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Sample records for acid glycoprotein agp

  1. Characterization of hepatic cellular uptake of α1-acid glycoprotein (AGP), part 2: involvement of hemoglobin β-chain on plasma membranes in the uptake of human AGP by liver parenchymal cells.

    PubMed

    Komori, Hisakazu; Nishi, Koji; Uehara, Nao; Watanabe, Hiroshi; Shuto, Tsuyoshi; Suenaga, Ayaka; Maruyama, Toru; Otagiri, Masaki

    2012-04-01

    Human α(1) -acid glycoprotein (AGP), a lipocalin family member, serves as a carrier for basic drugs and endogenous hormones. It is mainly distributed in the liver and also has anti-inflammatory effects. We previously discovered a protein in liver parenchymal cells that interacts with AGP and it was identified as hemoglobin β-chain (HBB). The purpose of this study was to clarify the role of HBB in the hepatic cellular uptake of AGP. Ligand blotting experiments showed that the interaction of (125) I-AGP with hemoglobin was saturable and was significantly suppressed in the presence of excess unlabeled AGP. In addition, the cellular uptake of fluorescein isothiocianate-AGP by HepG2 cells was saturable and temperature dependent. This uptake was inhibited by fillipin and methyl-β-cyclodextrin, but not chlorpromazine, suggesting that AGP is taken up via caveolae/lipid rafts endocytic pathway. Immunostaining showed that HBB and caveolin-1, exclusively expressed in caveolae, were partially colocalized on the plasma membranes of HepG2 cells. HBB knockdown with siRNA decreased the uptake of AGP by HepG2 cells by 40%, and exogenous hemoglobin inhibited the uptake by 40%-50%. These findings indicate that HBB is located on the liver plasma membrane and that it contributes to the intracellular uptake of AGP.

  2. Localization and possible function of nrF-AGP, an alpha-1-acid glycoprotein-like protein in viviparous fish Neoditrema ransonnetii (Perciformes, Embiotocidae).

    PubMed

    Nakamura, Osamu; Watabe, Yuki; Matsumoto, Naoko; Takasugi, Osamu; Watanabe, Ayako; Tsutsui, Shigeyuki

    2014-12-01

    The nrF-AGP, a 51-kDa acidic glycoprotein found in surfperch (Neoditrema ransonnetii; Perciformes, Embiotocidae), is a member of the lipocalin superfamily. nrF-AGP is the major component in ovarian cavity fluid (OCF), but not in plasma of pregnant females, which suggests its potential relevance in pregnancy. However, its production in the liver, irrespective of reproductive cycle and sex, indicates that the protein also has physiological functions other than its contribution to reproduction. In the present study, Western blot analysis indicated that this protein is widely distributed in the cutaneous and intestinal mucosa, bile, and abdominal adipose tissue of fish, as well as plasma and OCF. Immunohistochemical staining of nrF-AGP was observed in hepatocytes, adipocytes, pancreatic cells, epidermal cells, and epithelial cells of ovigerous lamellae. Transcripts were detected in adipose tissue as well as hepatocytes by reverse transcription PCR analysis. This broad distribution of nrF-AGP suggests that this protein participates in various biological processes through its ability to bind to hydrophobes. After administration of biotinylated F-AGP into the ovarian cavity, the protein was detected in the cytoplasm of the intestinal epithelial cells of the fetus within 4 h. This suggests that nrF-AGP in the ovarian cavity acts as a transporter delivering maternal resources to the fetus.

  3. Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism.

    PubMed

    Matsunaga, Hisami; Sadakane, Yutaka; Haginaka, Jun

    2003-08-01

    Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.

  4. Reverse lectin ELISA for detecting fucosylated forms of α1-acid glycoprotein associated with hepatocellular carcinoma

    PubMed Central

    Stål, Per; Zenlander, Robin; Edenvik, Pia; Alexandersson, Catharina; Haglund, Mats; Rydén, Ingvar; Påhlsson, Peter

    2017-01-01

    Altered fucosylation of glycoproteins is associated with development of hepatocellular carcinoma (HCC). Lectins have been commonly used to assay changes in fucosylation of plasma glycoproteins. In the present study a recombinantly engineered form of the fucose binding lectin Aleuria aurantia (AAL) consisting of a single binding site for fucose (S2), was used to construct a reverse lectin ELISA method. Microtiter plates coated with the S2 lectin were used to capture glycoproteins from plasma samples followed by antibody detection of S2-bound fucosylated α1-acid glycoprotein (S2-bound AGP). The method was used to compare the level of S2-bound AGP in serum samples from a small cohort of patients with hepatitis, cirrhosis or HCC. Using the reverse S2 lectin ELISA it was shown that the levels of S2-bound AGP was significantly higher in HCC patients compared to non-cancer patients and that there was also a significant elevation of S2-bound AGP in HCC patients compared to cirrhosis patients. There was no correlation between the level of S2-bound AGP and total AGP concentration. The performance of S2-bound AGP in differentiating HCC from cirrhosis samples or hepatitis samples were compared to other markers. A combination of S2-bound AGP, α-fetoprotein and AGP concentration showed performances giving area under receiver operating curves of 0.87 and 0.95 respectively. PMID:28296934

  5. a1-acid glycoprotein inhibits lipogenesis in neonatal swine adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serum a1-acid glycoprotein (AGP) is elevated during late gestation and at birth in the pig and rapidly declines postnatally. In contrast, the pig is born with minimal lipid stores in the adipose tissue, but rapidly accumulates lipid during the first week. The present study examined if AGP can affe...

  6. 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...

  7. 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 ...

  8. Down-regulatory effect of alpha 1-acid glycoprotein on bovine neutrophil degranulation.

    PubMed

    Miranda-Ribera, Alba; Lecchi, Cristina; Bronzo, Valerio; Scaccabarozzi, Licia; Sartorelli, Paola; Franciosi, Federica; Ceciliani, Fabrizio

    2010-07-01

    In this paper, the possible involvement of the acute phase protein alpha(1)-acid glycoprotein (AGP) in the local immunomodulation of inflammation was investigated. The dose response of bovine neutrophils to AGP as to mobilization of primary and secondary granules was studied. It was found that AGP fulfils a protective role against spontaneous exocytosis of secondary, but not primary, granules. This downregulatory effect is much more evident when degranulation is challenged with Zymosan activated serum (ZAS). AGP activity is dose-dependent, the acute phase concentration being more active than the physiological one. Carbohydrate moiety of AGP was found to be critical, since experimentally desialylated protein does not maintain its exocytosis-modulatory activity. The fact that AGP may modulate the degranulation of neutrophils confirms the hypothesis that AGP is heavily involved in the fine tuning of neutrophil activity in the inflammatory environment.

  9. The immune system modulator a1-acid glycoprotein inhibits insulin and IGF1 induced protein synthesis in C2C12 myotubes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha-1 acid glycoprotein (AGP) has previously been demonstrated by our laboratory to be negatively correlated with growth rate in newborn piglets. However, a mechanism of action for AGP in growth has not been identified. Previous research has demonstrated that AGP can modify adipose tissue metabo...

  10. O-Glycosylation of α-1-Acid Glycoprotein of Human Milk Is Lactation Stage Related

    PubMed Central

    Berghausen-Mazur, Marta; Hirnle, Lidia; Kątnik-Prastowska, Iwona

    2015-01-01

    Abstract Background: Human milk provides a multitude of glycoproteins, including highly glycosylated α-1-acid glycoprotein (AGP), which elicits anti-inflammatory and immunomodulatory properties. The milk AGP glycoforms may provide the breastfed infant with a wide range of biological benefits. Here, we analyzed the reactivity of O-linked sugar-specific lectins with human milk AGP over the process of lactation and compared the results with those of the lactating mother's plasma. Materials and Methods: Relative amounts of human skim milk AGP O-glycans were analyzed in early colostrum, colostrum, and transitional and mature milk samples of 127 healthy mothers by lectin–AGP enzyme-linked immunosorbent assay using sialyl T (sialyl-α2,3/α2,6 Galβ1,3GalNAc-), asialyl T (Galβ1,3GalNAc-), and Tn (GalNAc-) antigen-specific biotinylated Artocarpus integrifolia (Jacalin), Arachis hypogaea (PNA), and Vicia villosa (VVA) lectins, respectively. Results: Milk AGP elicited high expression of Jacalin- and PNA-reactive glycotopes and low expression of VVA-reactive glycotopes, which were absent on plasma AGP of lactating mothers and healthy individuals. The expression of sialyl, asialyl T, and Tn glycotopes of human milk AGP was lactation stage related. The relative amount of Jacalin-reactive AGP glycotope was highest in the colostrum samples and then decreased starting from Day 8 of lactation. In contrast, an increase of the relative amount of PNA-reactive glycotope with milk maturation was observed. The relative amount of VVA-reactive glycotope remained almost constant over the development of lactation. Conclusions: Milk AGP differs from mother's plasma AGP by the presence of O-linked sialylated and asialylated T as well as Tn antigens. The variation of the expression of sialylated and asialylated T and Tn antigens on AGP is associated with milk maturation. PMID:26057552

  11. Inositol phosphatase activity of the Escherichia coli agp-encoded acid glucose-1-phosphatase.

    PubMed

    Cottrill, Michael A; Golovan, Serguei P; Phillips, John P; Forsberg, Cecil W

    2002-09-01

    When screening an Escherichia coli gene library for myo-inositol hexakisphosphate (InsP6) phosphatases (phytases), we discovered that the agp-encoded acid glucose-1-phosphatase also possesses this activity. Purified Agp hydrolyzes glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6 with pH optima, 6.5, 3.5, and 4.5, respectively, and was stable when incubated at pH values ranging from 3 to 10. Glucose-1-phosphate was hydrolyzed most efficiently at 55 degrees C. while InsP6 and p-nitrophenyl phosphate were hydrolyzed maximally at 60 degrees C. The Agp exhibited Km values of (0.39 mM, 13 mM, and 0.54 mM for the hydrolysis of glucose-1-phosphate, p-nitrophenyl phosphate, and InsP6, respectively. High-pressure liquid chromatography (HPLC) analysis of inositol phosphate hydrolysis products of Agp demonstrated that the enzyme catalyzes the hydrolysis of phosphate from each of InsP6, D-Ins(1,2,3,4,5)P5, Ins(1,3,4,5,6)P5, and Ins(1,2,3,4,6)P5, producing D/L-Ins(1,2,4,5,6)P5. D-Ins(1,2,4,5)P4, D/L-Ins(1,4,5,6)P4 and D/L-Ins(1,2,4,6)P4, respectively. These data support the contention that Agp is a 3-phosphatase.

  12. Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition.

    PubMed

    Sadakane, Yutaka; Matsunaga, Hisami; Nakagomi, Kazuya; Hatanaka, Yasumaru; Haginaka, Jun

    2002-07-19

    Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.

  13. Alpha(1)-acid glycoprotein is contained in bovine neutrophil granules and released after activation.

    PubMed

    Rahman, Mizanur M D; Miranda-Ribera, Alba; Lecchi, Cristina; Bronzo, Valerio; Sartorelli, Paola; Franciosi, Federica; Ceciliani, Fabrizio

    2008-09-15

    The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein alpha(1)-acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50-60kDa) AGP isoform (PMN-AGP), as determined by Western blotting and confirmed by fluorescence microscopy. The presence of AGP in bovine neutrophils has been confirmed by fluorescence immunocytometry. In addition, bovine neutrophils contain also a 42-45kDa isoform, which has the same MW as plasma-, liver-delivered, AGP. cDNA sequence of plasma- and PMN-AGP revealed that (i) the two proteins are products of the same gene; (ii) the differences in molecular weight are due do different post-translational modifications. This result was confirmed by deglycosylation of the two glycoforms. Exocytosis studies showed that isolated neutrophils exposed to several challengers, including Zymosan activated serum (ZAS) and phorbol 12-myristate 13-acetate (PMA), which mimic the inflammatory activation, released PMN-AGP as early as 15min. AGP's mRNA is physiologically expressed by mature resting neutrophils. Real-time PCR on LPS, ZAS and PMA challenged cells revealed that the level of expression apparently does not increase after inflammatory activation. Collectively, the findings reported in this paper proved that PMN-AGP: (i) is a hyperglycosylated glycoform of plasma AGP, (ii) is stored in granules, and (iii) is released by neutrophils in response to activation. Due to its anti-inflammatory activity, PMN-AGP may work as a fine tuning of the neutrophils functions in the inflammatory focus, i.e. it can reduce the damages caused by an excess of inflammatory response.

  14. An Acute-phase Protein as a Regulator of Sperm Survival in the Bovine Oviduct: Alpha 1-acid-glycoprotein Impairs Neutrophil Phagocytosis of Sperm In Vitro

    PubMed Central

    LIU, Jinghui; MAREY, Mohamed A.; KOWSAR, Rasoul; HAMBRUCH, Nina; SHIMIZU, Takashi; HANEDA, Shingo; MATSUI, Motozumi; SASAKI, Motoki; HAYAKAWA, Hiroyuki; PFARRER, Christiane; MIYAMOTO, Akio

    2014-01-01

    We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct. PMID:24931131

  15. Lactation Stage-Related Expression of Sialylated and Fucosylated Glycotopes of Human Milk α-1-Acid Glycoprotein

    PubMed Central

    Hirnle, Lidia; Berghausen-Mazur, Marta; Kątnik-Prastowska, Iwona M.

    2014-01-01

    Abstract Background: Because terminal sugars of α-1-acid glycoprotein (AGP) are reported to be involved in anti-inflammatory and immunomodulatory processes, their expressions might have an influence on the proper function of immune system of newborns. Here, relative amounts of sialylated and fucosylated glycotopes on human milk AGP over normal lactation were investigated. Materials and Methods: AGP concentration and relative amounts of its sialylated and fucosylated glycovariants were analyzed in early colostrum, colostrum, and transitional and mature milk samples of 127 healthy mothers by lectin–AGP enzyme-linked immunosorbent assay using α2,3- and α2,6-sialic acid and α1,2-, α1,3-, and α1,6-fucose specific biotinylated Maackia amurensis, Sambucus nigra, Ulex europaeus, Tetragonolobus purpureus, and Lens culinaris lectins, respectively. Results: AGP concentration in human milk was about 30 times lower than in plasma of lactating mothers and decreased gradually over lactation. Milk AGP showed significantly higher expression of sialylated and fucosylated glycotopes in comparison with those of plasma AGP. Milk AGP glycovariants containing α2,6-sialylated and α1,6- and α1,2-fucosylated glycotopes showed the highest relative amounts in early colostrums. With progression of lactation, the expressions of glycotopes α1,2-fucosylated decreased starting from Day 4 and those of α2,6-sialylated and α1,6-fucosylated from Day 8 of lactation, whereas the level of α2,3-sialyl-glycotope was almost constant over 45 days of lactation. In contrast, the expression of α1,3-linked fucose on AGP was low in colostrums and significantly higher in transitional and mature milk. Conclusions: The relative amounts of sialylated and fucosylated glycovariants of human hindmilk AGP significantly varied between Days 2 and 45 of normal lactation. PMID:24892765

  16. The degree of branching of the glycans of alpha(1)-acid glycoprotein in asthma. A correlation with lung function and inflammatory parameters.

    PubMed

    Van Den Heuvel, M M; Poland, D C; De Graaff, C S; Hoefsmit, E C; Postmus, P E; Beelen, R H; Van Dijk, W

    2000-06-01

    Alpha(1)-acid glycoprotein (AGP) is a plasma protein belonging to the group of acute-phase proteins. It contains five N-linked glycans which, depending on pathophysiologic state, differ in their degree of branching (i.e., in the relative proportions of di-, tri-, and tetraantennary glycans). Changes in the degree of branching of these glycans have been shown to affect various immunomodulatory properties of AGP. We wanted to investigate whether changes occur in the branching of AGP glycans in plasma and in bronchoalveolar lavage fluid (BALF) in asthma. For this purpose, we selected three groups of patients for study: patients with atopic asthma (AA), atopic nonasthmatic patients, and a group of patients with various interstitial lung diseases (ILDs). The plasma AGP concentration was normal in both atopic study groups, but was increased in ILD patients. In contrast, the branching of glycans of AGP was altered in subjects with AA, whereas it was normal in the other study groups. The presence of asthma symptoms correlated with the increased glycan branching of AGP in both plasma and BALF. Additionally, the degree of branching of AGP in BALF was related to FEV(1), to the provocative dose of histamine causing a 20% decrease in FEV (PD(20)), and to the number of eosinophils. In conclusion, asthma is accompanied by changes in the branching of AGP glycans that indicate an inflammatory reaction that differs markedly from a normal acute-phase response, in which decreased branching of AGP occurs.

  17. Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS.

    PubMed

    Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Gun Wook; Hwang, Heeyoun; Jeong, Hoi Keun; Yun, Ki Na; Ji, Eun Sun; Kim, Kwang Hoe; Kim, Jun Seok; Kim, Jong Won; Yun, Sung Ho; Choi, Chi-Won; Kim, Seung Il; Lim, Jong-Sun; Jeong, Seul-Ki; Paik, Young-Ki; Lee, Soo-Youn; Park, Jisook; Kim, Su Yeon; Choi, Young-Jin; Kim, Yong-In; Seo, Jawon; Cho, Je-Yoel; Oh, Myoung Jin; Seo, Nari; An, Hyun Joo; Kim, Jin Young; Yoo, Jong Shin

    2016-12-02

    Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.

  18. The glycosylation of AGP and its associations with the binding to methadone.

    PubMed

    Behan, Jennifer L; Cruickshank, Yvonne E; Matthews-Smith, Gerri; Bruce, Malcolm; Smith, Kevin D

    2013-01-01

    Methadone remains the most common form of pharmacological therapy for opioid dependence; however, there is a lack of explanation for the reports of its relatively low success rate in achieving complete abstinence. One hypothesis is that in vivo binding of methadone to the plasma glycoprotein alpha-1-acid glycoprotein (AGP), to a degree dependent on the molecular structure, may render the drug inactive. This study sought to determine whether alterations present in the glycosylation pattern of AGP in patients undergoing various stages of methadone therapy (titration < two weeks, harm reduction < one year, long-term > one and a half years) could affect the affinity of the glycoprotein to bind methadone. The composition of AGP glycosylation was determined using high pH anion exchange chromatography (HPAEC) and intrinsic fluorescence analysed to determine the extent of binding to methadone. The monosaccharides galactose and N-acetyl-glucosamine were elevated in all methadone treatment groups indicating alterations in AGP glycosylation. AGP from all patients receiving methadone therapy exhibited a greater degree of binding than the normal population. This suggests that analysing the glycosylation of AGP in patients receiving methadone may aid in determining whether the therapy is likely to be effective.

  19. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture.

    PubMed

    Caperna, T J; Shannon, A E; Stoll, M; Blomberg, L A; Ramsay, T G

    2015-07-01

    Alpha-1 acid glycoprotein (AGP, orosomucoid, 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 species, and its circulating concentration appears to be associated with growth rate. The purpose of the present study was to investigate the regulation of AGP synthesis in hepatocytes prepared from suckling piglets and to provide a framework to compare its regulation with that of haptoglobin (HP), a positive acute-phase protein. Hepatocytes were isolated from preweaned piglets and maintained in serum-free monolayer culture for up to 72 h. The influences of hormones, cytokines, and redox modifiers on the expression and secretion of AGP and HP were determined by relative polymerase chain reaction and by measuring the concentration of each protein secreted into culture medium. The messenger RNA abundance and/or secretion of AGP protein was enhanced by interleukin (IL)-17a, IL-1, and resveratrol and inhibited by tumor necrosis factor-α (TNF), oncostatin M, and thyroid hormone (P < 0.05). HP expression and synthesis were upregulated by oncostatin M, IL-6, and dexamethasone and downregulated by TNF (P < 0.01). The overall messenger RNA expression at 24 h was in agreement with the secreted protein patterns confirming that control of these proteins in hepatocytes is largely transcriptional. Moreover, these data support the consideration that AGP is a negative acute-phase reactant and appears to be regulated by cytokines (with the exception of TNF) and hormones primarily in a manner opposite to that of the positive acute-phase protein, HP.

  20. Investigation of the retention behavior of structurally diverse drugs on alpha(1) acid glycoprotein column: insight on the molecular factors involved and correlation with protein binding data.

    PubMed

    Chrysanthakopoulos, Marios; Vallianatou, Theodosia; Giaginis, Costas; Tsantili-Kakoulidou, Anna

    2014-08-18

    Retention of 49 structurally diverse drugs on alpha1 acid glycoprotein column was investigated under different chromatographic conditions. Acetonitrile and 2-propanol were used as organic modifiers at different percentages and the pH was adjusted at 7.0 using PBS. Analysis of extrapolated and isocratic retention in terms of lipophilicity and electrostatic interactions revealed significant effect of the nature and percentage of organic modifier, which was attributed to the different shielding degree of the charged sites on the stationary phase by the buffer constituents. AGP retention factors were compared to HSA retention factors analyzed previously. Application of LSER analysis, extended to incorporate fractions ionized, demonstrated hydrogen bond acidity, dipolarity/polarizability and excess molar refraction as the most significant parameters for all AGP chromatographic indices, elucidating the differentiation of AGP retention from octanol-water partitioning and HSA retention. An attempt to correlate AGP chromatographic indices to AGP association constants, available in literature, supported the importance of stationary shielding in retention mechanism. Thus, isocratic retention factors logk10(ACN)(AGP) show a moderate but still better performance than lipophilicity in the case of A variant and may be a useful tool for the estimation of relevant association constants. For F1/S binding simulation lower stationary phase shielding is needed to obtain a significant two term regression equation, where logk20(ACN)(AGP) exerts a secondary contribution next to the most important bulk effect expressed by molecular weight.

  1. Critical assessment of the diagnostic value of feline alpha1-acid glycoprotein for feline infectious peritonitis using the likelihood ratios approach.

    PubMed

    Paltrinieri, Saverio; Saverio, Paltrinieri; Giordano, Alessia; Alessia, Giordano; Tranquillo, Vito; Vito, Tranquillo; Guazzetti, Stefano; Stefano, Guazzetti

    2007-05-01

    Alpha-1-acid glycoprotein (AGP) increases in the blood of cats with feline infectious peritonitis (FIP), a lethal disease caused by feline coronavirus (FCoV). However, the diagnostic potential of AGP might be limited because AGP also increases in pathophysiological conditions other than FIP. In this retrospective study, the diagnostic potential of serum AGP concentration was evaluated on the basis of the pretest probability of disease, according to the Bayesian approach. Serum AGP levels from cats with FIP (group 1; n = 58) and without FIP (group 2; n = 104) were evaluated. Non-FIP cats were further subgrouped as follows: 2a) inflammation (n = 26), 2b) asymptomatic FCoV infection (n = 49), 2c) injection-site sarcoma (n = 19), 2d) postvaccination (n = 7), and 2e) specific pathogen free (n = 3). Standard descriptive analyses by group and empirical receiver-operating characteristic (ROC) curve estimation were performed. Ordinary logistic regression analysis was performed to derive an estimate of the continuous likelihood ratio to produce the posttest probability of disease for any combination of pretest probability and serum AGP value. The comparison of serum AGP levels in the different groups and the analysis of the ROC curve confirmed that serum AGP is a powerful discriminating marker for FIP. The Bayesian approach demonstrated that when the pretest probability of FIP is high, based on history and clinical signs (groups 1 or 2a), moderate serum AGP levels (1.5-2 mg/ml) can discriminate cats with FIP from others, while only high serum AGP levels (>3 mg/ml) can support a diagnosis of FIP in cats with a low pretest probability of disease (groups 2b to 2e).

  2. Inflammation-induced expression of sialyl Lewis X-containing glycan structures on alpha 1-acid glycoprotein (orosomucoid) in human sera

    PubMed Central

    1993-01-01

    The glycosylation of the acute phase glycoprotein alpha 1-acid glycoprotein (AGP) in human sera is subject to marked changes during acute inflammation as a result of the cytokine-induced hepatic acute phase reaction. The changes described thus far comprise alterations in the type of branching of the carbohydrate structures as revealed by increased reactivity of AGP with concanavalin A. We now report on acute inflammation-induced increases in alpha 1-->3-fucosylated AGP molecules, as detected by the reactivity of AGP towards the fucose- binding Aleuria aurantia lectin (AAL) in crossed affino- immunoelectrophoresis of human sera. Laparotomy of women, for the removal of benign tumors of the uterus, was used as a model for the development of the hepatic acute phase response. Hugh increases were detected in the amounts of strongly AAL-reactive fractions of AGP, presumably containing three or more fucosylated N-acetyllactosamine units. At least part of these Lewis X-type glycans (Gal beta 1-->[Fuc alpha 1-->3]GlcNAc-R) appeared to be substituted also with an alpha 2-- >3-linked sialic acid residue. This was revealed by the laparotomy- induced abundant staining of AGP with an antisialyl Lewis X monoclonal antibody (CSLEX-1) on blots of sodium dodecyl sulfate-polyacrylamide gels containing AGP isolated from the sera of a patient at various days after operation. It is concluded that acute inflammation induces a strong increase in sialyl Lewis X-substituted AGP molecules that persists at a high level throughout the inflammatory period. We postulate that these changes represent a physiological feedback response on the interaction between leukocytes and inflamed endothelium, which is mediated via sialylated Lewis X structures and the selectin endothelial-leukocyte adhesion molecule 1. PMID:7679706

  3. Analysis of Free Drug Fractions in Serum by Ultrafast Affinity Extraction and Two-Dimensional Affinity Chromatography using α1-Acid Glycoprotein Microcolumns

    PubMed Central

    Bi, Cong; Zheng, Xiwei; Hage, David S.

    2016-01-01

    In the circulatory system, many drugs are reversibly bound to serum proteins such as human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), resulting in both free and protein-bound fractions for these drugs. This report examined the use of microcolumns containing immobilized AGP for the measurement of free drug fractions by ultrafast affinity extraction and a two-dimensional affinity system. Several drugs known to bind AGP were used as models to develop and evaluate this approach. Factors considered during the creation of this method included the retention of the drugs on the microcolumns, the injection flow rate, the microcolumn size, and the times at which a second AGP column was placed on-line with the microcolumn. The final system had residence times of only 110–830 ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10–20 min when using only 3–10 µL of sample per injection. This method was used to measure the free fractions of the model drugs at typical therapeutic levels in serum, giving good agreement with the results obtained by ultrafiltration. This approach was also used to estimate the binding constants for each drug with AGP in serum, even for drugs that had significant interactions with both AGP and HSA in such samples. These results indicated that AGP microcolumns could be used with ultrafast affinity extraction to measure free drug fractions in a label-free manner and to study the binding of drugs with AGP in complex samples such as serum. PMID:26797422

  4. The external amino acid signaling pathway promotes activation of Stp1 and Uga35/Dal81 transcription factors for induction of the AGP1 gene in Saccharomyces cerevisiae.

    PubMed Central

    Abdel-Sater, Fadi; Iraqui, Ismaïl; Urrestarazu, Antonio; André, Bruno

    2004-01-01

    Yeast cells respond to the presence of amino acids in their environment by inducing transcription of several amino acid permease genes including AGP1, BAP2, and BAP3. The signaling pathway responsible for this induction involves Ssy1, a permease-like sensor of external amino acids, and culminates with proteolytic cleavage and translocation to the nucleus of the zinc-finger proteins Stp1 and Stp2, the lack of which abolishes induction of BAP2 and BAP3. Here we show that Stp1-but not Stp2-plays an important role in AGP1 induction, although significant induction of AGP1 by amino acids persists in stp1 and stp1 stp2 mutants. This residual induction depends on the Uga35/Dal81 transcription factor, indicating that the external amino acid signaling pathway activates not only Stp1 and Stp2, but also another Uga35/Dal81-dependent transcriptional circuit. Analysis of the AGP1 gene's upstream region revealed that Stp1 and Uga35/Dal81 act synergistically through a 21-bp cis-acting sequence similar to the UAS(AA) element previously found in the BAP2 and BAP3 upstream regions. Although cells growing under poor nitrogen-supply conditions display much higher induction of AGP1 expression than cells growing under good nitrogen-supply conditions, the UAS(AA) itself is totally insensitive to nitrogen availability. Nitrogen-source control of AGP1 induction is mediated by the GATA factor Gln3, likely acting through adjacent 5'-GATA-3' sequences, to amplify the positive effect of UAS(AA). Our data indicate that Stp1 may act in combination with distinct sets of transcription factors, according to the gene context, to promote induction of transcription in response to external amino acids. The data also suggest that Uga35/Dal81 is yet another transcription factor under the control of the external amino acid sensing pathway. Finally, the data show that the TOR pathway mediating global nitrogen control of transcription does not interfere with the external amino acid signaling pathway. PMID

  5. Specific glycosylation of α1-acid glycoprotein characterises patients with familial Mediterranean fever and obligatory carriers of MEFV

    PubMed Central

    Poland, D; Drenth, J; Rabinovitz, E; Livneh, A; Bijzet, J; van het, Hof B; van Dijk, W

    2001-01-01

    BACKGROUND—Familial Mediterranean fever (FMF) is a periodic febrile disorder, characterised by fever and serositis. The acute phase response during attacks of FMF results from the release of cytokines, which in turn induce increased expression and changed glycosylation of acute phase proteins. A recent study indicated that attacks in FMF are accompanied by a rise of plasma concentrations of serum amyloid A (SAA) and C reactive protein (CRP), which remain significantly raised during remission relative to healthy controls. Another study suggested that obligatory heterozygotes also display an inflammatory acute phase response.
OBJECTIVE—To determine the state of inflammation in homozygotic and heterozygotic MEFV genotypes.
METHODS—CRP and SAA were studied by enzyme linked immunosorbent assay (ELISA). The glycosylation of the acute phase protein, α1-acid glycoprotein (AGP), was visualised with crossed affinoimmunoelectrophoresis with concanavalin A as diantennary glycan-specific component and Aleuria aurantia lectin as fucose-specific affinity component.
RESULTS—FMF attacks were associated with an increase (p<0.05) in the serum inflammation parameters CRP, SAA, and AGP. The glycosylation of AGP showed an increase (p<0.05) in fucosylated AGP glycoforms, whereas the branching of the glycans remained unaffected. The glycosylation of AGP in the MEFV carrier group, compared with that in a healthy control group, was characterised by a significant increase (p<0.05) in branching of the glycans, whereas the fucosylation remained unaffected.
CONCLUSION—The findings suggest an FMF-specific release of cytokines, resulting in a different glycosylation of AGP between a homozygotic and heterozygotic MEFV genotype.

