and receptor. PMID:891745

  7. 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

  8. The DNA sequence of the equine herpesvirus 4 gene encoding glycoprotein gp17/18, the homologue of herpes simplex virus glycoprotein gD.

    PubMed

    Cullinane, A A; Neilan, J; Wilson, L; Davison, A J; Allen, G

    1993-09-01

    The nucleotide sequence of the gene to the left of the gI gene of equine herpesvirus 4 (EHV-4) was determined. The gene encodes a peptide of 402 amino acids with an unprocessed M(r) of 45,323. The predicted polypeptide has several features of a glycoprotein including a hydrophobic signal sequence, a membrane spanning domain and four potential N-linked glycosylation sites within the proposed external domain. The predicted amino acid sequence of EHV-4 gD shows 83% identity with that of equine herpesvirus 1 gD. Conservation of the tertiary structure is suggested by the alignment of six cysteine residues with those of the gD of six other alphaherpesviruses. Screening a lambda gt11/EHV-4 expression library with monoclonal antibodies against several of the most abundant EHV-4 glycoproteins unequivocally identified the protein encoded by the EHV-4 gD gene as gp17/18. PMID:8397286

  9. 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.

  10. The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

    PubMed Central

    Leser, G P; Ector, K J; Lamb, R A

    1996-01-01

    The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway. Images PMID:8741847

  11. 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

  12. 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. PMID:26611586

  13. ProGlycProt: a repository of experimentally characterized prokaryotic glycoproteins

    PubMed Central

    Bhat, Aadil H.; Mondal, Homchoru; Chauhan, Jagat S.; Raghava, Gajendra P. S.; Methi, Amrish; Rao, Alka

    2012-01-01

    ProGlycProt (http://www.proglycprot.org/) is an open access, manually curated, comprehensive repository of bacterial and archaeal glycoproteins with at least one experimentally validated glycosite (glycosylated residue). To facilitate maximum information at one point, the database is arranged under two sections: (i) ProCGP—the main data section consisting of 95 entries with experimentally characterized glycosites and (ii) ProUGP—a supplementary data section containing 245 entries with experimentally identified glycosylation but uncharacterized glycosites. Every entry in the database is fully cross-referenced and enriched with available published information about source organism, coding gene, protein, glycosites, glycosylation type, attached glycan, associated oligosaccharyl/glycosyl transferases (OSTs/GTs), supporting references, and applicable additional information. Interestingly, ProGlycProt contains as many as 174 entries for which information is unavailable or the characterized glycosites are unannotated in Swiss-Prot release 2011_07. The website supports a dedicated structure gallery of homology models and crystal structures of characterized glycoproteins in addition to two new tools developed in view of emerging information about prokaryotic sequons (conserved sequences of amino acids around glycosites) that are never or rarely seen in eukaryotic glycoproteins. ProGlycProt provides an extensive compilation of experimentally identified glycosites (334) and glycoproteins (340) of prokaryotes that could serve as an information resource for research and technology applications in glycobiology. PMID:22039152

  14. 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

  15. 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

  16. Detection of Autoantibodies Against Myelin Oligodendrocyte Glycoprotein in Multiple Sclerosis and Related Diseases.

    PubMed

    Spadaro, Melania; Meinl, Edgar

    2016-01-01

    Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) occur in a proportion of patients with different inflammatory demyelinating diseases of the central nervous system, such as childhood multiple sclerosis (MS), acute disseminated encephalomyelitis (ADEM), and neuromyelitis optica spectrum disorders (NMOSD). We describe here in detail a sensitive cell-based assay that allows the identification of autoantibodies against MOG in serum. PMID:25814289

  17. 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...

  18. B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

    PubMed Central

    Pancino, G; Chappey, C; Saurin, W; Sonigo, P

    1993-01-01

    In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines. Images PMID:7678301

  19. 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 ...

  20. 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...

  1. 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.

  2. 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…

  3. HCMV Encoded Glycoprotein M (UL100) Interacts with Rab11 Effector Protein FIP4

    PubMed Central

    Krzyzaniak, Magdalena A.; Mach, Michael; Britt, William J.

    2009-01-01

    The envelope of human cytomegalovirus (HCMV) consists of a large number of glycoproteins. The most abundant glycoprotein in the HCMV envelope is the glycoprotein M (UL100) which together with glycoprotein N (UL73) form the gM/gN protein complex. Using yeast two hybrid screening, we found that the gM carboxy-terminal cytoplasmic tail (gM-CT) interacts with FIP4, a Rab11-GTPase effector protein. Depletion of FIP4 expression in HCMV infected cells resulted in a decrease of infectious virus production that was also associated with an alteration of the HCMV assembly compartment (AC) phenotype. A similar phenotype was also observed in HCMV infected cells that expressed dominant negative Rab11(S25N). Recently, it has been shown that FIP4 interactions with Rab11 and additionally with Arf6/Arf5 are important for the vesicular transport of proteins in the endosomal recycling compartment (ERC) and during cytokinesis. Surprisingly, FIP4 interaction with gM-CT limited binding of FIP4 with Arf5/Arf6, however, FIP4 interaction with gM-CT did not prevent recruitment of Rab11 into the ternary complex. These data argued for a contribution of the ERC during cytoplasmic envelopment of HCMV and revealed a novel FIP4 function independent of Arf5 or Arf6 activity. PMID:19761540

  4. Enzymatic glycoprotein synthesis: Preparation of ribonuclease glycoforms via enzymatic glycopeptide condensation and glycosylation

    SciTech Connect

    Witte, K.; Sears, P.; Martin, R.; Wong, C.H.

    1997-03-05

    In order to study the effects carbohydrates have on glycoprotein structure and funciton, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in vivo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a `tag` to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN`. These fragments were religated by subtilisin 8397 to the full length RNase by addition glycerol; this method points to a new chemical-enzymatic process for the synthesis of glycoproteins using synthetic peptides and glycopeptides as substrates for enzymatic ligation followed by further enzymatic glycosylations. 29 refs., 6 figs.

  5. Association of erosive esophagitis with Helicobacter pylori eradication: a role of salivary bicarbonate and glycoprotein secretion.

    PubMed

    Namiot, D B; Namiot, Z; Markowski, A R; Leszczyńska, K; Bucki, R; Kemona, A; Gołebiewska, M

    2009-01-01

    In some populations, Helicobacter pylori eradication is associated with development of erosive esophagitis. The aim of this study was to evaluate the contribution of salivary bicarbonate and glycoprotein secretion to the pathogenesis of erosive esophagitis developing after H. pylori eradication. Gastroscopy and saliva collection were performed at recruitment and 12 months after completion of eradication therapy. Eighty-eight patients with duodenal ulcer were recruited to the study. Erosive esophagitis was found in 13 patients (grade A, 8 patients; grade B, 4 patients; grade C, 1 patient). Among the 74 subjects who completed the study, erosive esophagitis was detected in 21 patients (grade A, 15 patients; grade B, 6 patients); they all were successfully eradicated. Bicarbonate and glycoprotein secretion was not found to differ significantly between the subjects with and without erosive esophagitis both before and 1 year after H. pylori eradication. However, it was lower in H. pylori-infected (baseline) than in H. pylori-noninfected erosive esophagitis subjects (1 year after successful eradication) (bicarbonate 2.34 [1.29-3.40)]vs. 3.64 [2.70-4.58]micromol/min and glycoprotein 0.23 [0.15-0.31]vs. 0.35 [0.28-0.43] mg/min, P= 0.04 and P= 0.04, respectively). We conclude that changes in salivary bicarbonate and glycoprotein secretion related to H. pylori eradication do not promote the development of erosive esophagitis in duodenal ulcer patients. PMID:19222537

  6. Separation of bivalent anti-T cell immunotoxin from Pichia pastoris glycoproteins by borate anion exchange.

    PubMed

    Woo, Jung Hee; Neville, David M

    2003-08-01

    A major problem encountered in the large-scale purification of the bivalent anti-T cell immunotoxin, A-dmDT390-bisFv(G4S), from Pichia pastoris supernatants was the presence of host glycoproteins exhibiting similar charge, size, and hydrophobicity characteristics. We overcame this problem by employing borate anion exchange chromatography. The borate anion has an affinity for carbohydrates and imparts negative charges to these structures. We found that at a concentration of sodium borate between 50 and 100 mM, the nonglycosylated immunotoxin did not bind to Poros 50 HQ anion exchanger resin, but glycoproteins, including aggregates related to the immunotoxin, did. By using this property of the immunotoxin in the presence of sodium borate, we successfully developed a 3-step purification procedure: (i) Butyl-650M hydrophobic interaction chromatography, (ii) Poros 50 HQ anion exchange chromatography in the presence of borate, and (iii) HiTrap Q anion exchange chromatography. The final preparation exhibited a purity of greater than 98% and a yield of greater than 50% from the supernatant. Previously, boronic acid resins have been used to separate glycoproteins from proteins. However, combining borate anion with conventional anion exchange resins accomplishes the separation of the immunotoxin from glycoproteins and eliminates the need to evaluate nonstandard resins with respect to good manufacturing practice guidelines. PMID:12951782

  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. Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products.

    PubMed

    Anderson, Carrie L; Wang, Yang; Rustandi, Richard R

    2012-06-01

    CE-based methods have increasingly been applied to the analysis of a variety of different type proteins. One of those techniques is imaged capillary isoelectric focusing (icIEF), a method that has been used extensively in the field of protein-based drug development as a tool for product identification, stability monitoring, and characterization. It offers many advantages over the traditional labor-intensive IEF slab gel method and even standard cIEF with on-line detection technologies with regard to method development, reproducibility, robustness, and speed. Here, specific examples are provided for biopharmaceutical glycoprotein products such as mAbs, erythropoietin (EPO), and recombinant Fc-fusion proteins, though the technique can be adapted for many other therapeutic proteins. Applications of iCIEF using a Convergent Bioscience instrument (Toronto, Canada) with whole-field imaging technology are presented and discussed. These include a quick method to establish an identity test for many protein-based products, product release, and stability evaluation of glycoproteins with respect to charge heterogeneity under accelerated temperature stress, different pH conditions, and in different formulations. Finally, characterization of glycoproteins using this iCIEF technology is discussed with respect to biosimilar development, clone selection, and antigen binding. The data presented provide a "taste'' of what icIEF method can do to support the development of biopharmaceutical glycoprotein products from early clone screening for better product candidates to characterization of the final commercial products. PMID:22736354

  9. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion.

    PubMed

    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

  10. 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

  11. Ion Mobility-Mass Correlation Trend Line Separation of Glycoprotein Digests without Deglycosylation

    PubMed Central

    Li, Hongli; Bendiak, Brad; Siems, William F.; Gang, David R.; Hill, Herbert H.

    2013-01-01

    A high-throughput ion mobility mass spectrometer (IMMS) was used to rapidly separate and analyze peptides and glycopeptides derived from glycoproteins. Two glycoproteins, human α-1-acid glycoprotein and antithrombin III were digested with trypsin and subjected to electrospray traveling wave IMMS analysis. No deglycosylation steps were performed; samples were complex mixtures of peptides and glycopeptides. Peptides and glycosylated peptides with different charge states (up to 4 charges) were observed and fell on distinguishable trend lines in 2-D IMMS spectra in both positive and negative modes. The trend line separation patterns matched between both modes. Peptide sequence was identified based on the corresponding extracted mass spectra and collision induced dissociated (CID) experiments were performed for selected compounds to prove class identification. The signal-to-noise ratio of the glycopeptides was increased dramatically with ion mobility trend line separation compared to non-trend line separation, primarily due to selection of precursor ion subsets within specific mobility windows. In addition, isomeric mobility peaks were detected for specific glycopeptides. IMMS demonstrated unique capabilities and advantages for investigating and separating glycoprotein digests in this study and suggests a novel strategy for rapid glycoproteomics studies in the future. PMID:23914139

  12. 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

  13. IMMUNOHISTOCHEMICAL DETECTION OF P-GLYCOPROTEIN IN TELEOST TISSUES USING MAMMALIAN POLYCLONAL AND MONOCLONAL ANTIBODIES

    EPA Science Inventory

    Mammalian P-glycoprotein is a highly conserved 170 kD integral plasma membrane protein functioning as an energy dependent efflux pump of exogenous and endogenous lipophilic aromatic compounds entering the cell by diffusion. n this study, the tissue specificity of one polyclonal (...

  14. Expeditious Chemoenzymatic Synthesis of Homogeneous N-Glycoproteins Carrying Defined Oligosaccharide Ligands

    PubMed Central

    Ochiai, Hirofumi; Huang, Wei; Wang, Lai-Xi

    2009-01-01

    An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the transglycosylation activity of the endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A catalyzed transglycosylation using ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserves the natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon transglycosylation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily introduced at the N-pentasaccharide (Man3GlcNAc2) core using azido-containing Man3GlcNAc oxazoline as the donor substrate. The introduction of azido functionality permits further site-specific modifications of the resulting glycoproteins, as demonstrated by the successful attachment of two copies of αGal epitopes to ribonuclease B. This study reveals a broad substrate specificity of Endo-A for transglycosylation, and the chemoenzymatic method described here points to a new avenue for a quick access to various homogeneous N-glycoproteins for structure-activity relationship studies and for biomedical applications. PMID:18803385

  15. Development of Recombinant Newcastle Disease Viruses Expressing the Glycoprotein (G) of Avian Metapneumovirus as Bivalent Vaccines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using reverse genetics technology, Newcastle disease virus (NDV) LaSota strain-based recombinant viruses were engineered to express the glycoprotein (G) of avian metapneumovirus (aMPV), subtype A, B or C, as bivalent vaccines. These recombinant viruses were slightly attenuated in vivo, yet maintaine...

  16. Mutating Conserved Cysteines in the Alphavirus E2 Glycoprotein Causes Virus-Specific Assembly Defects

    PubMed Central

    Snyder, Anthony J.; Sokoloski, Kevin J.