 PMID:11454642

  6. Fucosylated Glycans in α1-Acid Glycoprotein for Monitoring Treatment Outcomes and Prognosis of Cancer Patients

    PubMed Central

    Yazawa, Shin; Takahashi, Ryo; Yokobori, Takehiko; Sano, Rie; Mogi, Akira; Saniabadi, Abby R.; Kuwano, Hiroyuki; Asao, Takayuki

    2016-01-01

    One standard treatment option for advanced-stage cancer is surgical resection of malignant tumors following by adjuvant chemotherapy and chemoradiotherapy. Additionally, neoadjuvant chemotherapy may be applied if required. During the time course of treatments, patients are generally followed by computed tomography (CT) surveillance, and by tumor marker diagnosis. However, currently, early evidence of recurrence and/or metastasis of tumors with a clinically relevant biomarker remains a major therapeutic challenge. In particular, there has been no validated biomarker for predicting treatment outcomes in therapeutic settings. Recently, we have looked at glycoforms of serum α1-acid glycoprotein (AGP) by using a crossed affinoimmunoelectrophoresis with two lectins and an anti-AGP antibody. The primary glycan structures of AGP were also analyzed by a mass spectrometer and a novel software in a large number of patients with various cancers. Accordingly, the relative abundance of α1,3fucosylated glycans in AGP (FUCAGP) was found to be significantly high in cancer patients as compared with the healthy controls. Further, strikingly elevated levels of FUCAGP were found in patients with poor prognosis but not in patients with good prognosis. In the current study, levels of FUCAGP in serum samples from various cancer patients were analyzed and 17 patients including 13 who had undergone chemotherapy were followed for several years post operation. FUCAGP level determined diligently by using a mass spectrometer was found to change along with disease prognosis as well as with responses to treatments, in particular, to various chemotherapies. Therefore, FUCAGP levels measured during following-up of the patients after operation appeared to be clinically relevant biomarker of treatment intervention. PMID:27295180

  7. cap alpha. -D-Mannopyranosylmethyl-P-nitrophenyltriazene effects on the degradation and biosynthesis of N-linked oligosaccharide chains on. cap alpha. /sub 1/-acid glycoprotein by liver cells

    SciTech Connect

    Docherty, P.A.; Aronson, N.N. Jr.

    1986-05-01

    The effects of ..cap alpha..-D-mannopyranosylmethyl-p-nitrophenyltriazene (..cap alpha..-ManMNT) on the degradation and processing of oligosaccharide chains on ..cap alpha../sub 1/-acid glycoprotein (AGP) were studied. Addition of the triazene to a perfused liver blocked the complete degradation of endocytosed N-acetyl (/sup 14/C)glucosamine-labeled asialo-AGP and caused the accumulation of Man/sub 2/GlcNAc/sub 1/ fragments in the lysosome-enriched fraction of the liver homogenate. This compound also reduced the reincorporation of lysosomally-derived (/sup 14/C)GlcNAc into newly secreted glycoproteins. Cultured hepatocytes treated with the inhibitor synthesized and secreted fully-glycosylated AGP. However, the N-linked oligosaccharide chains on AGP secreted by the ..cap alpha..-ManMNT-treated hepatocytes remained sensitive to digestion with endoglycosidase H, were resistant to neuraminidase, and consisted of Man/sub 9-7/GlcNAc/sub 2/ structures as analyzed by high resolution Bio-Gel P-4 chromatography. As measured by their resistance to cleavage by endoglycosidase H, the normal processing of all six carbohydrate chains on AGP to the complex form did not completely resume until nearly 24 h after triazene treatment. Since ManMNT is likely to irreversibly inactivate ..cap alpha..-D-mannosidases, the return of AGP to secretory forms with complex chains after 24 h probably resulted from synthesis of new processing enzymes.

  8. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  9. The glucocorticoid receptor regulates the binding of C/EPBbeta on the alpha-1-acid glycoprotein promoter in vivo.

    PubMed

    Savoldi, G; Fenaroli, A; Ferrari, F; Rigaud, G; Albertini, A; Di Lorenzo, D

    1997-12-01

    A complex interaction between the Glucocorticoid Receptor (GR), C/EBPbeta, and other transcription factors activate the Alpha-1 Acid Glycoprotein (AGP) promoter in HTC(JZ-1) rat hepatoma culture cells. This effect is mediated by the so-called Steroid Responsive Unit (SRU) of the AGP promoter that contains several binding sites for C/EBP transcription factors, some of which overlap with the Glucocorticoid Responsive Element (GRE). Our in vivo footprinting experiments revealed that the GRE- and the C/EBP-binding sites were already occupied glucocorticoid dependently in HTC(JZ-1) cells 10 min after dexamethasone administration (10(-6) M). Furthermore, local changes in the chromatine structure shown by the appearance of DNAse I hypersensitive sites (HS sites) also took place. These changes were probably dependent on a tissue-specific organization of the chromatine at the SRU because they were not detectable in a different glucocorticoid-responsive cell line (PC12) that did not express AGP. Here, we have also shown that withdrawal of dexamethasone or addition of the anti-glucocorticoid RU486 were able to revert the pattern induced by dexamethasone in vivo. The disappearance of the protected region and the hypersensitive sites, typical of the hormone activated promoter, confirmed the necessity of the GR to be bound by the agonist and the inability of the GR-antagonist complex to bind the DNA. By functional assays, we showed that the occupancy of the SRU by these transcriptional proteins in vivo correlated with the activation of the AGP gene transcription. With these results, we have shown that one of the functions of the GR to activate transcription of the AGP gene is to recruit C/EBPbeta and to maintain it bound at its target DNA sequences (SRU). This process was not accomplished by RU486.

  10. Plasma α1-Acid Glycoprotein Can Be Used to Adjust Inflammation-Induced Hyporetinolemia in Vitamin A-Sufficient, but Not Vitamin A-Deficient or -Supplemented Rats12

    PubMed Central

    Gieng, Sin H.; Rosales, Francisco J.

    2006-01-01

    We examined the association between α1-acid glycoprotein (AGP), all-trans-retinol (retinol), and albumin concentrations in a longitudinal animal model of IL-6–induced inflammation. Vitamin A–sufficient (VAS) male Sprague-Dawley rats were administered recombinant human IL-6 [n = 4, 65 μg/(kg·d)] or PBS (n = 4) continuously for 7 d via osmotic minipumps. Plasma samples were obtained daily and concentrations of retinol, AGP, albumin, and total protein were measured. Compared with both baseline and controls, retinol and albumin decreased (P < 0.05), AGP increased (P < 0.05), and total protein concentrations were unaffected in IL-6–treated rats. In vitamin A–deficient (VAD) rats, AGP concentrations were significantly lower at all time points and increased only to one-third of that in VAS rats. The AGP cut-off value indicative of inflammation was 0.11 g/L (i.e., 95% upper limit of baseline concentrations). After 20.5 h, there was an inverse linear correlation between AGP concentrations and the relative change in retinol to baseline (y = −0.18x + 0.48, r = −0.84, P < 0.001). However, changes in AGP and albumin were not correlated (P = 0.94). The application of this function to retinol concentrations in rats from separate experiments showed that hyporetinolemia cannot be adjusted using plasma AGP in VAD or vitamin A–supplemented rats. In conclusion, correcting inflammation-induced hyporetinolemia using an acute-phase protein requires longitudinally derived data, knowledge of vitamin A status, and a common underlying mechanism of change. PMID:16772457

  11. Multiple ligand-binding properties of the lipocalin member chicken alpha1-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: the essential role of the conserved tryptophan residue.

    PubMed

    Zsila, Ferenc; Matsunaga, Hisami; Bikádi, Zsolt; Haginaka, Jun

    2006-08-01

    Multiple ligand-binding properties of the 30-kDa chicken alpha(1)-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K(a) approximately 10(5)-10(6) M(-1)) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, L-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via pi-pi stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the pi-pi* transitions of the stacked indole ring-ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core beta-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.

  12. Use of boronic acid nanoparticles in glycoprotein enrichment.

    PubMed

    Xu, Yawei; Zhang, Lijuan; Lu, Haojie

    2013-01-01

    Glyco-specific enrichment methods for mass spectrometry pretreatment are invaluable for the detection of low abundant glycoproteins or glycopeptides. For example, boronic acid can specifically interact with glycans in nonaqueous or basic aqueous solutions. Here, we describe a glyco-specific enrichment method which uses a boronic acid-functionalized "core-satellite" composite nanoparticle to isolate glycoproteins or glycopeptides from complex biological samples. Furthermore, we also demonstrate detection limit improvements and show how to evaluate the percent recovery from the glycoprotein or glycopeptide enrichment process via SDS-PAGE and (16)O/(18)O labeling strategies.

  13. Inhibition of neutrophil activation by alpha1-acid glycoprotein.

    PubMed Central

    Costello, M J; Gewurz, H; Siegel, J N

    1984-01-01

    We report that alpha1-acid glycoprotein (AAG), a naturally occurring human plasma protein and acute phase reactant of uncertain biological function, inhibits human neutrophil aggregation and superoxide anion generation induced by a variety of stimuli including zymosan treated serum, formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate. Inhibition was transient, directly proportional to the glycoprotein concentration and inversely proportional to the concentration of the stimulus added. Desialyzation, resulting in the removal of a substantial portion of the molecule's negative charge, did not alter the effectiveness of AAG. Removal of the penultimate galactose residues from desialyzed AAG resulted in a slight but significant reversal of inhibition, suggesting that the heteropolysaccharide units of AAG may be important for inhibition of cellular function. We therefore suggest that the acute phase glycoprotein AAG may be a significant modulator of neutrophil as well as platelet and lymphocyte function during inflammation. PMID:6321072

  14. Tomato LeAGP-1 arabinogalactan-protein purified from transgenic tobacco corroborates the Hyp contiguity hypothesis.

    PubMed

    Zhao, Zhan Dong; Tan, Li; Showalter, Allan Marshall; Lamport, Derek Thomas Anthony; Kieliszewski, Marcia Jane

    2002-08-01

    Functional analysis of the hyperglycosylated arabinogalactan-proteins (AGPs) attempts to relate biological roles to the molecular properties that result largely from O-Hyp glycosylation putatively coded by the primary sequence. The Hyp contiguity hypothesis predicts contiguous Hyp residues as attachment sites for arabino-oligosaccharides (arabinosides) and clustered, non-contiguous Hyp residues as arabinogalactan polysaccharide sites. Although earlier tests of naturally occurring hydroxyproline-rich glycoproteins (HRGPs) and HRGPs designed by synthetic genes were consistent with a sequence-driven code, the predictive value of the hypothesis starting from the DNA sequences of known AGPs remained untested due to difficulties in purifying a single AGP for analysis. However, expression in tobacco (Nicotiana tabacum) of the major tomato (Lycopersicon esculentum) AGP, LeAGP-1, as an enhanced green fluorescent protein fusion glycoprotein (EGFP)-LeAGP-1, increased its hydrophobicity sufficiently for chromatographic purification from other closely related endogenous AGPs. We also designed and purified two variants of LeAGP-1 for future functional analysis: one lacking the putative glycosylphosphatidylinositol (GPI)-anchor signal sequence; the other lacking a 12-residue internal lysine-rich region. Fluorescence microscopy of plasmolysed cells confirmed the location of LeAGP-1 at the plasma membrane outer surface and in Hechtian threads. Hyp glycoside profiles of the fusion glycoproteins gave ratios of Hyp-polysaccharides to Hyp-arabinosides plus non-glycosylated Hyp consistent with those predicted from DNA sequences by the Hyp contiguity hypothesis. These results demonstrate a route to the purification of AGPs and the use of the Hyp contiguity hypothesis for predicting the Hyp O-glycosylation profile of an HRGP from its DNA sequence.

  15. Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl-Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation.

    PubMed

    Zaazaa, Hala E; Salama, Nahla N; Abd El Halim, Lobna M; Salem, Maissa Y; Abd El Fattah, Laila E

    2016-01-01

    Rapid and simple isocratic high-performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of αl -acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (ά) and the stereochemical resolution factor (Rs ) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S-hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1-10 µg mL(-1) and 0.5-5 µg mL(-1) for S-hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S-hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process.

  16. Effect of inositol and phytases on hematological indices and α-1 acid glycoprotein levels in laying hens fed phosphorus-deficient corn-soybean meal-based diets.

    PubMed

    Zyła, K; Grabacka, M; Pierzchalska, M; Duliński, R; Starzyńska-Janiszewska, A

    2013-01-01

    The effects of feeding low nonphytate phosphorus (NPP) corn-soybean meal-based diets supplemented with myo-inositol at 0.1%, or with phytase B at 1,300 acid phosphatase units/kg, or with phytase B enriched in 6-phytase A at 300 phytase units/kg on the hematological indices and the α-1 acid glycoprotein (AGP) concentrations in the blood of Bovans Brown laying hens were investigated. The experimental design comprised also a negative control diet and an internal control diet that had the NPP content adjusted by the addition of 0.304 g of monocalcium phosphate per kg to reach the NPP level similar to that resulting from the combined action of both phytases. A total of sixty 50-wk-old hens were randomly assigned to the dietary treatments with 12 cage replicates of 1 hen, and fed the experimental diets until wk 62, when the blood samples were taken and analyzed for basic hematological indices and for AGP concentrations in sera. The hematological indices from all the experimental groups remained in a normal range; nevertheless, the statistically significant effects of diet on hemoglobin concentration (P = 0.003), erythrocyte counts (P = 0.035), the percentage of lymphocytes (P = 0.020), heterophils (P = 0.002), eosinophils (P = 0.023), and basophils (P = 0.001) in the leucocyte population, as well as on the heterophil to lymphocyte ratio (P = 0.003), were observed. The highest erythrocyte counts were characteristic for hens fed the diet supplemented with both phytase A and phytase B. The highest heterophil to lymphocyte ratios were found in blood of hens fed the diet supplemented with phytase B, whereas the highest basophil percentages and the highest AGP concentrations occurred in birds fed the negative control diet. A highly significant correlation was observed between AGP concentrations in sera and BW losses determined previously. The results indicate that the low-NPP corn soybean meal-based diets increased acute phase protein level in laying hens. Phytase B alone

  17. Distribution of primaquine in human blood: Drug-binding to alpha 1-glycoprotein

    SciTech Connect

    Kennedy, E.; Frischer, H. )

    1990-12-01

    To clarify the distribution of the antimalarial primaquine in human blood, we measured the drug separately in the liquid, cellular, and ultrafiltrate phases. Washed red cells resuspended at a hematocrit of 0.4 were exposed to a submaximal therapeutic level of 250 ng/ml of carbon 14-labeled primaquine. The tracer was recovered quantitatively in separated plasma and red cells. Over 75% of the total labeled drug was found in red cells suspended in saline solution, but only 10% to 30% in red cells suspended in plasma. The plasma effect was not mediated by albumin. Studies with alpha 1-acid glycoprotein (AGP), tris(2-butoxyethyl)phosphate, an agent that displaces AGP-bound drugs, and cord blood known to have decreased AGP established that primaquine binds to physiologic amounts of the glycoprotein in plasma. Red cell primaquine concentration increased linearly as AGP level fell and as the free drug fraction rose. We suggest that clinical blood levels of primaquine include the red cell fraction or whole blood level because (1) erythrocytic primaquine is a sizable and highly variable component of the total drug in blood; (2) this component reflects directly the free drug in plasma, and inversely the extent of binding to AGP; (3) the amount of free primaquine may influence drug transport into specific tissues in vivo; and (4) fluctuations of AGP, an acute-phase reactant that increases greatly in patients with malaria and other infections, markedly affect the partition of primaquine in blood. Because AGP binds many basic drugs, unrecognized primaquine-drug interactions may exist.

  18. Immunohistochemical analysis of cell wall hydroxyproline-rich glycoproteins in the roots of resistant and susceptible wax gourd cultivars in response to Fusarium oxysporum f. sp. Benincasae infection and fusaric acid treatment.

    PubMed

    Xie, Dasen; Ma, Li; Samaj, Jozef; Xu, Chunxiang

    2011-08-01

    Hydroxyproline-rich glycoproteins (HRGPs) play a defensive role in host-pathogen interactions. However, specific roles of individual HRGPs in plant defense against pathogen are poorly understood. Changes in extracellular distribution and abundance of individual cell wall HRGPs were investigated on root sections of two wax gourd (Benincasa hispida Cogn.) cultivars (Fusarium wilt resistant and susceptible, respectively), which were analyzed by immunolabelling with 20 monoclonal antibodies recognizing different epitopes of extensins and arabinogalactan proteins (AGPs) after being inoculated with Fusarium oxysporum f. sp. Benincasae or treated with fusaric acid (FA). These analyses revealed the following: (1) The levels of JIM11 and JIM20 interacting extensins were higher in the resistant cultivar. Either treatment caused a dramatic decrease in signal in both cultivars, but some new signal appeared in the rhizodermis. (2) The AGPs or rhamnogalacturonan containing CCRCM7-epitope were enhanced in the resistant cultivar, but not in the susceptible one by either treatment. (3) Either treatment caused a slight increase in the levels of the AGPs recognized by LM2 and JIM16, but there were no differences between two cultivars. (4) The MAC204 signal nearly disappeared after FA treatment, but this was not the case with pathogen attack. (5) The LM14 signal slightly decreased after both treatments in both cultivars, but a less decrease was observed with the resistant cultivar. These results indicate that the CCRCM7 epitope likely contributed to the resistance of wax gourd to this pathogen, and JIM11 and JIM20 interacting extensins as well as LM2, LM14, MAC204 and JIM16 interacting AGPs were involved in the host-pathogen interaction.

  19. Analytical and preparative enantioseparation of DL-penicillamine and DL-cysteine by high-performance liquid chromatography on alpha-acid glycoprotein and beta-cyclodextrin columns using ninhydrin as a reversible tagging reagent.

    PubMed

    Bhushan, Ravi; Kumar, Rajender

    2009-04-10

    Two sulfur-containing amino acids, DL-cysteine (Cys) and DL-penicillamine (PenA), were condensed with ninhydrin to form their spirothiazolidine derivatives. These were separated by HPLC using alpha-acid glycoprotein (AGP) and beta-cyclodextrin (beta-CD) columns. The resolution conditions were optimized and the results were compared. Since the method provided resolution greater than 2 it was also applied to preparative separation. After separation, each of them was detagged using Zn dust and 10% aqueous trifluoroacetic acid. For analytical purposes dinitrophenyl (DNP) derivatives of DL-Cys and DL-PenA were also prepared and were resolved on both the columns. The detection was carried out using photodiode array detection system at 231 nm. The limits of detection were found to be 0.01% and 0.004% for spirothiazolidine carboxylic acid and DNP derivatives, respectively.

  20. Discovery of Inhibitors for the Ether Lipid-Generating Enzyme AGPS as Anti-Cancer Agents.

    PubMed

    Piano, Valentina; Benjamin, Daniel I; Valente, Sergio; Nenci, Simone; Marrocco, Biagina; Mai, Antonello; Aliverti, Alessandro; Nomura, Daniel K; Mattevi, Andrea

    2015-11-20

    Dysregulated ether lipid metabolism is an important hallmark of cancer cells. Previous studies have reported that lowering ether lipid levels by genetic ablation of the ether lipid-generating enzyme alkyl-glycerone phosphate synthase (AGPS) lowers key structural and oncogenic ether lipid levels and alters fatty acid, glycerophospholipid, and eicosanoid metabolism to impair cancer pathogenicity, indicating that AGPS may be a potential therapeutic target for cancer. In this study, we have performed a small-molecule screen to identify candidate AGPS inhibitors. We have identified several lead AGPS inhibitors and have structurally characterized their interactions with the enzyme and show that these inhibitors bind to distinct portions of the active site. We further show that the lead AGPS inhibitor 1a selectively lowers ether lipid levels in several types of human cancer cells and impairs their cellular survival and migration. We provide here the first report of in situ-active pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy.

  1. Characterization of the Arabinogalactan Protein 31 (AGP31) of Arabidopsis thaliana

    PubMed Central

    Hijazi, May; Durand, Jessica; Pichereaux, Carole; Pont, Frédéric; Jamet, Elisabeth; Albenne, Cécile

    2012-01-01

    Proteins are important actors in plant cell walls because they contribute to their architecture and their dynamics. Among them, hydroxyproline (Hyp)-rich glycoproteins constitute a complex family of O-glycoproteins with various structures and functions. In this study, we characterized an atypical Hyp-rich glycoprotein, AGP31 (arabinogalactan protein 31), which displays a multidomain organization unique in Arabidopsis thaliana, consisting of a short arabinogalactan protein (AGP) motif, a His stretch, a Pro-rich domain, and a C-terminal PAC (PRP-AGP containing Cys) domain. The use of various mass spectrometry strategies was innovative and powerful: it permitted us to locate Hyp residues, to demonstrate the presence of carbohydrates, and to refine their distribution over the Pro-rich domain. Most Hyp were isolated within repeated motifs such as KAOV, KSOV, K(PO/OP)T, K(PO/OP)V, T(PO/OP)V, and Y(PO/OP)T. A few extensin-like motifs with contiguous Hyp (SOOA and SOOT) were also found. The Pro-rich domain was shown to carry Gal residues on isolated Hyp but also Ara residues. The existence of new type Hyp-O-Gal/Ara-rich motifs not recognized by the β-glucosyl Yariv reagent but interacting with the peanut agglutinin lectin was proposed. In addition, the N-terminal short AGP motif was assumed to be substituted by arabinogalactans. Altogether, AGP31 was found to be highly heterogeneous in cell walls because arabinogalactans could be absent, Hyp-O-Gal/Ara-rich motifs of different sizes were observed, and truncated forms missing the C-terminal PAC domain were found, suggesting degradation in muro and/or partial glycosylation prior to secretion. PMID:22270363

  2. 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.

  3. 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

  4. Serum sialic acid and glycoprotein levels in some Libyan cancer patients.

    PubMed

    Balo, N N; Ishaq, M

    1991-01-01

    Sialic acid is a common conjugate of some serum glycoproteins and glycolipids. Elevated levels of serum sialic acid and alterations in serum glycoproteins have been observed in certain types of cancer. In this study sialic acid concentration in the sera of patients with various types of cancer was determined. In addition to this, serum glycoproteins were also analysed by electrophoretic method. Our results indicate that serum sialic acid levels are generally raised in all types of cancer studied. This increase was more pronounced in case of lung, bronchogenic, intestinal and breast cancer. Some alterations in the serum glycoprotein profiles were also observed, particularly in bronchogenic and gall bladder cancer where an additional band in the low molecular weight region was present and in lung, breast and lymphoma where a band in the middle molecular weight region was found missing when compared with normals.

  5. Interaction of serum proteins with CYP isoforms in human liver microsomes: inhibitory effects of human and bovine albumin, alpha-globulins, alpha-1-acid glycoproteins and gamma-globulins on CYP2C19 and CYP2D6.

    PubMed

    Xu, Bang Qian; Ishii, Mikio; Ding, Li Rong; Fischer, Nancy E; Inaba, Tadanobu

    2003-03-14

    The effects of serum proteins on the in vitro hydroxylation pathways of mephenytoin (CYP2C19) and debrisoquine (CYP2D6) were studied to enhance the predictability of in vivo drug metabolism from in vitro assays. Both CYP substrates are known to be weakly bound to albumin and the applicability of the free drug hypothesis was further appraised. Since bovine serum albumin (BSA) is used widely in in vitro assays, a comparison between human and bovine proteins was made. Four major serum proteins were studied: albumin, alpha1-acid glycoprotein (AGP), alpha- and gamma-globulins. Human serum albumin (HSA) inhibited both CYP activities about 20% more than BSA. The addition of human alpha-globulins, but not the bovine protein, resulted in marked reduction of 86% and 41% in CYP2C19 and CYP2D6 activities, respectively. This reduction of activity was strikingly greater than the fraction bound (14 and 22%, respectively). The inhibition was of the competitive type and the Ki values of human alpha-globulins on CYP2C19 and CYP2D6 were found to be 0.45% (4.5 mg/ml) and 3.5% (35 mg/ml), respectively. The effect of both human and bovine gamma-globulins on CYP isoforms was negligible. The Ki values of human and bovine AGP for CYP2C19 were 1.84% (420 microM) and 0.93% (210 microM), respectively. For HSA, human alpha-globulins and human and bovine AGP, the strongly decreased CYP activities in vitro cannot be explained by the free drug hypothesis. A direct interaction of these serum proteins with CYP enzymes is postulated. Differential effects of bovine and human serum proteins and CYP specific inhibition were observed.

  6. Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

    PubMed

    Albani, J R; Plancke, Y D

    1999-05-31

    Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.

  7. Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins.

    PubMed

    Lentz, T L; Wilson, P T; Hawrot, E; Speicher, D W

    1984-11-16

    Evidence was presented earlier that a host-cell receptor for the highly neurotropic rabies virus might be the acetylcholine receptor. The amino acid sequence of the glycoprotein of rabies virus was compared by computer analysis with that of snake venom curaremimetic neurotoxins, potent ligands of the acetylcholine receptor. A statistically significant sequence relation was found between a segment of the rabies glycoprotein and the entire sequence of long neurotoxins. The greatest identity occurs with residues considered most important in neurotoxicity, including those interacting with the acetylcholine binding site of the acetylcholine receptor. Because of the similarity between the glycoprotein and the receptor-binding region of the neurotoxins, this region of the viral glycoprotein may function as a recognition site for the acetylcholine receptor. Direct binding of the rabies virus glycoprotein to the acetylcholine receptor could contribute to the neurotropism of this virus.

  8. 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

  9. A family of abundant plasma membrane-associated glycoproteins related to the arabinogalactan proteins is unique to flowering plants

    PubMed Central

    1989-01-01

    We have identified a family of abundant peripheral plasma membrane glycoproteins that is unique to flowering plants. They are identified by a monoclonal antibody, MAC 207, that recognizes an epitope containing L-arabinose and D-glucuronic acid. Immunofluorescence and immunogold labeling studies locate the MAC 207 epitope to the outer surface of the plasma membrane both in protoplasts and in intact tissues. In some cells MAC 207 also binds to the vacuolar membrane, probably reflecting the movement of the plasma membrane glycoproteins in the endocytic pathway. The epitope recognized by MAC 207 is also present on a distinct soluble proteoglycan secreted into the growth medium by carrot (Daucus carota) suspension culture cells. Biochemical evidence identifies this neutral proteoglycan as a member of the large class of arabinogalactan proteins (AGPs), and suggests a structural relationship between it and the plasma membrane glycoproteins. AGPs have the property of binding to beta-glycans, and we therefore propose that one function of the AGP-related, plasma membrane-associated glycoproteins may be to act as cell surface attachment sites for cell wall matrix polysaccharides. PMID:2469683

  10. [Incidence of varying factors on the immunochemical behavior of alpha 1-acid glycoprotein].

    PubMed

    Biou, D; Durand, G; Feger, J; Agneray, J

    1980-02-01

    Electroimmunodiffusion methods of Laurell and radial immunodiffusion method of Mancini are compared for the qualitative and quantitative analysis of native and desialylated alpha 1-acid glycoprotein. Samples are incubated under different conditions at decreasing pH (3.5 to 0.5 pH units), with increasing ionic strength and with neuraminidase during different time intervals. Results show a pronounced decrease in electrophoretic mobility of alpha 1-acid glycoprotein treated either with acidic reagents or with neuraminidase (ionic strength has no effect). Such a procedure might involve chemical or enzymatic hydrolysis by which sialyl residues are removed. This hydrolysis implicates lower results in the estimation of the desialylated glycoprotein by electroimmunodiffusion. On the other hand, the amounts of alpha 1-acid glycoprotein evaluated by radial immunodiffusion are not modified after incubation. This is expected since diffusion and antigenic properties are not related to the sialic acid content. The data suggest that radial immunodiffusion, less accurate and sensitive than electroimmunodiffusion, is nevertheless more adequate for estimating native and desialylated alpha 1-acid glycoprotein.

  11. Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy.

    PubMed

    Silva Miranda, Mayra; Rodríguez, Kendy Wek; Martínez Cordero, Erasmo; Rojas-Espinosa, Oscar

    2006-12-01

    Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression.