    2012-01-01

    There are 80 trimeric, glycoprotein spikes that cover the surface of an alphavirus particle. The spikes, which are composed of three E2 and E1 glycoprotein heterodimers, are responsible for receptor binding and mediating fusion between the viral and host-cell membranes during entry. In addition, the cytoplasmic domain of E2 interacts with the nucleocapsid core during the last stages of particle assembly, possibly to aid in particle stability. During assembly, the spikes are nonfusogenic until the E3 glycoprotein is cleaved from E2 in the trans-Golgi network. Thus, a mutation in E2 potentially has effects on virus entry, spike assembly, or spike maturation. E2 is a highly conserved, cysteine-rich transmembrane glycoprotein. We made single cysteine-to-serine mutations within two distinct regions of the E2 ectodomain in both Sindbis virus and Ross River virus. Each of the E2 Cys mutants produced fewer infectious particles than wild-type virus. Further characterization of the mutant viruses revealed differences in particle morphology, fusion activity, and polyprotein cleavage between Sindbis and Ross River virus mutants, despite the mutations being made at corresponding positions in E2. The nonconserved assembly defects suggest that E2 folding and function is species dependent, possibly due to interactions with a virus-specific chaperone. PMID:22238319

  17. Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase

    SciTech Connect

    Yamaguchi, Yoshiki; Hirao, Takeshi; Sakata, Eri; Kamiya, Yukiko; Kurimoto, Eiji; Yoshida, Yukiko; Suzuki, Tadashi; Tanaka, Keiji; Kato, Koichi

    2007-10-26

    Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF{sup Fbs1} ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man{sub 3}GlcNAc{sub 2} by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol.

  18. Glycoprotein gene truncation in avian metapneumovirus subtype C isolates from the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The published glycoprotein (G) gene sequences of Avian metapneumovirus subtype-C (aMPV-C) isolated from domestic turkeys and wild bids in the United States (1996-2003) remain controversial in length. To explore the relationship between G gene size variation and the year of isolation and cell cultur...

  19. Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tunicamycins, streptovirudins, and corynetoxins are natural products that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg**2+ complex established as a transition state analog for hexosamine-1-phosphate:pren...

  20. 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

  1. Delayed Infection after Immunization with a Peptide from the Transmembrane Glycoprotein of the Feline Immunodeficiency Virus

    PubMed Central

    Richardson, J.; Moraillon, A.; Crespeau, F.; Baud, S.; Sonigo, P.; Pancino, G.

    1998-01-01

    Recent advances in the quantitative assessment of viral burden, by permitting the extension of criteria applied to assess the efficacy of vaccines from all-or-none protection to diminution of the viral burden, may allow the identification of original immunogens of value in combined vaccines. Peptides corresponding to three domains of the envelope glycoproteins of feline immunodeficiency virus that are recognized during natural infection were used to immunize cats. After challenge with a primary isolate of feline immunodeficiency virus, the development of acute infection was monitored by quantitative assessment of the viral burden in plasma and tissues by competitive reverse transcription-PCR, by measurement of the humoral response developed to viral components, and by lymphocyte subset analysis. Whereas immunization with two peptides derived from the surface glycoprotein had no effect on the early course of infection, immunization with a peptide derived from the transmembrane glycoprotein delayed infection, as reflected by a diminished viral burden in the early phase of primary infection and delayed seroconversion. This peptide, located in the membrane-proximal region of the extracellular domain, has homology to an epitope of human immunodeficiency virus type 1 recognized by a broadly neutralizing monoclonal antibody. These results suggest that lentivirus transmembrane glycoproteins share a determinant in the juxtamembrane ectodomain which could be of importance in the design of vaccines against AIDS. PMID:9499101

  2. Function and 3D Structure of the N-Glycans on Glycoproteins

    PubMed Central

    Nagae, Masamichi; Yamaguchi, Yoshiki

    2012-01-01

    Glycosylation is one of the most common post-translational modifications in eukaryotic cells and plays important roles in many biological processes, such as the immune response and protein quality control systems. It has been notoriously difficult to study glycoproteins by X-ray crystallography since the glycan moieties usually have a heterogeneous chemical structure and conformation, and are often mobile. Nonetheless, recent technical advances in glycoprotein crystallography have accelerated the accumulation of 3D structural information. Statistical analysis of “snapshots” of glycoproteins can provide clues to understanding their structural and dynamic aspects. In this review, we provide an overview of crystallographic analyses of glycoproteins, in which electron density of the glycan moiety is clearly observed. These well-defined N-glycan structures are in most cases attributed to carbohydrate-protein and/or carbohydrate-carbohydrate interactions and may function as “molecular glue” to help stabilize inter- and intra-molecular interactions. However, the more mobile N-glycans on cell surface receptors, the electron density of which is usually missing on X-ray crystallography, seem to guide the partner ligand to its binding site and prevent irregular protein aggregation by covering oligomerization sites away from the ligand-binding site. PMID:22942711

  3. Stable rescue of a glycoprotein gII deletion mutant of pseudorabies virus by glycoprotein gI of bovine herpesvirus 1.

    PubMed

    Kopp, A; Mettenleiter, T C

    1992-05-01

    Glycoproteins homologous to glycoprotein B (gB) of herpes simplex virus constitute the most highly conserved group of herpesvirus glycoproteins. This strong conservation of amino acid sequences might be indicative of a common functional role. Indeed, gB homologs have been implicated in the processes of viral entry and virus-mediated cell-cell fusion. Recently, we showed that pseudorabies virus (PrV) lacking the essential gB-homologous glycoprotein gII could be propagated on a cell line expressing the gB homolog of bovine herpesvirus 1, gI(BHV-1), leading to a phenotypic complementation of the gII defect (I. Rauh, F. Weiland, F. Fehler, G. Keil, and T.C. Mettenleiter, J. Virol. 65:621-631, 1991). However, this pseudotypic virus could still replicate only on complementing cell lines, thereby limiting experimental approaches to analyze the effects of the gB exchange in detail. We describe here the construction and isolation of a PrV recombinant, 9112C2, that lacks gII(PrV) but instead stably carries and expresses the gene encoding gI(BHV-1). The recombinant is able to replicate on noncomplementing cells with growth kinetics and final titers similar to those of its gII-positive wild-type PrV parent. Neutralization tests and immunoprecipitation analyses demonstrated incorporation of gI(BHV-1) into 9112C2 virions with concomitant absence of gII(PrV). Analysis of in vitro host ranges of wild-type PrV, BHV-1, and recombinant 9112C2 showed that in cells of pig, rabbit, canine, monkey, or human origin, the plating efficiency of 9112C2 was similar to that of its PrV parent. Exchange of gII(PrV) for gI(BHV-1) in recombinant 9112C2 or by phenotypic complementation of gII- PrV propagated on gI(BHV-1)-expressing cell lines resulted in penetration kinetics intermediate between those of wild-type PrV and BHV-1. In conclusion, we report the first isolation of a viral recombinant in which a lethal glycoprotein mutation has been rescued by a homologous glycoprotein of a different

  4. Modulation of a Glycoprotein Recognition System on Rat Hepatic Endothelial Cells by Glucose and Diabetes Mellitus

    PubMed Central

    Summerfield, John A.; Vergalla, John; Jones, E. Anthony

    1982-01-01

    The cellular location and carbohydrate specificities of a glycoprotein recognition system on rat hepatic sinusoidal cells have been determined. Purified preparations of endothelial, Kupffer, and parenchymal cells were prepared by collagenase liver perfusion, centrifugation on Percoll gradients, and centrifugal elutriation. 125I-labeled agalactoorosomucoid, an N-acetylglucosamine-terminated glycoprotein, was selectively taken up in vitro by endothelial cells. Uptake was shown to be protein dependent, calcium ion dependent, and saturable, and could be described by Michaelis-Menten kinetics (apparent Km 0.29 μM; apparent maximum velocity 4.8 pmol/h per 5 × 106 cells). Uptake was inhibited not only by N-acetylglucosamine, mannose, and mannan but also by glucose, fructose, and a glucose-albumin conjugate. Inhibition by glucose was competitive over a wide range of concentrations and was almost 100% at a glucose concentration of 56 mM. Fasting and the induction of diabetes mellitus prior to isolation of cells was associated with 60% reductions in the recovery of endothelial cells. Uptake by cells isolated from fasted rats was enhanced (apparent maximum velocity 14.3 pmol/h per 5 × 106 cells without change in the apparent Km). These observations suggest that fasting is associated with a marked increase in the mean number of glycoprotein receptors per endothelial cell isolated from normal rats. This effect of fasting could be due to upregulation of glycoprotein receptors on endothelial cells or to the selective isolation of a subpopulation of endothelial cells from fasted animals that bears more glycoprotein receptors per cell than does another subpopulation of these cells. In addition, in vivo studies of the fate of intravenously administered 125I-agalactoorosomucoid indicated that its rate of disappearance from plasma, hepatic accumulation, and catabolism were slower in diabetic than in normal rats. The results suggest that modulation of a carbohydrate

  5. 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

  6. 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

  7. 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.

  8. Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity.

    PubMed Central

    Cartron, J P; Blanchard, D

    1982-01-01

    Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins. Images Fig. 1. Fig. 2. PMID:6187337

  9. Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins.

    PubMed Central

    Veit, M; Reverey, H; Schmidt, M F

    1996-01-01

    We report remarkable differences in the fatty acid content of thioester-type acylated glycoproteins of enveloped viruses from mammalian cells. The E2 glycoprotein of Semliki Forest virus contains mainly palmitic acid like most other palmitoylated proteins analysed so far. However, the other glycoprotein (E1) of the same virus, as well as the HEF (haemagglutinin esterase fusion) glycoprotein of influenza C virus, are unique in this respect because they are acylated primarily with stearic acid. Comparative radiolabelling of uninfected cells with different fatty acids suggests that stearate may also be the prevailing fatty acid in some cellular acylproteins. To look for further differences between palmitoylated and stearoylated glycoproteins we characterized stearoylation in more detail. We identified the acylation site of HEF as a cysteine residue located at the boundary between the transmembrane region and the cytoplasmic tail. The attachment of stearate to HEF and E1 occurs post-translationally in a pre-Golgi compartment. Thus, stearoylated and palmitoylated proteins cannot be discriminated on the basis of the fatty acid linkage site or the intracellular compartment, where acylation occurs. However, stearoylated acylproteins contain a very short, positively charged cytoplasmic tail, whereas in palmitoylated proteins this molecular region is longer. Replacing the short cytoplasmic tail of stearoylated HEF with the long influenza A virus haemagglutinin (HA) tail in an HEF-HA chimera, and subsequent vaccinia T7 expression in CV-1 cells, yielded proteins with largely palmitic acid bound. The reverse chimera, HA-HEF with a short cytoplasmic tail was not fatty acylated at all during expression, indicating that conformational or topological constraints control fatty acid transfer. PMID:8761467

  10. 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

  11. 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

  12. 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.

  13. Putative glycoprotein and glycolipid polymorphonuclear leukocyte receptors for the Actinomyces naeslundii WVU45 fimbrial lectin.

    PubMed Central

    Sandberg, A L; Ruhl, S; Joralmon, R A; Brennan, M J; Sutphin, M J; Cisar, J O

    1995-01-01

    Recognition of receptors on sialidase-treated polymorphonuclear leukocytes (PMNs) by the Gal/GalNAc lectin associated with the type 2 fimbriae of certain strains of actinomyces results in activation of the PMNs, phagocytosis, and destruction of the bacteria. In the present study, plant lectins were utilized as probes to identify putative PMN receptors for the actinomyces lectin. The Gal-reactive lectin from Ricinus communis (RCAI), the Gal/GalNAc-reactive lectins from R. communis (RCAII) and Bauhinia purpurea (BPA), as well as the Gal beta 1-3GalNAc-specific lectins from Arachis hypogaea (PNA) and Agaricus bisporus (ABA) inhibited killing of Actinomyces naeslundii WVU45 by sialidase-treated PMNs. These five lectins detected a 130-kDa surface-labeled glycoprotein on nitrocellulose transfers of PMN extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This glycoprotein was revealed only after treatment of the transfers with sialidase, a condition analogous to the sialidase dependence of the lectin-mediated biological responses of the PMNs to the actinomyces. The mannose-reactive lectin concanavalin A did not inhibit killing of the actinomyces and failed to detect the 130-kDa glycoprotein but did block PMN-dependent killing of Escherichia coli B, a bacterium that possesses mannose-sensitive fimbriae. Therefore, the PMN glycoprotein receptor for A. naeslundii is clearly distinct from those recognized by E. coli. Two major putative glycolipid receptors were also identified by actinomyces and RCAI overlays on sialidase-treated thin-layer chromatograms of PMN gangliosides. Thus, both a 130-kDa glycoprotein and certain gangliosides are implicated in the attachment of the actinomyces to PMNs. PMID:7790078

  14. Preliminary characterization of an epitope involved in neutralization and cell attachment that is located on the major bovine rotavirus glycoprotein.

    PubMed Central

    Sabara, M; Gilchrist, J E; Hudson, G R; Babiuk, L A

    1985-01-01

    The 38,200-molecular weight (unreduced)/41,900-molecular-weight (reduced) glycoprotein of bovine rotavirus, isolate C486, was identified as the major neutralizing antigen. This glycoprotein as well as the corresponding glycoprotein of another bovine rotavirus serotype also specifically attached to cell monolayers under normal conditions for virus adsorption in vitro. Further support for this glycoprotein being directly responsible for virus attachment to cells was that (i) infectious virus of both serotypes could compete with the C486 glycoprotein for cell surface receptors, and (ii) neutralizing monospecific antiserum and neutralizing monoclonal antibodies directed toward the glycoprotein could block this virus-cell interaction. Preliminary epitope mapping of the glycoprotein with monoclonal antibodies further localized the neutralization-adsorption domain to a peptide with an approximate molecular weight of 14,000. The effect of two protein modifications, glycosylation and disulfide bridging, on the reactivity of this peptide with antibodies and cell surface receptors was investigated. It was demonstrated that, whereas glycosylation did not appear to affect these reactivities, disulfide bridging seemed to be essential. Images PMID:2578197

  15. Aberrant trafficking of hepatitis B virus glycoproteins in cells in which N-glycan processing is inhibited

    PubMed Central

    Lu, Xuanyong; Mehta, Anand; Dadmarz, Mitra; Dwek, Raymond; Blumberg, Baruch S.; Block, Timothy M.

    1997-01-01

    The role of N-glycan trimming in glycoprotein fate and function is unclear. We have recently shown that hepatitis B virus (HBV) DNA is not efficiently secreted from cells in which α-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidase-inhibited cells, viral DNA, accompanied by envelope and core proteins, most likely accumulate within lysosomal compartments. Pulse–chase experiments show that although the viral glycoproteins (L, M, and S) are dysfunctional, in the sense that they do not mediate virion egress and are not efficiently secreted from the cell, they all still leave the endoplasmic reticulum (ER). Surprisingly, however, the glycoproteins retained within the cell were not rapidly degraded, appearing as aggregates, enriched for L and M, with intracellular half-lives exceeding 20 h. Moreover, by 24 h after synthesis, a substantial fraction of the detained glycoproteins appeared to return to the ER, although a considerable amount was also found in the lysosomes. To our knowledge, this is the first report that shows, as a consequence of inhibiting glycosylation processing, certain glycoproteins (i) become dysfunctional and aggregate, yet still depart from the ER, and (ii) have extended rather than shortened half-lives. Taken together, these data suggest that proper intracellular routing of HBV glycoproteins requires ER glucosidase function. It is hypothesized that failure to process N-glycan causes HBV glycoproteins to aggregate and that impaired protein–protein interactions and trafficking are the result of misfolding. PMID:9122203

  16. 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

  17. Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells

    PubMed Central

    Chang, Haigang; Song, Shanshan; Chen, Zhongcan; Wang, Yaxiao; Yang, Lujun; Du, Mouxuan; Ke, Yiquan; Xu, Ruxiang; Jin, Baozhe; Jiang, Xiaodan

    2014-01-01

    Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells. PMID:25206849

  18. The measles virus (MV) glycoproteins interact with cellular chaperones in the endoplasmic reticulum and MV infection upregulates chaperone expression.

    PubMed

    Bolt, G

    2001-01-01

    The present study examines the coprecipitation of measles virus (MV) glycoproteins with host cell endoplasmic reticulum (ER) chaperone proteins. Both the haemagglutinin (H) and fusion (F) glycoproteins interacted with calnexin and GRP78, whereas interaction with calreticulin was only demonstrated for the H glycoprotein. The alpha-glucosidase inhibitor castanospermine reduced and delayed the association of F proteins with calnexin. We have previously shown that alpha-glucosidase activity is important for the functionality and antigenicity of the MV F glycoprotein and for release of MV particles from infected cells. Thus, interaction with calnexin appears vital for processing of nascent MV F protein into its functional conformation. In contrast to many other viral glycoproteins, a substantial proportion of the pulsed MV glycoproteins remained associated with ER chaperones for more than 2(1/2) h. Thus, the slow and incomplete migration of MV glycoproteins to the cell surface may result from their retention by ER chaperones, probably due to malfolding. MV infection upregulated the cellular expression of calreticulin and GRP78 and also increased their presence at the cell surface. The chaperone proteins are involved in a wide range of cellular processes, and their induction by MV may play a role for the pathogenesis of measles and its sequelae. PMID:11765911

  19. 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

  20. 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.

  1. Pulse-chase analysis of N-linked sugar chains from glycoproteins in mammalian cells.

    PubMed

    Avezov, Edward; Ron, Efrat; Izenshtein, Yana; Adan, Yosef; Lederkremer, Gerardo Z

    2010-01-01

    Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-(3)H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2

  2. Association and release of the major intrinsic membrane glycoprotein from peripheral nerve myelin.

    PubMed Central

    Poduslo, J F; Yao, J K

    1985-01-01

    Hypo-osmotic homogenization of the endoneurium from the adult-rat sciatic nerve and subsequent evaluation of the 197 000 g aqueous supernatant by sodium dodecyl sulphate pore-gradient electrophoresis (SDS-p.g.e.) revealed a release of the major glycoprotein (P0) (29 000 Mr) from peripheral nerve myelin. Immunological verification of the presence of this asparagine-linked glycoprotein in the aqueous supernatant was obtained by immune overlay after SDS-p.g.e. and electrophoretic transfer to nitrocellulose using anti-P0 gamma-globulin followed by autoradiographic detection with 125I-protein A. A comparison of successive hypo- and iso-osmotic extractions of the endoneurium revealed that the hypo-osmotic extraction released increasing amounts of P0 into the supernatant fraction, whereas the iso-osmotic treatment revealed lower levels of P0 extracted from the myelin and lesser amounts with each successive extraction. Three successive hypo-osmotic extractions resulted in a 2.0-, 2.9-, and 9.5-fold increase in the amount of P0 released compared with the successive iso-osmotic extractions. Although these results suggest that this major myelin glycoprotein has properties similar to those of extrinsic membrane proteins, temperature-dependent phase-partitioning experiments with Triton X-114 revealed that this glycoprotein is recovered in the detergent-enriched lower phase. These results indicate that this major myelin glycoprotein is an amphipathic integral membrane protein with a distinct hydrophobic domain and yet has solubility characteristics typical of an extrinsic membrane protein. P0 labelled in vitro with [3H]mannose could be immunoprecipitated from the aqueous supernatant with anti-P0 gamma-globulin by centrifugation at 197000g without the addition of second antibody or protein A. Analysis of such an immune precipitate after incorporation in vitro with [14C]acetate to label endoneurial lipids revealed that all major endoneurial lipid classes contained radioactive

  3. Effects of freezing on the estimated amounts of Tamm--Horsfall glycoprotein in urine, as determined by radioimmunoassay.

    PubMed Central

    Goodall, A A; Marshall, R D

    1980-01-01

    Freeze-drying or freezing of salt-free solutions of human Tamm--Horsfall glycoprotein appeared to lead to changes in the structure of the latter, changes that increased its ability to bind with antibody raised, in rabbits, against it. This alteration in avidity of the glycoprotein was observed irrespective of whether antiserum was raised against freeze-dried or non-frozen antigen. The implications of this finding for the radioimmunoassay of the glycoprotein in urine samples were studied. Appropriate treatment for urine samples, before assay, was devised. The amount of Tamm--Horsfall glycoprotein excreted was shown to range from 30 to 138 mg in normal males and 43 to 126 mg in normal females per 24 h. PMID:7213344

  4. 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

  5. The effect of ammonium chloride and tunicamycin on the glycoprotein content and infectivity of herpes simplex virus type 1.

    PubMed

    Kousoulas, K G; Bzik, D J; DeLuca, N; Person, S

    1983-03-01

    Infectious virions of MP, a syncytial strain of herpes simplex virus type 1, are formed in the presence of 50 mM NH4Cl. Underglycosylated virion glycoproteins are synthesized in infected cells and are incorporated into virions in the presence of the same concentration of NH4Cl. We conclude that fully glycosylated glycoproteins are not required for viral infectivity. Virus particles, deficient in glycosylated glycoproteins, are assembled in the presence of tunicamycin but they are not infectious. The decrease in infectivity could be due to the decreased amount of the gB or possibly other peptides and/or to the lack of the high-mannose saccharides of precursor glycoproteins. PMID:6301148

  6. Effect of ammonium chloride and tunicamycin on the glycoprotein content and infectivity of herpes simplex virus type 1

    SciTech Connect

    Kousoulas, K.G.; Bzik, D.J.; DeLuca, N.; Person, S.

    1983-01-01

    Infectious virions of MP, a syncytial strain of herpes simplex virus type 1, are formed in the presence of 50 mM NH/sub 4/Cl. Underglycosylated virion glycoproteins are synthesized in infected cells and are incorporated into virions in the presence of the same concentration of NH/sub 4/Cl. We conclude that fully glycosylated glycoproteins are not required for viral infectivity. Virus particles, deficient in glycosylated glycoproteins, are assembled in the presence of tunicamycin but they are not infectious. The decrease in infectivity could be due to the decreased amount of the gB or possibly other peptides and/or to the lack of the high-mannose saccharides of precursor glycoproteins. 32 references, 4 figures.

  7. Pregnancy - associated glycoproteins as a new diagnostic tool in cattle reproduction.

    PubMed

    Gajewski, Z; Pertajitis, M; Sousa, N; Beckers, J; Pawliński, B; Janett, F

    2009-12-01

    Pregnancy-associated glycoproteins (PAGs) are powerful pregnancy markers in domestic cattle. These proteins are expressed in mono- and binucleate trophoblast cells from the first days of gestation until calving. Different molecules were identified as being expressed at various stages of pregnancy. However, up to date, their functions and activities during pregnancy were not yet established. Specific RIA tests were developed (classic and alternative RIA) and used to measure the concentration of these glycoproteins in blood during gestation and the postpartum period in cattle. In maternal blood, PAGs rise to detectable levels from days 24 to 28 after fertilization. A recent study indicated that PAGs can also be detected in milk samples. However, concentrations in milk are much lower when compared to those of plasma. PMID:19946848

  8. 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

  9. The morphology of porcine oocytes is associated with zona pellucida glycoprotein transcript contents.

    PubMed

    Jackowska, Marta; Kempisty, Bartosz; Antosik, Paweł; Bukowska, Dorota; Budna, Joanna; Lianeri, Margarita; Rosińska, Ewa; Woźna, Magdalena; Jagodziński, Paweł P; Jaśkowski, Jedrzej M

    2009-03-01

    We hypothesized that oocyte morphology may be associated with the accumulation of specific mRNAs encoding proteins responsible for the gamete fertilization ability. Therefore, the aim of the study was to evaluate the transcript levels of porcine zona pellucida (pZP1, pZP2, pZP3 and pZP4) glycoproteins in oocytes classified by a four-grade morphological scale (I-IV) accounting for either a homogeneous cytoplasm and a complete cumulus oophorus (grade I) or a heterogenous cytoplasm and decreased number of cumulus layers in the other grades (II, III and IV). We observed a significant increase of all investigated pZP glycoprotein mRNAs in grade I oocytes as compared to other grades (p<0.05). Our observations suggest that porcine oocyte morphology is associated with pZP transcript contents and may be related to an increased fertilization ability of higher quality oocytes. PMID:19352420

  10. Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carteri

    PubMed Central

    Wenzl, Stephan; Thym, Detlef; Sumper, Manfred

    1984-01-01

    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 1̇1̇2). 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. ImagesFig. 2.Fig. 3.Fig. 4.Fig. 6.Fig. 7. PMID:16453512

  11. A 138-kDa glycoprotein from Dictyostelium membranes with folate deaminase and folate binding activity.

    PubMed

    Greiner, R A; Jacobs-Krahnen, D; Mutzel, R; Malchow, D; Wurster, B

    1992-03-15

    A 138-kDa glycoprotein comprising folate deaminase activity was purified to apparent homogeneity from membranes of Dictyostelium discoideum. Deaminase activity could be effectively inhibited by p-chloromercuriphenylsulfonate. This treatment protected folate from deamination and thus allowed investigation of folate binding to deaminase fractions. Two types of folate binding sites, differing in affinity and specificity, were detected on the folate deaminase glycoprotein. One type displays high affinity and binds folate stronger than N10-methylfolate. This binding site appears to be identical with the catalytic site of folate deaminase. The other type of binding site shows lower affinity but prefers N10-methylfolate relative to folate. A similar preference for N10-methylfolate was observed in chemotaxis tests pointing to the possibility that the second type of binding site is involved in chemotactic perception of folate compounds. Folate perception and deamination could thus be performed by activities residing on the same polypeptide. PMID:1544893

  12. Finding the sweet spot: Assembly and Glycosylation of the Dystrophin-Associated Glycoprotein Complex

    PubMed Central

    Townsend, DeWayne

    2014-01-01

    The dystrophin-associated glycoprotein complex (DGC) is a collection of glycoproteins that are essential for the normal function of striated muscle and many other tissues. Recent genetic studies have implicated the components of this complex in over a dozen forms of muscular dystrophy. Furthermore, disruption of the DGC has been implicated in many forms of acquired disease. This review aims to summarize the current state of knowledge regarding the processing and assembly of dystrophin associated proteins with a focus primarily on the dystroglycan heterodimer and the sarcoglycan complex. These proteins form the transmembrane portion of the DGC and undergo a complex multi-step processing with proteolytic cleavage, differential assembly, and both N- and O-glycosylation. The enzymes responsible for this processing and a model describing the sequence and subcellular localization of these events are discussed. PMID:25125182

  13. Simultaneous assay of neutral sugars and amino sugars by an automatic sugar analyzer: applications to glycoproteins.

    PubMed

    Kellich, G; Ziegler, D

    1975-04-01

    The simultaneous assay of neutral sugars and amino sugars commonly found in glycoproteins is described. The automatic sugar analyzer used for the determination is based on the ion-exchange chromatography of sugar-borate complexes on a strong anion-exchange resin. The sugars are identified with the orcinol/sulfuric acid reagent. While less than 40 nmol of mannose, fucose, galactose, glucose, xylose, or arabinose is sufficient for analysis at least 200 nmol mannosamine, glucosamine, or galactosamine is required; acidic monosaccharides cannot be determined. The technique of sugar analysis is applied to structural studies on natural compounds, e.g. the monosaccharide composition of lichenan and the carbohydrate moiety of the glycoproteins ovomucoid and Collocalia mucoid. PMID:1150155

  14. [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

  15. Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

    PubMed Central

    Letarte-Muirhead, M; Barclay, A N; Williams, A F

    1975-01-01

    The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight. Images PLATE 4 PLATE 1 PLATE 2 PLATE 3 PMID:56177

  16. Perspectives on the future of platelet glycoprotein IIb/IIIa blockade therapy.

    PubMed Central

    Tcheng, J E

    1998-01-01

    During the past 2 decades, advances in molecular and cellular biology have greatly expanded our understanding of the critical role of platelets in the pathogenesis of acute coronary syndromes. This work has suggested that aggressive platelet inhibition might reduce cardiovascular morbidity and mortality rates beyond the reductions already achieved with aspirin and other conventional therapies. Researchers in basic science laboratories have identified a specific platelet receptor, the glycoprotein IIb/IIIa integrin, which serves as the mediator, or final common pathway, leading to platelet aggregation. This glycoprotein receptor is a logical target for the development of antagonists to inhibit thrombosis. A number of these antagonists have been tested in clinical trials involving coronary intervention, unstable angina, and acute myocardial infarction. Herein, I present some of the rationale behind the investigation of these agents, as well as some issues to be considered with regard to the future of this exciting new class of drugs. PMID:9566064

  17. 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

  18. Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species.

    PubMed Central

    Bruschke, C J; Hulst, M M; Moormann, R J; van Rijn, P A; van Oirschot, J T

    1997-01-01

    Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses. PMID:9261392

  19. 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. PMID:21693135

  20. Evidence for mucin-like glycoproteins that tether sporozoites of Cryptosporidium parvum to the inner surface of the oocyst wall.