  12. Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy

    PubMed Central

    Silva Miranda, Mayra; Rodríguez, Kendy Wek; Martínez Cordero, Erasmo; Rojas-Espinosa, Oscar

    2006-01-01

    Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression. PMID:17222216

  13. Bile acid transport in sister of P-glycoprotein (ABCB11) knockout mice.

    PubMed

    Lam, Ping; Wang, Renxue; Ling, Victor

    2005-09-20

    In vertebrates, bile flow is essential for movement of water and solutes across liver canalicular membranes. In recent years, the molecular motor of canalicular bile acid secretion has been identified as a member of the ATP binding cassette transporter (ABC) superfamily, known as sister of P-glycoprotein (Spgp) or bile salt export pump (Bsep, ABCB11). In humans, mutations in the BSEP gene are associated with a very low level of bile acid secretion and severe cholestasis. However, as reported previously, because the spgp(-)(/)(-) knockout mice do not express severe cholestasis and have substantial bile acid secretion, we investigated the "alternative transport system" that allows these mice to be physiologically relatively normal. We examined the expression levels of several ABC transporters in spgp(-)(/)(-) mice and found that the level of multidrug resistance Mdr1 (P-glycoprotein) was strikingly increased while those of Mdr2, Mrp2, and Mrp3 were increased to only a moderate extent. We hypothesize that an elevated level of Mdr1 in the spgp(-)(/)(-) knockout mice functions as an alternative pathway to transport bile acids and protects hepatocytes from bile acid-induced cholestasis. In support of this hypothesis, we showed that plasma membrane vesicles isolated from a drug resistant cell line expressing high levels of P-glycoprotein were capable of transporting bile acids, albeit with a 5-fold lower affinity compared to Spgp. This finding is the first direct evidence that P-glycoprotein (Mdr1) is capable of transporting bile acids.

  14. 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.

  15. 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

  16. Metrology for AGP - Astrometric Gravitation Probe

    NASA Astrophysics Data System (ADS)

    Gai, Mario; et al.

    2015-08-01

    The Astrometric Gravitation Probe (AGP) is a concept of space mission aimed at tests of Fundamental Physics in the Solar system, using Fizeau interferometry and coronagraphy techniques to implement differential astrometry among superposed stellar fields. The main goal is verification of the General Relativity (GR) and competing gravitation theories in the weak field of the Solar System by high precision measurement of the light deflection in the vicinity of the Sun at < 10-7 and of the main and minor planet dynamics at the microarcsec/year level. The AGP payload concept is based on a single main telescope (1.15 m diameter) implementing a multi-aperture Fizeau interferometer, for simultaneous observation of four regions close to the Solar limb and in opposition; coronagraphic techniques are applied on the elementary sub-apertures. The star displacement due to light deflection is derived by differential astrometry on images taken in different deflection conditions (e.g. ON and OFF). The instrument design is focused on systematic error control through multiple field simultaneous observation and calibration. The metrology system requirements related to the science goals are discussed, and the technical aspects of possible implementations are investigated. The potential benefit of auto-collimation and cophasing techniques derives from monitoring comparably large sections of the optical system common to the stellar beams. The performance at microarcsec level is verified by simulation.

  17. Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein.

    PubMed

    Albani, J R

    2006-11-06

    Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels.

  18. An analysis of amino acid sequences surrounding archaeal glycoprotein sequons.

    PubMed

    Abu-Qarn, Mehtap; Eichler, Jerry

    2007-05-01

    Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N-glycosylation made by sequon-bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N-glycosylation.

  19. Purification, characterization and functional expression of a new peptide with an analgesic effect from Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1).

    PubMed

    Wang, Yu; Wang, Li; Cui, Yong; Song, Yong-Bo; Liu, Yan-Feng; Zhang, Rong; Wu, Chun-Fu; Zhang, Jing-Hai

    2011-07-01

    In this study, a new peptide named BmK AGP-SYPU1 with an analgesic effect was purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK) through a four-step chromatographic process. The mouse twisting test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU1 was further qualified by RP-HPLC and HPCE. The molecular mass determined by the MALDI-4800-TOF/TOF MS for BmK AGP-SYPU1 was 7544 Da. Its primary structure of the N-terminal was obtained using Edman degradation. The gene sequence of BmK AGP-SYPU1 was cloned from the cDNA pool and genomic of scorpion glands, respectively, and then expressed in Escherichia coli. The sequence determination showed that BmK AGP-SYPU1 was composed of 66 amino acid residues with a new primary structure. The metal chelating affinity column and cation exchange chromatography were used to purify the recombinant BmK AGP-SYPU1. Consequently, the native and recombinant BmK AGP-SYPU1 showed similar analgesic effects on mice as assayed using a mouse twisting model. These results suggested that BmK AGP-SYPU1 is a new analgesic component found in the Chinese scorpion Buthus martensi Karsch.

  20. Malonic acid suppresses mucin-type O-glycan degradation during hydrazine treatment of glycoproteins.

    PubMed

    Goso, Yukinobu

    2016-03-01

    Hydrazine treatment is frequently used for releasing mucin-type O-glycans (O-glycans) from glycoproteins because the method provides O-glycans that retain a reducible GalNAc at their reducing end, which is available for fluorescent labeling. However, many O-glycans are degraded by "peeling" during this treatment. In the current study, it was found that malonic acid suppressed O-glycan degradation during hydrazine treatment of bovine fetuin or porcine gastric mucin in both the gas and liquid phases. This is paradoxical because the release of O-glycans from glycoproteins occurs under alkaline conditions. However, malonic acid seems to prevent the degradation through its acidic property given that other weak acids also prevented the degradation. Accordingly, disodium malonate did not suppress O-glycan degradation. Application of this method to rat gastric mucin demonstrated that the majority of the major O-glycans obtained in the presence of malonic acid were intact, whereas those obtained in the absence of malonic acid were degraded. These results suggest that hydrazine treatment in the presence of malonic acid would allow glycomic analysis of native mucin glycoproteins.

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

    PubMed

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

    2012-03-09

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

  2. AGP (Astrometric Gravitation Probe) optical design report

    NASA Astrophysics Data System (ADS)

    Riva, Alberto; Gai, Mario; Landini, Federico; Lazzarini, Paolo; Gallieni, Daniele; Tintori, Matteo; Anselmi, Alberto; Cesare, Stefano; Busonero, Deborah; Lattanzi, Mario Gilberto; Vecchiato, Alberto

    2016-08-01

    This paper describes the current opto-mechanical design of AGP, a mission designed for astrometric verification of General Relativity (GR) and competing gravitation theories by means of precise determination of light deflection on field stars, and of orbital parameters of selected Solar System objects. The optical concept includes a planar rear-view mirror for simultaneous imaging on the CCD mosaic detector of fields of view also from the direction opposite to the Sun, affected by negligible deflection, for the sake of real time calibration. The precision of astrometric measurements on individual stars will be of order of 1 mas, over two fields separated by few degrees around the Sun and observed simultaneously. We describe the optical design characteristics, with particular reference to manufacturing and tolerancing aspects, evidencing the preservation of very good imaging performance over the range of expected operating conditions.

  3. Biopartitioning micellar chromatography with sodium dodecyl sulfate as a pseudo α(1)-acid glycoprotein to the prediction of protein-drug binding.

    PubMed

    Hadjmohammadi, Mohammadreza; Salary, Mina

    2013-01-01

    A simple and fast method is of urgent need to measure protein-drug binding affinity in order to meet the rapid development of new drugs. Biopartitioning micellar chromatography (BMC), a mode of micellar liquid chromatography (MLC) using micellar mobile phases in adequate experimental conditions, can be useful as an in vitro system in mimicking the drug-protein interactions. In this study, sodium dodecyl sulfate-micellar liquid chromatography (SDS-MLC) was used for the prediction of protein-drug binding based on the similar property of SDS micelles to α(1)-acid glycoprotein (AGP). The relationships between the BMC retention data of a heterogeneous set of 14 basic and neutral drugs and their plasma protein binding parameter were studied and the predictive ability of models was evaluated. Modeling of logk(BMC) of these compounds was established by multiple linear regression (MLR) and second-order polynomial models obtained in two different concentrations (0.07 and 0.09M) of SDS. The developed MLR models were characterized by both the descriptive and predictive ability (R(2)=0.882, R(CV)(2)=0.832 and R(2)=0.840, R(CV)(2)=0.765 for 0.07 and 0.09M SDS, respectively). The p values <0.01 also indicated that the relationships between the protein-drug binding and the logk(BMC) values were statistically significant at the 99% confidence level. The standard error of estimation showed the standard deviation of the regression to be 11.89 and 13.87 for 0.07 and 0.09M, respectively. The application of the developed model to a prediction set demonstrated that the model was also reliable with good predictive accuracy. The external and internal validation results showed that the predicted values were in good agreement with the experimental value.

  4. Infection with Listeria monocytogenes impairs sialic acid addition to host cell glycoproteins

    PubMed Central

    1994-01-01

    Listeria monocytogenes is a facultative intracellular bacterium that causes severe disease in neonates and immunocompromised adults. Although entry, multiplication, and locomotion of Listeria in the cytosol of infected cells are well described, the impact of such infection on the host cell is unknown. In this report, we investigate the effect of L. monocytogenes infection on MHC class I synthesis, processing, and intracellular trafficking. We show that L. monocytogenes infection interferes with normal processing of N-linked oligosaccharides on the major histocompatibility complex (MHC) class I heavy chain molecule, H-2Kd, resulting in a reduced sialic acid content. The glycosylation defect is more pronounced as the infection progresses and results from interference with the addition of sialic acid rather than its removal by a neuraminidase. The effect is found in two different cell lines and is not limited to MHC class I molecules since CD45, a surface glycoprotein, and LGP120, a lysosomal glycoprotein, are similarly affected by L. monocytogenes infection. The glycosylation defect is specific for infection by L. monocytogenes since neither Trypanosoma cruzi nor Yersinia enterocolitica, two other intracellular pathogens, reproduces the effect. The resultant hyposialylation of H-2Kd does not impair its surface expression in infected cells. Diminished sialic acid content of surface glycoproteins may enhance host-defense by increasing susceptibility to lysis and promoting clearance of Listeria-infected cells. PMID:7964488

  5. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket.

    PubMed

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Glycoprotein synthesis

    SciTech Connect

    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.

  13. 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.

  14. 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.

  15. 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.

  16. Simple boric acid-based fluorescent focusing for sensing of glucose and glycoprotein via multipath moving supramolecular boundary electrophoresis chip.

    PubMed

    Dong, Jingyu; Li, Si; Wang, Houyu; Meng, Qinghua; Fan, Liuyin; Xie, Haiyang; Cao, Chengxi; Zhang, Weibing

    2013-06-18

    Boric acid-based fluorescent complex probe of BBV-HPTS (boronic acid-based benzyl viologen (BBV) and hydroxypyrene trisulfonic acid trisodium salt (HPTS)) was rarely used for sensitive sensing of saccharide (especially glycoprotein) via electrophoresis. We proposed a novel model of moving supramolecular boundary (MSB) formed with monosaccharide or glycoprotein in microcolumn and the complex probe of BBV-HPTS in the cathodic injection tube, developed a method of MSB fluorescent focusing for sensitive recognition of monosaccharide and glycoprotein, and designed a special multipath capillary electrophoresis (CE) chip for relative experiments. As a proof of concept, glucose and hemoglobin A1c (HbA1c) were respectively used as the mode saccharide and glycoprotein for the relevant demonstration. The experiments revealed that (i) the complex of BBV-HPTS could interact with free glucose or bound one in glycoprotein; (ii) the fluorescent signal was a function of glucose or glycoprotein content approximately; and (iii) interestingly the fluorescent band motion was dependent on glucose content. The developed method had the following merits: (i) low cost; (ii) low limit of detection (down to 1.39 pg/mL for glucose and 2.0 pg per capillary HbA1c); and (iii) high throughput (up to 12 runs or more per patch) and speed (less than 5 min). The developed method has potential use for sensitive monitoring of monosaccharide and glycoprotein in biomedical samples.

  17. Chemical synthesis of glycoproteins with the specific installation of gradient enriched 15N-labeled amino acids for getting insight into glycoprotein behavior.

    PubMed

    Kajihara, Yasuhiro; Nguyen, Minh Hien; Izumi, Masayuki; Sato, Hajime; Okamoto, Ryo

    2017-03-09

    We propose a novel partially 15N-labelling method for the amide backbone of a synthetic glycoprotein. By use of a chemical approach utilizing SPPS and NCL, we inserted thirteen 15N-labeled amino acids at specific positions of the protein backbone, while intentionally varying the enrichment of 15N atoms. This idea enables us to discriminate even the same type of amino acid based on the intensities of 1H-15N HSQC signals, thus allowing us to understand the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied CD spectra and T1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure.

  18. The adsorption and lubrication behavior of synovial fluid proteins and glycoproteins on the bearing-surface materials of hip replacements.

    PubMed

    Roba, Marcella; Naka, Marco; Gautier, Emanuel; Spencer, Nicholas D; Crockett, Rowena

    2009-04-01

    The selectivity of synovial fluid protein adsorption onto ultra-high molecular weight polyethylene (UHMWPE) and alumina (Al(2)O(3)), and in particular the ability of glycoproteins to adsorb in the presence of all the other synovial fluid proteins, was investigated by means of fluorescence microscopy and gel electrophoresis (SDS-PAGE). The non-specific nature of protein adsorption from synovial fluid indicated that the lubrication of artificial hip-joint materials may not be attributable to a single protein as has been frequently suggested. The friction behavior of polyethylene (PE) sliding against Al(2)O(3) in solutions of bovine serum albumin (BSA), alpha-1-acid glycoprotein (AGP) and alpha-1-antitrypsin (A1AT) was investigated by means of colloidal probe atomic force microscopy. BSA was shown to be a poorer boundary lubricant than the phosphate buffered saline used as a control. This was attributed to denaturation of the BSA upon adsorption, which provided a high-shear-strength layer at the interface, impairing the lubrication. Interestingly, both the glycoproteins AGP and A1AT, despite their low concentrations, improved lubrication. The lubricating properties of AGP and A1AT were attributed to adsorption via the hydrophobic backbone, allowing the hydrophilic carbohydrate moieties to be exposed to the aqueous solution, thus providing a low-shear-strength fluid film that lubricated the system. The amount of glycoprotein adsorbed on hydrophobic surfaces was determined by means of optical waveguide lightmode spectroscopy (OWLS), allowing conclusions to be drawn about the conformation of the glycan residues following adsorption.

  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

    2014-06-07

    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.

  20. Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study.

    PubMed

    Albani, J R; Sillen, A; Plancke, Y D; Coddeville, B; Engelborghs, Y

    2000-07-24

    Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor

  1. Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue.

    PubMed

    Matsubara, Naoki; Oiwa, Kei; Hohsaka, Takahiro; Sadamoto, Reiko; Niikura, Kenichi; Fukuhara, Norio; Takimoto, Akio; Kondo, Hirosato; Nishimura, Shin-Ichiro

    2005-11-18

    The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.

  2. Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein.

    PubMed

    Albani, Jihad R

    2003-05-01

    We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.

  3. 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.

  4. Deoxycholic acid derivatives as inhibitors of P-glycoprotein-mediated multidrug efflux.

    PubMed

    Rocheblave, Luc; de Ravel, Marc Rolland; Monniot, Elodie; Tavenard, Jeremy; Cuilleron, Claude-Yves; Grenot, Catherine; Radix, Sylvie; Matera, Eva-Laure; Dumontet, Charles; Walchshofer, Nadia

    2016-12-01

    Deoxycholic acid derivatives were designed as P-glycoprotein (Pgp, ABCB1) inhibitors. Thus the synthesis and the biological activity of methyl deoxycholate derivatives 5-10 and their ether analogs 15-20 have been reported. The potency of these compounds to modulate Pgp-mediated MDR was evaluated through daunorubicin accumulation and potentiation of doxorubicin cytotoxicity in K562/R7 multidrug resistant cells overexpressing Pgp. In parallel, their intrinsic toxicity was appreciated on K562 sensitive cells. Methyl 12α-[(2R or 2S) tetrahydro-2H-pyran-2-yloxy]-3-oxo-5β-cholan-24-oate 9b has shown a good efficiency as a Pgp inhibitor and a low intrinsic toxicity. Therefore, this derivative constitutes a new lead compound which can be used as a starting point to improve the design of non-toxic Pgp modulators.

  5. 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.

  6. Mass spectrometry signal amplification for ultrasensitive glycoprotein detection using gold nanoparticle as mass tag combined with boronic acid based isolation strategy.

    PubMed

    Liu, Minbo; Zhang, Lijuan; Xu, Yawei; Yang, Pengyuan; Lu, Haojie

    2013-07-25

    We describe a novel method for rapid and ultrasensitive detection of intact glycoproteins without enzymatic pretreatment which was commonly used in proteomic research. This method is based on using gold nanoparticle (AuNP) as signal tag in laser desorption/ionization mass spectrometry (LDI-MS) analysis combined with boronic acid assisted isolation strategy. Briefly speaking, target glycoproteins were firstly isolated from sample solution with boronic acid functionalized magnetic microparticles, and then the surface modified gold nanoparticles were added to covalently bind to the glycoproteins. After that, these AuNP tagged glycoproteins were eluted from magnetic microparticles and applied to LDI-MS analysis. The mass signal of AuNP rather than that of glycoprotein was detected and recorded in this strategy. Through data processing of different standard glycoproteins, we have demonstrated that the signal of AuNP could be used to quantitatively represent glycoprotein. This method allows femtomolar detection of intact glycoproteins. We believe that the successful validation of this method on three different kinds of glycoproteins suggests the potential use for tracking trace amount of target glycoproteins in real biological samples in the near future.

  7. Glycoprotein 340 and sialic acid in minor-gland and whole saliva of children, adolescents, and adults.

    PubMed

    Sonesson, Mikael; Ericson, Dan; Kinnby, Bertil; Wickström, Claes

    2011-12-01

    Glycoprotein 340 (gp-340) is a bacterial-binding glycoprotein found in major-gland and minor-gland saliva. Sialic acid, a common terminal structure of salivary glycoproteins, interacts with microorganisms and host ligands, as well as with free radicals. This study investigated the contents of gp-340 and sialic acid in minor-gland saliva and whole saliva of children (3 yr of age), adolescents (14 yr of age), and adults (20-25 yr of age). Labial-gland saliva and buccal-gland saliva were collected on filter paper, and unstimulated whole saliva was collected by draining into a tube. The relative amount of gp-340 and sialic acid was determined by ELISA and by enzyme-linked lectin assay (ELLA), respectively. In minor-gland saliva, no statistically significant differences in gp-340 and sialic acid were seen between the age-groups. Among adults, significantly lower amounts of gp-340 and sialic acid were seen in labial saliva compared with buccal saliva. In whole saliva, the amount of gp-340 was significantly lower among adults compared with children. No differences between genders were seen. Stable content of gp-340 and sialic acid in minor-gland saliva across the age-groups, and a higher content of gp-340 in the whole saliva of the youngest age-group (3-yr-olds) compared with the adult group, may reflect that those components are vital innate factors of immunity in children's saliva.

  8. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    PubMed

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-07

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection.

  9. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  10. Contribution of mdr1b-type P-glycoprotein to okadaic acid resistance in rat pituitary GH3 cells.

    PubMed

    Ritz, V; Marwitz, J; Sieder, S; Ziemann, C; Hirsch-Ernst, K I; Quentin, I; Steinfelder, H J

    1999-08-01

    Okadaic acid as well as other, structurally different, inhibitors of serine/threonine phosphatases 1 and 2A induce apoptosis in pituitary GH3 cells. Incubation with stepwise raised concentrations of okadaic acid resulted in the isolation of cells that were increasingly less sensitive to the cytotoxic effect of this agent. After about 18 months cells were selected that survived at 300 nM okadaic acid, which is about 30 times the initially lethal concentration. This study revealed that a major pharmacokinetic mechanism underlying cell survival was the development of a P-glycoprotein-mediated multidrug resistance (MDR) phenotype. The increase in mRNA levels of the mdr1b P-glycoprotein isoform correlated with the extent of drug resistance. Functional assays revealed that increasing drug resistance was paralleled by a decreased accumulation of rhodamine 123, a fluorescent dye which is a substrate of mdr1-mediated efflux activity. Resistance could be abolished by structurally different chemosensitizers of P-glycoprotein function like verapamil and reserpine but not by the leukotriene receptor antagonist MK571 which is a modulator of the multidrug resistance-associated protein (MRP). Okadaic acid resistance included cross-resistance to other cytotoxic agents that are substrates of mdr1-type P-glycoproteins, like doxorubicin and actinomycin D, but not to non-substrates of mdr1, e.g. cytosine arabinoside. Thus, functional as well as biochemical features support the conclusion that okadaic acid is a substrate of the mdr1-mediated efflux activity in rat pituitary GH3 cells. Maintenance of resistance after withdrawal of okadaic acid as well as metaphase spreads of 100 nM okadaic acid-resistant cells suggested a stable MDR genotype without indications for the occurrence of extrachromosomal amplifications, e.g. double minute chromosomes.

  11. Implications of the presence of N-glycolylneuraminic Acid in Recombinant Therapeutic Glycoproteins

    PubMed Central

    Ghaderi, Darius; Taylor, Rachel E.; Padler-Karavani, Vered; Diaz, Sandra; Varki, Ajit

    2011-01-01

    Recombinant glycosylated biotherapeutic agents are usually produced in non-human mammalian cell lines, which can synthesize and/or metabolically incorporate the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). This contamination was previously known but ignored, as normal humans were thought to not react against Neu5Gc. However, recent findings indicate that humans have variable spectra of sometimes high levels of circulating anti-Neu5Gc antibodies. We studied two monoclonal antibodies in clinical use (Cetuximab and Panitumumab), and show covalently-bound Neu5Gc on Cetuximab. Anti-Neu5Gc antibodies from normal humans interact with Cetuximab in a Neu5Gc-specific manner and generate immune complexes in vitro. Mice with a human-like defect in Neu5Gc synthesis generate anti-Neu5Gc antibodies upon injection with Cetuximab. Circulating anti-Neu5Gc antibodies enhance Cetuximab clearance. These findings have potential relevance to half-life, efficacy and immune reactions in patients given such drugs. Finally, we show a method to reduce the Neu5Gc content of cultured cell lines and their secreted glycoproteins. PMID:20657583

  12. Glycomic analysis of sialic acid linkages in glycans derived from blood serum glycoproteins.

    PubMed

    Alley, William R; Novotny, Milos V

    2010-06-04

    A number of alterations to the normal glycomic profile have been previously described for a number of diseases and disorders, thus underscoring the medical importance of studying the glycans associated with proteins present in biological samples. An important alteration in cancer progression is an increased level of alpha2,6-sialylation, which aids in increasing the metastatic potential of tumor cells. Here we report a glycomic method that selectively amidates alpha2,6-linked sialic acids, while those that are alpha2,3-linked undergo spontaneous lactonization. Following subsequent permethylation, MALDI-TOF MS analysis revealed that many sialylated glycans present on glycoproteins found in blood serum featured increased levels of alpha2,6-sialylation in breast cancer samples. On the basis of the altered ratios of alpha2,3-linked to alpha2,6-linked sialic acids, many of these glycans became diagnostically relevant when they did not act as such indicators when based on traditional glycomic profiling alone.

  13. In silico Analysis for Predicting Fatty Acids of Black Cumin Oil as Inhibitors of P-Glycoprotein

    PubMed Central

    Ali, Babar; Jamal, Qazi Mohd. Sajid; Mir, Showkat R.; Shams, Saiba; Al-Wabel, Naser A.; Kamal, Mohammad A.

    2015-01-01

    Background: Black cumin oil is obtained from the seeds of Nigella sativa L. which belongs to family Ranunculaceae. The seed oil has been reported to possess antitumor, antioxidant, antibacterial, anti-inflammatory, hypoglycemic, central nervous system depressant, antioxidant, and immunostimulatory activities. These bioactivities have been attributed to the fixed oil, volatile oil, or their components. Seed oil consisted of 15 saturated fatty acids (17%) and 17 unsaturated fatty acids (82.9%). Long chain fatty acids and medium chain fatty acids have been reported to increase oral bioavailability of peptides, antibiotics, and other important therapeutic agents. In earlier studies, permeation enhancement and bioenhancement of drugs has been done with black cumin oil. Objective: In order to recognize the mechanism of binding of fatty acids to P-glycoprotein (P-gp), linoleic acid, oleic acid, margaric acid, cis-11, 14-eicosadienoic acid, and stearic acid were selected for in silico studies, which were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. Materials and Methods: Template search with BLAST and HHblits has been performed against the SWISS-MODEL template library. The target sequence was searched with BLAST against the primary amino acid sequence of P-gp from Rattus norvegicus. Results: The amount of energy needed by linoleic acid, oleic acid, eicosadienoic acid, margaric acid, and stearic acid to bind with P-gp were found to be − 10.60, −10.48, −9.95, −11.92, and − 10.37 kcal/mol, respectively. The obtained data support that all the selected fatty acids have contributed to inhibit P-gp activity thereby enhances the bioavailability of drugs. Conclusion: This study plays a significant role in finding hot spots in P-gp and may offer the further scope of designing potent and specific inhibitors of P-gp. SUMMARY Generation of 3D structure of fatty acid compounds from Black cumin oil and 3D homology modeling of Rat P

  14. Inhibitory effect of isothiocyanate derivant targeting AGPS by computer-aid drug design on proliferation of glioma and hepatic carcinoma cells.

    PubMed

    Zhu, Yu; Li, Wen-Ming; Zhang, Ling; Xue, Jing; Zhao, Meng; Yang, Ping

    2015-01-01

    Lipids metabolism was involved in the process of many types of tumor and alkylglycerone phosphate synthase (AGPS) was considered implicated in tumor process. Benzyl isothiocyanate (BITC) showed the inhibitory effect of tumor and AGPS activity, therefore, we screened a group of small molecular compound based on BITC by computer-aid design targeting AGPS and the results showed that the derivants could suppress the proliferation, the expression of tumor related genes such as survivin and Bcl-2, and the level of ether lipids such as lysophosphatidic acid ether (LPAe) and platelet activating factor ether (PAFe); however, the activity of caspase-3/8 was improved in glioma U87MG and hepatic carcinoma HepG2 cells in vitro.

  15. Facile synthesis of red emitting 3-aminophenylboronic acid functionalized copper nanoclusters for rapid, selective and highly sensitive detection of glycoproteins.

    PubMed

    Li, Xin-Ge; Zhang, Fei; Gao, Ya; Zhou, Qing-Meng; Zhao, Ye; Li, Yan; Huo, Jian-Zhong; Zhao, Xiao-Jun

    2016-12-15

    As an emerging class of fluorescent probes, copper nanoclusters (Cu NCs) have been considered as an intriguing candidate for detecting biomoleculars due to their outstanding fluorescent properties, excellent biocompatibility and low cost. Herein, we fabricated bovine serum albumin (BSA) protected Cu NCs (BSA-Cu NCs) and further functionalized them with 3-aminophenylboronic acid (APBA) for selectively discerning glycoproteins. In aqueous solution, Cu(2+) ions were directly reduced into BSA-Cu NCs by hydrazine hydrate (N2H4·H2O) at room-temperature using BSA as the capping agent. The synthetic process was very rapid, simple and easy for controlling due to the lack of any other complicated procedure such as heating and adjusting the pH value of the reactive mixture. The APBA-Cu NCs showed strong fluorescent emission at 630nm in the red range. So it can effectively avoid the disturbance of auto-fluorescence in biosamples. The fluorescence of the APBA-Cu NCs was obviously quenched by glycoprotein samples. Then, the APBA-Cu NCs were employed as a probe for selective capture and sensitive detection of glycoproteins with a wide linear range of 5-220nM and a low detection limit of 2.60nM owing to the covalent reaction between the boric acid group of APBA and the cis-glycol groups of the glycoproteins. The developed method was also successfully applied to determine glycoproteins in egg white of chickens and human urine samples with quantitative spike recoveries from 95% to 104%.

  16. A Rapid Approach for Fabricating Boronic Acid-Functionalized Plates for On-Probe Detection of Glycoprotein and Glycopeptide

    PubMed Central

    Liu, Yu-Ching; Chen, Chao-Jung

    2017-01-01

    We developed a rapid and simple approach without using complex mechanical or chemical protocols to fabricate boronic acid-functionalized plates for glycoprotein or glycopeptide enrichment and mass spectrometry (MS) analysis. By coating the boronic acid-functionalized silica particles on a polydimethylsiloxane (PDMS)-coated matrix-assisted laser desorption/ionization (MALDI) plate, these particles can form a firmly monolayer of particles on PDMS membrane for sample handling without peeling off. The boronic acid particles-coated PDMS plate (BP plate) was successfully applied to the enrichment of horseradish peroxidase (HRP) protein and their digested glycopeptides. PMID:28337401

  17. New insights in the conformation of α 1-acid glycoprotein (orosomucoid). Quenching resolved emission anisotropy studies

    NASA Astrophysics Data System (ADS)

    Albani, J. R.

    1999-09-01

    Quenching resolved emission anisotropy method was applied to study the dynamics of the two classes of Trp residues of human α 1-acid glycoprotein (orosomucoid), in the absence and presence of progesterone. In the absence of progesterone, the values of the anisotropies of the surface and buried Trp residues are 0.155 and 0.178, respectively. These values lower than the limiting anisotropy (0.267) indicate that both classes of Trp residues display residual motions. In the presence of progesterone, the values of the anisotropies decrease from 0.155 to 0.146 and from 0.178 to 0.167. Thus, binding of progesterone to orosomucoid increases the internal dynamics of the protein. Also, the fact that in the absence or in the presence of progesterone, the anisotropies of both classes are close, means that the amplitudes of the motions of the two classes are not significantly different. From our data and from the well-known position of the carbohydrate residues on orosomucoid, we suggest the presence of a hydrophobic pocket within the protein and where the 'buried' Trp residues can be found.

  18. Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

    PubMed Central

    Konrad, Zvia; Eichler, Jerry

    2002-01-01

    Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane. PMID:12069685

  19. Abscisic acid- and stress-induced highly proline-rich glycoproteins regulate root growth in rice.

    PubMed

    Tseng, I-Chieh; Hong, Chwan-Yang; Yu, Su-May; Ho, Tuan-Hua David

    2013-09-01

    In the root of rice (Oryza sativa), abscisic acid (ABA) treatment, salinity, or water deficit stress induces the expression of a family of four genes, REPETITIVE PROLINE-RICH PROTEIN (RePRP). These genes encode two subclasses of novel proline-rich glycoproteins with highly repetitive PX₁PX₂ motifs, RePRP1 and RePRP2. RePRP orthologs exist only in monocotyledonous plants, and their functions are virtually unknown. Rice RePRPs are heavily glycosylated with arabinose and glucose on multiple hydroxyproline residues. They are significantly different from arabinogalactan proteins that have glycan chains composed of arabinose and galactose. Transient and stable expressions of RePRP-green fluorescent protein reveal that a fraction of this protein is localized to the plasma membrane. In rice roots, ABA treatment increases RePRP expression preferentially in the elongation zone. Overexpression of RePRP in transgenic rice reduces root cell elongation in the absence of ABA, similar to the effect of ABA on wild-type roots. Conversely, simultaneous knockdown of the expression of RePRP1 and RePRP2 reduces the root sensitivity to ABA, indicating that RePRP proteins play an essential role in ABA/stress regulation of root growth and development. Moreover, rice RePRPs specifically interact with a polysaccharide, arabinogalactan, in a dosage-dependent manner. It is suggested that RePRP1 and RePRP2 are functionally redundant suppressors of root cell expansion and probably act through interactions with cell wall components near the plasma membrane.

  20. C-reactive protein and alpha 1-acid glycoprotein levels in dogs infected with Ehrlichia canis.

    PubMed Central

    Rikihisa, Y; Yamamoto, S; Kwak, I; Iqbal, Z; Kociba, G; Mott, J; Chichanasiriwithaya, W

    1994-01-01

    To elucidate whether acute-phase protein responses occur in dogs infected with Ehrlichia canis, C-reactive protein (CRP) and alpha 1-acid glycoprotein (AAG) levels were serially measured in the plasma of five dogs experimentally inoculated with E. canis and 10 sham-inoculated or noninoculated control dogs. The CRP concentration was measured by a canine-specific capture enzyme-linked immunosorbent assay, and the AAG concentration was measured by a canine-specific radial immunodiffusion method. In all E. canis-inoculated dogs, a 3.3- to 6.5-fold increase in the plasma CRP concentration and a 1.9- to 8.6-fold increase in the plasma AAG concentration over the preinoculation level occurred at days 4 to 6 postexposure. Despite the persistence of E. canis and high antibody titers, both CRP and AAG concentrations gradually declined to preexposure levels by day 34 postexposure. E. canis-infected dogs had mild and transient clinical signs which resolved without treatment by day 14 postexposure. The CRP and AAG concentrations in control inoculated or nontreated dogs remained within the normal range throughout the experimental period. Of 12 dogs naturally infected with E. canis, 75% had greater than 50 micrograms of CRP per ml and 83% had greater than 500 micrograms of AAG per ml. All of these 12 dogs had chronic and severe clinical signs of canine ehrlichiosis. Thus, elevations in the levels of acute-phase proteins occur in both acute and chronic canine ehrlichiosis. Determination of CRP and AAG concentrations may help in assessing the severity of inflammatory damage in dogs with E. canis infections. PMID:8027343

  1. Protective efficacy of ellagic acid on glycoproteins, hematological parameters, biochemical changes, and electrolytes in myocardial infarcted rats.

    PubMed

    Kannan, M Mari; Quine, S Darlin; Sangeetha, T

    2012-07-01

    The cardioprotective property of ellagic acid in rats has been reported previously. The present study reveals the protective role of ellagic acid in biochemical parameters including serum iron, plasma iron binding capacity, uric acid, glycoprotein, and electrolytes along with hematological parameters. Rats were subcutaneously injected with isoproterenol (ISO) (100 mg/kg) for 2 days to induce myocardial infarction. ISO-induced rats showed a significant increase in their levels of serum iron, serum uric acid, and blood glucose, and a significant decrease in their levels of plasma iron binding capacity, serum total protein, albumin/globulin ratio, and heart glycogen, when compared with normal control rats. The altered hematological parameters were also observed in ISO-induced rats when compared with normal control rats. Pretreatment with ellagic acid at doses of 7.5 and 15 mg/kg produced significant beneficial effect by returning all the above-mentioned biochemical and hematological parameters to near normal levels.

  2. Effects of Eimeria acervulina infection severity on growth performance, apparent ileal amino acid digestibility, and plasma concentrations of amino acids, carotenoids, and α1-acid glycoprotein in broilers.

    PubMed

    Rochell, S J; Parsons, C M; Dilger, R N

    2016-07-01

    An experiment was conducted to evaluate growth performance, apparent ileal digestibility (AID) of amino acids, and plasma concentrations of amino acids, carotenoids, and α1-acid glycoprotein, an acute-phase protein, in broilers inoculated with graded doses of E. acervulina oocysts. Ross 308 male broilers (400 total) were housed in battery cages from 1 to 21 d post-hatch and received common corn-soybean meal-based diets throughout the experiment. At 9 d post-hatch, birds were individually weighed and allotted to 4 treatment groups with 10 replicate cages of 10 birds per cage. At 15 d post-hatch, all birds were inoculated with 1 mL of distilled water that contained 0, 2.5 × 10(5), 5.0 × 10(5), or 1.0 × 10(6) sporulated E. acervulina oocysts. At 21 d, birds were euthanized for collection of blood and ileal digesta. Body weight gain and feed efficiency decreased linearly (P < 0.05) with increasing E. acervulina dose. With the exception of Trp and Gly, AID values decreased (P < 0.05) linearly or quadratically for all amino acids by an average of 2.6 percentage units for birds inoculated with 1.0 × 10(6) oocysts compared with uninfected birds. Infection with E. acervulina caused a quadratic decrease (P < 0.05) in plasma carotenoid concentrations. Plasma concentrations of Arg and Tyr decreased linearly (P < 0.05) with increasing E. acervulina inoculation dose and plasma Gln and Asn decreased quadratically (P < 0.01). Linear increases (P < 0.05) were observed for plasma Lys, Leu, Ile, Val, Pro, and Orn as E. acervulina inoculation dose increased. Plasma α1-acid glycoprotein of broilers was not influenced (P > 0.05) by E. acervulina infection. In conclusion, E. acervulina challenge adversely impacted growth performance, plasma carotenoids, and AID of amino acids in a dose-dependent manner. However, plasma amino acid responses to graded E. acervulina inoculation doses varied considerably among amino acids. Thus, these results indicated that alterations

  3. A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization

    PubMed Central

    Silva, Laurie A.; Khomandiak, Solomiia; Ashbrook, Alison W.; Weller, Romy; Heise, Mark T.; Morrison, Thomas E.

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a reemerging arbovirus responsible for outbreaks of infection throughout Asia and Africa, causing an acute illness characterized by fever, rash, and polyarthralgia. Although CHIKV infects a broad range of host cells, little is known about how CHIKV binds and gains access to the target cell interior. In this study, we tested whether glycosaminoglycan (GAG) binding is required for efficient CHIKV replication using CHIKV vaccine strain 181/25 and clinical isolate SL15649. Preincubation of strain 181/25, but not SL15649, with soluble GAGs resulted in dose-dependent inhibition of infection. While parental Chinese hamster ovary (CHO) cells are permissive for both strains, neither strain efficiently bound to or infected mutant CHO cells devoid of GAG expression. Although GAGs appear to be required for efficient binding of both strains, they exhibit differential requirements for GAGs, as SL15649 readily infected cells that express excess chondroitin sulfate but that are devoid of heparan sulfate, whereas 181/25 did not. We generated a panel of 181/25 and SL15649 variants containing reciprocal amino acid substitutions at positions 82 and 318 in the E2 glycoprotein. Reciprocal exchange at residue 82 resulted in a phenotype switch; Gly82 results in efficient infection of mutant CHO cells but a decrease in heparin binding, whereas Arg82 results in reduced infectivity of mutant cells and an increase in heparin binding. These results suggest that E2 residue 82 is a primary determinant of GAG utilization, which likely mediates attenuation of vaccine strain 181/25. IMPORTANCE Chikungunya virus (CHIKV) infection causes a debilitating rheumatic disease that can persist for months to years, and yet there are no licensed vaccines or antiviral therapies. Like other alphaviruses, CHIKV displays broad tissue tropism, which is thought to be influenced by virus-receptor interactions. In this study, we determined that cell-surface glycosaminoglycans are

  4. BAD-lectins: boronic acid-decorated lectins with enhanced binding affinity for the selective enrichment of glycoproteins.

    PubMed

    Lu, Ying-Wei; Chien, Chih-Wei; Lin, Po-Chiao; Huang, Li-De; Chen, Chang-Yang; Wu, Sz-Wei; Han, Chia-Li; Khoo, Kay-Hooi; Lin, Chun-Cheng; Chen, Yu-Ju

    2013-09-03

    The weak and variable binding affinities exhibited by lectin-carbohydrate interactions have often compromised the practical utility of lectin in capturing glycoproteins for glycoproteomic applications. We report here the development and applications of a new type of hybrid biomaterial, namely a boronic acid-decorated lectin (BAD-lectin), for efficient bifunctional glycoprotein labeling and enrichment. Our binding studies showed an enhanced affinity by BAD-lectin, likely to be mediated via the formation of boronate ester linkages between the lectin and glycan subsequent to the initial recognition process and thus preserving its glycan-specificity. Moreover, when attached to magnetic nanoparticles (BAD-lectin@MNPs), 2 to 60-fold improvement on detection sensitivity and enrichment efficiency for specific glycoproteins was observed over the independent use of either lectin or BA. Tested at the level of whole cell lysates for glycoproteomic applications, three different types of BAD-lectin@MNPs exhibited excellent specificities with only 6% overlapping among the 295 N-linked glycopeptides identified. As many as 236 N-linked glycopeptides (80%) were uniquely identified by one of the BAD-lectin@MNPs. These results indicated that the enhanced glycan-selective recognition and binding affinity of BAD-lectin@MNPs will facilitate a complementary identification of the under-explored glycoproteome.

  5. Purification, characterization and cDNA cloning of an analgesic peptide from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU2).

    PubMed

    Zhang, R; Yang, Z; Liu, Y F; Cui, Y; Zhang, J H

    2011-01-01

    In this study an analgesic peptide was purified through five continuous chromatographic steps. The mouse twisting model test was used to identify the target peptides in every separation step. The purified BmK AGP-SYPU2 was further qualified by Reverse Phase-High Performance Liquid Chromatography and High Performance Capillary Electrophoresis. The molecular weight, isoelectric point, and N-terminal sequence of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was cloned by rapid amplification of the cDNA ends from the cDNA pool of scorpion glands. Sequence determination showed that the mature BmK AGP-SYPU2 peptide is composed of 66 amino acid residues, and BmK AGP-SYPU2 is identical to BmK alpha2 (GenBank Acc. No. AF288608) and BmK alphaTX11 (GenBank Acc. No. AF155364). We report herein a purification procedure that yields substantial amounts of natural BmK AGP-SYPU2 with high analgesic activity.

  6. Effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

    SciTech Connect

    Barraud, B.; Balavoine, S.; Feldmann, G.; Lardeux, B.

    1996-04-01

    While the effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of {alpha}{sub 1}-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of {alpha}{sub 1}-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1{beta}, interleukin-6 and tumor necrosis factor {alpha}) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify {alpha}{sub 1}-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of {alpha}{sub 1}-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines. 49 refs., 2 figs., 2 tabs.

  7. Glycoprotein biosynthesis in calf kidney. Glycoprotein sialyltransferase activities towards serum glycoproteins and calf Tamm-Horsfall glycoprotein.

    PubMed

    van Dijk, W; Lasthuis, A M; van den Eijnden, D H

    1979-04-18

    CMP-AcNeu:glycoprotein sialyltransltransltransltransltransferase of calf kidney cortex was characterized using serum glycoproteins and Tamm-Horsfall glycoprotein, obtained from calf urine, as acceptors. Native calf Tamm-Horsfall glycoprotein showed the best acceptor properties, followed by desialylated calf fetuin and desialylated human alpha 1-acid glycoprotein exhibiting V values of, respectively, 114, 63 and 41 nmol/h per g wet wt. of kidney cortex and Km values of 0.12, 0.16 and 0.26 mM glycoprotein acceptor. Desialylated ovine submaxillary mucine appeared to be a very poor acceptor. Tamm-Horsfall glycoprotein sialyltransferase could be distinguished from serum glycoprotein sialyltransferase by competition studies. In addition the two glycoprotein sialyltransferase activities showed different distributions over the three regions of the calf kidney: the ratios of the Tamm-Horsfall to serum glycoprotein sialyltransferase activities decreased from 3.3 in the cortex to 0.8 and 0.4 in the medulla and the papilla, respectively. It was concluded that in calf kidney at least two different sialyltransferases exist. The high cortical Tamm-Horsfall glycoprotein sialyltransferases activity corresponds markedly to the origin of the urinary Tamm-Horsfall glycoprotein, namely the distal part of the kidney tubule. Inactivation of glycoprotein sialyltransferase activity by preincubation at various temperatures and during storage at 0 degree C, could be reduced by the addition of CMP-AcNeu. The possible relevance towards the in vivo sialylation of this finding is discussed.

  8. [Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence].

    PubMed

    Starodubova, E S; Kuzmenko, Y V; Latanova, A A; Preobrazhenskaya, O V; Karpov, V L

    2016-01-01

    An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.

  9. Identifying the differences in mechanisms of mycophenolic acid controlling fucose content of glycoproteins expressed in different CHO cell lines.

    PubMed

    Zhang, An; Tsang, Valerie Liu; Markely, Lam R; Kurt, Lutfiye; Huang, Yao-Ming; Prajapati, Shashi; Kshirsagar, Rashmi

    2016-11-01

    In the biopharmaceutical industry, glycosylation is a critical quality attribute that can modulate the efficacy of a therapeutic glycoprotein. Obtaining a consistent glycoform profile is desired because molecular function can be defined by its carbohydrate structures. Specifically, the fucose content of oligosaccharides in glycoproteins is one of the most important attributes that can significantly affect antibody-dependent cellular cytotoxicity (ADCC) activity. It is therefore important to understand the fucosylation pathway and be able to control fucosylation at the desired level to match predecessor materials in late stage and biosimilar programs. Several strategies were explored in this study and mycophenolic acid (MPA) was able to finely modulate the fucose content with the least undesired side effects. However, the response was significantly different between CHO cell lines of different lineages. Further experiments were then performed for a deeper understanding of the mechanism of fucosylation in different CHO cell lines. Results indicated that changes in the intracellular nucleotide involved in fucosylation pathway after MPA treatment are the main cause of the differences in fucosylation level response in different CHO cell lines. Differences in MPA metabolism in the various CHO cell lines directly resulted in different levels of afucosylation measured in antibodies produced by the CHO cell lines. Biotechnol. Bioeng. 2016;113: 2367-2376. © 2016 Wiley Periodicals, Inc.

  10. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike

    PubMed Central

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K.; Schlie, Katrin; Siebert, C. Alistair; Garten, Wolfgang; Bowden, Thomas A.; Strecker, Thomas; Huiskonen, Juha T.

    2016-01-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits. PMID:26849049

  11. Acidic pH-Induced Conformations and LAMP1 Binding of the Lassa Virus Glycoprotein Spike.

    PubMed

    Li, Sai; Sun, Zhaoyang; Pryce, Rhys; Parsy, Marie-Laure; Fehling, Sarah K; Schlie, Katrin; Siebert, C Alistair; Garten, Wolfgang; Bowden, Thomas A; Strecker, Thomas; Huiskonen, Juha T

    2016-02-01

    Lassa virus is an enveloped, bi-segmented RNA virus and the most prevalent and fatal of all Old World arenaviruses. Virus entry into the host cell is mediated by a tripartite surface spike complex, which is composed of two viral glycoprotein subunits, GP1 and GP2, and the stable signal peptide. Of these, GP1 binds to cellular receptors and GP2 catalyzes fusion between the viral envelope and the host cell membrane during endocytosis. The molecular structure of the spike and conformational rearrangements induced by low pH, prior to fusion, remain poorly understood. Here, we analyzed the three-dimensional ultrastructure of Lassa virus using electron cryotomography. Sub-tomogram averaging yielded a structure of the glycoprotein spike at 14-Å resolution. The spikes are trimeric, cover the virion envelope, and connect to the underlying matrix. Structural changes to the spike, following acidification, support a viral entry mechanism dependent on binding to the lysosome-resident receptor LAMP1 and further dissociation of the membrane-distal GP1 subunits.

  12. Mechanism of ligand binding to alpha 1-acid glycoprotein (orosomucoid): correlated thermodynamic factors and molecular parameters of polarity.

    PubMed Central

    Urien, S; Giroud, Y; Tsai, R S; Carrupt, P A; Brée, F; Testa, B; Tillement, J P

    1995-01-01

    Eight ligands were used in this study, four basic, three neutral and one acidic. Their binding to serum alpha 1-acid glycoprotein (orosomucoid) was measured at several temperatures, and the data were analysed together by a general model with three unknowns, number of binding sites, delta H0 and delta S0. The partition coefficients of the ligands were measured in octanol/water and heptane/water systems (log Poct. and log Phep.), and their molecular volumes were calculated by molecular modelling techniques. These structural properties allow determination of polarity parameters (delta log Poct.-hep., lambda oct. and lambda hep.) which encode in different proportions the various polar interactions between the solute and the aqueous and organic phases, i.e. hydrogen-bonding capacity and dipolarity/polarizability. This study shows that good correlations exist between delta H0 or delta S0 and polarity parameters, such that the enthalpic contribution to binding increases with increasing polarity of the ligands, mainly hydrogen-bond-donor acidity, whereas their entropic contribution to binding decreases. PMID:7887909

  13. Dietary Hizikia fusiformis glycoprotein-induced IGF-I and IGFBP-3 associated to somatic growth, polyunsaturated fatty acid metabolism, and immunity in juvenile olive flounder Paralichthys olivaceus.

    PubMed

    Choi, Youn Hee; Kim, Kang-Woong; Han, Hyon-Sob; Nam, Taek Jeong; Lee, Bong-Joo

    2014-01-01

    This study was aimed to examine the effect of dietary glycoprotein extracted from the sea mustard Hizikia fusiformis (Phaeophyceae: Sargassaceae) as a dietary supplement on growth performance in association with somatotropin level, proximate compositions, and immunity in juvenile olive flounder Paralichthys olivaceus. Water-ethanol extracted glycoprotein from H. fusiformis was supplemented to three fishmeal-based diets at the concentration of 0, 5, and 10gkg(-1) diet (designated as H0, H5, and H10, respectively). After a 12week-long feeding trial, growth performance and biochemical responses were analyzed including proximate composition, and whole body amino acids and fatty acids. We also measured plasma insulin like growth factor (IGF), IGF-binding protein (IGFBP) and interleukin (IL). The fish fed H5 showed the greatest weight gain among the dietary treatments. In parallel with the growth, the fish fed the diets containing H. fusiformis glycoprotein showed an increased plasma IGF-I activity and increased expression of 43-kDa IGFBP-3 compared to that in the control, whereas an opposite trend was observed for 34-kDa IGFBP-1. Although no differences were found in the level of whole body linoleic acid (C18:2n-6) and linolenic acid (C18:3n-3) among treatments, increases in arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were observed in fish fed H5 compared to control. IL-2 and -6 levels increased significantly in fish fed H10 compared to those in the control indicating increased immunity. These results suggest that supplementation of H. fusiformis glycoprotein in fish diet may be beneficial for fish growth and immunity in juvenile olive flounder.

  14. 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

  15. A single-amino-acid substitution eliminates the stringent carbohydrate requirement for intracellular transport of a viral glycoprotein.

    PubMed

    Pitta, A M; Rose, J K; Machamer, C E

    1989-09-01

    In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the two consensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G protein required at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at 37 degrees C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differences between these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major role in determining the stringency for the carbohydrate requirement. The rates at which the glycosylated and nonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smaller fraction of the nonglycosylated protein was transported. These results suggest that the carbohydrate does not promote intracellular transport directly but influences a polypeptide folding or oligomerization step which is critical for transport.

  16. The classical arabinogalactan protein AGP18 mediates megaspore selection in Arabidopsis.

    PubMed

    Demesa-Arévalo, Edgar; Vielle-Calzada, Jean-Philippe

    2013-04-01

    Female gametogenesis in most flowering plants depends on the predetermined selection of a single meiotically derived cell, as the three other megaspores die without further division or differentiation. Although in Arabidopsis thaliana the formation of the functional megaspore (FM) is crucial for the establishment of the gametophytic generation, the mechanisms that determine the specification and fate of haploid cells remain unknown. Here, we show that the classical arabinogalactan protein 18 (AGP18) exerts an active regulation over the selection and survival of megaspores in Arabidopsis. During meiosis, AGP18 is expressed in integumentary cells located in the abaxial region of the ovule. Overexpression of AGP18 results in the abnormal maintenance of surviving megaspores that can acquire a FM identity but is not sufficient to induce FM differentiation before meiosis, indicating that AGP18 positively promotes the selection of viable megaspores. We also show that all four meiotically derived cells in the ovule of Arabidopsis are competent to differentiate into a gametic precursor and that the function of AGP18 is important for their selection and viability. Our results suggest an evolutionary role for arabinogalactan proteins in the acquisition of monospory and the developmental plasticity that is intrinsic to sexual reproduction in flowering plants.

  17. Effect of binding of Calcofluor White on the carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) on the structure and dynamics of the protein moiety. A fluorescence study.

    PubMed

    Albani, J R

    2001-08-23

    Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously studied at low and high concentrations of Calcofluor compared to that of the protein. alpha1-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. At equimolar concentrations of Calcofluor and alpha1-acid glycoprotein, the fluorophore displays free motions [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94], while at high concentration of Calcofluor, its surrounding microenvironment is rigid, inducing the rigidity of the fluorophore itself [Albani, J. R.; Sillen, A.; Plancke, Y. D.; Coddeville, B.; Engelborghs, Y. Carbohydr. Res. 2000, 327, 333-340]. In the present work, red-edge excitation spectra and steady-state anisotropy studies performed on Trp residues in the presence of Calcofluor, showed that the apparent dynamics of Trp residues are not modified. However, deconvoluting the emission spectra with two different methods into different components, reveals that the structure of the protein matrix has been disrupted in the presence of high Calcofluor concentrations.

  18. Cloning and characterization of an androgen-dependent acidic epididymal glycoprotein/CRISP1-like protein from the monkey.

    PubMed

    Sivashanmugam, P; Richardson, R T; Hall, S; Hamil, K G; French, F S; O'Rand, M G

    1999-01-01

    A cDNA encoding an acidic epididymal glycoprotein (AEG)-like, CRISP1 (cysteine-rich secretory protein) protein from the monkey (Macaca mullata) epididymis has been cloned and sequenced. The monkey AEG (mAEG) has an open reading frame that encodes a protein containing 249 amino acids with a deduced molecular mass of 28 kDa. The mAEG protein sequence is 85% identical to human and 44% identical to mouse CRISP1, including all 16 conserved cysteine residues. mAEG also shows a significant amino acid homology with other CRISP proteins, rat AEG/DE, human TPX1/CRISP2, and guinea pig acrosomal autoantigen 1 (AA1). In addition, mAEG shows somewhat less homology to a toxin from the Mexican beaded lizard and to a human glioma pathogenesis-related protein. Northern blot analysis shows that the mRNA for mAEG is expressed in all the regions of the epididymis except the caput and was not detected in the testis, prostate, seminal vesicle, and brain. In castrated animals, mAEG gene expression in the epididymis is significantly diminished; however, testosterone enanthate replacement restored the normal level of expression, demonstrating that expression of mAEG is androgen dependent. Western blot analysis of monkey epididymal regions using mouse antirecombinant human AEG identified a 28-kDa protein only in the caudal region. Immunohistochemical analysis identified mAEG only in the principal cells of the cauda epididymal epithelium. Immunofluorescence analysis identified mAEG on the principal piece of the sperm tail and as small patches over the middle piece and head regions. The results described in the present study suggest that mAEG (CRISP1) is secreted in the monkey epididymis, regulated by androgens and present on epididymal spermatozoa.

  19. [Serum protein binding of fentanyl. The effect of postoperative acute phase reaction with elevated alpha 1-acid glycoprotein and methodologic problems in determination by equilibrium dialysis].

    PubMed

    Wiesner, G; Taeger, K; Peter, K

    1996-04-01

    Numerous basic drugs are extensively bound to alpha 1-acid glycoprotein. Fentanyl, with a pKa value of 8.43, is also a basic drug. Protein binding studies have yielded contradictory results concerning binding of fentanyl to alpha 1-acid glycoprotein. In this study we investigated time courses of serum protein concentrations and serum protein binding of fentanyl during postoperative acute phase reaction, assuming that an increase of alpha 1-acid glycoprotein is accompanied by an increase of serum protein binding, if fentanyl is extensively bound to alpha 1-acid glycoprotein. Fentanyl protein binding measurements using equilibrium dialysis can be affected by volume shifts and pH changes. Therefore, volume shifts from buffer to serum and the influence of various phosphate buffers on increasing pH due to loss of CO2 were also evaluated. METHODS. Thirteen patients with no history of renal or hepatic disease undergoing an operation with a significant acute phase reaction were studied. Preoperatively and on the first 3 postoperative days serum concentrations of alpha 1-acid glycoprotein, albumin, total protein and apolipoprotein A and B were determined by rocket immunoeolectrophoresis, biuret method and laser nephelometry, respectively. Corresponding serum protein binding of fentanyl was measured by adding 40 ng of fentanyl to 1 ml serum followed by equilibrium dialysis at 37 degrees C for 4 h. A 0.167 M phosphate buffer (pH 7.27), which gave a final pH of 7.40, was used. Volume shifts from buffer to serum were measured. Fentanyl concentration in serum before dialysis (FS) was determined by gas chromatography, and fentanyl concentration in buffer after dialysis (FB) was determined by radioimmunoassay. Serum protein binding (SPB) was calculated by the formula: SPB = (FS - FB - FB*c)/(FS - FB) where c is a correction factor. Ten randomly selected patient sera were dialyzed against four phosphate buffers of different pH values and molarities, and the serum pH at the end of

  20. Interaction of anticancer drug clofarabine with human serum albumin and human α-1 acid glycoprotein. Spectroscopic and molecular docking approach.

    PubMed

    Ajmal, Mohammad Rehan; Nusrat, Saima; Alam, Parvez; Zaidi, Nida; Khan, Mohsin Vahid; Zaman, Masihuz; Shahein, Yasser E; Mahmoud, Mohamed H; Badr, Gamal; Khan, Rizwan Hasan

    2017-02-20

    The binding interaction between clofarabine, an important anticancer drug and two important carrier proteins found abundantly in human plasma, Human Serum Albumin (HSA) and α-1 acid glycoprotein (AAG) was investigated by spectroscopic and molecular modeling methods. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of HSA and AAG is quenched by clofarabine and the static mode of fluorescence quenching is operative. UV-vis spectroscopy deciphered the formation of ground state complex between anticancer drug and the two studied proteins. Clofarabine was found to bind at 298K with both AAG and HSA with the binding constant of 8.128×10(3) and 4.120×10(3) for AAG and HSA, respectively. There is stronger interaction of clofarabine with AAG as compared to HSA. The Gibbs free energy change was found to be negative for the interaction of clofarabine with AAG and HSA indicating that the binding process is spontaneous. Binding of clofarabine with HSA and AAG induced ordered structures in both proteins and lead to molecular compaction. Clofarabine binds to HSA near to drug site II. Hydrogen bonding and hydrophobic interactions were the main bonding forces between HSA-clofarabine and AAG-clofarabine as revealed by docking results. This study suggests the importance of binding of anticancer drug to AAG spatially in the diseases like cancers where the plasma concentration of AAG increases many folds. Design of drug dosage can be adjusted accordingly to achieve optimal treatment outcome.

  1. Dynamics of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White.

    PubMed

    Albani, J R; Sillen, A; Coddeville, B; Plancke, Y D; Engelborghs, Y

    1999-11-23

    Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated alpha 1-acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated alpha 1-acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated alpha 1-acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.

  2. Nucleic acids encoding modified human immunodeficiency virus type 1 (HIV-1) group M consensus envelope glycoproteins

    DOEpatents

    Haynes, Barton F [Durham, NC; Gao, Feng [Durham, NC; Korber, Bette T [Los Alamos, NM; Hahn, Beatrice H [Birmingham, AL; Shaw, George M [Birmingham, AL; Kothe, Denise [Birmingham, AL; Li, Ying Ying [Hoover, AL; Decker, Julie [Alabaster, AL; Liao, Hua-Xin [Chapel Hill, NC

    2011-12-06

    The present invention relates, in general, to an immunogen and, in particular, to an immunogen for inducing antibodies that neutralizes a wide spectrum of HIV primary isolates and/or to an immunogen that induces a T cell immune response. The invention also relates to a method of inducing anti-HIV antibodies, and/or to a method of inducing a T cell immune response, using such an immunogen. The invention further relates to nucleic acid sequences encoding the present immunogens.