    PubMed

    Chatterjee, Anirban; Banerjee, Sulagna; Steffen, Martin; O'Connor, Roberta M; Ward, Honorine D; Robbins, Phillips W; Samuelson, John

    2010-01-01

    Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls. PMID:19949049

  1. The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line

    PubMed Central

    Hudson, James E.; Johnson, Terry C.

    1977-01-01

    When monolayer cultures of neuroblastoma N2a cells were prelabelled with [3H]fucose to steady state, and then reincubated in complete medium in the presence of unlabelled 40mm-l-fucose, there was a rapid metabolism of fucosylated cellular macromolecules and the specific radioactivity of the acid-insoluble material decreased by 22% within 2h. After this period of time the remaining radioactive glycoproteins appeared to be more stable and the rate of loss of specific radioactivity markedly decreased. Since fucose is known to be associated predominantly with plasma-membrane components, the analysis of fucosylated glycoproteins was characterized in plasma-membrane fractions by polyacrylamide-gel electrophoresis. Two experimental approaches were used to measure glycoprotein degradation and turnover in the cell-surface membranes. In one set of experiments, with a similar incubation procedure to that used with intact cells, three membrane components were rapidly degraded (150000, 130000 and 48000 daltons), but another surface glycoprotein (68000 daltons) appeared to be more slowly metabolized than the mean rate of glycoprotein degradation. The relationship of the degradation of membrane glycoproteins to their turnover was analysed by dual-label experiments that used both [14C]fucose and [3H]fucose. Glycoproteins of the surface membrane of neuroblastoma cells were found to turn over at heterogeneous rates. The components mentioned above that exhibited significantly rapid rates of degradation, were also shown to turn over more rapidly than the average surface component. In addition to the membrane components detected by the use of only [3H]fucose, dual-label experiments illustrated that numerous surface glycoproteins were metabolized more rapidly or slowly than most of the cell-surface constituents. PMID:911319

  2. Sub 2-μm macroporous silica particles derivatized for enhanced lectin affinity enrichment of glycoproteins.

    PubMed

    Mann, Benjamin F; Mann, Amanda K P; Skrabalak, Sara E; Novotny, Milos V

    2013-02-01

    A new, mechanically stable silica microparticle with macrosized internal pores (1.6 μm particles with 100 nm pores) has been developed for chromatography. The particles are characterized by an extensive network of interconnected macropores with a high intraparticle void volume, as observed by transmission electron microscopy (TEM). They are synthesized by an aerosol assembly technique called ultrasonic spray pyrolysis (USP). The particles have a high surface area for a macroporous material, ∼200 m(2)/g, making them suitable for large biomolecular separations. To demonstrate their potential for bioseparations, they have been functionalized with lectins for affinity enrichment of glycoproteins. The material was derivatized with two lectins, Concanavalin A (Con A) and Aleuria aurantia lectin (AAL), and binding properties were tested with standard glycoproteins. The columns exhibited excellent binding capacities for microaffinity enrichment: Con A was able to bind 75 μg of a standard glycoprotein in a 50 × 1 mm column. Following initial tests, the lectin microcolumns were utilized for enrichment of glycoproteins from 1 μL volumes of blood serum samples, performed in triplicate for each lectin. The enriched serum fractions were subjected to side-by-side glycomic and glycoproteomic profiling analyses with mass spectrometry to show that the new particles offer excellent sensitivity for microscale analyses of precious biological sample materials. The unique combination of the macroporous architecture and small particle diameter suggests the material may have advantages for conventional modes of chromatographic separation of macromolecules in an ultra-high-pressure liquid chromatography (UHPLC) format. PMID:23278114

  3. Encoding Asymmetry of the N-Glycosylation Motif Facilitates Glycoprotein Evolution

    PubMed Central

    Williams, Ryan; Mohammad, Naveed; Ho, Chi Yip; Li, Carey F.; Chang, Belinda S. W.; Demetriou, Michael; Dennis, James W.

    2014-01-01

    Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions) and site multiplicity (backup and status quo) might be revealed by a phylogenic examination of glycoproteins and NXS/T(X≠P) N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A)-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A) content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A) is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A)-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have contributed to

  4. A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells

    PubMed Central

    Mabashi-Asazuma, Hideaki; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

    2014-01-01

    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. PMID:24362443

  5. 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.

  6. Glycoprotein biomarker panel for pancreatic cancer discovered by quantitative proteomics analysis.

    PubMed

    Nie, Song; Lo, Andy; Wu, Jing; Zhu, Jianhui; Tan, Zhijing; Simeone, Diane M; Anderson, Michelle A; Shedden, Kerby A; Ruffin, Mack T; Lubman, David M

    2014-04-01

    Pancreatic cancer is a lethal disease where specific early detection biomarkers would be very valuable to improve outcomes in patients. Many previous studies have compared biosamples from pancreatic cancer patients with healthy controls to find potential biomarkers. However, a range of related disease conditions can influence the performance of these putative biomarkers, including pancreatitis and diabetes. In this study, quantitative proteomics methods were applied to discover potential serum glycoprotein biomarkers that distinguish pancreatic cancer from other pancreas related conditions (diabetes, cyst, chronic pancreatitis, obstructive jaundice) and healthy controls. Aleuria aurantia lectin (AAL) was used to extract fucosylated glycoproteins and then both TMT protein-level labeling and label-free quantitative analysis were performed to analyze glycoprotein differences from 179 serum samples across the six different conditions. A total of 243 and 354 serum proteins were identified and quantified by label-free and TMT protein-level quantitative strategies, respectively. Nineteen and 25 proteins were found to show significant differences in samples between the pancreatic cancer and other conditions using the label-free and TMT strategies, respectively, with 7 proteins considered significant in both methods. Significantly different glycoproteins were further validated by lectin-ELISA and ELISA assays. Four candidates were identified as potential markers with profiles found to be highly complementary with CA 19-9 (p < 0.001). Obstructive jaundice (OJ) was found to have a significant impact on the performance of every marker protein, including CA 19-9. The combination of α-1-antichymotrypsin (AACT), thrombospondin-1 (THBS1), and haptoglobin (HPT) outperformed CA 19-9 in distinguishing pancreatic cancer from normal controls (AUC = 0.95), diabetes (AUC = 0.89), cyst (AUC = 0.82), and chronic pancreatitis (AUC = 0.90). A marker panel of AACT, THBS1, HPT, and CA 19

  7. Autophagy and the Effects of Its Inhibition on Varicella-Zoster Virus Glycoprotein Biosynthesis and Infectivity

    PubMed Central

    Buckingham, Erin M.; Carpenter, John E.; Jackson, Wallen

    2014-01-01

    Autophagy and the effects of its inhibition or induction were investigated during the entire infectious cycle of varicella-zoster virus (VZV), a human herpesvirus. As a baseline, we first enumerated the number of autophagosomes per cell after VZV infection compared with the number after induction of autophagy following serum starvation or treatment with tunicamycin or trehalose. Punctum induction by VZV was similar in degree to punctum induction by trehalose in uninfected cells. Treatment of infected cells with the autophagy inhibitor 3-methyladenine (3-MA) markedly reduced the viral titer, as determined by assays measuring both cell-free virus and infectious foci (P < 0.0001). We next examined a virion-enriched band purified by density gradient sedimentation and observed that treatment with 3-MA decreased the amount of VZV gE, while treatment with trehalose increased the amount of gE in the same band. Because VZV gE is the most abundant glycoprotein, we selected gE as a representative viral glycoprotein. To further investigate the role of autophagy in VZV glycoprotein biosynthesis as well as confirm the results obtained with 3-MA inhibition, we transfected cells with ATG5 small interfering RNA to block autophagosome formation. VZV-induced syncytium formation was markedly reduced by ATG5 knockdown (P < 0.0001). Further, we found that both expression and glycan processing of VZV gE were decreased after ATG5 knockdown, while expression of the nonglycosylated IE62 tegument protein was unchanged. Taken together, our cumulative results not only documented abundant autophagy within VZV-infected cells throughout the infectious cycle but also demonstrated that VZV-induced autophagy facilitated VZV glycoprotein biosynthesis and processing. PMID:24198400

  8. Zinc supplementation ameliorates glycoprotein components and oxidative stress changes in the lung of streptozotocin diabetic rats.

    PubMed

    Sacan, Ozlem; Turkyilmaz, Ismet Burcu; Bayrak, Bertan Boran; Mutlu, Ozgur; Akev, Nuriye; Yanardag, Refiye

    2016-04-01

    Zinc (Zn) is a component of numerous enzymes that function in a wide range of biological process, including growth, development, immunity and intermediary metabolism. Zn may play a role in chronic states such as cardiovascular disease and diabetes mellitus. Zn acts as cofactor and for many enzymes and proteins and has antioxidant, antiinflammatory and antiapoptotic effects. Taking into consideration that lung is a possible target organ for diabetic complications, the aim of this study was to investigate the protective role of zinc on the glycoprotein content and antioxidant enzyme activities of streptozotocin (STZ) induced diabetic rat tissues. Female Swiss albino rats were divided into four groups. Group I, control; Group II, control + zinc sulfate; Group III, STZ-diabetic; Group IV, diabetic + zinc sulfate. Diabetes was induced by intraperitoneal injection of STZ (65 mg/kg body weight). Zinc sulfate was given daily by gavage at a dose of 100 mg/kg body weight every day for 60 days to groups II and IV. At the last day of the experiment, rats were sacrificed, lung tissues were taken. Also, glycoprotein components, tissue factor (TF) activity, protein carbonyl (PC), advanced oxidative protein products (AOPP), hydroxyproline, and enzyme activities in lung tissues were determined. Glycoprotein components, TF activity, lipid peroxidation, non enzymatic glycation, PC, AOPP, hydroxyl proline, lactate dehydrogenase, catalase, superoxide dismutase, myeloperoxidase, xanthine oxidase, adenosine deaminase and prolidase significantly increased in lung tissues of diabetic rats. Also, glutathione levels, paraoxonase, arylesterase, carbonic anhydrase, and Na(+)/K(+)- ATPase activities were decreased. Administration of zinc significantly reversed these effects. Thus, the study indicates that zinc possesses a significantly beneficial effect on the glycoprotein components and oxidant/antioxidant enzyme activities. PMID:26817646

  9. Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei

    PubMed Central

    Zarschler, Kristof; Janesch, Bettina; Kainz, Birgit; Ristl, Robin; Messner, Paul; Schäffer, Christina

    2015-01-01

    The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051T possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95 kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051T cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix. PMID:20513375

  10. Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei.

    PubMed

    Zarschler, Kristof; Janesch, Bettina; Kainz, Birgit; Ristl, Robin; Messner, Paul; Schäffer, Christina

    2010-07-01

    The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T) possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051(T) cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix. PMID:20513375

  11. Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit.

    PubMed

    Reid, P E; Walker, D C; Terpin, T; Owen, D A

    1988-10-01

    Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundant O-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars with vicinal diols whose periodate oxidation is prevented by an O-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisable vicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have an O-acyl substituent located at position C-8 or C-9 (or which have two or three side chain O-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative. PMID:2464561

  12. Effect of fluoride exposure on serum glycoprotein pattern and sialic acid level in rabbits.

    PubMed

    Ciftci, Gulay; Cenesiz, Sena; Yarim, Gul Fatma; Nisbet, Ozlem; Nisbet, Cevat; Cenesiz, Metin; Guvenc, Dilek

    2010-01-01

    This study describes the effects of fluoride exposure on the protein profile, glycoprotein pattern, and total sialic acid concentration of serum in rabbits. For this aim; 20 healthy New Zealand rabbits were used. The rabbits were divided into two equal groups each with ten animals according to their weighing: control group and experimental group. The rabbits in control group were given drinking tap water containing 0.29 mg/l sodium fluoride and experimental group received the same tap water to which was added 40 mg/l sodium fluoride for 70 days. Blood samples were taken from each rabbit on day 70. Serum fluoride concentrations were measured by a fluoride-specific ion electrode in serum. The fluoride levels in the serum were found as 18.4 (+/-1.58) microg/L in control and 301.3 (+/-52.18) microg/L in fluoride exposed rabbits. The sialic acid levels were found as 69.2 (+/-0.32) mg/dL in control and 43.4 (+/-0.13) mg/dL in fluoride exposed group. The electrophoretic patterns of serum proteins, glycoproteins, and total sialic acid concentration were determined. Fifteen different protein fractions with molecular weights ranging from 22 to 249 kDa were displayed in the serum protein electrophoretic gel of both groups. The raw concentrations of the protein fractions decreased in fluoride exposed rabbits as compared with the control rabbits. The serum glycoprotein pattern revealed seven major protein bands from 47 to 167 kDa in experimental and control groups. The slight decrease of raw concentration of the protein bands in glycoprotein pattern of serum was observed in fluoride toxication comparing to control. The results suggest that serum TSA determination and serum protein electrophoresis can be used to evaluate prognosis of fluoride exposure as a supplementary laboratory test in combination with clinical and other laboratory findings of fluorosis. PMID:19904501

  13. 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.

  14. Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates

    PubMed Central

    Benyair, Ron; Ogen-Shtern, Navit; Mazkereth, Niv; Shai, Ben; Ehrlich, Marcelo; Lederkremer, Gerardo Z.

    2015-01-01

    Endoplasmic reticulum α1,2 mannosidase I (ERManI), a central component of ER quality control and ER-associated degradation (ERAD), acts as a timer enzyme, modifying N-linked sugar chains of glycoproteins with time. This process halts glycoprotein folding attempts when necessary and targets terminally misfolded glycoproteins to ERAD. Despite the importance of ERManI in maintenance of glycoprotein quality control, fundamental questions regarding this enzyme remain controversial. One such question is the subcellular localization of ERManI, which has been suggested to localize to the ER membrane, the ER-derived quality control compartment (ERQC), and, surprisingly, recently to the Golgi apparatus. To try to clarify this controversy, we applied a series of approaches that indicate that ERManI is located, at the steady state, in quality control vesicles (QCVs) to which ERAD substrates are transported and in which they interact with the enzyme. Both endogenous and exogenously expressed ERManI migrate at an ER-like density on iodixanol gradients, suggesting that the QCVs are derived from the ER. The QCVs are highly mobile, displaying dynamics that are dependent on microtubules and COP-II but not on COP-I vesicle machinery. Under ER stress conditions, the QCVs converge in a juxtanuclear region, at the ERQC, as previously reported. Our results also suggest that ERManI is turned over by an active autophagic process. Of importance, we found that membrane disturbance, as is common in immunofluorescence methods, leads to an artificial appearance of ERManI in a Golgi pattern. PMID:25411339

  15. 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. PMID:21960768

  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. 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.