  3. Interaction of the recently approved anticancer drug nintedanib with human acute phase reactant α 1-acid glycoprotein

    NASA Astrophysics Data System (ADS)

    Abdelhameed, Ali Saber; Ajmal, Mohammad Rehan; Ponnusamy, Kalaiarasan; Subbarao, Naidu; Khan, Rizwan Hasan

    2016-07-01

    A comprehensive study of the interaction of the newly approved tyrosine kinase inhibitor, Nintedanib (NTB) and Alpha-1 Acid Glycoprotein (AAG) has been carried out by utilizing UV-Vis spectroscopy, fluorescence spectroscopy, circular dichroism, dynamic light scattering and molecular docking techniques. The obtained results showed enhancement of the UV-Vis peak of the protein upon binding to NTB with the fluorescence intensity of AAG is being quenched by NTB via the formation of ground state complex (i.e. Static quenching). Forster distance (Ro) obtained from fluorescence resonance energy transfer (FRET) is found to be 2.3 nm. The calculated binding parameters from the modified Stern-Volmer equation showed that NTB binds to AAG with a binding constant in the order of 103. Conformational alteration of the protein upon its binding to NTB was confirmed by the circular dichroism. Dynamic light scattering results showed that the binding interaction of NTB leads to the reduction in hydrodynamic radii of AAG. Dynamic molecular docking results showed that the NTB fits into the central binding cavity in AAG and hydrophobic interaction played the key role in the binding process also the docking studies were performed with methotrexate and clofarabine drugs to look into the common binding regions of these drugs on AAG molecule, it was found that five amino acid residues namely Phe 113, Arg 89, Tyr 126, Phe 48 and Glu 63 were common among the binding regions of three studied drugs this phenomenon of overlapping binding regions may influence the drug transport by the carrier molecule in turn affecting the metabolism of the drug and treatment outcome.

  4. Expression and distribution of extensins and AGPs in susceptible and resistant banana cultivars in response to wounding and Fusarium oxysporum

    PubMed Central

    Wu, Yunli; Fan, Wei; Li, Xiaoquan; Chen, Houbin; Takáč, Tomáš; Šamajová, Olga; Fabrice, Musana Rwalinda; Xie, Ling; Ma, Juan; Šamaj, Jozef; Xu, Chunxiang

    2017-01-01

    Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is soil-borne disease of banana (Musa spp.) causing significant economic losses. Extensins and arabinogalactan proteins (AGPs) are cell wall components important for pathogen defence. Their significance for Foc resistance in banana was not reported so far. In this study, two banana cultivars differing in Foc sensitivity were used to monitor the changes in transcript levels, abundance and distribution of extensins and AGPs after wounding and Foc inoculation. Extensins mainly appeared in the root cap and meristematic cells. AGPs recognized by JIM13, JIM8, PN16.4B4 and CCRC-M134 antibodies located in root hairs, xylem and root cap. Individual AGPs and extensins showed specific radial distribution in banana roots. At the transcript level, seven extensins and 23 AGPs were differentially expressed between two banana cultivars before and after treatments. Two extensins and five AGPs responded to the treatments at the protein level. Most extensins and AGPs were up-regulated by wounding and pathogen inoculation of intact plants but down-regulated by pathogen attack of wounded plants. Main components responsible for the resistance of banana were MaELP-2 and MaPELP-2. Our data revealed that AGPs and extensins represent dynamic cell wall components involved in wounding and Foc resistance. PMID:28218299

  5. Resistance to neutralization by broadly reactive antibodies to the human immunodeficiency virus type 1 gp120 glycoprotein conferred by a gp41 amino acid change.

    PubMed Central

    Thali, M; Charles, M; Furman, C; Cavacini, L; Posner, M; Robinson, J; Sodroski, J

    1994-01-01

    A neutralization-resistant variant of human immunodeficiency virus type 1 (HIV-1) that emerged during in vitro propagation of the virus in the presence of neutralizing serum from an infected individual has been described. A threonine-for-alanine substitution at position 582 in the gp41 transmembrane envelope glycoprotein of the variant virus was responsible for the neutralization-resistant phenotype (M.S. Reitz, Jr., C. Wilson, C. Naugle, R. C. Gallo, and M. Robert-Guroff, Cell 54:57-63, 1988). The mutant virus also exhibited reduced sensitivity to neutralization by 30% of HIV-1-positive sera that neutralized the parental virus, suggesting that a significant fraction of the neutralizing activity within these sera can be affected by the amino acid change in gp41 (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. Robert-Guroff, J. Virol. 64:3240-3248, 1990). It is shown here that the change of alanine 582 to threonine specifically confers resistance to neutralizing by antibodies directed against both groups of discontinuous, conserved epitopes related to the CD4 binding site on the gp120 exterior envelope glycoprotein. Only minor differences in binding of these antibodies to wild-type and mutant envelope glycoproteins were observed. Thus, the antigenic structure of gp120 can be subtly affected by an amino acid change in gp41, with important consequences for sensitivity to neutralization. Images PMID:7507184

  6. Incorporation of Precursors into Ribonucleic Acid, Protein, Glycoprotein, and Lipoprotein of Avian Myeloblastosis Virions

    PubMed Central

    Baluda, M. A.; Nayak, D. P.

    1969-01-01

    Freshly explanted leukemic myeloblasts produce avian myeloblastosis virus (AMV) at a constant rate without any obvious cytopathic effect; therefore, subviral components are continually synthesized at a steady rate. The incorporation of various radioactive precursors into virions was monitored by determination of radioactivity in purified virus after density equilibrium sedimentation in preformed sucrose gradients. The kinetics of incorporation of 3H-uridine have shown that there is an average time interval of 3 to 4 hr (half-life) between the time viral ribo-nucleic acid (RNA) is synthesized and the time it is released as a mature virus particle; this represents the average time interval spent by AMV-RNA in an intracellular pool. Studies with 14C-phenylalanine have revealed that some protein synthesis takes place at or near the cell surface immediately prior to maturation and release of virus. 14C-glucosamine also appears to be incorporated into the outer viral envelope shortly before maturation. On the other hand, there is an average lag of about 16 to 20 hr before 14C-ethanolamine incorporated into intracellular lipoprotein appears in free virions; this probably reflects the kinetics of replacement of cellular surface membrane. Actinomycin D inhibits AMV-RNA within 30 min but permits the maturation of AMV to continue for at least 2 hr. AMV released in the presence of actinomycin D contains AMV-RNA synthesized before the addition of the drug. PMID:4311791

  7. Evidence for differences in the binding of drugs to the two main genetic variants of human alpha 1-acid glycoprotein.

    PubMed Central

    Herve, F; Gomas, E; Duche, J C; Tillement, J P

    1993-01-01

    1. Human alpha 1-acid glycoprotein (AAG), a plasma transport protein, has three main genetic variants. F1. S and A. Native commercial AAG (a mixture of almost equal proportions of these three variants) has been separated by chromatography into variants which correspond to the proteins of the two genes which code for AAG in humans: the A variant and a mixture of the F1 and S variants (60% F1 and 40% S). Their binding properties towards imipramine, warfarin and mifepristone were studied by equilibrium dialysis. 2. The F1S variant mixture strongly bound warfarin and mifepristone with an affinity of 1.89 and 2.06 x 10(6) l mol-1, respectively, but had a low affinity for imipramine. Conversely, the A variant strongly bound imipramine with an affinity of 0.98 x 10(6) l mol-1. The low degree of binding of warfarin and mifepristone to the A variant sample was explained by the presence of protein contaminants in this sample. These results indicate specific drug transport roles for each variant, with respect to its separate genetic origin. 3. Control binding experiments performed with (unfractionated) commercial AAG and with AAG isolated from individuals with either the F1/A or S/A phenotypes, agreed with these findings. The results for the binding of warfarin and mifepristone by the AAG samples were similar to those obtained with the F1S mixture: the mean high-affinity association constant of the AAG samples for each drug was of the same order as that of the F1S mixture: the decrease in the number of binding sites of the AAG samples, as compared with the F1S mixture, was explained by the smaller proportion of variants F1 and/or S in these samples. Conversely, results of the imipramine binding study with the AAG samples concurred with those for the binding of this basic drug by the A variant, with respect to the proportion of the A variant in these samples. Images Figure 1 PMID:9114911

  8. Evidence for differences in the binding of drugs to the two main genetic variants of human alpha 1-acid glycoprotein.

    PubMed

    Herve, F; Gomas, E; Duche, J C; Tillement, J P

    1993-09-01

    1. Human alpha 1-acid glycoprotein (AAG), a plasma transport protein, has three main genetic variants. F1. S and A. Native commercial AAG (a mixture of almost equal proportions of these three variants) has been separated by chromatography into variants which correspond to the proteins of the two genes which code for AAG in humans: the A variant and a mixture of the F1 and S variants (60% F1 and 40% S). Their binding properties towards imipramine, warfarin and mifepristone were studied by equilibrium dialysis. 2. The F1S variant mixture strongly bound warfarin and mifepristone with an affinity of 1.89 and 2.06 x 10(6) l mol-1, respectively, but had a low affinity for imipramine. Conversely, the A variant strongly bound imipramine with an affinity of 0.98 x 10(6) l mol-1. The low degree of binding of warfarin and mifepristone to the A variant sample was explained by the presence of protein contaminants in this sample. These results indicate specific drug transport roles for each variant, with respect to its separate genetic origin. 3. Control binding experiments performed with (unfractionated) commercial AAG and with AAG isolated from individuals with either the F1/A or S/A phenotypes, agreed with these findings. The results for the binding of warfarin and mifepristone by the AAG samples were similar to those obtained with the F1S mixture: the mean high-affinity association constant of the AAG samples for each drug was of the same order as that of the F1S mixture: the decrease in the number of binding sites of the AAG samples, as compared with the F1S mixture, was explained by the smaller proportion of variants F1 and/or S in these samples. Conversely, results of the imipramine binding study with the AAG samples concurred with those for the binding of this basic drug by the A variant, with respect to the proportion of the A variant in these samples.

  9. Comparison of α1-Antitrypsin, α1-Acid Glycoprotein, Fibrinogen and NOx as Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis)

    PubMed Central

    Guha, Anirban; Guha, Ruby; Gera, Sandeep

    2013-01-01

    Mastitis set apart as clinical and sub clinical is a disease complex of dairy cattle, with sub clinical being the most important economically. Of late, laboratories showed interest in developing biochemical markers to diagnose sub clinical mastitis (SCM) in herds. Many workers reported noteworthy alternation of acute phase proteins (APPs) and nitric oxide, (measured as nitrate+nitrite = NOx) in milk due to intra-mammary inflammation. But, the literature on validation of these parameters as indicators of SCM, particularly in riverine milch buffalo (Bubalus bubalis) milk is inadequate. Hence, the present study focused on comparing several APPs viz. α1- anti trypsin, α1- acid glycoprotein, fibrinogen and NOx as indicators of SCM in buffalo milk. These components in milk were estimated using standardized analytical protocols. Somatic cell count (SCC) was done microscopically. Microbial culture was done on 5% ovine blood agar. Of the 776 buffaloes (3,096 quarters) sampled, only 347 buffaloes comprising 496 quarters were found positive for SCM i.e. milk culture showed growth in blood agar with SCC≥2×105 cells/ml of milk. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. It was observed that α1- anti trypsin and NOx had a highly significant (p<0.01) increase in SCM milk, whereas, the increase of α1- acid glycoprotein in infected milk was significant (p<0.05). Fibrinogen was below detection level in both healthy and SCM milk. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×105 cells/ml of milk as the benchmark. Udder profile correlation coefficient was also used. Allowing for statistical and epidemiological analysis, it was concluded that α1- anti trypsin indicates SCM irrespective of etiology, whereas α1- acid glycoprotein better diagnosed SCM caused by gram positive bacteria. NOx did not prove to be a good

  10. Comparison of α1-Antitrypsin, α1-Acid Glycoprotein, Fibrinogen and NOx as Indicator of Subclinical Mastitis in Riverine Buffalo (Bubalus bubalis).

    PubMed

    Guha, Anirban; Guha, Ruby; Gera, Sandeep

    2013-06-01

    Mastitis set apart as clinical and sub clinical is a disease complex of dairy cattle, with sub clinical being the most important economically. Of late, laboratories showed interest in developing biochemical markers to diagnose sub clinical mastitis (SCM) in herds. Many workers reported noteworthy alternation of acute phase proteins (APPs) and nitric oxide, (measured as nitrate+nitrite = NOx) in milk due to intra-mammary inflammation. But, the literature on validation of these parameters as indicators of SCM, particularly in riverine milch buffalo (Bubalus bubalis) milk is inadequate. Hence, the present study focused on comparing several APPs viz. α1- anti trypsin, α1- acid glycoprotein, fibrinogen and NOx as indicators of SCM in buffalo milk. These components in milk were estimated using standardized analytical protocols. Somatic cell count (SCC) was done microscopically. Microbial culture was done on 5% ovine blood agar. Of the 776 buffaloes (3,096 quarters) sampled, only 347 buffaloes comprising 496 quarters were found positive for SCM i.e. milk culture showed growth in blood agar with SCC≥2×10(5) cells/ml of milk. The cultural examination revealed Gram positive bacteria as the most prevalent etiological agent. It was observed that α1- anti trypsin and NOx had a highly significant (p<0.01) increase in SCM milk, whereas, the increase of α1- acid glycoprotein in infected milk was significant (p<0.05). Fibrinogen was below detection level in both healthy and SCM milk. The percent sensitivity, specificity and accuracy, predictive values and likelihood ratios were calculated taking bacterial culture examination and SCC≥2×10(5) cells/ml of milk as the benchmark. Udder profile correlation coefficient was also used. Allowing for statistical and epidemiological analysis, it was concluded that α1- anti trypsin indicates SCM irrespective of etiology, whereas α1- acid glycoprotein better diagnosed SCM caused by gram positive bacteria. NOx did not prove to be a

  11. Recent Progress in Electrochemical Biosensors for Glycoproteins

    PubMed Central

    Akiba, Uichi; Anzai, Jun-ichi

    2016-01-01

    This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements. PMID:27916961

  12. Effect of 9-cis retinoic acid and all-trans retinoic acid in combination with verapamil on P-glycoprotein expression in L1210 cells.

    PubMed

    Breier, A; Stetka, J; Bohacova, V; Macejova, D; Brtko, J; Sulova, Z

    2014-01-01

    The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

  13. CXCL14 enhances proliferation and migration of NCI-H460 human lung cancer cells overexpressing the glycoproteins containing heparan sulfate or sialic acid.

    PubMed

    Park, Cho Rong; You, Dong-Joo; Kim, Dong-Kyu; Moon, Mi Jin; Lee, Cheolju; Oh, Seung-Hyun; Ahn, Curie; Seong, Jae Young; Hwang, Jong-Ik

    2013-05-01

    CXCL14 is a chemokine family member that is involved in various cellular responses in addition to immune cell activation. Although constitutive CXCL14 expression in normal epithelial cells may help protect against infection by activating immune systems, its expression in cancer cells has raised controversy regarding its possible role in tumorigenesis. However, the underlying mechanisms for this disparity remain unknown. Investigation of cellular CXCL14 binding properties might increase our understanding of the peptide's roles in tumorigenesis. In the present study, we found that CXCL14 binds to various cell types. Interestingly, binding to NCI-H460 cells was prevented by heparan sulfate and N-acetyl neuraminic acid. Next, we examined effect of CXCL14 binding in NCI-H460 and NCI-H23. CXCL14 enhanced proliferation and migration in NCI-H460 but had no effect on NCI-H23. A reporter gene assay with various transcription factor response elements revealed that only nuclear factor-κB (NF-κB) signaling was activated by CXCL14 in NCI-H460 cells, which was blocked by BAPTA-AM, TPCA-1, and brefeldin A. Exogenous expression of some glycoproteins such as syndecan-4, podoplanin, and CD43 in these cells enhanced CXCL14 binding and NF-κB activity. Collectively, these results demonstrate that CXCL14 binding to glycoproteins harboring heparan sulfate proteoglycans and sialic acids leads proliferation and migration of some cancer cells.

  14. Identification of amino acid changes in the envelope glycoproteins of bovine viral diarrhea viruses isolated from alpaca that may be involved in host adaptation.

    PubMed

    Neill, John D; Dubovi, Edward J; Ridpath, Julia F

    2015-09-30

    Bovine viral diarrhea viruses (BVDV) are most commonly associated with infections of cattle. However, BVDV are often isolated from closely related ruminants with a number of BVDV-1b viruses being isolated from alpacas that were both acutely and persistently infected. The complete nucleotide sequence of the open reading frame of eleven alpaca-adapted BVDV isolates and the region encoding the envelope glycoproteins of an additional three isolates were determined. With the exception of one, all alpaca isolates were >99.2% similar at the nucleotide level. The Hercules isolate was more divergent, with 95.7% sequence identity to the other viruses. Sequence similarity of the 14 viruses indicated they were isolates of a single BVDV strain that had adapted to and were circulating through alpaca herds. Hercules was a more distantly related strain that has been isolated only once in Canada and represented a separate adaptation event that possessed the same adaptive changes. Comparison of amino acid sequences of alpaca and bovine-derived BVDV strains revealed three regions with amino acid sequences unique to all alpaca isolates. The first contained two small in-frame deletions near the N-terminus of the E2 glycoprotein. The second was found near the C-terminus of the E2 protein where four altered amino acids were located within a 30 amino acid domain that participates in E2 homodimerization. The third region contained three variable amino acids in the C-terminus of the E(rns) within the amphipathic helix membrane anchor. These changes were found in the polar side of the amphipathic helix and resulted in an increased charge within the polar face. Titration of bovine and alpaca viruses in both bovine and alpaca cells indicated that with increased charge in the amphipathic helix, the ability to infect alpaca cells also increased.

  15. Förster energy-transfer studies between Trp residues of alpha1-acid glycoprotein (orosomucoid) and the glycosylation site of the protein.

    PubMed

    Albani, Jihad R

    2003-10-10

    Energy-transfer studies between Trp residues of alpha(1)-acid glycoprotein and the fluorescent probe Calcofluor White were performed. Calcofluor White interacts with carbohydrate residues of the protein, while the three Trp residues are located at the surface (Trp-160) and in hydrophobic domains of the protein (Trp-25 and Trp-122). Binding of Calcofluor to the protein induces a decrease in the fluorescence intensity of the Trp residues accompanied by an increase of that of Calcofluor White. Efficiency (E) of Trp fluorescence quenching was determined to be equal to 45%, and the Förster distance R(o), at which the efficiency of energy transfer is 50%, was calculated to be 18.13 A. This low distance and the value of the efficiency clearly indicate that energy transfer between Trp residues and Calcofluor White is weak.

  16. Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75- and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum.

    PubMed

    Gorski, J P; Griffin, D; Dudley, G; Stanford, C; Thomas, R; Huang, C; Lai, E; Karr, B; Solursh, M

    1990-09-05

    Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.

  17. Quercetin-glutamic acid conjugate with a non-hydrolysable linker; a novel scaffold for multidrug resistance reversal agents through inhibition of P-glycoprotein.

    PubMed

    Kim, Mi Kyoung; Kim, Yunyoung; Choo, Hyunah; Chong, Youhoon

    2017-02-01

    Previously, we have reported remarkable effect of a quercetin-glutamic acid conjugate to reverse multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer agents through inhibition of P-glycoprotein (Pgp)-mediated drug efflux. Due to the hydrolysable nature, MDR-reversal activity of the quercetin conjugate was attributed to its hydrolysis product, quercetin. However, several lines of evidence demonstrated that the intact quercetin-glutamic acid conjugate has stronger MDR-reversal activity than quercetin. In order to evaluate this hypothesis and to identify a novel scaffold for MDR-reversal agents, we prepared quercetin conjugates with a glutamic acid attached at the 7-O position via a non-hydrolysable linker. Pgp inhibition assay, Pgp ATPase assay, and MDR-reversal activity assay were performed, and the non-hydrolysable quercetin conjugates showed significantly higher activities compared with those of quercetin. Unfortunately, the quercetin conjugates were not as effective as verapamil in Pgp-inhibition and thereby reversing MDR, but it is worth to note that the structurally modified quercetin conjugates with a non-cleavable linker showed significantly improved MDR-reversal activity compared with quercetin. Taken together, the quercetin conjugates with appropriate structural modifications were shown to have a potential to serve as a scaffold for the design of novel MDR-reversal agents.

  18. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    SciTech Connect

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  19. Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues.

    PubMed

    Albani, Jihad René

    2004-01-01

    We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.

  20. Examination of the biological role of the alpha(2-->6)-linked sialic acid in gangliosides binding to the myelin-associated glycoprotein (MAG).

    PubMed

    Schwardt, Oliver; Gäthje, Heiko; Vedani, Angelo; Mesch, Stefanie; Gao, Gan-Pan; Spreafico, Morena; von Orelli, Johannes; Kelm, Sørge; Ernst, Beat

    2009-02-26

    The tetrasaccharide 1, a substructure of ganglioside GQ1b alpha, shows a remarkable affinity for the myelin-associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program. In our search for structurally simplified and pharmacokinetically improved mimics of 1, modifications of the core disaccharide, the alpha(2-->3)- and the alpha(2-->6)-linked sialic acid were synthesized. Biphenylmethyl and (S)-lactate were identified as suitable replacements for the alpha(2-->6)-linked sialic acid. Combined with a core modification and the earlier found aryl amide substituent in the 9-position of the alpha(2-->3)-linked sialic acid, high affinity MAG antagonists were identified. All mimics were tested in a competitive target-based binding assay, providing relative inhibitory potencies (rIP). Compared to the reference tetrasaccharide 1, the rIPs of the most potent antagonists 59 and 60 are enhanced nearly 400-fold. Their K(D)s determined in surface plasmon resonance experiments are in the low micromolar range. These results are in semiquantitative agreement with molecular modeling studies. This new class of glycomimetics will allow to validate the role of MAG in the axon regeneration process.

  1. Circular dichroism of erythrocyte membrane glycoproteins.

    PubMed

    Decker, R V; Carraway, K L

    1975-03-28

    The circular dichroism spectra were obtained for purified equine, human and bovine membrane glycoproteins, which have 40, 55 and 70% carbohydrate, respectively. The spectra in aqueous buffer show similar shapes, maxima and minima but somewhat different peak amplitudes. Analysis of the spectra indicated that the glycoproteins can be pictured as existing primarily in an unordered form in dilute aqueous buffer with small amounts of alpha-helix (13-23%) present. In 2-chloroethanol, a helix-promoting solvent, the amount of alpha-helix is increased to 60-70%. The glycoproteins underwent denaturation in guanidine hydrochloride, although evidence of some residual structure did remain. The spectra of the glycoproteins change relatively little on going from aqueous buffer to dodecylsulfate solutions. Removal of 60% of the sialic acid does not induce significant conformational alterations. The anomalous behavior of the glycoproteins during molecular weight determinations does not appear to be related primarily to conformational restrictions on the polypeptide chain.

  2. [Biological role of heterogeneous glycoprotein structures].

    PubMed

    Jakab, Lajos

    2016-07-01

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

  3. Both α2,3- and α2,6-Linked Sialic Acids on O-Linked Glycoproteins Act as Functional Receptors for Porcine Sapovirus

    PubMed Central

    Alfajaro, Mia Madel; Kim, Ji-Yun; Park, Jun-Gyu; Son, Kyu-Yeol; Ryu, Eun-Hye; Sorgeloos, Frederic; Kwon, Hyung-Jun; Park, Su-Jin; Lee, Woo Song; Cho, Duck; Kwon, Joseph; Choi, Jong-Soon; Kang, Mun-Il; Goodfellow, Ian; Cho, Kyoung-Oh

    2014-01-01

    Sapovirus, a member of the Caliciviridae family, is an important cause of acute gastroenteritis in humans and pigs. Currently, the porcine sapovirus (PSaV) Cowden strain remains the only cultivable member of the Sapovirus genus. While some caliciviruses are known to utilize carbohydrate receptors for entry and infection, a functional receptor for sapovirus is unknown. To characterize the functional receptor of the Cowden strain of PSaV, we undertook a comprehensive series of protein-ligand biochemical assays in mock and PSaV-infected cell culture and/or piglet intestinal tissue sections. PSaV revealed neither hemagglutination activity with red blood cells from any species nor binding activity to synthetic histo-blood group antigens, indicating that PSaV does not use histo-blood group antigens as receptors. Attachment and infection of PSaV were markedly blocked by sialic acid and Vibrio cholerae neuraminidase (NA), suggesting a role for α2,3-linked, α2,6-linked or α2,8-linked sialic acid in virus attachment. However, viral attachment and infection were only partially inhibited by treatment of cells with sialidase S (SS) or Maackia amurensis lectin (MAL), both specific for α2,3-linked sialic acid, or Sambucus nigra lectin (SNL), specific for α2,6-linked sialic acid. These results indicated that PSaV recognizes both α2,3- and α2,6-linked sialic acids for viral attachment and infection. Treatment of cells with proteases or with benzyl 4-O-β-D-galactopyranosyl-β-D-glucopyranoside (benzylGalNAc), which inhibits O-linked glycosylation, also reduced virus binding and infection, whereas inhibition of glycolipd synthesis or N-linked glycosylation had no such effect on virus binding or infection. These data suggest PSaV binds to cellular receptors that consist of α2,3- and α2,6-linked sialic acids on glycoproteins attached via O-linked glycosylation. PMID:24901849

  4. Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein.

    PubMed

    Villar, Victor Hugo; Vögler, Oliver; Barceló, Francisca; Gómez-Florit, Manuel; Martínez-Serra, Jordi; Obrador-Hevia, Antònia; Martín-Broto, Javier; Ruiz-Gutiérrez, Valentina; Alemany, Regina

    2014-04-01

    The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10-100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3 ± 1.11% and 68.8 ± 1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5-10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.

  5. Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids.

    PubMed

    Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay J; Kwatra, Deep; Paturi, Kalyani D; Mitra, Ashim K

    2010-04-05

    HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.

  6. Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain.

    PubMed

    Radian, R; Bendahan, A; Kanner, B I

    1986-11-25

    Using the reconstitution conditions developed recently (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865) we have now purified the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain to apparent homogeneity. A partially purified transporter preparation was passed over wheat germ agglutinin-Sepharose 6MB and non-bound proteins were washed away. The transport activity, as expressed upon reconstitution of the protein into liposomes, was eluted by a solution containing Triton X-100 and N-acetylglucosamine. The specific transport activity was increased almost 400-fold over that of the crude extract. Taking into account an approximately 2.5-fold inactivation during the lectin column chromatography, the actual purification is about 1000-fold. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the active fractions revealed one band of 80 kDa and small amounts of a band which ran at an apparent molecular mass of 160 kDa. The ratio between the two could be experimentally changed such as, for instance, by lyophilization. Polyclonal antibodies were prepared against the 80-kDa band which also cross-reacted with the 160-kDa band, indicating that the latter apparently represents a dimer form of the first. Using Protein A-Sepharose Cl-4B and the antibody against the 80-kDa band, we were able to quantitatively immunoprecipitate the potential gamma-aminobutyric acid transport activity from a crude transporter preparation. The pure transporter preparation exhibited the same features of the transporter in synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogeneity, affinity, and efflux and exchange properties. We conclude that the 80-kDa band represents the gamma-aminobutyric acid transporter.

  7. Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids

    PubMed Central

    Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay J.; Kwatra, Deep; Paturi, Kalyani D.; Mitra, Ashim. K.

    2010-01-01

    HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the cellular washing and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of PappB-A to PappA-B in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake, elevation of cyclosporine uptake in the presence of 5 μM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories. PMID:20163160

  8. Novel mutations in Marburg virus glycoprotein associated with viral evasion from antibody mediated immune pressure.

    PubMed

    Kajihara, Masahiro; Nakayama, Eri; Marzi, Andrea; Igarashi, Manabu; Feldmann, Heinz; Takada, Ayato

    2013-04-01

    Marburg virus (MARV) and Ebola virus, members of the family Filoviridae, cause lethal haemorrhagic fever in humans and non-human primates. Although the outbreaks are concentrated mainly in Central Africa, these viruses are potential agents of imported infectious diseases and bioterrorism in non-African countries. Recent studies demonstrated that non-human primates passively immunized with virus-specific antibodies were successfully protected against fatal filovirus infection, highlighting the important role of antibodies in protective immunity for this disease. However, the mechanisms underlying potential evasion from antibody mediated immune pressure are not well understood. To analyse possible mutations involved in immune evasion in the MARV envelope glycoprotein (GP) which is the major target of protective antibodies, we selected escape mutants of recombinant vesicular stomatitis virus (rVSV) expressing MARV GP (rVSVΔG/MARVGP) by using two GP-specific mAbs, AGP127-8 and MGP72-17, which have been previously shown to inhibit MARV budding. Interestingly, several rVSVΔG/MARVGP variants escaping from the mAb pressure-acquired amino acid substitutions in the furin-cleavage site rather than in the mAb-specific epitopes, suggesting that these epitopes are recessed, not exposed on the uncleaved GP molecule, and therefore inaccessible to the mAbs. More surprisingly, some variants escaping mAb MGP72-17 lacked a large proportion of the mucin-like region of GP, indicating that these mutants efficiently escaped the selective pressure by deleting the mucin-like region including the mAb-specific epitope. Our data demonstrate that MARV GP possesses the potential to evade antibody mediated immune pressure due to extraordinary structural flexibility and variability.