  18. 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

  19. Purification and physical chemical characterization of 23S glycoprotein from sea urchin (Anthocidaris crassispina) eggs.

    PubMed

    Giga, Y; Ikai, A

    1985-07-01

    A large glycoprotein with a sedimentation coefficient, S(0)20,w, of 23.3S was purified to homogeneity from sea urchin eggs (Anthocidaris crassispina) by gel filtration on Sepharose CL-4B and ion-exchange chromatography on DEAE-cellulose. The molecular weight of the protein was 700,000 as determined by sedimentation equilibrium. On polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS) it showed a single band with an apparent molecular weight of 180,000 or 360,000 in the presence or absence of 2-mercaptoethanol, respectively. The protein consisted of four polypeptides of equal molecular weight, which were disulfide bonded in pairs. Its carbohydrate content as determined by the phenol-sulfuric acid method was 20% of the total weight. The amino acid and carbohydrate compositions, circular dichroic spectrum and electron microscopic image are also presented. The protein showed many structural similarities with the previously purified major glycoprotein (MCP) in the coelomic fluid of the same animal in addition to being immunologically cross reactive with it. However, the two proteins were distinct glycoproteins. Their biological functions have not been identified. PMID:4044553

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. 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. PMID:25296505

  5. Virion envelope glycoproteins as epidermiological markers of Venezuelan encephalitis virus isolates.

    PubMed Central

    Wiebe, M E; Scherer, W F

    1980-01-01

    Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations. PMID:7372798

  6. Recent Advances in the Mass Spectrometric Analysis of Glycoproteins: Theoretical Considerations

    PubMed Central

    Lazar, Iulia M.; Lazar, Alexandru C.; Cortes, Diego F.; Kabulski, Jarod L.

    2011-01-01

    Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large-scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large-scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in mass spectrometry (MS) instrumentation, and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow-up to this work, we review innovative capillary and microfluidic-MS workflows for the identification, sequencing, and characterization of glycoconjugates. PMID:21171109

  7. 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

  8. Enhancement of feline immunodeficiency virus infection after immunization with envelope glycoprotein subunit vaccines.

    PubMed Central

    Siebelink, K H; Tijhaar, E; Huisman, R C; Huisman, W; de Ronde, A; Darby, I H; Francis, M J; Rimmelzwaan, G F; Osterhaus, A D

    1995-01-01

    Cats were immunized three times with different recombinant feline immunodeficiency virus (FIV) candidate vaccines. Recombinant vaccinia virus (rVV)-expressed envelope glycoprotein with (vGR657) or without (vGR657 x 15) the cleavage site and an FIV envelope bacterial fusion protein (beta-Galactosidase-Env) were incorporated into immune-stimulating complexes or adjuvanted with Quil A. Although all immunized cats developed antibodies against the envelope protein, only the cats vaccinated with the rVV-expressed envelope glycoproteins developed antibodies which neutralized FIV infection of Crandell feline kidney cells. These antibodies failed to neutralize infection of thymocytes with a molecularly cloned homologous FIV. After the third immunization the cats were challenged with homologous FIV. Two weeks after challenge the cell-associated viral load proved to be significantly higher in the cats immunized with vGR657 and vGR657 x 15 than in the other cats. The cats immunized with vGR657 and vGR657 x 15 also developed antibodies against the Gag proteins more rapidly than the cats immunized with beta-Galactosidase-Env or the control cats. This suggested that immunization with rVV-expressed glycoprotein of FIV results in enhanced infectivity of FIV. It was shown that the observed enhancement could be transferred to naive cats with plasma collected at the day of challenge. PMID:7745719

  9. Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins.

    PubMed

    Hammad, Loubna A; Saleh, Marwa M; Novotny, Milos V; Mechref, Yehia

    2009-06-01

    A simple, sensitive, and rapid quantitative LC-MS/MS assay was designed for the simultaneous quantification of free and glycoprotein bound monosaccharides using a multiple reaction monitoring (MRM) approach. This study represents the first example of using LC-MS/MS methods to simultaneously quantify all common glycoprotein monosaccharides, including neutral and acidic monosaccharides. Sialic acids and reduced forms of neutral monosaccharides are efficiently separated using a porous graphitized carbon column. Neutral monosaccharide molecules are detected as their alditol acetate anion adducts [M + CH(3)CO(2)](-) using electrospray ionization in negative ion MRM mode, while sialic acids are detected as deprotonated ions [M - H](-). The new method exhibits very high sensitivity to carbohydrates with limits of detection as low as 1 pg for glucose, galactose, and mannose, and below 10 pg for other monosaccharides. The linearity of the described approach spans over three orders of magnitudes (pg to ng). The method effectively quantified monosaccharides originating from as little as 1 microg of fetuin, ribonuclease B, peroxidase, and alpha(1)-acid glycoprotein human (AGP) with results consistent with literature values and with independent CE-LIF measurements. The method is robust, rapid, and highly sensitive. It does not require derivatization or postcolumn addition of reagents. PMID:19318280

  10. 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

  11. 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

  12. 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

  13. 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

  14. A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins.

    PubMed

    Pfeiffer, Anett; Stephanowitz, Heike; Krause, Eberhard; Volkwein, Corinna; Hirsch, Christian; Jarosch, Ernst; Sommer, Thomas

    2016-06-01

    A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins. PMID:27053108

  15. 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

  16. Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein.

    PubMed Central

    Schuurhuis, G. J.; Broxterman, H. J.; de Lange, J. H.; Pinedo, H. M.; van Heijningen, T. H.; Kuiper, C. M.; Scheffer, G. L.; Scheper, R. J.; van Kalken, C. K.; Baak, J. P.

    1991-01-01

    Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance. Images Figure 1 PMID:1681887

  17. Identification of glycoprotein storage diseases by lectins: a new diagnostic method.

    PubMed

    Alroy, J; Orgad, U; Ucci, A A; Pereira, M E

    1984-12-01

    The specific diagnosis of glycoprotein storage diseases is made by demonstrating a deficiency in enzyme activity or an elevation of undegraded oligosaccharides in cells or body fluids. Prospective sampling and expensive specialized biochemistry, which is also time consuming, are required for such studies. We used lectin reagents on paraffin-embedded tissue sections to identify the specific sugars in undegraded stored substances. We studied 22 cases of glycoprotein storage diseases and differentiated histochemically between alpha- and beta-mannosidosis, fucosidosis, and sialisidosis. Cells affected with alpha-mannosidosis stained with Concanavalia ensiformis (Con A), Triticum vulgaris (WGA), and succinyl-WGA (S-WGA), while beta-mannosidosis cells did not stain with any of the lectins used. In fucosidosis the affected cells stained with Ulex europeus-I (UEA-I), while sialisidosis-affected cells stained with WGA, and in three cases with Arachis hypogea (PNA). This study indicates that lectin histochemistry provides a reliable specific diagnostic pattern for some glycoprotein storage diseases using a simple and inexpensive method. PMID:6501863

  18. 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

  19. 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.

  20. Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

    PubMed Central

    Mardones, Gonzalo A.; Bonifacino, Juan S.

    2014-01-01

    Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons. PMID:24831812

  1. 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

  2. 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

  3. 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

  4. Involvement of divalent cations in the complex between the platelet glycoproteins IIb and IIIa.

    PubMed

    Hangen, I; Bjerrum, O J; Gogstad, G; Korsmo, R; Solum, N O

    1982-02-01

    The major immunoprecipitate (No. 16) seen on crossed immunoelectrophoresis of Triton X-100-solubilized platelet proteins against whole platelet antibodies represents a complex containing the membrane glycoproteins IIb and IIIa. When EDTA is present during the solubilization, immunoprecipitate 16 as such is not observed, and two new arcs, termed 16a and 16b, appear. As with 16 these immunoprecipitates become radioactively labelled on lactoperoxidase-catalyzed iodination of platelets. Immunoprecipitate 16a showed partial immunochemical identity with 16, and was precipitated by an antibody raised against immunoprecipitate 16. The areas covered by immunoprecipitates 16, 16a and 16b were strongly reduced compared to normal with platelets from a patient with Glanzmann's thrombasthenia type II. Such platelets are known to contain reduced amounts of glycoproteins IIb and IIIa. The new arcs appearing when divalent cations are chelated by EDTA thus represent proteins derived from the immunoprecipitate 16 proteins, and divalent cations seem to be necessary to preserve the protein complex containing glycoprotein IIb and IIIa. The different complex formations between the components of immunoprecipitate 16 may reflect biochemical alterations of functional importance. PMID:6173070

  5. A multimethodological approach to identification of glycoproteins from the proteome of Francisella tularensis, an intracellular microorganism

    PubMed Central

    Balonova, Lucie; Hernychova, Lenka; Mann, Benjamin F.; Link, Marek; Bilkova, Zuzana; Novotny, Milos V.; Stulik, Jiri

    2010-01-01

    It appears that most glycoproteins found in pathogenic bacteria are associated with virulence. Despite the recent identification of novel virulence factors, the mechanisms of virulence in Francisella tularensis are poorly understood. In spite of its importance, questions about glycosylation of proteins in this bacterium and its potential connection with bacterial virulence have not been answered yet. In the present study, several putative Francisella tularensis glycoproteins were characterized through the combination of carbohydrate-specific detection and lectin affinity with highly sensitive mass spectrometry utilizing the bottom-up proteomic approach. The protein PilA that was recently found as being possibly glycosylated, as well as other proteins with designation as novel factors of virulence, were among the proteins identified in this study. The reported data compile the list of potential glycoproteins that may serve as a take-off platform for a further definition of proteins modified by glycans, faciliting a better understanding of the function of protein glycosylation in pathogenicity of Francisella tularensis. PMID:20175567

  6. Photoaffinity labeling of the multidrug-resistance-related P-glycoprotein with photoactive analogs of verapamil

    SciTech Connect

    Safa, A.R. )

    1988-10-01

    Verapamil, a phenylalkylamine calcium channel blocker, has been shown to reverse multidrug resistance in tumor cells, possibly by increasing drug retention through interaction with an outward drug transporter of the resistant cells. In this study two photoactive radioactive analogs of verapamil, N-(p-azido(3,5-{sup 3}H)benzoyl)aminomethyl verapamil and N-(p-azido(3-{sup 125}I)salicyl)aminomethyl verapamil, were synthesized and used to identify the possible biochemical target(s) for verapamil in multidrug-resistance DC-3F/VCRd-5L Chinese hamster lung cells selected for resistance to vincristine. The results show that a specifically labeled 150- to 180-kDa membrane protein in resistant cells was immunoprecipitated with a monoclonal antibody specific for P-glycoprotein. Phenylalkylamine binding specificity was established by competitive blocking of specific photolabeling with the nonradioactive photoactive analogs as well as with verapamil. Photoaffinity labeling was also inhibited by 50 {mu}M concentrations of the calcium channel blockers nimodipine, nifedipine, nicardipine, azidopine, bepridil, and diltiazem and partially by prenylamine. Moreover, P-glycoprotein labeling was inhibited in a dose-dependent manner by vinblastine with half-maximal inhibition at 0.2 {mu}M compared to that by verapamil at 8 {mu}M. These data provide direct evidence that P-glycoprotein has broad drug recognition capacity and that it serves as a molecular target for calcium channel blocker action in reversing multidrug resistance.

  7. Intestinal mucins from normal subjects and patients with cystic fibrosis. Variable contents of the disulphide-bound 118 kDa glycoprotein and different reactivities with an anti-(118 kDa glycoprotein) antibody.

    PubMed Central

    Mantle, M; Stewart, G

    1989-01-01

    1. A specific antibody was developed against the disulphide-bound 118 kDa glycoprotein of human intestinal mucin and used to establish an e.l.i.s.a. Fourteen purified mucins [eight normal (N) and six cystic fibrosis (CF)] had the same affinity for the antibody in the e.l.i.s.a., but their relative immunoreactivities varied widely (approx. 100,000-fold). In general, CF mucins were more antigenic than N mucins. 2. Variations (approx. 10-fold) were detected in the 118 kDa glycoprotein content of both N and CF mucins (assessed from Coomassie Blue-stained polyacrylamide gels), but these did not appear to be responsible for the differences in mucin immunoreactivity. 3. Variations (approx. 6-fold) were also observed in the size of the 118 kDa peak produced by N and CF mucins on Western blots. These were mostly due to differences in the 118 kDa glycoprotein content of mucins, although a small proportion resulted from changes in the number of antigenic determinants within individual 118 kDa glycoproteins. 4. After concanavalin A affinity chromatography of four reduced mucins (two N and two CF), purified 118 kDa glycoprotein was recovered in the bound fractions from the column, specifically eluted by methyl alpha-mannoside. 5. The amounts of 118 kDa glycoprotein isolated from the four mucins varied as predicted from the size of their 118 kDa bands on Coomassie Blue-stained gels. 6. Three 118 kDa glycoproteins (one N and two CF) showed almost identical reactivity in the e.l.i.s.a.; the fourth had fewer antigenic determinants. 7. Since differences in 118 kDa glycoprotein content and in the number of antigenic determinants within the 118 kDa glycoprotein did not account for variations in the reactivity of native mucins in the e.l.i.s.a., it appeared that accessibility of the 118 kDa glycoprotein to antibody binding may be critical in determining mucin immunoreactivity. This suggests that the three-dimensional conformation of CF mucins may differ from that of N mucins, leading

  8. Anti-β2 glycoprotein I antibodies in complex with β2 glycoprotein I induce platelet activation via two receptors: apolipoprotein E receptor 2' and glycoprotein I bα.