  9. Recombinant sialidase NanA (rNanA) cleaves α2-3 linked sialic acid of host cell surface N-linked glycoprotein to promote Edwardsiella tarda infection.

    PubMed

    Chigwechokha, Petros Kingstone; Tabata, Mutsumi; Shinyoshi, Sayaka; Oishi, Kazuki; Araki, Kyosuke; Komatsu, Masaharu; Itakura, Takao; Shiozaki, Kazuhiro

    2015-11-01

    Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, α2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in α2-3 sialo-linked glycoproteins, but not in glycolipids and α2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that α2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of α2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of α2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAKS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing

  10. St. John's wort may ameliorate 2,4,6-trinitrobenzenesulfonic acid colitis off rats through the induction of pregnane X receptors and/or P-glycoproteins.

    PubMed

    Sehirli, A O; Cetinel, S; Ozkan, N; Selman, S; Tetik, S; Yuksel, M; Dulger, F G A

    2015-04-01

    It is reported that deficiencies of the pregnane X receptor (PXR) and P-glycoprotein (P-gp), the latter of which is encoded by the MDR1 gene, are important factors in the pathogenesis of inflammatory bowel disease (IBD). It is also known that the activation of PXR is protective of IBD due to the mutual repression between PXR and nuclear factor kappa B (NF-κB) expression and because NF-κB was reported to play a pivotal role in the pathogenesis of ulcerative colitis. The goal of this study was to investigate whether St. John's wort (SJW) and spironolactone (SPL), both known to have strong inducing effects on cytochrome P 450 (CYP) enzymes as well as PXR and P-gp, have ameliorating effects on 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis of rats through induction of PXR and/or P-gp. Wistar albino rats (250 - 300 g) were divided into control and TNBS-colitis groups. Each group was then divided into a) control (saline), b) SJW (300 mg/kg p.o. bid), and c) SPL (80 mg/kg p.o.) groups. Drugs were given for 7 days. Both treatments ameliorated the clinical hallmarks of colitis, as determined by body weight loss and assessment of diarrhea, colon length, and bowel histology. Plasma levels of NF-κB, tumour necrosis factor-alpha (TNF-α) and tissue myeloperoxidase (MPO) activity, as well as the oxidative stress markers that increased during colitis, decreased significantly after both treatments. The PXR and P-gp expression in the intestinal tissues was diminished in the colitis group but increased after drug treatments. Both drugs appeared to have significant antioxidant and anti-inflammatory effects and ameliorated the TNBS colitis of the rats, most likely through their PXR- and P-gp-inducing properties.

  11. Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism.

    PubMed

    Dufrasne, François E; Lombard, Catherine; Goubau, Patrick; Ruelle, Jean

    2016-10-14

    BST-2 or tetherin is a host cell restriction factor that prevents the budding of enveloped viruses at the cell surface, thus impairing the viral spread. Several countermeasures to evade this antiviral factor have been positively selected in retroviruses: the human immunodeficiency virus type 2 (HIV-2) relies on the envelope glycoprotein (Env) to overcome BST-2 restriction. The Env gp36 ectodomain seems involved in this anti-tetherin activity, however residues and regions interacting with BST-2 are not clearly defined. Among 32 HIV-2 ROD Env mutants tested, we demonstrated that the asparagine residue at position 659 located in the gp36 ectodomain is mandatory to exert the anti-tetherin function. Viral release assays in cell lines expressing BST-2 showed a loss of viral release ability for the HIV-2 N659D mutant virus compared to the HIV-2 wild type virus. In bst-2 inactivated H9 cells, those differences were lost. Subtilisin treatment of infected cells demonstrated that the N659D mutant was more tethered at the cell surface. Förster resonance energy transfer (FRET) experiments confirmed a direct molecular link between Env and BST-2 and highlighted an inability of the mutant to bind BST-2. We also tested a virus presenting a truncation of 109 amino acids at the C-terminal part of Env, a cytoplasmic tail partial deletion that is spontaneously selected in vitro. Interestingly, viral release assays and FRET experiments indicated that a full Env cytoplasmic tail was essential in BST-2 antagonism. In HIV-2 infected cells, an efficient Env-mediated antagonism of BST-2 is operated through an intermolecular link involving the asparagine 659 residue as well as the C-terminal part of the cytoplasmic tail.

  12. Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach.

    PubMed

    Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

    2016-04-15

    In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 10(4). With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300 K was calculated as -5.234 kcal mol(-1) for CBZ-AAG interaction and -6.237 kcal mol(-1) for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are -9.553 kcal mol(-1) and -14.618 cal mol(-1) K(-1) respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol(-1) and 7.206 cal mol(-1) K(-1) respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

  13. Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach

    NASA Astrophysics Data System (ADS)

    Ajmal, Mohammad Rehan; Abdelhameed, Ali Saber; Alam, Parvez; Khan, Rizwan Hasan

    2016-04-01

    In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 104. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as - 5.234 kcal mol- 1 for CBZ-AAG interaction and - 6.237 kcal mol- 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are - 9.553 kcal mol- 1 and - 14.618 cal mol- 1K- 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol- 1 and 7.206 cal mol- 1K- 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

  14. Enhanced gene delivery efficiency of cationic liposomes coated with PEGylated hyaluronic acid for anti P-glycoprotein siRNA: a potential candidate for overcoming multi-drug resistance.

    PubMed

    Ran, Rui; Liu, Yayuan; Gao, Huile; Kuang, Qifang; Zhang, Qianyu; Tang, Jie; Huang, Kai; Chen, Xiaoxiao; Zhang, Zhirong; He, Qin

    2014-12-30

    RNA interference is an effective method to achieve highly specific gene regulation. However, the commonly used cationic liposomes have poor biocompatibility, which may lead to systematic siRNA delivery of no avail. PEGylation is a good strategy in shielding the positive charge of cationic liposomes, but the enhanced serum stability is often in company with compromised cellular uptake and endosome escape. In this study, PEG was covalently linked to negatively charged hyaluronic acid and it was used to coat the liposome-siRNA nanoparticles. The resulting PEG-HA-NP complex had a diameter of 188.6 ± 10.8 nm and a dramatically declined zeta-potential from +34.9 ± 4.0 mV to -18.2 ± 2.2 mV. Owing to the reversed surface charge, PEG-HA-NP could remain stable in fetal bovine serum (FBS) to up to 24h. In contrast with normal PEGylation, hyaluronic acid and PEG co-modified PEG-HA-NP provided comparable cellular uptake and P-glycoprotein downregulation efficacy in MCF-7/ADR cells compared with Lipofectamine RNAiMAX and naked NP regardless of its anionic charged surface. Because of its good biocompatibility in serum, PEG-HA-NP possessed the best tumor accumulation, cellular uptake and subsequently the strongest P-glycoprotein silencing capability in tumor bearing mice compared with naked NP and HA-NP after i.v. injection, with a 34% P-glycoprotein downregulation. Therefore, PEG-HA coated liposomal complex was demonstrated to be a promising siRNA delivery system in adjusting solid tumor P-glycoprotein expression, which may become a potential carrier in reversing MDR for breast cancer therapy.

  15. Bioinformatics Prediction and Evolution Analysis of Arabinogalactan Proteins in the Plant Kingdom

    PubMed Central

    Ma, Yuling; Yan, Chenchao; Li, Huimin; Wu, Wentao; Liu, Yaxue; Wang, Yuqian; Chen, Qin; Ma, Haoli

    2017-01-01

    Arabinogalactan proteins (AGPs) are a family of extracellular glycoproteins implicated in plant growth and development. With a rapid growth in the number of genomes sequenced in many plant species, the family members of AGPs can now be predicted to facilitate functional investigation. Building upon previous advances in identifying Arabidopsis AGPs, an integrated strategy of systematical AGP screening for “classical” and “chimeric” family members is proposed in this study. A Python script named Finding-AGP is compiled to find AGP-like sequences and filter AGP candidates under the given thresholds. The primary screening of classical AGPs, Lys-rich classical AGPs, AGP-extensin hybrids, and non-classical AGPs was performed using the existence of signal peptides as a necessary requirement, and BLAST searches were conducted mainly for fasciclin-like, phytocyanin-like and xylogen-like AGPs. Then glycomodule index and partial PAST (Pro, Ala, Ser, and Thr) percentage are adopted to identify AGP candidates. The integrated strategy successfully discovered AGP gene families in 47 plant species and the main results are summarized as follows: (i) AGPs are abundant in angiosperms and many “ancient” AGPs with Ser-Pro repeats are found in Chlamydomonas reinhardtii; (ii) Classical AGPs, AG-peptides, and Lys-rich classical AGPs first emerged in Physcomitrella patens, Selaginella moellendorffii, and Picea abies, respectively; (iii) Nine subfamilies of chimeric AGPs are introduced as newly identified chimeric subfamilies similar to fasciclin-like, phytocyanin-like, and xylogen-like AGPs; (iv) The length and amino acid composition of Lys-rich domains are largely variable, indicating an insertion/deletion model during evolution. Our findings provide not only a powerful means to identify AGP gene families but also probable explanations of AGPs in maintaining the plant cell wall and transducing extracellular signals into the cytoplasm. PMID:28184232

  16. The primary structure of a procaryotic glycoprotein. Cloning and sequencing of the cell surface glycoprotein gene of halobacteria.

    PubMed

    Lechner, J; Sumper, M

    1987-07-15

    The hexagonally patterned surface layer of halobacteria consists of a true glycoprotein. This procaryotic glycoprotein has recently been shown to exhibit novel features with respect to saccharide structure and saccharide biosynthesis. The primary structure and the location of glycosylation sites were determined by cloning and sequencing of the glycoprotein gene of Halobacterium halobium. According to the predicted amino acid sequence, the glycoprotein is synthesized with a N-terminal leader sequence of 34 amino acid residues reminiscent of eucaryotic and procaryotic signal peptides. A hydrophobic stretch of 21 amino acid residues at the C terminus probably serves as a transmembrane domain. 14 threonine residues are clustered adjacent to this membrane anchor and linked to these threonines are all the disaccharides of the cell surface glycoprotein. 12 N-glycosylation sites are distributed over the polypeptide chain.

  17. The sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter confers high-level expression of the GUS reporter gene in the potato tuber.

    PubMed

    Kim, Tae-Won; Goo, Young-Min; Lee, Cheol-Ho; Lee, Byung-Hyun; Bae, Jung-Myung; Lee, Shin-Woo

    2009-10-01

    Molecular farming refers to the process of creating bioengineered plants with the capability of producing potentially valuable products, such as drugs, vaccines, and chemicals. We have investigated the potential of the sweet potato ADP-glucose pyrophosphorylase gene (ibAGP1) promoter and its transit peptide (TP) as an expression system for the mass production of foreign proteins in potato. The ibAGP1 promoter and its TP sequence were transformed into potato along with beta-glucuronidase (GUS) as a reporter gene, and GUS activity was subsequently analyzed in the transgenic potato plants. In tuber tissues, GUS activity in transgenic plants carrying only the ibAGP1 promoter (ibAGP1::GUS) increased up to 15.6-fold compared with that of transgenic plants carrying only the CaMV35S promoter (CaMV35S::GUS). GUS activity in transgenic plants was further enhanced by the addition of the sweetpotato TP to the recombinant vector (ibAGP1::TP::GUS), with tuber tissues showing a 26-fold increase in activity compared with that in the CaMV35S::GUS-transgenic lines. In leaf tissues, the levels of GUS activity found in ibAGP1::GUS-transgenic lines were similar to those in CaMV35S::GUS-lines, but they were significantly enhanced in ibAGP1::TP::GUS-lines. GUS activity gradually increased with increasing tuber diameter in ibAGP1::GUS-transgenic plants, reaching a maximum level when the tuber was 35 mm in diameter. In contrast, extremely elevated levels of GUS activity - up to about 10-fold higher than that found in CaMV35S::GUS-lines - were found in ibAGP1::TP::GUS-transgenic lines at a much earlier stage of tuber development (diameter 4 mm), and these higher levels were maintained throughout the entire tuber developmental stage. These results suggest that the sweetpotato ibAGP1 promoter and its TP are a potentially strong foreign gene expression system that can be used for molecular farming in potato plants.

  18. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    E2, along with E^rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions including cell attachment, host range susceptibility and virulence in natural hosts. In infected cells, E2 forms homodimers as well as heterodimers with E1, media...

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

    PubMed

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

    2014-02-21

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

  20. Pregnane X Receptor Not Nuclear Factor-kappa B Up-regulates P-glycoprotein Expression in the Brain of Chronic Epileptic Rats Induced by Kainic Acid.

    PubMed

    Yu, Nian; Zhang, Yan-Fang; Zhang, Kang; Cheng, Yong-Fei; Ma, Hai-Yan; Di, Qing

    2017-03-16

    Drug-resistance epilepsy (DRE) is attributed to the brain P-glycoprotein (P-gp) overexpression. We previously reported that nuclear factor-kappa B (NF-κB) played a critical role in regulating P-gp expression at the brain of the acute seizure rats. This study was extended further to investigate the interaction effect of NF-κB and pregnane X receptor (PXR) on P-gp expression at the brain of chronic epileptic rats treated with carbamazepine (CBZ). The chronic epileptic models were induced by the micro-injection of kainic acid (KA) into rats' hippocampus. Subsequently, the successful models were treated with different intervention agents of CBZ; PMA(a non-specific PXR activity inhibitor) or PDTC(a specific NF-κB activity inhibitor) respectively. The expression levels of P-gp and its encoded gene mdr1a/b were significantly up-regulated on the brain of KA-induced chronic epilepsy rats or the epilepsy rats treated with CBZ for 1 week, meanwhile with a high expression of PXR. The treatment of PMA dramatically reduced both PXR and P-gp expressions at the protein and mRNA levels in the chronic epilepsy brain. By compared to the epilepsy model group, the P-gp expression was not markedly attenuated by the inhibition of NF-κB activity with PDTC treatment, nevertheless with a decrease of NF-κB expression in this intervention group. Higher levels of proinflammatory cytokines(IL-1β, IL-6, TNF-α) were found both in the brain tissue and the serum in the epilepsy rats of each group. There was a declined trend of the pro-inflammatory cytokines expression of the PDTC treatment group but with no statistical significance. This study demonstrates for the first time that P-gp up-regulation is due to increase PXR expression in the chronic phase of epilepsy, differently from that NF-κB signaling may induce the P-gp expression in the acute seizure phase. Our results offer insights into the mechanism underlying the development of DRE using or not using CBZ treatment.

  1. Roles of P-glycoprotein and multidrug resistance protein in transporting para-aminosalicylic acid and its N-acetylated metabolite in mice brain

    PubMed Central

    Hong, Lan; Xu, Cong; O'Neal, Stefanie; Bi, Hui-chang; Huang, Min; Zheng, Wei; Zeng, Su

    2014-01-01

    Aim: Para-aminosalicylic acid (PAS) is effective in the treatment of manganism-induced neurotoxicity (manganism). In this study we investigated the roles of P-glycoprotein (MDR1a) and multidrug resistance protein (MRP) in transporting PAS and its N-acetylated metabolite AcPAS through blood-brain barrier. Methods: MDR1a-null or wild-type mice were intravenously injected with PAS (200 mg/kg). Thirty minutes after the injection, blood samples and brains were collected, and the concentrations of PAS and AcPAS in brain capillaries and parenchyma were measured using HPLC. Both MDCK-MDR1 and MDCK-MRP1 cells that overexpressed P-gp and MRP1, respectively, were used in two-chamber Transwell transport studies in vitro. Results: After injection of PAS, the brain concentration of PAS was substantially higher in MDR1a-null mice than in wild-type mice, but the brain concentration of AcPAS had no significant difference between MDR1a-null mice and wild-type mice. Concomitant injection of PAS with the MRP-specific inhibitor MK-571 (50 mg/kg) further increased the brain concentration of PAS in MDR1a-null mice, and increased the brain concentration of AcPAS in both MDR1a-null mice and wild-type mice. Two-chamber Transwell studies with MDCK-MDR1 cells demonstrated that PAS was not only a substrate but also a competitive inhibitor of P-gp, while AcPAS was not a substrate of P-gp. Two-chamber Transwell studies with the MDCK-MRP1 cells showed that MRP1 had the ability to transport both PAS and AcPAS across the BBB. Conclusion: P-gp plays a major role in the efflux of PAS from brain parenchyma into blood in mice, while MRP1 is involved in both PAS and AcPAS transport in the brain. PMID:25418377

  2. Extracellular glycoprotein from virulent and avirulent Cryptococcus species.

    PubMed Central

    Ross, A; Taylor, I E

    1981-01-01

    Two virulent strains of Cryptococcus neoformans and two nonvirulent forms (C. albidus and C. laurentii) were grown in liquid culture to produce maximal capsule formation. A glycoprotein was isolated from the culture medium and was homogeneous as determined by cellulose acetate electrophoresis and anion-exchange chromatography. The amino acid, neutral sugar, amino sugar, uronic acid, and O-acetyl compositions and the infrared spectra of the glycoprotein were determined. The product of the C. neoformans strains contained more mannose and uronic acid than did that from the nonpathogenic strains. O-acetyl groups were absent from glycoprotein of the two nonpathogens. PMID:7228406

  3. 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.

  4. Affinity Capillary Electrophoresis for Selective Control of Electrophoretic Mobility of Sialic Acid Using Lanthanide-Hexadentate Macrocyclic Polyazacarboxylate Complexes.

    PubMed

    Goto, Daiki; Ouchi, Kazuki; Shibukawa, Masami; Saito, Shingo

    2015-01-01

    It is difficult to control the electrophoretic mobility in order to obtain high resolution among saccharides in complex samples. We report herein on a new affinity capillary electrophoresis (ACE) method for an anionic monosaccharide, N-acetylneuraminic acid (Neu5Ac), which is important in terms of pathological diagnosis, using lanthanide-hexadentate macrocyclic polyazacarboxylate complexes (Ln-NOTA) as affinity reagents. It was shown that Ln-NOTA complexes increased the anionic mobility of Neu5Ac by approximately 40% through selective complexation with Neu5Ac. The extent of change in the mobility strongly depended on the type of central metal ion of Ln-NOTA. The stability constant (K) of Lu-NOTA with Neu5Ac was determined by ACE to be log Kb = 3.62 ± 0.04, which is the highest value among artificial receptors for Neu5Ac reported so far. Using this ACE, the Neu5Ac content in a glycoprotein sample, α1-acid glycoprotein (AGP), was determined after acid hydrolysis. Complete separation between Neu5Ac and hydrolysis products was successful by controlling the mobility to determine the concentration of Neu5Ac.

  5. Paromomycin Derived from Streptomyces sp. AG-P 1441 Induces Resistance against Two Major Pathogens of Chili Pepper.

    PubMed

    Balaraju, Kotnala; Kim, Chang-Jin; Park, Dong-Jin; Nam, Ki-Woong; Zhang, Kecheng; Sang, Mee Kyung; Park, Kyungseok

    2016-09-28

    This is the first report that paromomycin, an antibiotic derived from Streptomyces sp. AG-P 1441 (AG-P 1441), controlled Phytophthora blight and soft rot diseases caused by Phytophthora capsici and Pectobacterium carotovorum, respectively, in chili pepper (Capsicum annum L.). Chili pepper plants treated with paromomycin by foliar spray or soil drenching 7 days prior to inoculation with P. capsici zoospores showed significant (p < 0.05) reduction in disease severity (%) when compared with untreated control plants. The disease severity of Phytophthora blight was recorded as 8% and 50% for foliar spray and soil drench, respectively, at 1.0 ppm of paromomycin, compared with untreated control, where disease severity was 83% and 100% by foliar spray and soil drench, respectively. A greater reduction of soft rot lesion areas per leaf disk was observed in treated plants using paromomycin (1.0 μg/ml) by infiltration or soil drench in comparison with untreated control plants. Paromomycin treatment did not negatively affect the growth of chili pepper. Furthermore, the treatment slightly promoted growth; this growth was supported by increased chlorophyll content in paromomycin-treated chili pepper plants. Additionally, paromomycin likely induced resistance as confirmed by the expression of pathogenesis-related (PR) genes: PR-1, β-1,3-glucanase, chitinase, PR-4, peroxidase, and PR-10, which enhanced plant defense against P. capsici in chili pepper. This finding indicates that AG-P 1441 plays a role in pathogen resistance upon the activation of defense genes, by secretion of the plant resistance elicitor, paromomycin.

  6. Molecular basis of viral persistence: a single amino acid change in the glycoprotein of lymphocytic choriomeningitis virus is associated with suppression of the antiviral cytotoxic T-lymphocyte response and establishment of persistence.

    PubMed Central

    Salvato, M; Borrow, P; Shimomaye, E; Oldstone, M B

    1991-01-01

    Isolates of lymphocytic choriomeningitis virus (LCMV) that elicit a cytotoxic T-lymphocyte response (CTL+) have been compared with isolates that suppress the CTL response (CTL-) in an effort to map this phenotype. A single amino acid change in the glycoprotein of the LCMV Armstrong (ARM) strain is consistently associated with the CTL- trait and the ability of the virus to persist (P+). The CTL+ P- parental strain spontaneously gives rise to CTL- P+ variants within lymphoid tissues of mice persistently infected from birth. To map the structural basis of the phenotype, the complete RNA sequence of LCMV ARM 53b (CTL+) was compared with that of its variant ARM clone 13 (CTL-). Differences in 5 of 10,600 nucleotides were found. Three changes are noted in the large L RNA segment, and two are noted in the small S RNA segment. Only two of the changes distinguishing CTL+ from CTL- isolates affect amino acid coding: lysine to glutamine at amino acid 1079 of the polymerase protein, and phenylalanine to leucine at amino acid 260 of the envelope glycoprotein (GP). We also analyzed two additional CTL- variants and four spontaneous CTL+ revertants. All three CTL- variants differ from the original CTL+ parental strain at GP amino acid 260, indicating that this amino acid change is consistently associated with the CTL- phenotype. By contrast the other four mutations in LCMV are not associated with the CTL- phenotype. Sequence analysis of the coding regions of four CTL+ revertants of ARM clone 13 did not reveal back mutations at the GP 260 locus. This finding indicates that the GP 260 mutation is necessary but not sufficient for a CTL- P+ phenotype and that the reversion to CTL+ P- is likely either due to secondary mutations in other regions of the viral genome or to quasispecies within the revertant population that make significant contributions to the phenotype. Images PMID:1840619

  7. Relation between proteins tertiary structure, tryptophan fluorescence lifetimes and tryptophan S(o)→(1)L(b) and S(o)→(1)L(a) transitions. Studies on α1-acid glycoprotein and β-lactoglobulin.

    PubMed

    Albani, Jihad René

    2011-05-01

    We measured fluorescence lifetimes and fluorescence spectra (excitation and emission) of tryptophan residues of α(1)-acid glycoprotein (three Trp residues) and β-lactoglobulin (two Trp residues) in absence and presence of 450 μM progesterone. Progesterone binds only to α(1)-acid glycoprotein. In absence of progesterone, each of the two proteins displays three fluorescence lifetimes. Addition of progesterone induces a partial inhibition of the S(o) → (1)L(a) transition without affecting fluorescence lifetimes. The same experiments performed in presence of denatured proteins in 6 M guanidine show that addition of progesterone inhibits partially the S(o) → (1)L(a) transition and its peak is 15 nm shifted to the red compared to that obtained for native proteins. However, the S(o) → (1)L(b) transition position peak is not affected by protein denaturation. Thus, the tertiary structure of the protein plays an important role by modulating the tryptophan electronic transitions. Fluorescence emission decay recorded in absence and presence of progesterone yields three fluorescence lifetimes whether proteins are denatured or not. Thus, protein tertiary structure is not responsible for the presence of three fluorescence lifetimes. These characterize tryptophan substructures reached at the excited states and which population (pre-exponential values) depend on the tryptophan residues interaction with their microenvironment(s) and thus on the global conformation of the protein.

  8. Ammonia transport in the kidney by Rhesus glycoproteins.

    PubMed

    Weiner, I David; Verlander, Jill W

    2014-05-15

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

  9. Facile fabrication of high-quality Ag/PS coaxial nanocables based on the mixed mode of soft/hard templates.

    PubMed

    Wan, Mimi; Zhao, Wenbo; Peng, Fang; Wang, Qi; Xu, Ping; Mao, Chun; Shen, Jian

    2016-08-01

    A new kind of high-quality Ag/PS coaxial nanocables can be facilely synthesized by using soft/hard templates method. In order to effectively introduce Ag sources into porous polystyrene (PS) nanotubes which were trapped in porous anodic aluminum oxide (AAO) hard template, Pluronic F127 (F127) was used as guiding agent, soft template and reductant. Meanwhile, ethylene glycol solution was also used as solvent and co-reducing agent to assist in the formation of silver nanowires. The influences of concentration of F127 and reducing reaction time on the formation of Ag/PS coaxial nanocables were discussed. Results indicated that the high-quality Ag/PS coaxial nanocables can be obtained by the mixed mode of soft/hard templates under optimized conditions. This strategy is expected to be extended to design more metal/polymer coaxial nanocables for the benefit of creation of complex and functional nanoarchitectures and components.

  10. Facile fabrication of high-quality Ag/PS coaxial nanocables based on the mixed mode of soft/hard templates

    PubMed Central

    Wan, Mimi; Zhao, Wenbo; Peng, Fang; Wang, Qi; Xu, Ping; Mao, Chun; Shen, Jian

    2016-01-01

    A new kind of high-quality Ag/PS coaxial nanocables can be facilely synthesized by using soft/hard templates method. In order to effectively introduce Ag sources into porous polystyrene (PS) nanotubes which were trapped in porous anodic aluminum oxide (AAO) hard template, Pluronic F127 (F127) was used as guiding agent, soft template and reductant. Meanwhile, ethylene glycol solution was also used as solvent and co-reducing agent to assist in the formation of silver nanowires. The influences of concentration of F127 and reducing reaction time on the formation of Ag/PS coaxial nanocables were discussed. Results indicated that the high-quality Ag/PS coaxial nanocables can be obtained by the mixed mode of soft/hard templates under optimized conditions. This strategy is expected to be extended to design more metal/polymer coaxial nanocables for the benefit of creation of complex and functional nanoarchitectures and components. PMID:27477888

  11. Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1.

    PubMed

    Seibeck, Sven; Borucki, Berthold; Otto, Harald; Inomata, Katsuhiko; Khawn, Htoi; Kinoshita, Hideki; Michael, Norbert; Lamparter, Tilman; Heyn, Maarten P

    2007-11-27

    The bacteriophytochrome Agp1 was reconstituted with a locked 5Zs-biliverdin in which the C(4)=C(5) and C(5)-C(6) bonds of the methine bridge between rings A and B are fixed in the Z and syn configuration/conformation, respectively. In Agp1-5Zs the photoconversion proceeds via the Lumi-R intermediate to Meta-R(A), but the following millisecond-transition to Meta-R(C) is blocked. Consistently, no transient proton release was detected. The photoconversion of Agp1-5Zs is apparently arrested in a Meta-R(A)-like intermediate, since the subsequent syn to anti rotation around the C(5)-C(6) bond is prevented by the lock. The Meta-R(A)-like photoproduct was characterized by its distinctive CD spectrum suggesting a reorientation of ring D.

  12. 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.

  13. Involvement of the alpha2,8-polysialyltransferases II/STX and IV/PST in the biosynthesis of polysialic acid chains on the O-linked glycoproteins in rainbow trout ovary.

    PubMed

    Asahina, Shinji; Sato, Chihiro; Matsuno, Midori; Matsuda, Tsukasa; Colley, Karen; Kitajima, Ken

    2006-11-01

    Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing alpha2,8-linked polysialic acid (polySia) on its O-linked glycans. Biosynthesis of the polySia chains is developmentally regulated and only occurs at later stage of oogenesis. Two alpha2,8-polysialyltransferases (alpha2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), responsible for the biosynthesis of polySia on N-glycans of glycoproteins, are known in mammals. However, nothing has been known about which alpha2,8-polySTs are involved in the biosynthesis of polySia on O-linked glycans in any glycoproteins. We thus sought to identify cDNA encoding the alpha2,8-polyST involved in polysialylation of PSGP. A clone for PST orthologue, rtPST, and two clones for the STX orthologue, rtSTX-ov and rtSTX-em, were identified in rainbow trout. The deduced amino acid sequence of rtPST shows a high identity (72-77%) to other vertebrate PSTs, while that of rtSTX-ov shows 92% identity with rtSTX-em and a significant identity (63-76%) to other vertebrate STXs. The rtPST exhibited the in vivo alpha2,8-polyST activity, although its in vitro activity was low. However, the rtSTXs showed no in vivo and very low in vitro activities. Interestingly, co-existence of rtPST and rSTX-ov in the reaction mixture synergistically enhanced the alpha2,8-polyST activity. During oogenesis, rtPST was constantly expressed, while the expression of rtSTX-ov was not increased until polySia chain is abundantly biosynthesized in the later stage. rtSTX-em was not expressed in ovary. These results suggest that the enhanced expression of rtSTX-ov under the co-expression with rtPST may be important for the biosynthesis of polySia on O-linked glycans of PSGP.

  14. "Clickable" affinity ligands for effective separation of glycoproteins.

    PubMed

    Suksrichavalit, Thummaruk; Yoshimatsu, Keiichi; Prachayasittikul, Virapong; Bülow, Leif; Ye, Lei

    2010-06-04

    In this paper, we present a new modular approach to immobilize boronic acid ligands that can offer effective separation of glycoproteins. A new "clickable" boronic acid ligand was synthesized by introducing a terminal acetylene group into commercially available 3-aminophenyl boronic acid. The clickable ligand, 3-(prop-2-ynyloxycarbonylamino)phenylboronic acid (2) could be easily coupled to azide-functionalized hydrophilic Sepharose using Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction under mild condition. Compared to other boronic acid affinity gels, the new affinity gel displayed superior effectiveness in separating model glycoproteins (ovalbumin and RNase B) from closely related bovine serum albumin and RNase A in the presence of crude Escherichia coli proteins. Because of the simplicity of the immobilization through "click chemistry", the new ligand 2 is expected to not only offer improved glycoprotein separation in other formats, but also act as a useful building block to develop new chemical sensors for analysis of other glycan compounds.

  15. Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins

    PubMed Central

    1978-01-01

    Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the

  16. 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.

  17. Effects of L- and D-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and HIV and SIV replication.

    PubMed Central

    Bahbouhi, Bouchaib; Chazal, Nathalie; Seidah, Nabil Georges; Chiva, Cristina; Kogan, Marcelo; Albericio, Fernando; Giralt, Ernest; Bahraoui, Elmostafa

    2002-01-01

    The aim of the present study was to evaluate the capacity of synthetic l- and d-peptides encompassing the HIV-1(BRU) gp160 REKR cleavage site to interfere with HIV and simian immuno-deficiency virus (SIV) replication and maturation of the envelope glycoprotein (Env) precursors. To facilitate their penetration into cells, a decanoyl (dec) group was added at the N-terminus. The sequences synthesized included dec5d or dec5l (decREKRV), dec9d or dec9l (decRVVQREKRV) and dec14d or dec14l (TKAKRRVVQREKRV). The peptide dec14d was also prepared with a chloromethane (cmk) group as C-terminus. Because l-peptides exhibit significant cytotoxicity starting at 35 microM, further characterization was conducted mostly with d-peptides, which exhibited no cytotoxicity at concentrations higher than 70 microM. The data show that only dec14d and dec14dcmk could inhibit HIV-1(BRU), HIV-2(ROD) and SIV(mac251) replication and their syncytium-inducing capacities. Whereas peptides dec5d and dec9d were inactive, dec14dcmk was at least twice as active as peptide dec14d. At the molecular level, our data show a direct correlation between anti-viral activity and the ability of the peptides to interfere with maturation of the Env precursors. Furthermore, we show that when tested in vitro the dec14d peptide inhibited PC7 with an inhibition constant K(i)=4.6 microM, whereas the peptide dec14l preferentially inhibited furin with a K(i)=28 microM. The fact that PC7 and furin are the major prohormone convertases reported to be expressed in T4 lymphocytes, the principal cell targets of HIV, suggests that they are involved in the maturation of HIV and SIV Env precursors. PMID:12071862

  18. 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.

  19. The Role of HIV-1 gp41 Glycoprotein in Infectious Tropism Inferred from Physico-Chemical Properties of its Amino Acid Sequence

    NASA Astrophysics Data System (ADS)

    Figueroa, E.; Villarreal, C.; Huerta, L.; Cocho, G.

    2006-09-01

    We performed a statistical analysis of the amino acid sequence of the gp41 ectodomain of the Human Immunodeficiency Virus type 1. We found strong correlations between physicochemical properties of highly variable residues and the viral infectious tropism.

  20. The trail of my studies on glycoproteins from enterokinase to tumor markers.

    PubMed

    Yamashina, Ikuo

    2010-01-01

    This review describes the results of the author's studies on glycoproteins which have been carried out for more than 50 years. Starting from the elucidation of basic structures of glycoproteins, i.e. the structure of the linkage between an amino acid and a sugar and the occurrence of the beta-mannosidic linkage as the common structure of glycoproteins, the author became interested in the cell membrane glycoproteins focused on the comparison of cancer cells versus normal cells. These studies were then extended to the establishment of sugar-directed and cancer-associated monoclonal antibodies. Some of the monoclonal antibodies are useful for cancer diagnosis.

  1. The trail of my studies on glycoproteins from enterokinase to tumor markers

    PubMed Central

    YAMASHINA, Ikuo

    2010-01-01

    This review describes the results of the author’s studies on glycoproteins which have been carried out for more than 50 years. Starting from the elucidation of basic structures of glycoproteins, i.e. the structure of the linkage between an amino acid and a sugar and the occurrence of the β-mannosidic linkage as the common structure of glycoproteins, the author became interested in the cell membrane glycoproteins focused on the comparison of cancer cells versus normal cells. These studies were then extended to the establishment of sugar-directed and cancer-associated monoclonal antibodies. Some of the monoclonal antibodies are useful for cancer diagnosis. PMID:20551595

  2. A juxta-membrane amino acid sequence of P-selectin glycoprotein ligand-1 is involved in moesin binding and ezrin/radixin/moesin-directed targeting at the trailing edge of migrating lymphocytes.

    PubMed

    Serrador, Juan M; Urzainqui, Ana; Alonso-Lebrero, Jose L; Cabrero, J Román; Montoya, Maria C; Vicente-Manzanares, Miguel; Yáñez-Mó, María; Sánchez-Madrid, Francisco

    2002-06-01

    P-selectin glycoprotein ligand 1 (PSGL-1) is an adhesion receptor localized on the tips of microvilli that is involved in the rolling of neutrophils on activated endothelium. We found that PSGL-1 was concentrated at the uropod of chemokine-stimulated lymphoid cells. Dynamic fluorescence videomicroscopy analyses of migrating lymphocytes demonstrated that PSGL-1 and moesin redistributed towards the cellular uropod at the trailing edge of these cells, where activated ezrin/radixin/moesin (ERM) proteins were located. An eighteen amino acid sequence in the juxta-membrane region of the PSGL-1 cytoplasmic tail was found to be critical for uropod targeting and moesin binding. Substitution of S336, S348, and the basic cluster R337K338 by alanines within this region significantly impaired both moesin binding and PSGL-1 polarization. These results underline the role of moesin in the subcellular redistribution of PSGL-1 in lymphoid cells and make evident the importance of specific serine residues within the cytoplasmic tail of PSGL-1 for this process.

  3. Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

    PubMed Central

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

    1985-01-01

    The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1. Images PMID:2987829

  4. Glycoprotein Degradation in the Blind Loop Syndrome

    PubMed Central

    Prizont, Roberto

    1981-01-01

    Contents obtained from jejunum of normal controls, self-emptying and self-filling blind loop rats were analyzed for the presence of glycoprotein-degrading glycosidases. The blind loop syndrome was documented by the increased fat excretion and slower growth rate of self-filling blind loop rats 6 wk after surgery. With p-nitrophenylglycosides as substrate, the specific activity of α-N-acetylgalactosaminidase, a potential blood group A destroying glycosidase, was 0.90±0.40 mU/mg of protein. This level was 23-fold higher than the specific activity of normal controls. In partially purified self-filling blind loop contents, the activity of α-N-acetylgalactosaminidase was 9- to 70-fold higher than activities of self-emptying and normal controls. Antibiotic treatment with chloromycetin and polymyxin decreased 24-fold the glycosidase levels in self-filling blind loops. In experiments with natural substrate, the blood group A titer of a20,000g supernate from normal jejunal homogenates decreased 128-fold after 24-h incubation with blind loop contents. Normal contents failed to diminish the blood group reactivity of the natural substrate. Furthermore, blind loop contents markedly decreased the blood group A titer of isolated brush borders. Incubation between blind loop bacteria and mucosal homogenates or isolated brush borders labeled with d-[U-14C]glucosamine revealed increased production of labeled ether extractable organic acids. Likewise, intraperitoneal injection of d-[U-14C]glucosamine into self-filling blind loop rats resulted in incorporation of the label into luminal short chain fatty acids. These results suggest that glycosidases may provide a mechanism by which blind loop bacteria obtain sugars from intestinal glycoproteins. The released sugars are used and converted by bacteria into energy and organic acids. This use of the host's glycoproteins would allow blind loop bacteria to grow and survive within the lumen independent of exogenous sources. PMID:6257760

  5. Subunit structure of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins.

    PubMed

    Sangadala, S; Kim, D; Brewer, J M; Mendicino, J

    1991-03-27

    The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3 degrees and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures. The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous. The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate

  6. Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein.

    PubMed Central

    Elango, N; Coligan, J E; Jambou, R C; Venkatesan, S

    1986-01-01

    The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5. Images PMID:3003381

  7. Regulation of cytokine gene expression by orosomucoid in neonatal swine adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adipose tissue has been reported to express a1-acid glycoprotein (AGP) mRNA, a protein with inflammatory and immunomodulatory properties. The present study was designed to determine if AGP can regulate pro-inflammatory or anti-inflammatory cytokine expression in neonatal porcine subcutaneous adipos...

  8. Amino acid sequence and molecular modelling of glycoprotein IIb-IIIa and fibronectin receptor iso-antagonists from Trimeresurus elegans venom.

    PubMed Central

    Scaloni, A; Di Martino, E; Miraglia, N; Pelagalli, A; Della Morte, R; Staiano, N; Pucci, P

    1996-01-01

    Low-molecular-mass Arg-Gly-Asp (RGD)-containing polypeptides were isolated from the venom of Trimeresurus elegans by a simple two-step procedure consisting of membrane filtration and reverse-phase HPLC. A combination of electrospray MS, fast-atom bombardment MS and Edman degradation allowed us to ascertain the presence in the venom of different isoforms and to determine their primary structures. The amino acid sequences resembled the structure of elegantin, the only disintegrin previously reported from the T. elegans venom [Williams, Rucinski, Holt and Niewiarowski (1990) Biochim. Biophys, Acta 1039, 81-89]. MS analyses indicated the occurrence of differential proteolytic processing at both the N-terminus and the C-termins of the polypeptide chains. The amino acid sequence alignment of the elegantin isoforms with known components of the disintegrin family demonstrated the complete conservation of the 12 cysteine residues involved in disulphide bridges. Molecular modelling of elegantins predicted an overall folding of these molecules quite similar to that reported for the kistrin solution structure. The newly identified polypeptide isoforms strongly inhibited ADP-induced aggregation in both human and canine platelet-rich plasma but showed a different species-dependent specificity. These molecules were also able to inhibit B16-BL6 murine melanoma cell adhesion to immobilized fibronectin. The comparison of the structures and biological activities of elegantin isoforms and kistrin allowed us to highlight some structural features that, in addition to the RGD locus might be involved in the interaction of these snake-venom polypeptides with the integrin receptors on the platelet and cell surface. PMID:8920980

  9. Envelope glycoprotein of arenaviruses.

    PubMed

    Burri, Dominique J; da Palma, Joel Ramos; Kunz, Stefan; Pasquato, Antonella

    2012-10-17

    Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.

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

    SciTech Connect

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

    1990-04-05

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

  11. Nuclear Factor-Kappa B Activity Regulates Brain Expression of P-Glycoprotein in the Kainic Acid-Induced Seizure Rats

    PubMed Central

    Yu, Nian; Di, Qing; Liu, Hao; Hu, Yong; Jiang, Ying; Yan, Yu-kui; Zhang, Yan-fang; Zhang, Ying-dong

    2011-01-01

    This study was aimed to investigate the effect of NF-κB activity on the seizure susceptibility, brain damage, and P-gp expression in kainic acid- (KA-) induced seizure rats. Male SD rats were divided into saline control group (NS group), KA induced epilepsy group (EP group), and epilepsy group intervened with NF-κB inhibitor-pyrrolidine dithiocarbamate salt (PDTC group) or with dexamethasone (DEX group). No seizures were observed in the rats of NS group. Compared with NS group, increased P-gp expression and NF-κB activation in the rat brain of the EP group were observed after KA micro-injection. Both PDTC and DEX pre-treatment significantly increased the latency to grade III or V seizure onset compared to EP group but failed to show neuron-protective effect as the number of survival neurons didn't significantly differ from that in EP group. Furthermore, PDTC pre-treatment significantly decreased P-gp expression along with NF-κB activation in the hippocampus CA3 area and amygdala complex of rats compared with the EP group, implying that NF-κB activation involved in the seizure susceptibility and seizure induced brain P-gp over-expression. Additionally, DEX pre-treatment only decreased P-gp expression level without inhibition of NF-κB activation, suggesting NF-κB independent pathway may also participate in regulating seizure induced P-gp over-expression. PMID:21403895

  12. Fractionation of the genetic variants of human alpha 1-acid glycoprotein in the native form by chromatography on an immobilized copper(II) affinity adsorbent. Heterogeneity of the separate variants by isoelectrofocusing and by concanavalin A affinity chromatography.

    PubMed

    Hervé, F; Gomas, E; Duché, J C; Tillement, J P

    1993-05-19

    Fractionation of the three main genetic variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), in their native (sialylated) form, by chromatography on immobilized copper(II) affinity adsorbent was investigated. This chromatographic method had been previously developed to fractionate the desialylated protein variants. For that purpose, the three main AAG phenotypes samples (F1S/A, F1/A and S/A), which had been previously isolated from individual human plasma samples, and an AAG sample from commercial source (a mixture of the phenotypes) were used in the native form. Affinity chromatography of these different samples on an iminodiacetate Sepharose-copper(II) gel at pH 7 resolved two protein peaks, irrespective of the origin of the native AAG sample used. The unbound peak 1 was found to consist of the F1, the S or both variants, depending on the phenotype of the AAG sample used in the chromatography. The bound peak 2 was found to consist of the A variant in a pure form. The fractionation results obtained with native AAG were found to be the same as those originally yielded by the desialylated protein. However, comparison of the interactions of native and desialylated AAG with immobilized copper(II) ions, using an affinity chromatographic method and a non-chromatographic equilibrium binding technique, respectively, showed that desialylation increased the non-specific interactions of the protein with immobilized copper(II) ions. The AAG variants were not fractionated when affinity chromatography was performed using immobilized zinc, nickel or cobalt(II) ions, instead of copper. After purification of each variant in the sialylated form (F1, S and A), their respective heterogeneity was studied by analytical isoelectrofocusing with carrier ampholytes in the pH range 2.5-4.5. In addition, the lectin-binding behaviour of the separate sialylated AAG variants was investigated by affinity chromatography on immobilized concanavalin A.

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

    PubMed

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

    2014-03-10

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

  14. Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry.

    PubMed

    Weed, Darin J; Pritchard, Suzanne M; Gonzalez, Floricel; Aguilar, Hector C; Nicola, Anthony V

    2017-03-01

    Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions.IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a

  15. Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin,. cap alpha. /sub 1/-acid glycoprotein and albumin

    SciTech Connect

    de Vera, N.; Cristofol, R.M.; Farre, R.

    1988-01-01

    The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37/sup 0/C. At 1.7 nM L-(/sup 3/H)-NE binding was approx. 25%. The binding was rapid and was not influenced by the incubation time. (/sup 3/H)-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37/sup 0/C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles x mg/sup -1/ protein, and low-affinity sites with a Kd of 2.7 ..mu..M and a Bmax of 3.3 pmoles x mg/sup -1/ protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. Ne binding to pure human plasma proteins was also studied using ultrafiltration.

  16. Conserved ion and amino acid transporters identified as phosphorylcholine-modified N-glycoproteins by metabolic labeling with propargylcholine in Caenorhabditis elegans cells.

    PubMed

    Snodgrass, Casey J; Burnham-Marusich, Amanda R; Meteer, John C; Berninsone, Patricia M

    2015-04-01

    Phosphorylcholine (PC) modification of proteins by pathogens has been implicated in mediating host-pathogen interactions. Parasitic nematodes synthesize PC-modified biomolecules that can modulate the host's antibody and cytokine production to favor nematode survival, contributing to long-term infections. Only two nematode PC-modified proteins (PC-proteins) have been unequivocally identified, yet discovering the protein targets of PC modification will be paramount to understanding the role(s) that this epitope plays in nematode biology. A major hurdle in the field has been the lack of techniques for selective purification of PC-proteins. The nonparasitic nematode Caenorhabditis elegans expresses PC-modified N-linked glycans, offering an attractive model to study the biology of PC-modification. We developed a robust method to identify PC-proteins by metabolic labeling of primary embryonic C. elegans cells with propargylcholine, an alkyne-modified choline analog. Cu(I)-catalyzed cycloaddition with biotin-azide enables streptavidin purification and subsequent high-throughput LC-MS identification of propargyl-labeled proteins. All proteins identified using stringent criteria are known or predicted to be membrane or secreted proteins, consistent with the model of a Golgi-resident, putative PC-transferase. Of the 55 PC-N-glycosylation sites reported, 33 have been previously observed as N-glycosylation sites in high-throughput screens of C. elegans. Several identified PC-proteins are nematode-specific proteins, but 10 of the PC-proteins are widely conserved ion transporters and amino acid transporters, while eight are conserved proteins involved in synaptic function. This finding suggests a functional role for PC-modification beyond immunomodulation. The approach presented in this study provides a method to identify PC-proteins in C. elegans and related nematodes.

  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.

  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. 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

  20. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody.

    PubMed

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J S Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941-50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

  1. Substitution at Aspartic Acid 1128 in the SARS Coronavirus Spike Glycoprotein Mediates Escape from a S2 Domain-Targeting Neutralizing Monoclonal Antibody

    PubMed Central

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J. S. Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941–50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111–1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus. PMID:25019613

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

    PubMed Central

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

    2015-01-01

    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

  3. Purification and characterization of a soluble glycoprotein from garlic (Allium sativum) and its in vitro bioactivity.

    PubMed

    Wang, Yan; Zou, Tingting; Xiang, Minghui; Jin, Chenzhong; Zhang, Xuejiao; Chen, Yong; Jiang, Qiuqing; Hu, Yihong

    2016-10-02

    A soluble glycoprotein was purified to homogeneity from ripe garlic (Allium sativum) bulbs using ammonium sulfate precipitation, Sephadex G-100 gel filtration, and diethylaminoethyl-52 cellulose anion-exchange chromatography. A native mass of 55.7 kDa estimated on gel permeation chromatography and a molecular weight of 13.2 kDa observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis supported that the glycoprotein is a homotetramer. β-Elimination reaction result suggested that the glycoprotein is an N-linked type. Fourier-transform infrared spectroscopy proved that it contains sugar. Gas chromatography-mass spectrometer analysis showed that its sugar component was galactose. The glycoprotein has 1,1-diphenyl-2-picrylhydrazil free radical scavenging activity and the peroxidation inhibition ability to polyunsaturated fatty acid. These results indicated that the glycoprotein has potential for food additives, functional foods, and even biotechnological and medical applications.

  4. 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

  5. Arginine residues in the C-terminal and their relationship with the analgesic activity of the toxin from the Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1).

    PubMed

    Wang, Yu; Song, Yong-Bo; Yang, Guang-Zhao; Cui, Yong; Zhao, Yong-Shan; Liu, Yan-Feng; Ma, Yan; Wu, Chun-Fu; Zhang, Jing-Hai

    2012-09-01

    In this study, we investigated the functional role of arginines in the C-terminal (65-67) of BmK AGP-SYPU1, an analgesic peptide from the Chinese scorpion Buthus martensii Karsch. Using site-directed mutagenesis, arginines at the C-terminal (65-66) were deleted or added to the C-terminal (67). The genes for three mutants of BmK AGP-SYPU1 were obtained by PCR. An analgesic activity assay was used to evaluate the role of arginine residues in the analgesic activity. The three-dimensional structure of BmK AGP-SYPU1 was established by homology modeling. As a result, we showed that the arginines in the C-terminal are crucial for the analgesic activity and may be located at analgesic functional sites. Our work has implications for further modification of scorpion toxins to obtain new analgesic peptides with enhanced activity.

  6. 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

  7. 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.

  8. 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.

  9. Differential mode of interaction of ThioflavinT with native β structural motif in human α 1-acid glycoprotein and cross beta sheet of its amyloid: Biophysical and molecular docking approach

    NASA Astrophysics Data System (ADS)

    Ajmal, Mohammad Rehan; Nusrat, Saima; Alam, Parvez; Zaidi, Nida; Badr, Gamal; Mahmoud, Mohamed H.; Rajpoot, Ravi Kant; Khan, Rizwan Hasan

    2016-08-01

    The present study details the interaction mechanism of Thioflavin T (ThT) to Human α1-acid glycoprotein (AAG) applying various spectroscopic and molecular docking methods. Fluorescence quenching data revealed the binding constant in the order of 104 M-1 and the standard Gibbs free energy change value, ΔG = -6.78 kcal mol-1 for the interaction between ThT and AAG indicating process is spontaneous. There is increase in absorbance of AAG upon the interaction of ThT that may be due to ground state complex formation between ThT and AAG. ThT impelled rise in β-sheet structure in AAG as observed from far-UV CD spectra while there are minimal changes in tertiary structure of the protein. DLS results suggested the reduction in AAG molecular size, ligand entry into the central binding pocket of AAG may have persuaded the molecular compaction in AAG. Isothermal titration calorimetric (ITC) results showed the interaction process to be endothermic with the values of standard enthalpy change ΔH0 = 4.11 kcal mol-1 and entropy change TΔS0 = 10.82 kcal.mol- 1. Moreover, docking results suggested hydrophobic interactions and hydrogen bonding played the important role in the binding process of ThT with F1S and A forms of AAG. ThT fluorescence emission at 485 nm was measured for properly folded native form and for thermally induced amyloid state of AAG. ThT fluorescence with native AAG was very low, while on the other hand with amyloid induced state of the protein AAG showed a positive emission peak at 485 nm upon the excitation at 440 nm, although it binds to native state as well. These results confirmed that ThT binding alone is not responsible for enhancement of ThT fluorescence but it also required beta stacked sheet structure found in protein amyloid to give proper signature signal for amyloid. This study gives the mechanistic insight into the differential interaction of ThT with beta structures found in native state of the proteins and amyloid forms, this study reinforce

  10. DNA Methylation at the Neonatal State and at the Time of Diagnosis: Preliminary Support for an Association with the Estrogen Receptor 1, Gamma-Aminobutyric Acid B Receptor 1, and Myelin Oligodendrocyte Glycoprotein in Female Adolescent Patients with OCD

    PubMed Central

    Nissen, Judith Becker; Hansen, Christine Søholm; Starnawska, Anna; Mattheisen, Manuel; Børglum, Anders Dupont; Buttenschøn, Henriette Nørmølle; Hollegaard, Mads

    2016-01-01

    Obsessive–compulsive disorder (OCD) is a neuropsychiatric disorder. Non-genetic factors and their interaction with genes have attracted increasing attention. Epigenetics is regarded an important interface between environmental signals and activation/repression of genomic responses. Epigenetic mechanisms have not previously been examined in OCD in children and adolescents. The aim of the present study was to examine the DNA methylation profile of selected genes in blood spots from neonates later diagnosed with OCD and in the same children/adolescents at the time of diagnosis compared with age- and sex-matched controls. Furthermore, we wanted to characterize the association of the differential methylation profiles with the severity of OCD and treatment outcome. Dried and new blood spot samples were obtained from 21 female children/adolescents with verified OCD and 12 female controls. The differential methylation was analyzed using a linear model and the correlation with the severity of OCD and treatment outcome was analyzed using the Pearson correlation. We evaluated selected Illumina Infinium HumanMethylation450 BeadChip probes within and up to 100,000 bp up- and downstream of 14 genes previously associated with OCD (SLC1A1, SLC25A12, GABBR1, GAD1, DLGAP1, MOG, BDNF, OLIG2, NTRK2 and 3, ESR1, SL6A4, TPH2, and COMT). The study found no significantly differential methylation. However, preliminary support for a difference was found for the gamma-aminobutyric acid (GABA) B receptor 1 (cg10234998, cg17099072) in blood samples at birth and for the estrogen receptor 1 (ESR1) (cg10939667), the myelin oligodendrocyte glycoprotein (MOG) (cg16650906), and the brain-derived neurotrophic factor (BDNF) (cg14080521) in blood samples at the time of diagnosis. Preliminary support for an association was observed between the methylation profiles of GABBR1 and MOG and baseline severity, treatment effect, and responder status; and between the methylation profile of ESR1 and baseline

  11. 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.

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

    PubMed

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

    2015-10-06

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

  13. 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

  14. Role of sialidase in glycoprotein utilization by Tannerella forsythia.

    PubMed

    Roy, Sumita; Honma, Kiyonobu; Douglas, C W Ian; Sharma, Ashu; Stafford, Graham P

    2011-11-01

    The major bacterial pathogens associated with periodontitis include Tannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth of T. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host-pathogen interactions in vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing that T. forsythia can utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with a nanH mutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of the nanH mutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a key in vivo nutrient source for T. forsythia when growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.

  15. The Crystal Structures of the N-terminal Photosensory Core Module of Agrobacterium Phytochrome Agp1 as Parallel and Anti-parallel Dimers.

    PubMed

    Nagano, Soshichiro; Scheerer, Patrick; Zubow, Kristina; Michael, Norbert; Inomata, Katsuhiko; Lamparter, Tilman; Krauß, Norbert

    2016-09-23

    Agp1 is a canonical biliverdin-binding bacteriophytochrome from the soil bacterium Agrobacterium fabrum that acts as a light-regulated histidine kinase. Crystal structures of the photosensory core modules (PCMs) of homologous phytochromes have provided a consistent picture of the structural changes that these proteins undergo during photoconversion between the parent red light-absorbing state (Pr) and the far-red light-absorbing state (Pfr). These changes include secondary structure rearrangements in the so-called tongue of the phytochrome-specific (PHY) domain and structural rearrangements within the long α-helix that connects the cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) and the PHY domains. We present the crystal structures of the PCM of Agp1 at 2.70 Å resolution and of a surface-engineered mutant of this PCM at 1.85 Å resolution in the dark-adapted Pr states. Whereas in the mutant structure the dimer subunits are in anti-parallel orientation, the wild-type structure contains parallel subunits. The relative orientations between the PAS-GAF bidomain and the PHY domain are different in the two structures, due to movement involving two hinge regions in the GAF-PHY connecting α-helix and the tongue, indicating pronounced structural flexibility that may give rise to a dynamic Pr state. The resolution of the mutant structure enabled us to detect a sterically strained conformation of the chromophore at ring A that we attribute to the tight interaction with Pro-461 of the conserved PRXSF motif in the tongue. Based on this observation and on data from mutants where residues in the tongue region were replaced by alanine, we discuss the crucial roles of those residues in Pr-to-Pfr photoconversion.

  16. 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

  17. Improved tryptic digestion assisted with an acid-labile anionic surfactant for the separation and characterization of glycopeptide glycoforms of a proteolytic-resistant glycoprotein by capillary electrophoresis time-of-flight mass spectrometry.

    PubMed

    Barroso, Albert; Giménez, Estela; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2016-04-01

    Certain glycoproteins are rather difficult to digest due to compacted tertiary or quaternary structures. In a previous study, a capillary LC coupled to TOF-MS (μLC-TOF-MS) method was developed for the detection and characterization of the glycopeptide glycoforms of human transferrin (Tf), a proteolytic resistant glycoprotein, in serum samples. After immunoaffinity purification, Tf was digested with trypsin in the presence of RapiGest(®) and μLC-TOF-MS analyses permitted to detect the N413 and N611 glycopeptide glycoforms. Conversely, the use of this surfactant, albeit mandatory to quantitatively digest the isolated Tf, proved detrimental to CE-TOF-MS analysis due to its interaction with the inner surface of the silica capillary walls. As CE is usually regarded as an interesting alternative to other separation techniques (low consumption of reagents, excellent separation efficiency, and reduced analysis times), in this work, the undesirable interferences of the surfactant have been removed to allow the correct separation and detection of Tf glycoforms by CE-TOF-MS. Moreover, the digestion protocol described by the RapiGest(®) manufacturer has been modified to minimize desialylation of Tf glycopeptide glycoforms. The new developed CE-TOF-MS methodology has been then compared with the former μLC-TOF-MS by means of sensitivity and separation efficiency of Tf glycopeptide glycoforms in the standard glycoprotein. Additionally, Tf glycopeptide glycoforms from serum of healthy volunteers and patients with congenital disorders of glycosylation have also been analyzed following the developed methodology.

  18. Developmental changes in microheterogeneity of foetal plasma glycoproteins of mice

    PubMed Central

    Gustine, David L.; Zimmerman, Ernest F.

    1973-01-01

    Changes in microheterogeneity of foetal plasma glycoproteins during development of mouse embryos were investigated. Analysis of foetal plasma by polyacrylamide-gel electrophoresis indicated three major zones of proteins: (1) transferrins, (2) α-foetoproteins and (3) albumin. Three transferrins (Tr1, Tr2, Tr3) and five α-foetoproteins (Fp1, Fp2, Fp3, Fp4, Fp5) were resolved. Evidence for the presence of transferrins was the binding of 59Fe to the three electrophoretic variants. By day 15.5 of gestation, there was a marked increase in the more-acidic components (Tr3, Fp4, Fp5) and a decrease in the less-acidic ones (Tr1, Tr2, Fp1, Fp2, Fp3). Treatment of foetal plasma with neuraminidase at this time of development converted the more acidic components into Tr1 and Tr2 and Fp1, Fp2 and Fp3. Furthermore, it was shown that early in development (day 12.5) only the less-acidic components of transferrin and α-foetoprotein were synthesized; at the later time in development (day 14.5) new synthesis of the acidic components of both groups occurred. That these more-acidic components of α-foetoprotein (Fp4, Fp5) were in fact electrophoretic variants of the less-acidic α-foetoproteins was shown by the immunoprecipitation of labelled Fp4 and Fp5 with anti-Fp1, anti-Fp2 and anti-Fp3. From these results it is postulated that the plasma glycoproteins that are synthesized later in development contain increased amounts of sialic acid and that the observed changes in microheterogeneity of these proteins represent regulation of glycoprotein biosynthesis at the level of carbohydrate attachment. PMID:4353382

  19. 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.