    PubMed

    Zhang, Wenjing; Gao, Fei; Lu, Donghe; Sun, Na; Yin, Xiaoxue; Jin, Meili; Liu, Yanhong

    2016-03-01

    Anti-β2 glycoprotein I (anti-β2GPI ) antibodies are important contributors to thrombosis, especially in patients with antiphospholipid syndrome (APS). However, the mechanism by which anti-β2GPI antibodies are involved in the pathogenesis of thrombosis is not fully understood. In this report, we investigated the role of anti- β2GPI antibodies in complexes with β2GPI as mediators of platelet activation, which can serve as a potential source contributing to thrombosis. We examined the involvement of the apolipoprotein E receptor 2' (apoER2') and glycoprotein I ba (GP I ba) in platelet activation induced by the anti-β2GPI /β2GPI complex. The interaction between the anti-β2GPI /β2GPI complex and platelets was examined using in vitro methods, in which the Fc portion of the antibody was immobilized using protein A coated onto a microtiter plate. Platelet activation was assessed by measuring GPII b/III a activation and P-selectin expression and thromboxane B2 production as well as p38 mitogen-activated protein kinase phosphorylation. Our results revealed that the anti-β2GPI /β2GPI complex was able to activate platelets, and this activation was inhibited by either the anti-GP I bα antibody or the apoER2' inhibitor. Results showed that the anti-β2GPI /β2GPI complex induced platelet activation via GPI ba and apoER2', which may then contribute to the prothrombotic tendency in APS patients. PMID:26620053

  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. Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuronal gene transfer

    PubMed Central

    Federici, Thais; Kutner, Robert; Zhang, Xian-Yang; Kuroda, Hitoshi; Tordo, Noël; Boulis, Nicholas M; Reiser, Jakob

    2009-01-01

    Background The delivery of therapeutic genes to the central nervous system (CNS) using viral vectors represents an appealing strategy for the treatment of nerve injury and disorders of the CNS. Important factors determining CNS targeting include tropism of the viral vectors and retrograde transport of the vector particles. Retrograde transport of equine anemia virus (EIAV)-based lentiviral vectors pseudotyped with the glycoprotein derived from the Rabies virus RabERA strain from peripheral muscle to spinal motor neurons (MNs) was previously reported. Despite therapeutic effects achieved in mouse models of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), the efficiency of this approach needs to be improved for clinical translation. To date there has not been a quantitative assessment of pseudotyped HIV-1-based lentiviral vectors to transduce MNs. Here, we describe quantitative tests to analyze the retrograde transport capacity of HIV-1 vectors pseudotyped with the G glycoprotein derived from Rabies and Rabies-related viruses (Lyssaviruses). Methods With a view toward optimizing the retrograde transport properties of HIV-1-based lentiviral vectors, we compared the glycoproteins from different enveloped viruses belonging to the Rhabdoviridae family, genus Lyssavirus, and evaluated their ability to transduce specific cell populations and promote retrograde axonal transport. We first tested the transduction performance of these pseudotypes in vitro in SH-SY5Y neuroblastoma cells, NSC-34 neuroblastoma-spinal cord hybrid cells, and primary mixed spinal cord and pure astrocyte cultures. We then analyzed the uptake and retrograde transport of these pseudotyped vectors in vitro, using Campenot chambers. Finally, intraneural injections were performed to evaluate the in vivo retrograde axonal transport of these pseudotypes. Results Both the in vitro and in vivo studies demonstrated that lentiviral vectors pseudotyped with the glycoprotein derived from the

  12. 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

  13. 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

  14. 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

  15. Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C.

    PubMed

    Chakravarty, Sanchita; Suraj, K; Gupta, Satish Kumar

    2005-05-01

    To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway. PMID:15805145

  16. Galectin Binding to Neo-Glycoproteins: LacDiNAc Conjugated BSA as Ligand for Human Galectin-3

    PubMed Central

    Böcker, Sophia; Laaf, Dominic; Elling, Lothar

    2015-01-01

    Carbohydrate-lectin interactions are relatively weak. As they play an important role in biological recognition processes, multivalent glycan ligands are designed to enhance binding affinity and inhibitory potency. We here report on novel neo-glycoproteins based on bovine serum albumin as scaffold for multivalent presentation of ligands for galectins. We prepared two kinds of tetrasaccharides (N-acetyllactosamine and N,N-diacetyllactosamine terminated) by multi-step chemo-enzymatic synthesis utilizing recombinant glycosyltransferases. Subsequent conjugation of these glycans to lysine groups of bovine serum albumin via squaric acid diethyl ester yielded a set of 22 different neo-glycoproteins with tuned ligand density. The neo-glycoproteins were analyzed by biochemical and chromatographic methods proving various modification degrees. The neo-glycoproteins were used for binding and inhibition studies with human galectin-3 showing high affinity. Binding strength and inhibition potency are closely related to modification density and show binding enhancement by multivalent ligand presentation. At galectin-3 concentrations comparable to serum levels of cancer patients, we detect the highest avidities. Selectivity of N,N-diacetyllactosamine terminated structures towards galectin-3 in comparison to galectin-1 is demonstrated. Moreover, we also see strong inhibitory potency of our scaffolds towards galectin-3 binding. These novel neo-glycoproteins may therefore serve as selective and strong galectin-3 ligands in cancer related biomedical research. PMID:26213980

  17. Functional role of the heterodimeric glycoprotein hormone, GPA2/GPB5, and its receptor, LGR1: An invertebrate perspective.

    PubMed

    Rocco, David A; Paluzzi, Jean-Paul V

    2016-08-01

    In vertebrates, follicle-stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid-stimulating hormone (TSH) are glycoprotein hormones that play central roles in metabolism, reproduction and development. Recently, a novel heterodimeric glycoprotein hormone, called GPA2/GPB5, was discovered in humans; however, contrary to its vertebrate glycoprotein hormone relatives, the physiological role of GPA2/GPB5 has not yet been fully elucidated in any vertebrate or invertebrate. Moreover, it is unclear as to whether GPA2/GPB5 functions as a heterodimer or as individual GPA2 and GPB5 monomers in these organisms. GPA2- and GPB5-like subunits have been identified or predicted in a wide array of animal phyla including the nematodes, chordates, hemichordates, arthropods, molluscs, echinoderms and annelids. So far, molecular studies on transcript expression of the GPA2/GPB5 subunits and its putative receptor, the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1), suggests this glycoprotein hormone system plays a developmental role and may also function in hydromineral balance in invertebrates. This mini-review summarizes the current state of knowledge on the physiological actions and activity of this evolutionarily ancient heterodimeric glycoprotein hormone with a particular focus on its known functions in the invertebrates. PMID:26704853

  18. Induction of apoptosis and reversal of permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM by ginsenoside Rh2

    PubMed Central

    Zhang, Hui; Gong, Jian; Zhang, Huilai; Kong, Di

    2015-01-01

    Multidrug resistance is a phenomenon that cancer cells develop a cross-resistant phenotype against several unrelated drugs, and permeability glycoprotein derived from the overexpression of multidrug resistance gene 1 has been taken as the most significant cause of multidrug resistance. In the present study, ginsenoside Rh2 was used to reverse permeability glycoprotein-mediated multidrug resistance of MCF-7/ADM cell line. Effects of ginsenoside Rh2 on the apoptotic process and caspase-3 activity of MCF-7 and MCF-7/ADM cell lines were determined using flow cytometry and microplate reader. Methyl thiazolyl tetrazolium test was conducted to assess the IC50 values of ginsenoside Rh2 and adriamycin on MCF-7 and MCF-7/ADM cultures; Rhodamin 123 assay was used to assess the retention of permeability glycoprotein after ginsenoside Rh2 treatment; flow cytometry and real time polymerase chain reaction were used to determine the expression levels of permeability glycoprotein and multidrug resistance gene 1 in drug-resistant cells and their parental cells after exposure to ginsenoside Rh2. The results showed that ginsenoside Rh2, except for inducing apoptosis, had the ability to reverse multidrug resistance in MCF-7/ADM cell line without changing the expression levels of permeability glycoprotein and multidrug resistance gene 1. Our findings provided some valuable information for the application of ginsenoside Rh2 in cancer therapy, especially for multidrug resistance reversal in clinic. PMID:26191135

  19. 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.

  20. Effect of an aqueous extract of Scoparia dulcis on plasma and tissue glycoproteins in streptozotocin induced diabetic rats.

    PubMed

    Latha, M; Pari, L

    2005-02-01

    The influence of Scoparia dulcis, a traditionally used plant for the treatment of diabetes mellitus, was examined in streptozotocin diabetic rats on dearrangement in glycoprotein levels. Diabetes was induced in male Wistar rats by a single intraperitoneal injection of streptozotocin. An aqueous extract of Scoparia dulcis plant was administered orally for 6 weeks. The effect of the Scoparia dulcis extract on blood glucose, plasma insulin, plasma and tissue glycoproteins studied was in comparison to glibenclamide. The levels of blood glucose and plasma glycoproteins were increased significantly whereas the level of plasma insulin was significantly decreased in diabetic rats. There was a significant decrease in the level of sialic acid and elevated levels of hexose, hexosamine and fucose in the liver and kidney of streptozotocin diabetic rats. Oral administration of Scoparia dulcis plant extract (SPEt) to diabetic rats led to decreased levels of blood glucose and plasma glycoproteins. The levels of plasma insulin and tissue sialic acid were increased whereas the levels of tissue hexose, hexosamine and fucose were near normal. The present study indicates that Scoparia dulcis possesses a significant beneficial effect on glycoproteins in addition to its antidiabetic effect. PMID:15739907

  1. Drosophila UDP-glucose:glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins.

    PubMed Central

    Parker, C G; Fessler, L I; Nelson, R E; Fessler, J H

    1995-01-01

    A Drosophila UDP-glucose:glycoprotein glucosyltransferase was isolated, cloned and characterized. Its 1548 amino acid sequence begins with a signal peptide, lacks any putative transmembrane domains and terminates in a potential endoplasmic reticulum retrieval signal, HGEL. The soluble, 170 kDa glycoprotein occurs throughout Drosophila embryos, in microsomes of highly secretory Drosophila Kc cells and in small amounts in cell culture media. The isolated enzyme transfers [14C]glucose from UDP-[14C]Glc to several purified extracellular matrix glycoproteins (laminin, peroxidasin and glutactin) made by these cells, and to bovine thyroglobulin. These proteins must be denatured to accept glucose, which is bound at endoglycosidase H-sensitive sites. The unusual ability to discriminate between malfolded and native glycoproteins is shared by the rat liver homologue, previously described by A.J.Parodi and coworkers. The amino acid sequence presented differs from most glycosyltransferases. There is weak, though significant, similarity with a few bacterial lipopolysaccharide glycotransferases and a yeast protein Kre5p. In contrast, the 56-68% amino acid identities with partial sequences from genome projects of Caenorhabditis elegans, rice and Arabidopsis suggest widespread homologues of the enzyme. This glucosyltransferase fits previously proposed hypotheses for an endoplasmic reticular sensor of the state of folding of newly made glycoproteins. Images PMID:7729408

  2. Platelet-Derived Microparticles and the Potential of Glycoprotein IIb/IIIa Antagonists in Treating Acute Coronary Syndrome

    PubMed Central

    Li, Ximing; Cong, Hongliang

    2009-01-01

    Platelet glycoprotein IIb/IIIa receptors are major platelet membrane constituents. They are integral to the formation of the surface fibrinogen receptor on activated platelets, in which 73% of platelet-derived microparticles are positive for the glycoprotein IIa/IIIb receptor. Activated platelets can shed platelet-derived microparticles, especially during the course of an acute coronary syndrome. Data have shown that platelet-derived microparticles can bind to the endothelium, to leukocytes, and to the submatrix of vascular walls, and launch some signal-transduction pathways, such as the pertussis-toxin-sensitive G protein, extracellular signal-regulated kinase, and phosphoinositide 3-kinase pathways. One research group found that platelet-derived microparticles transfer glycoprotein IIb/IIIa receptors to isolated and whole-blood neutrophils. The receptors can co-localize with β2-integrins and cooperate in the activation of nuclear factor κB (NF-κB), which can be inhibited by glycoprotein IIb/IIIa receptor antagonists. Accordingly, it is possible that glycoprotein IIb/IIIa receptor antagonists produce a direct and marked effect on endothelial cells, smooth-muscle cells, and leukocytes through a platelet-derived microparticle pathway that will lead to a potential treatment for acute coronary syndrome. Herein, we review the medical literature and discuss the potential application of platelet-derived microparticles toward the treatment of acute coronary syndrome. PMID:19436807

  3. 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

  4. A Study of Anti Beta-2 Glycoprotein I and Anti-Prothrombin Antibodies in Patients with Unexplained Recurrent Pregnancy Losses.

    PubMed

    Singh, Angad; Nangia, Anita; Sharma, Sunita; Puri, Manju

    2016-06-01

    To compare the levels of IgG and IgM anti beta-2 glycoprotein I antibodies and IgG and IgM anti prothrombin antibodies among women with unexplained recurrent pregnancy losses and women with at least 2 live issues. To compare the prevalence of newer anti beta-2 glycoprotein I & anti prothrombin antibodies with conventional Lupus anticoagulant & anticardiolipin antibodies. 50 women with recurrent pregnancy losses & 50 matched controls were evaluated for the presence of: Lupus anticoagulant-screened by LA sensitive aPTT& DRVV and confirmatory Staclot Assay. ELISA kits were used for detecting IgG & IgM anticardiolipin, anti beta-2 glycoprotein I & anti prothrombin antibodies. 11/50 (22 %) women in study group and none in control group had circulating antiphospholipid antibodies. 2 cases (4 %) had lupus anticoagulant. 1 case (2 %) had anticardiolipin antibody & 6 cases (12 %) were positive for anti beta-2 Glycoprotein I antibody (p value = 0.027). 3 cases (6 %) had anti prothrombin antibody. All were mutually exclusive except for one. Women with recurrent pregnancy losses should be tested for anti beta-2 Glycoprotein I antibodies & anti prothrombin antibodies in addition to conventional lupus anticoagulant and anticardiolipin antibodies. This approach can decrease the incidence of SNAP (seronegative antiphospholipid syndrome) cases while establishing the true prevalence of antiphospholipid syndrome. PMID:27065583

  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. 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

  7. Evaluation of immunohistochemical expression of P-glycoprotein in neoplasms of the mammary gland in bitches.

    PubMed

    Badowska-Kozakiewicz, A M; Malicka, E

    2010-01-01

    The aim of the study was to investigate the P-glycoprotein expression in correlation with other neoplasm traits such as: histological type, the differentiation grade, proliferative activity, expression of the cyclooxygenase-2. Material for the investigation comprised 50 tumours of the mammary gland collected from bitches during surgical procedures performed in Warsaw Veterinary Clinics and Small Animal Clinic of the Department of Clinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - SGGW. All together 8 adenomas, 22 complex carcinomas, 15 simple carcinomas and 5 solid carcinomas. In case of cancers, the degree of histological malignancy was established: 1st degree of histological malignancy--18 neoplasms, 2nd degree of histological malignancy--14 neoplasms and 3rd degree of histological malignancy--10 neoplasms. Evaluations were conducted with histopathological and immunohistochemical methods using suitable antibodies. Proliferative activity was highly dependent on type of the neoplasm and the degree of histological malignancy. The highest value of the mitotic index was characteristic for solid and simple cancers and neoplasms with the highest degree of histological malignancy. Results of expression of the nuclear antigen Ki-67 were similar. Expression of P-glycoprotein was revealed in all types of neoplasms. The expression of P-glycoprotein was identified in cytoplasm and cell membranes of neoplastic cells. Positive expression of P-gp was observed in 76% of cancers. Complex carcinomas were the biggest group among the cancer types which demonstrated positive reaction of P-gp. High expression of P-gp was also established in cancers with the highest degree of malignancy. In bitches aged 9 through 12 years, the cancers featuring a positive reaction of P-gp constituted the most numerous group (63.2%); on the other hand, this cancer type barely appeared in the oldest bitches (10.5%). PMID:20731191

  8. Dimeric Architecture of the Hendra Virus Attachment Glycoprotein: Evidence for a Conserved Mode of Assembly▿ †

    PubMed Central

    Bowden, Thomas A.; Crispin, Max; Harvey, David J.; Jones, E. Yvonne; Stuart, David I.

    2010-01-01

    Hendra virus is a negative-sense single-stranded RNA virus within the Paramyxoviridae family which, together with Nipah virus, forms the Henipavirus genus. Infection with bat-borne Hendra virus leads to a disease with high mortality rates in humans. We determined the crystal structure of the unliganded six-bladed β-propeller domain and compared it to the previously reported structure of Hendra virus attachment glycoprotein (HeV-G) in complex with its cellular receptor, ephrin-B2. As observed for the related unliganded Nipah virus structure, there is plasticity in the Glu579-Pro590 and Lys236-Ala245 ephrin-binding loops prior to receptor engagement. These data reveal that henipaviral attachment glycoproteins undergo common structural transitions upon receptor binding and further define the structural template for antihenipaviral drug design. Our analysis also provides experimental evidence for a dimeric arrangement of HeV-G that exhibits striking similarity to those observed in crystal structures of related paramyxovirus receptor-binding glycoproteins. The biological relevance of this dimer is further supported by the positional analysis of glycosylation sites from across the paramyxoviruses. In HeV-G, the sites lie away from the putative dimer interface and remain accessible to α-mannosidase processing on oligomerization. We therefore propose that the overall mode of dimer assembly is conserved for all paramyxoviruses; however, while the geometry of dimerization is rather closely similar for those viruses that bind flexible glycan receptors, significant (up to 60°) and different reconfigurations of the subunit packing (associated with a significant decrease in the size of the dimer interface) have accompanied the independent switching to high-affinity protein receptor binding in Hendra and measles viruses. PMID:20375167

  9. Epitope dampening monotypic measles virus hemagglutinin glycoprotein results in resistance to cocktail of monoclonal antibodies.

    PubMed

    Lech, Patrycja J; Tobin, Gregory J; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P; Russell, Stephen J; Nara, Peter L

    2013-01-01

    The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

  10. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc

    PubMed Central

    Barriga, Gonzalo P.; Villalón-Letelier, Fernando; Márquez, Chantal L.; Bignon, Eduardo A.; Acuña, Rodrigo; Ross, Breyan H.; Monasterio, Octavio; Mardones, Gonzalo A.; Vidal, Simon E.; Tischler, Nicole D.

    2016-01-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses

  11. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

    PubMed

    Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

    2016-07-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses

  12. Core Structure of S2 from the Human Coronavirus NL63 Spike Glycoprotein

    SciTech Connect

    Zheng,Q.; Deng, Y.; Liu, J.; van der Hoek, L.; Berkhout, B.; Lu, M.

    2006-01-01

    Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an {alpha}-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 {sup o}C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.

  13. The Alphavirus E3 Glycoprotein Functions in a Clade-Specific Manner

    PubMed Central

    Snyder, Anthony J.

    2012-01-01

    The 80 trimeric, glycoprotein spikes that cover the surface of alphavirus particles are required for mediating viral entry into a host cell. Spike assembly is a regulated process that requires interactions between five structural proteins, E3, E2, 6K and its translational frameshift product TF, and E1. E3 is a small, ∼65-amino-acid glycoprotein that has two known functions: E3 serves as the signal sequence for translocation of the E3-E2-6K-E1 polyprotein into the endoplasmic reticulum (ER), and cleavage of E3 from E2 is essential for virus maturation. Nonetheless, when E3 is replaced with an ER signal sequence, spikes do not form and infectious particles are not assembled, suggesting an additional role(s) for E3 in the viral life cycle. To further investigate the role of E3 in spike assembly, we made chimeric viruses in which E3 from one alphavirus species is replaced with E3 from another species. Our results demonstrate that when E3 is interchanged between alphavirus species that belong to the same virus clade, viral titers and particle morphologies and compositions are similar to what are observed for the parental virus. In contrast, for chimeras in which E3 is derived from a different clade than the parental virus, we observed reduced titers and the formation of particles with atypical morphologies and protein compositions. We further characterized the E3 chimeras using a combination of structure-function and revertant analyses. This work revealed two specific interactions between E3 and its cognate E2 glycoprotein that are important for regulating spike assembly. PMID:23035234

  14. Characterization of African bat henipavirus GH-M74a glycoproteins.

    PubMed

    Weis, Michael; Behner, Laura; Hoffmann, Markus; Krüger, Nadine; Herrler, Georg; Drosten, Christian; Drexler, Jan Felix; Dietzel, Erik; Maisner, Andrea

    2014-03-01

    In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we compared the biological functions of the surface glycoproteins G and F of the prototype African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a well-characterized pathogenic member of the henipavirus genus. Glycoproteins are central determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular ephrin receptors and functional expression of fusion-competent F proteins are indispensable prerequisites for virus entry and cell-to-cell spread. In this study, we analysed the ability of the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did not cause evident fusion in the presence of heterotypic NiV G protein. Pulse-chase and surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of GH-M74a showed a functional defect that is most likely caused by impaired trafficking leading to less efficient proteolytic activation and surface expression. PMID:24296468

  15. A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism

    PubMed Central

    Li, Gang; Nguyen, Christopher C.; Ryckman, Brent J.; Britt, William J.; Kamil, Jeremy P.

    2015-01-01

    Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by the UL148 ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted for UL148 showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction of UL148 into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes. PMID:25831500

  16. Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.

    PubMed

    Effantin, Grégory; Estrozi, Leandro F; Aschman, Nick; Renesto, Patricia; Stanke, Nicole; Lindemann, Dirk; Schoehn, Guy; Weissenhorn, Winfried

    2016-07-01

    Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae. PMID:27399201

  17. A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression.

    PubMed

    Abd Hamid, Umi M; Royle, Louise; Saldova, Radka; Radcliffe, Catherine M; Harvey, David J; Storr, Sarah J; Pardo, Maria; Antrobus, Robin; Chapman, Caroline J; Zitzmann, Nicole; Robertson, John F; Dwek, Raymond A; Rudd, Pauline M

    2008-12-01

    Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression. PMID:18818422

  18. Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture

    PubMed Central

    Effantin, Grégory; Estrozi, Leandro F.; Aschman, Nick; Renesto, Patricia; Stanke, Nicole; Lindemann, Dirk; Schoehn, Guy; Weissenhorn, Winfried

    2016-01-01

    Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae. PMID:27399201

  19. Features that affect secretion and assembly of zona pellucida glycoproteins during mammalian oogenesis.

    PubMed

    Jovine, Luca; Qi, Huayu; Williams, Zev; Litscher, Eveline S; Wassarman, Paul M

    2007-01-01

    For sperm to fertilize eggs, they must bind to and penetrate the zona pellucida (ZP) that surrounds the plasma membrane of all mammalian eggs. The ZP first appears during oocyte growth and increases in thickness as oocytes increase in diameter. The ZP is an extracellular matrix composed of long, crosslinked filaments. In mice, three glycoproteins, called mZP1-3, are synthesised and secreted by growing oocytes and assembled into a thick (-6.5 microm) extracellular coat over a 2-3 week period. Recently, we identified several regions of nascent ZP glycoproteins that affect their secretion and incorporation into the ZP (assembly) by growing oocytes. Among these are the ZP domain, the consensus furin cleavage site (CFCS) and the C-terminal propeptide (CTP) with its transmembrane domain (TMD), external hydrophobic patch (EHP), charged patch (CP), conserved cysteine (Cys) residue, and short cytoplasmic tail (CT). Particularly important is the ZP domain, a approximately 260 amino acid region with 8 conserved Cys residues that is common to a variety of extracellular proteins of diverse functions found in a wide range of multicellular eukaryotes. Our results show that the ZP domain functions as a polymerisation module and that its N-terminal half, including 4 conserved Cys residues, is largely responsible for this role. Additionally, two conserved hydrophobic sequences, one within the ZP domain (internal hydrophobic patch; IHP) and another within the CTP (EHP), apparently regulate polymerisation of nascent ZP glycoproteins. Collectively, our findings suggest a general mechanism for assembly of all ZP domain proteins based on coupling between proteolytic processing and polymerisation. PMID:17566273

  20. Structural characterization of the N-linked pentasaccharide decorating glycoproteins of the halophilic archaeon Haloferax volcanii.

    PubMed

    Kandiba, Lina; Lin, Chia-Wei; Aebi, Markus; Eichler, Jerry; Guerardel, Yann

    2016-07-01

    N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii, glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl-O-4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl-O-4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process. PMID:26863921

  1. Do drugs have access to the P-glycoprotein drug-binding pocket through gates?

    PubMed

    Ferreira, Ricardo J; Ferreira, Maria-José U; Dos Santos, Daniel J V A

    2015-10-13

    The P-glycoprotein efflux mechanism is being studied since its identification as a leading protagonist in multidrug resistance. Recently, it was suggested that drugs enter the drug-binding pocket (DBP) through gates located between the transmembrane domains. For both a substrate and a modulator, the potential of mean force curves along the reaction coordinate obtained with the WHAM approach were similar, with no activation energy required for crossing the gate. Moreover, drug transit from bulk water into the DBP was characterized as an overall free-energy downhill process. PMID:26574244

  2. Hemostatic Function, Survival, and Membrane Glycoprotein Changes in Young versus Old Rabbit Platelets

    PubMed Central

    Blajchman, Morris A.; Senyi, Andrew F.; Hirsh, Jack; Genton, Edward; George, James N.

    1981-01-01

    Although in vitro studies have demonstrated functional differences between young and old platelets, in vivo differences have not been precisely established. Therefore the in vivo hemostatic function of young and old platelets and the survival time have been examined in rabbits. The hemostatic function was measured by performing serial ear bleeding times in irradiation-induced thrombocytopenic rabbits. After irradiation with 930 rad the platelet count gradually diminished reaching a nadir (∼20 × 103/μl) at 10 d. The platelets present in the circulation, 7-10 d after irradiation, were considered old platelets, and the platelets present after recovery, 11-14 d postirradiation, young platelets. The measurement of platelet size was consistent with the hypothesis that platelets become smaller with age: the mean size was 3.84 μm3 for old platelets and 5.86 μm3 for young platelets. Regression analysis of the relationship between the bleeding time and the platelet count in 18 rabbits showed a significantly different slope for rabbits with predominantly old platelets compared with rabbits with predominantly young platelets (P < 0.001). Young platelets were more effective giving much shorter bleeding times than old platelets at comparable platelet counts. Survival times of young and old platelets were measured using platelets harvested on day 8 postirradiation (old platelets) and day 12 postirradiation (young platelets) that were labeled and then reinjected into normal recipient animals. The mean platelet survival time, calculated by gamma function, of old platelets was 28.8 h; of young platelets, 87.4 h; and of normally circulating heterogeneous platelets, (normal platelets) 53.0 h. Notably, the survival of old platelets was found to be exponential, and of young platelets, linear. Analysis of the membrane glycoproteins in young, old and normal platelets indicated that there was no qualitative difference amongst the young, normal, and old platelets. The relative

  3. Synthesis, processing, and function of N-glycans in N-glycoproteins

    PubMed Central

    Bieberich, Erhard

    2014-01-01

    Many membrane-resident and secrected proteins, including growth factors and their receptors are N-glycosylated. The initial N-glycan structure consists of 14 sugar residues (Glc3Man9GlcNAc2) that are first synthesized in the endoplasmic reticulum (ER) as a branched structure on a lipid anchor (dolicholpyrophosphate) and then co-translationally, “en bloc” transferred and linked via N-acetylglucosamine (GlcNAc) to asparagine within a specific N-glycosylation acceptor sequence (Asn-X-Ser/Thr) of the nascent recipient protein. In the ER and then the Golgi apparatus, the N-linked glycan structure is modified by hydrolytic removal of sugar residues (“trimming”) followed by re-glycosylation with additional sugar residues (“processing”) such as galactose, fucose or sialic acid in complex N-glycoproteins. While the sequence of the reactions leading to biosynthesis, “en bloc” transfer and processing of N-glycans is well investigated, it is still not completely understood how N-glycans affect the biological fate and function of N-glycoproteins. Initially, N-glycans have been found to be critical for proper protein folding and quality control by chaperones in the ER, a process now known as the “calnexin/calreticulin cycle” in the unfolded protein response and ER-assisted degradation (ERAD). More recently, N-glycans have been shown to modulate the function of many cell surface proteins involved in migration and adhesion, including those regulating myelination. Currently, the Golgi has emerged as an organelle that is intimately linked to editing the function of N-glycans in N-glycoprotein transport and sorting. For one, it has been shown that mutations in Golgi glycosyltransferases and transport proteins lead to defects in N-glycan processing that cause severe congenital disorders of glycosylation (CDG). On the other hand, it has been found that N-glycans affect transport of glycosylated proteins in the Golgi, including sorting of secreted proteins such as

  4. Studies on the Golgi apparatus. Cumulative inhibition of protein and glycoprotein secretion by d-galactosamine

    PubMed Central

    Bauer, Christian H.; Lukaschek, Rainer; Reutter, Werner G.

    1974-01-01

    1. The administration of d-galactosamine leads to inhibition of protein and glycoprotein secretion by rat liver. To test the secretory function, the secretion times for galactose-and fucose-containing glycoproteins were determined; they were lengthened from 6 to 9min and from 8 to 13min respectively. 2. The Golgi apparatus was enriched 100–120-fold relative to the homogenate. A new linked-assay system for the marker enzyme, UDP-galactose–N-acetyl-d-glucosamine galactosyltransferase, is presented. The activity of the enzyme was measured spectrophotometrically by following the formation of UDP coupled to nicotinamide nucleotide reduction. The Michaelis constants were calculated to be 0.11mm for UDP-galactose with N-acetyl-d-glucosamine as exogenous acceptor and 19mm for N-acetyl-d-glucosamine itself. 3. The physiological substrate of the galactosyltransferase, UDP-galactose, can be replaced by UDP-galactosamine, which accumulates after d-galactosamine administration. Under conditions in vitro the rate of d-galactosamine transfer to an endogenous acceptor protein of the Golgi fraction reaches 9% of that with d-galactose; this finding is noteworthy, because normally a non-acetylated amino sugar does not occur in glycoproteins. 4. The albumin content of the Golgi-rich fraction was diminished to 55% of the reference value 6h after the injection of 375mg of d-galactosamine hydrochloride/kg body wt. The transfer of d-[1-14C]galactose to an endogenous acceptor protein fell to 60% compared with Golgi-rich fractions from untreated animals. Analysis of the Golgi-rich fraction by polyacrylamide-gel electrophoresis showed a decrease or loss of several protein bands. 5. Protein synthesis can be restored by up to 80% if the UTP pool, decreased after d-galactosamine administration, is filled up by several injections of uridine. 6. From the results presented it can be concluded that the disturbed secretion of proteins and glycoproteins was due to a cumulative effect of

  5. Reduction of cerebral glucose utilization by the HIV envelope glycoprotein Gp-120

    SciTech Connect

    Kimes, A.S.; London, E.D.; Szabo, G.; Raymon, L.; Tabakoff, B. )

    1991-05-01

    Gp-120 is a glycoprotein constituent of the human immunodeficiency virus (HIV) envelope. The effects of gp-120 on cerebral glucose utilization in rats were studied by the quantitative 2-deoxy-D-(1-14C) glucose method. Intracerebroventricular injection of gp-120 significantly reduced glucose utilization in the lateral habenula and the suprachiasmatic nucleus and decreased the global cerebral metabolic rate for glucose. The findings suggest that gp-120 and closely related peptides can alter neuronal function, thereby contributing to the sequelae of HIV infection.

  6. Pediatric cerebellar stroke associated with elevated titer of antibodies to β2-glycoprotein.

    PubMed

    Spalice, Alberto; Del Balzo, Francesca; Perla, Francesco Massimo; Papetti, Laura; Nicita, Francesco; Ursitti, Fabiana; Properzi, Enrico

    2011-06-01

    Antibodies to 2-glycoprotein I (anti-2GPI) have been associated with recurrent thrombosis and pregnancy morbidity. However, the prevalence of anti-2GPI in children suffering from cerebral and cerebellar infarction is unknown. We report on a 10-month-old boy who had an ischemic cerebellar stroke, secondary to antiphospholipid syndrome with high titers of immunoglobulin G anti-2GPI (first titer: 132U) anticardiolipin antibodies and lupus anticoagulant tests were negative. All other causes of infarction were excluded. To our knowledge, this is the first reported case of childhood cerebellar ischemic stroke with only anti-2GPI but no antibodies detectable in standard antiphospholipid assays. PMID:21388749

  7. Anterograde Glycoprotein-Dependent Transport of Newly Generated Rabies Virus in Dorsal Root Ganglion Neurons

    PubMed Central

    Bauer, Anja; Nolden, Tobias; Schröter, Josephine; Römer-Oberdörfer, Angela; Gluska, Shani; Perlson, Eran

    2014-01-01

    ABSTRACT Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by live-cell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 μm/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 μm/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 μm/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCE Rabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and

  8. Myelin-associated glycoprotein gene mutation causes Pelizaeus-Merzbacher disease-like disorder.

    PubMed

    Lossos, Alexander; Elazar, Nimrod; Lerer, Israela; Schueler-Furman, Ora; Fellig, Yakov; Glick, Benjamin; Zimmerman, Bat-El; Azulay, Haim; Dotan, Shlomo; Goldberg, Sharon; Gomori, John M; Ponger, Penina; Newman, J P; Marreed, Hodaifah; Steck, Andreas J; Schaeren-Wiemers, Nicole; Mor, Nofar; Harel, Michal; Geiger, Tamar; Eshed-Eisenbach, Yael; Meiner, Vardiella; Peles, Elior

    2015-09-01

    Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by mutations or rearrangements in PLP1. It presents in infancy with nystagmus, jerky head movements, hypotonia and developmental delay evolving into spastic tetraplegia with optic atrophy and variable movement disorders. A clinically similar phenotype caused by recessive mutations in GJC2 is known as Pelizaeus-Merzbacher-like disease. Both genes encode proteins associated with myelin. We describe three siblings of a consanguineous family manifesting the typical infantile-onset Pelizaeus-Merzbacher disease-like phenotype slowly evolving into a form of complicated hereditary spastic paraplegia with mental retardation, dysarthria, optic atrophy and peripheral neuropathy in adulthood. Magnetic resonance imaging and spectroscopy were consistent with a demyelinating leukodystrophy. Using genetic linkage and exome sequencing, we identified a homozygous missense c.399C>G; p.S133R mutation in MAG. This gene, previously associated with hereditary spastic paraplegia, encodes myelin-associated glycoprotein, which is involved in myelin maintenance and glia-axon interaction. This mutation is predicted to destabilize the protein and affect its tertiary structure. Examination of the sural nerve biopsy sample obtained in childhood in the oldest sibling revealed complete absence of myelin-associated glycoprotein accompanied by ill-formed onion-bulb structures and a relatively thin myelin sheath of the affected axons. Immunofluorescence, cell surface labelling, biochemical analysis and mass spectrometry-based proteomics studies in a variety of cell types demonstrated a devastating effect of the mutation on post-translational processing, steady state expression and subcellular localization of myelin-associated glycoprotein. In contrast to the wild-type protein, the p.S133R mutant was retained in the endoplasmic reticulum and was subjected to endoplasmic reticulum-associated protein degradation by the

  9. Glycopeptide-functionalized gold nanoparticles for antibody induction against the tumor associated mucin-1 glycoprotein.

    PubMed

    Cai, Hui; Degliangeli, Federica; Palitzsch, Björn; Gerlitzki, Bastian; Kunz, Horst; Schmitt, Edgar; Fiammengo, Roberto; Westerlind, Ulrika

    2016-03-01

    We report the preparation of gold nanoparticle (AuNP)-based vaccine candidates against the tumor-associated form of the mucin-1 (MUC1) glycoprotein. Chimeric peptides, consisting of a glycopeptide sequence derived from MUC1 and the T-cell epitope P30 sequence were immobilized on PEGylated AuNPs and the ability to induce selective antibodies in vivo was investigated. After immunization, mice showed significant MHC-II mediated immune responses and their antisera recognized human MCF-7 breast cancer cells. Nanoparticles designed according to this report may become key players in the development of anticancer vaccines. PMID:26853835

  10. Development of recombinant canine adenovirus type-2 expressing the Gn glycoprotein of Seoul virus.

    PubMed

    Yuan, Ziguo; Zhang, Xiuxiang; Zhang, Shoufeng; Liu, Ye; Gao, Shengyan; Zhang, Fei; Xu, Huijuan; Wang, Xiaohu; Hu, Rongliang

    2008-05-01

    Seoul virus glycoprotein Gn is a major structural protein and candidate antigen of hantavirus that induces a highly immunogenic response for hantavirus vaccine. In this study, a replication-competent recombinant canine adenovirus type-2 expressing Gn was constructed by the in vitro ligation method. The Gn expression cassette, including the human cytomegalovirus (hCMV) promoter/enhancer and the SV40 early mRNA polyadenylation signal, was cloned into the SspI site of the E3 region which is not essential for proliferation of CAV-2. Expression of Gn was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. PMID:18249007

  11. Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

    PubMed Central

    Volz, Asisa; Kupke, Alexandra; Song, Fei; Jany, Sylvia; Fux, Robert; Shams-Eldin, Hosam; Schmidt, Jörg; Becker, Christin; Eickmann, Markus; Becker, Stephan

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8+ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine. PMID:26018172

  12. Platelet Glycoproteins and Fibrinogen in Recovery from Idiopathic Sudden Hearing Loss

    PubMed Central

    Gorzelniak, Kerstin; Bremer, Alexis; Rudack, Claudia; Walter, Michael

    2014-01-01

    Background The pathomechanism and location of idiopathic sudden sensorineural hearing loss (ISSHL) is unclear. In a previous case-control study, we found elevated fibrinogen concentrations and a higher prevalence of T allele carriers of the glycoprotein (Gp) Ia C807T polymorphism in ISSHL patients. Methodology 127 patients with ISSHL (mean age 53.3 years, 48.8% females), who underwent a standard therapy with high dose steroids, pentoxifyllin and sterofundine over 8 days were included. We examined the influence of GpIa genotype and fibrinogen (BclI-, A312-, HaeIII-) genotype and fibrinogen plasma levels on hearing recovery after 8 weeks (change from baseline: 0 dB  =  no recovery, >0 to 10 dB = moderate recovery, >10 dB = good recovery). In a subsample of 59 patients with ISSHL, we further studied the association of platelet glycoprotein GpIa, Ib and IIIa densities on hearing recovery as well as the possible effect-modification of platelet glycoproteins on hearing recovery by plasma fibrinogen. Results In univariate analysis, neither the GpIa genotype nor fibrinogen genotype (all p>0.1) but lower fibrinogen levels (p = 0.029), less vertigo (p = 0.002) and lower GpIIIa receptor density (p = 0.037, n = 59) were associated with hearing recovery. In multivariate analysis, fibrinogen significantly modified the effect of GPIa receptor density on good hearing recovery (effect-modification on multiplicative scale OR = 0.45 (95% confidence interval (0.21–0.94)), p = 0.03). GPIb receptor density below the mean was associated with a 2-fold increase in good hearing recovery both in patients with fibrinogen levels above (p = 0.04) as well as in patients with fibrinogen levels below the mean (p = 0.06). There was no indication for an effect-modification (p = 0.97). Conclusions The findings suggest a vascular/rheological origin of ISSHL with unique features of thrombosis in the inner ear artery that may include complex

  13. Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation.

    PubMed

    Omote, Hiroshi; Figler, Robert A; Polar, Mark K; Al-Shawi, Marwan K

    2004-04-01

    A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K(m) for colchicine and a lower K(m) for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this

  14. 5T4 oncotrophoblast glycoprotein: janus molecule in life and a novel potential target against tumors.

    PubMed

    Zhao, Yu; Wang, Yuxia

    2007-04-01

    5T4 oncotrophoblast glycoprotein is a transmembrane protein expressed on the embryonic tissue and various malignant tumor cell surfaces. It plays a vital role in the multiple biological and pathological processes including massive cellular migration during the embryogenesis, cell invasion associated with implantation, and neoplastic metastasis in the progression of tumorigenesis. Its restricted profile of expression stratifies criteria of tumor-associated antigen and makes it a new promising candidate for immunotherapy for cancer. Hence, illustrating this molecular function is necessary for discovering the principle of the tumor diffusion and aggravation and is helpful for developing novel and effective strategies of cancer therapy. PMID:17484803

  15. Genotyping of Korean isolates of infectious hematopoietic necrosis virus (IHNV) based on the glycoprotein gene

    USGS Publications Warehouse

    Kim, W.-S.; Oh, M.-J.; Nishizawa, T.; Park, J.-W.; Kurath, G.; Yoshimizu, M.

    2007-01-01

    Glycoprotein (G) gene nucleotide sequences of four Korean isolates of infectious hematopoietic necrosis virus (IHNV) were analyzed to evaluate their genetic relatedness to worldwide isolates. All Korean isolates were closely related to Japanese isolates of genogroup JRt rather than to those of North American and European genogroups. It is believed that Korean IHNV has been most likely introduced from Japan to Korea by the movement of contaminated fish eggs. Among the Korean isolates, phylogenetically distinct virus types were obtained from sites north and south of a large mountain range, suggesting the possibility of more than one introduction of virus from Japan. ?? 2007 Springer-Verlag.

  16. The myelin oligodendrocyte glycoprotein directly binds nerve growth factor to modulate central axon circuitry

    PubMed Central

    Mei, Feng; Greenfield, Ariele; Jahn, Sarah; Shen, Yun-An A.; Reid, Hugh H.; McKemy, David D.

    2015-01-01

    Myelin oligodendrocyte glycoprotein (MOG) is a central nervous system myelin-specific molecule expressed on the outer lamellae of myelin. To date, the exact function of MOG has remained unknown, with MOG knockout mice displaying normal myelin ultrastructure and no apparent specific phenotype. In this paper, we identify nerve growth factor (NGF) as a binding partner for MOG and demonstrate that this interaction is capable of sequestering NGF from TrkA-expressing neurons to modulate axon growth and survival. Deletion of MOG results in aberrant sprouting of nociceptive neurons in the spinal cord. Binding of NGF to MOG may offer widespread implications into mechanisms that underlie pain pathways. PMID:26347141

  17. Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

    SciTech Connect

    Zhou Qi; Schneider, Irene C.; Gallet, Manuela; Kneissl, Sabrina; Buchholz, Christian J.

    2011-05-10

    The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.

  18. Determination of Glycosylation Sites and Site-specific Heterogeneity in Glycoproteins

    PubMed Central

    An, Hyun Joo; Froehlich, John W.; Lebrilla, Carlito B.

    2009-01-01

    Summary of Recent Advances Glycosylation is one of the most common post-translational modifications (PTMs) of proteins. At least 50% of human proteins are glycosylated with some estimates being as high as 70%. Glycoprotein analysis requires determining both the sites of glycosylation as well as the glycan structures associated with each site. Recent advances have led to the development of new analytical methods that employ mass spectrometry extensively making it possible to obtain the glycosylation site and the site microheterogeneity. These tools will be important for the eventual development of glycoproteomics. PMID:19700364

  19. Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A.

    PubMed

    Strotmeier, Jasmin; Mahrhold, Stefan; Krez, Nadja; Janzen, Constantin; Lou, Jianlong; Marks, James D; Binz, Thomas; Rummel, Andreas

    2014-04-01

    Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (HC). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the HCC and HCN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties. PMID:24583011

  20. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    PubMed Central

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200