  20. Investigation of binding mechanism of novel 8-substituted coumarin derivatives with human serum albumin and α-1-glycoprotein.

    PubMed

    Yeggoni, Daniel Pushpa Raju; Manidhar, Darla Mark; Suresh Reddy, Cirandur; Subramanyam, Rajagopal

    2016-09-01

    Coumarin molecules have biological activities possessing lipid-controlling activity, anti-hepatitis C activity, anti-diabetic, anti-Parkinson activity, and anti-cancer activity. Here, we have presented an inclusive study on the interaction of 8-substituted-7-hydroxy coumarin derivatives (Umb-1/Umb-2) with α-1-glycoprotein (AGP) and human serum albumin (HSA) which are the major carrier proteins in the human blood plasma. Binding constants obtained from fluorescence emission data were found to be KUmb-1=3.1 ± .01 × 10(4) M(-1), KUmb-2 = 7 ± .01 × 10(4) M(-1), which corresponds to -6.1 and -6.5 kcal/mol of free energy for Umb-1 and Umb-2, respectively, suggesting that these derivatives bind strongly to HSA. Also these molecules bind to AGP with binding constants of KUmb-1-AGP=3.1 ± .01 × 10(3) M(-1) and KUmb-2-AGP = 4.6 ± .01 × 10(3) M(-1). Further, the distance, r between the donor (HSA) and acceptor (Umb-1/Umb-2) was calculated based on the Forster's theory of non-radiation energy transfer and the values were observed to be 1.14 and 1.29 nm in Umb-1-HSA and Umb-2-HSA system, respectively. The protein secondary structure of HSA was partially unfolded upon binding of Umb-1 and Umb-2. Furthermore, site displacement experiments with lidocaine, phenylbutazone (IIA), and ibuprofen (IIIA) proves that Umb derivatives significantly bind to subdomain IIIA of HSA which is further supported by docking studies. Furthermore, Umb-1 binds to LYS402 with one hydrogen bond distance of 2.8 Å and Umb-2 binds to GLU354 with one hydrogen bond at a distance of 2.0 Å. Moreover, these molecules are stabilized by hydrophobic interactions and hydrogen bond between the hydroxyl groups of carbon-3 of coumarin derivatives.

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

    PubMed

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

    2015-07-07

    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.

  2. Binding properties of monoclonal antibodies recognizing external epitopes of the human MDR1 P-glycoprotein.

    PubMed

    Schinkel, A H; Arceci, R J; Smit, J J; Wagenaar, E; Baas, F; Dollé, M; Tsuruo, T; Mechetner, E B; Roninson, I B; Borst, P

    1993-09-30

    Monoclonal antibodies (MAbs) recognizing external epitopes of the human MDR1 P-glycoprotein have been used both for the detection of multidrug-resistant cells and as specific inhibitors of P-glycoprotein-mediated multidrug resistance. Using a panel of recently developed transfected or transgenic cell lines containing variants of the human MDR1 and MDR3 P-glycoproteins, we have compared the specificity and binding properties of the previously isolated MAbs MRK16, HYB-241, UIC2 and 4E3, and of the newly isolated MAb 7G4. The removal of 1, 2 or all 3 of the N-glycosylation sites present in the first extracellular loop of MDR1 P-glycoprotein did not significantly affect the binding of these MAbs. In contrast, 20 amino acid deletion in the first extracellular loop of MDR1 P-glycoprotein completely abolished binding of UIC2, whereas the binding of all other MAbs was hardly affected. None of the MAbs tested bound detectably to cell lines containing a high level of the human MDR3 P-glycoprotein. The differences in the binding specificity between UIC2 and the other tested antibodies parallel the reported functional differences in the ability of these antibodies to inhibit P-glycoprotein-mediated drug efflux.

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

    PubMed

    Iwasaki, Yasuhiko; Matsunaga, Aki; Fujii, Shuetsu

    2014-09-17

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

  4. Expression and antigenicity of recombinant human respiratory syncytial virus glycoproteins having different affinity tags.

    PubMed

    Lee, Han Saem; Kim, A-Reum; Kim, Kisoon; Lee, Wan-Ji; Kim, Sung Soon; Kim, You-Jin

    2016-12-29

    Human respiratory syncytial virus (HRSV) is a main cause of lower respiratory tract infections in infants and the elderly. Glycoprotein (G) is major antigen on the viral surface, and plays a key role for virus entry. Therefore, purification of the glycoprotein of HRSV is critical for the development of HRSV vaccine and serological diagnosis. In this study, we report the design and characterization of glycoprotein engineered rationally to enhance the protein solubility and to facilitate efficient purification. We permuted HRSV glycoproteins with two tags: (i) an immunoglobulin (Ig) M signal peptide and a protein A B domain tag to render HRSV glycoprotein secret into the culture media and (ii) a foldon and 6 × histidine tag with or without transmembrane domain. Three recombinant baculoviruses were constructed: (i) transmembrane-truncated HRSV glycoprotein (amino acid positions 66-298) inserted with the N-terminal IgM signal peptide and protein A B domain (MG-GΔTM), (ii) truncated HRSV glycoprotein (amino acid positions 66-298) fused with a C-terminal foldon and 6 × histidine tag (GΔTM-FH), and (iii) full-length HRSV glycoprotein (amino acid positions 1-298) fused with a C-terminal foldon and 6 × histidine tag (G-FH). Highly soluble recombinant MG-GΔTM protein was clearly purified using one-step affinity chromatography with IgG-sepharose resin, whereas the recombinant G-FH protein and truncated GΔTM-FH were purified partially using nickel-resin. Although, the antigenicity of GΔTM-FH was stronger than highly mannose-rich MG-GΔTM protein, MG-GΔTM induced neutralizing antibodies efficiently in the mice to protect from infectious HRSV.

  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. A sweet code for glycoprotein folding.

    PubMed

    Caramelo, Julio J; Parodi, Armando J

    2015-11-14

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

  7. Sulphation of proteins secreted by a human hepatoma-derived cell line. Sulphation of N-linked oligosaccharides on alpha 2HS-glycoprotein.

    PubMed Central

    Hortin, G; Green, E D; Baenziger, J U; Strauss, A W

    1986-01-01

    Several human glycoproteins, including alpha 1-antitrypsin, alpha 1-acid glycoprotein, transferrin, caeruloplasmin and alpha 2HS-glycoprotein, synthesized by the hepatoma-derived cell line HepG2 were observed to contain covalently linked sulphate. These proteins were estimated to contain about 0.1 mol of sulphate/mol of protein. The most abundant of the sulphated glycoproteins, alpha 2HS-glycoprotein, was analysed in detail. All of the sulphate on this protein was attached to N-linked oligosaccharides which contained sialic acid and resisted release by endoglycosidase H. Several independent analytical approaches established that approx. 10% of the molecules of alpha 2HS-glycoprotein contained sulphate. Our results suggest that a number of human plasma proteins contain small amounts of sulphate linked to oligosaccharides. Images Fig. 1. Fig. 2. Fig. 3. PMID:3017304

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

    PubMed

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

    2014-04-01

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

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

    PubMed

    Kandiba, Lina; Guan, Ziqiang; Eichler, Jerry

    2013-03-01

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

  10. Facing extremes: archaeal surface-layer (glyco)proteins.

    PubMed

    Eichler, Jerry

    2003-12-01

    Archaea are best known in their capacities as extremophiles, i.e. micro-organisms able to thrive in some of the most drastic environments on Earth. The protein-based surface layer that envelopes many archaeal strains must thus correctly assemble and maintain its structural integrity in the face of the physical challenges associated with, for instance, life in high salinity, at elevated temperatures or in acidic surroundings. Study of archaeal surface-layer (glyco)proteins has thus offered insight into the strategies employed by these proteins to survive direct contact with extreme environments, yet has also served to elucidate other aspects of archaeal protein biosynthesis, including glycosylation, lipid modification and protein export. In this mini-review, recent advances in the study of archaeal surface-layer (glyco)proteins are discussed.

  11. A benzoboroxole-based affinity ligand for glycoprotein purification at physiological pH.

    PubMed

    Rowe, Laura; El Khoury, Graziella; Lowe, Christopher R

    2016-05-01

    Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre-enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio-pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one-pot, multi-component synthesis reaction (Ugi synthesis) and an amine-functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis-diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff-type boronic acid. The solid-phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate-dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid-functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid-based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold.

  12. High-Mr glycoprotein profiles in human milk serum and fat-globule membrane.

    PubMed

    Shimizu, M; Yamauchi, K; Miyauchi, Y; Sakurai, T; Tokugawa, K; McIlhinney, R A

    1986-02-01

    Gradient-polyacrylamide-gel electrophoresis of human milk serum separated three high-Mr glycoprotein bands. The properties of the components corresponding to the three bands (tentatively termed 'Components C, B and A' in their order of migration) were compared by staining with four monoclonal antibodies and lectins. Components B and C both reacted with the four antibodies, but Component A did not. Components B and C were stained with peanut (Arachis hypogaea) agglutinin (PNA) and wheat (Triticum)-germ agglutinin (WGA), Component A being stained with soya-bean (Glycine max) agglutinin as well as PNA and WGA. These results suggest that Components B and C were related molecules, whereas Component A was markedly different from them. The reactivities of Components B and C were the same as those of PAS-0, a high-Mr periodate/Schiff (PAS)-positive glycoprotein previously isolated from human milk fat-globule membrane (MFGM). Component C, whose electrophoretic mobility was the same as PAS-0, could have been a soluble form of PAS-0. A high-Mr glycoprotein having the same properties as Component A was also observed in MFGM. The amino acid composition of the isolated Component A was significantly different from that of Component C and PAS-0, high threonine and serine contents being characteristic of Component A. The carbohydrate content of Component A was 65-80%, and was much higher than that of Component C and PAS-0. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid were each detected as constituent sugars of Component A. Component A represents, therefore, a new high-Mr glycoprotein species in human milk serum and MFGM. Since these glycoproteins were high-Mr mucin-like glycoproteins, the names 'HM glycoprotein-A' and 'HM glycoprotein-C' were proposed for Component A and Component C (PAS-O) respectively.

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

    PubMed

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

    2009-05-01

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

  14. Immunological characterization of deglycosylated human and swine trachea and Cowper's gland mucin glycoproteins.

    PubMed

    Sangadala, S; Brewer, J M; Mendicino, J

    1991-04-01

    Antibodies were raised in rabbits against purified swine and human trachea and Cowper's gland mucin glycoproteins and their deglycosylated polypeptide chains. Three monospecific antibody fractions that recognize the carbohydrate, the deglycosylated or unglycosylated regions of the polypeptide chains in these glycoproteins, were isolated by immunoaffinity chromatography. The human and swine trachea mucin glycoproteins showed extensive immunological homology in both their carbohydrate and polypeptide chains. The carbohydrate chains and unglycosylated region of the polypeptide chain in Cowper's gland mucin glycoprotein showed little or no cross-reaction with comparable regions in respiratory mucin glycoproteins. However, the polypeptide chains in the deglycosylated regions of all three mucin glycoproteins showed extensive homology. Five bands with molecular masses ranging from 40 to 46 kDa that differed by a constant molecular mass of approximately 1.5 kDa were detected in hydrolysates of all of the polypeptide chains after treatment with S. aureus V8 protease. Monospecific antibodies to the deglycosylated region of these chains reacted with the peptides, whereas those directed against the unglycosylated region did not. The results suggest that these chains contain tandem repeating sequences of amino acids.

  15. 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.

  16. Platelet membrane glycoproteins and their function: an overview.

    PubMed

    Kunicki, T J

    1989-07-01

    The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.

  17. Glycoproteins as substrates for production of hydrogen and methane by colonic bacterial flora.

    PubMed

    Perman, J A; Modler, S

    1982-08-01

    Hydrogen and methane in human breath derive entirely from bacterial fermentation in the intestinal lumen. The sources of substrates utilized for these reactions have not been completely determined. Basal excretion of both gases occurs in the fasted state, while malabsorbed carbohydrate commonly results in increased hydrogen but not methane production. Using an in vitro fecal incubation system, we have studied hydrogen and methane production from glycoproteins of endogenous as well as dietary origin. All glycoproteins tested yielded hydrogen when incubated with fecal homogenates. Mean hydrogen production from substrates containing less than 3% sugar (human serum albumin, bovine serum albumin, and alpha-casein) averaged 2.2 +/- 0.9% of hydrogen production from equivalent amounts of glucose, while carbohydrate-rich mucin yielded 46.0 +/- 6.7% of hydrogen production from glucose. Glycoproteins of intermediate carbohydrate content, including transferrin and egg white, yielded intermediate values. Methane production from glycoproteins was optimal from carbohydrate-poor protein substrates in fecal homogenates which accumulated hydrogen and became rapidly acidic when incubated with pure carbohydrate. In contrast, methane production was comparable for essentially sugar-free proteins, glycoproteins, and glucose when hydrogen did not accumulate and neutral pH was maintained. We conclude that glycoproteins are substrates for hydrogen and methane production by colonic bacteria from healthy adults. In individuals with bacterial overgrowth syndromes and in protein-losing enteropathy, bacterial catabolism of endogenous glycoproteins may cause increased basal hydrogen and methane excretion. These findings also raise the possibility that measurement of hydrogen or methane after oral administration of dietary glycoproteins may be useful in detection of protein malabsorption.

  18. Activation of factor XII by tobacco glycoprotein.

    PubMed

    Becker, C G; Dubin, T

    1977-08-01

    A glycoprotein of mol wt ca. 18,000 daltons isolated from cured tobacco leaves (TGP-L) and from cigarette smoke condensate (TGP-CSC) activated factor XII in normal human plasma in vitro as measured by (a) shortening of the partial thromboplastin time, (b) shortening of the lysis time of euglobulin clots, and (c) generation of kinin activity. These effects were not demonstrable in plasma deficient in factor XII. The capacity of TGP-L and TGP-CSC to activate factor XII was shown to depend on the presence of rutin, a substance chemically similar to quercetin and ellagic acid, which are known activators of factor XII. Rutin and rutin coupled to bovine serum albumin, but not bovine serum albumin alone, were also demonstrated to activate factor XII. The presence in cigarette smoke of material that is both allergenic and capable of activating factor XII of the intrinsic pathway of coagulatin may be important to the pathogenesis of cardiovascular and pulmonary disease associated with cigarette smoking.

  19. Activation of factor XII by tobacco glycoprotein

    PubMed Central

    1977-01-01

    A glycoprotein of mol wt ca. 18,000 daltons isolated from cured tobacco leaves (TGP-L) and from cigarette smoke condensate (TGP-CSC) activated factor XII in normal human plasma in vitro as measured by (a) shortening of the partial thromboplastin time, (b) shortening of the lysis time of euglobulin clots, and (c) generation of kinin activity. These effects were not demonstrable in plasma deficient in factor XII. The capacity of TGP-L and TGP-CSC to activate factor XII was shown to depend on the presence of rutin, a substance chemically similar to quercetin and ellagic acid, which are known activators of factor XII. Rutin and rutin coupled to bovine serum albumin, but not bovine serum albumin alone, were also demonstrated to activate factor XII. The presence in cigarette smoke of material that is both allergenic and capable of activating factor XII of the intrinsic pathway of coagulatin may be important to the pathogenesis of cardiovascular and pulmonary disease associated with cigarette smoking. PMID:874423

  20. Cell surface expression of v-fms-coded glycoproteins is required for transformation.

    PubMed Central

    Roussel, M F; Rettenmier, C W; Look, A T; Sherr, C J

    1984-01-01

    The viral oncogene v-fms encodes a transforming glycoprotein with in vitro tyrosine-specific protein kinase activity. Although most v-fms-coded molecules remain internally sequestered in transformed cells, a minor population of molecules is transported to the cell surface. An engineered deletion mutant lacking 348 base pairs of the 3.0-kilobase-pair v-fms gene encoded a polypeptide that was 15 kilodaltons smaller than the wild-type v-fms gene product. The in-frame deletion of 116 amino acids was adjacent to the transmembrane anchor peptide located near the middle of the predicted protein sequence and 432 amino acids from the carboxyl terminus. The mutant polypeptide acquired N-linked oligosaccharide chains, was proteolytically processed in a manner similar to the wild-type glycoprotein, and exhibited an associated tyrosine-specific protein kinase activity in vitro. However, the N-linked oligosaccharides of the mutant glycoprotein were not processed to complex carbohydrate chains, and the glycoprotein was not detected at the cell surface. Cells expressing high levels of the mutant glycoprotein did not undergo morphological transformation and did not form colonies in semisolid medium. The transforming activity of the v-fms gene product therefore appears to be mediated through target molecules on the plasma membrane. Images PMID:6390182

  1. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    PubMed

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  2. Calnexin, calreticulin and the folding of glycoproteins.

    PubMed

    1997-05-01

    Calnexin and calreticulin are molecular chaperones in the endoplasmic reticulum (ERJ. They are lectins that interact with newly synthesized glycoproteins that have undergone partial trimming of their core N-linked oligosaccharides. Together with the enzymes responsible for glucose removal and a glucosyltransferase that re-glucosylates already-trimmed glycoproteins, they provide a novel mechanism for promoting folding, oligomeric assembly and quality control in the ER.

  3. Effect of reduced renal mass on renal ammonia transporter family, Rh C glycoprotein and Rh B glycoprotein, expression.

    PubMed

    Kim, Hye-Young; Baylis, Chris; Verlander, Jill W; Han, Ki-Hwan; Reungjui, Sirirat; Handlogten, Mary E; Weiner, I David

    2007-10-01

    Kidneys can maintain acid-base homeostasis, despite reduced renal mass, through adaptive changes in net acid excretion, of which ammonia excretion is the predominant component. The present study examines whether these adaptations are associated with changes in the ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg). We used normal Sprague-Dawley rats and a 5/6 ablation-infarction model of reduced renal mass; control rats underwent sham operation. After 1 wk, glomerular filtration rate, assessed as creatinine clearance, was decreased, serum bicarbonate was slightly increased, and Na(+) and K(+) were unchanged. Total urinary ammonia excretion was unchanged, but urinary ammonia adjusted for creatinine clearance, an index of per nephron ammonia metabolism, increased significantly. Although reduced renal mass did not alter total Rhcg protein expression, both light microscopy and immunohistochemistry with quantitative morphometric analysis demonstrated hypertrophy of both intercalated cells and principal cells in the cortical and outer medullary collecting duct that was associated with increased apical and basolateral Rhcg polarization. Rhbg expression, analyzed using immunoblot analysis, immunohistochemistry, and measurement of cell-specific expression, was unchanged. We conclude that altered subcellular localization of Rhcg contributes to adaptive changes in single-nephron ammonia metabolism and maintenance of acid-base homeostasis in response to reduced renal mass.

  4. Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase.

    PubMed Central

    Greene, T W; Woodbury, R L; Okita, T W

    1996-01-01

    As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production (T.W. Greene, S.E. Chantler, M.L. Khan, G.F. Barry, J. Preiss, T.W. Okita [1996] Proc Natl Acad Sci USA 93: 1509-1513). One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to l3 vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity was comparable to those of cells that expressed the wild-type recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711). The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP. PMID:8938421

  5. Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin.

    PubMed

    Higuchi, T; Xin, P; Buckley, M S; Erickson, D R; Bhavanandan, V P

    2000-07-01

    The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS-PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of approximately 50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

  6. Fluorescence studies on the nucleotide binding domains of the P-glycoprotein multidrug transporter.

    PubMed

    Liu, R; Sharom, F J

    1997-03-11

    One of the major causes of multidrug resistance in human cancers is expression of the P-glycoprotein multidrug transporter, which acts as an efflux pump for a diverse range of natural products, chemotherapeutic drugs, and hydrophobic peptides. In the present study, fluorescence techniques were used to probe the nucleotide binding domains (NBD) of P-glycoprotein. The transporter was labeled at two conserved cysteine residues, one within each NBD, using the thiol-reactive fluor 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS), and collisional quenching was used to assess solvent accessibility of the bound probe. Acrylamide was a poor quencher, which suggests that MIANS is buried in a relatively inaccessible region of the protein. Iodide ion was a highly effective quencher, whereas Cs+ was not, demonstrating the presence of a positive charge in the region close to the ATP binding site. The fluorescent nucleotide derivative 2'(3')-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) was hydrolysed slowly by P-glycoprotein, with a V(max) approximately 20-fold lower than that for unmodified ATP, and a K(M) of 81 microM. TNP-ATP and TNP-ADP inhibited P-glycoprotein ATPase activity, indicating that they interact with the NBD, whereas TNP-AMP was a very poor inhibitor. When TNP-nucleotides bound to P-glycoprotein, their fluorescence intensity was enhanced in a concentration-dependent manner. Both TNP-ATP and TNP-ADP bound to P-glycoprotein with substantially higher affinity than ATP, with K(d) values of 43 and 36 microM, respectively. Addition of ATP led to only partial displacement of TNP-ATP. Resonance energy transfer was observed between cysteine-bound MIANS and TNP-ATP/ADP, which indicated that the two fluorescent groups are located close to each other within the catalytic site of P-glycoprotein.

  7. 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.

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

    PubMed

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

    2015-11-23

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

  9. Myelin-associated glycoprotein (MAG): past, present and beyond.

    PubMed

    Quarles, Richard H

    2007-03-01

    The myelin-associated glycoprotein (MAG) is a type I transmembrane glycoprotein localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths where it functions in glia-axon interactions. It contains five immunoglobulin (Ig)-like domains and is in the sialic acid-binding subgroup of the Ig superfamily. It appears to function both as a ligand for an axonal receptor that is needed for the maintenance of myelinated axons and as a receptor for an axonal signal that promotes the differentiation, maintenance and survival of oligodendrocytes. Its function in the maintenance of myelinated axons may be related to its role as one of the white matter inhibitors of neurite outgrowth acting through a receptor complex involving the Nogo receptor and/or gangliosides containing 2,3-linked sialic acid. MAG is expressed as two developmentally regulated isoforms with different cytoplasmic domains that may activate different signal transduction pathways in myelin-forming cells. MAG contains a carbohydrate epitope shared with other glycoconjugates that is a target antigen in autoimmune peripheral neuropathy associated with IgM gammopathy and has been implicated in a dying back oligodendrogliopathy in multiple sclerosis.

  10. Refined structures of mouse P-glycoprotein

    PubMed Central

    Li, Jingzhi; Jaimes, Kimberly F; Aller, Stephen G

    2014-01-01

    The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpretations in the latter model. To address this concern, we generated an experimental electron density map from single-wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Å resolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug-free structure was refined to 3.8 Å resolution with a 9.4 and 8.1% decrease in Rwork and Rfree, respectively, (Rwork = 21.2%, Rfree = 26.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ∼95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was corrected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addition of three N-terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted >6 Å from their original positions. The drug translocation pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition. PMID:24155053

  11. 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.

  12. 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

    2014-06-30

    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.

  13. Radical scavenging glycoprotein inhibiting cyclooxygenase-2 and thromboxane A2 synthase from aloe vera gel.

    PubMed

    Yagi, A; Kabash, A; Mizuno, K; Moustafa, S M; Khalifa, T I; Tsuji, H

    2003-03-01

    An active glycoprotein fraction containing 58 % protein was isolated from Aloe vera gel by precipitation with 55 % ammonium sulfate followed by gel permeation using DEAE-Sephacel A-25, Sepharose 6B and Sephadex G-50 columns in a yield of 3 x 10 -3 %. The glycoprotein fraction showed a single band corresponding to a subunit of verectin at the same position when stained with both Coomassie brilliant blue and periodic acid-Schiff reagents on 18 % SDS-PAGE. The molecular weight (14 kDa) was confirmed by Sephadex G-50 column chromatography. The glycoprotein fraction showed a radical scavenging activity against superoxide anion generated by the xanthine-xanthine oxidase system as well as inhibition of cyclooxygenase-2 and reduction of thromboxane A 2 synthase level in vitro.

  14. HPLC-MS/MS analyses show that the near-Starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s) alternative to the pPGI-pPGM-AGP pathway.

    PubMed

    Bahaji, Abdellatif; Baroja-Fernández, Edurne; Sánchez-López, Angela María; Muñoz, Francisco José; Li, Jun; Almagro, Goizeder; Montero, Manuel; Pujol, Pablo; Galarza, Regina; Kaneko, Kentaro; Oikawa, Kazusato; Wada, Kaede; Mitsui, Toshiaki; Pozueta-Romero, Javier

    2014-01-01

    In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

  15. 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

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

    PubMed

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

    2014-09-22

    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 five conserved herpesvirus glycoproteins, a single unique glycoprotein, and two 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.

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

    PubMed

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

    2014-10-03

    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.

  18. A high boronate avidity monolithic capillary for the selective enrichment of trace glycoproteins.

    PubMed

    Li, Daojin; Li, Yang; Li, Xinglin; Bie, Zijun; Pan, Xianghua; Zhang, Qian; Liu, Zhen

    2015-03-06

    Boronate affinity materials, as effective sample enrichment sorbents for glycoproteomic analysis, have attracted increasing attention in recent years. However, most of boronate affinity materials suffer from an apparent limitation, limited binding strength. As a result, extraction of glycoproteins of trace concentration is rather difficult or impossible. In this study, we present a high boronate avidity monolithic capillary. Branched polyethyleneimine (PEI) was used as a scaffold to amplify the number of boronic acid moieties. While 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA), which exhibited ultrahigh affinity toward cis-diol-containing compounds, was employed as an affinity ligand. Due to the PEI-assisted synergistic multivalent binding, the monolithic column exhibited high boronate avidity toward glycoproteins, with binding constants of 10(-6)-10(-7)M. Such binding strength was the highest among already reported boronic acid-functionalized materials that can be used for glycoproteomic analysis. Besides, the boronate avidity monolithic column exhibited one additional beneficial feature, lowered binding pH (≥6.5). These features greatly favored the selective enrichment of trace glycoproteins from real samples. The feasibility for practical applications was demonstrated with the selective enrichment of trace glycoproteins in human saliva. As compared with other boronate avidity/affinity materials, the boronate avidity monolithic capillary exhibited the best performance.

  19. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

    PubMed

    Keck, Zhen-yong; Angus, Allan G N; Wang, Wenyan; Lau, Patrick; Wang, Yong; Gatherer, Derek; Patel, Arvind H; Foung, Steven K H

    2014-08-01

    A challenge for hepatitis C virus (HCV) vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs) against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc) were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape variants, which

  20. Glycoproteins histochemistry of the gills of Odontesthes bonariensis (Teleostei, Atherinopsidae).

    PubMed

    Díaz, A O; García, A M; Escalante, A H; Goldemberg, A L

    2010-11-01

    The histochemistry of glycoproteins (GP) in the mucous cells of the gills of the silverside Odontesthes bonariensis was identified with: (1) oxidizable vicinal diols; (2) sialic acid and some of their chain variants, carbon 7 ((7) C), carbon 8 ((8) C) or carbon 9 ((9) C); (3) sialic acid residues without O-acyl substitution and with O-acyl substitution at (7) C, (8) C or (9) C; (4) carboxyl groups and (5) sulphate groups. A battery of seven biotinylated lectins allowed GPs sugar residues to be distinguished. Mucous cells showed the presence of neutral, sulphated and sialylated GPs. Dolichos biflorus agglutinin (DBA) and Glycine max agglutinin (SBA) showed strong positive staining; Arachis hypogaea agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I) and Triticum vulgaris agglutinin (WGA) showed moderate staining, while Ulex europaeus agglutinin-I (UEA-I) was completely negative.

  1. Fast and efficient online release of N-glycans from glycoproteins facilitating liquid chromatography-tandem mass spectrometry glycomic profiling.

    PubMed

    Jmeian, Yazen; Hammad, Loubna A; Mechref, Yehia

    2012-10-16

    A novel online enzyme reactor incorporating peptide-N-glycosidase F (PNGase F) on a monolithic polymer support has been developed to allow the rapid simultaneous release of both neutral and acidic N-linked glycans from glycoproteins. The PNGase F monolithic reactor was fabricated in a fused silica using glycidyl methacrylate-co-ethylene dimethacrylate polymer. The reactor was coupled to a C8 trap and a porous graphitic carbon (PGC) HPLC-chip. This arrangement was interfaced to an ion trap mass spectrometer for liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The performance of the PNGase F reactor was optimized using the MS signal for the disialylated biantennary N-glycan derived from fetuin. Optimum conditions for glycan release were attained at room temperature using a loading flow rate of 2 μL/min and a reaction time of 6 min. The loading capacity of the reactor was determined to be around 2 pmol of glycoprotein. The online digestion and MS characterization experiments resulted in sensitivities as high as 100 fmol of glycoprotein and 0.1 μL of human blood serum. The enzyme reactor activity was also shown to remain stable after 1 month of continuous use. Both small and large glycoproteins as well as glycoproteins containing high-mannose glycans, fucolsylated glycans, sialylated glycans, and hybrid structures were studied. The model glycoproteins included ribonuclease B, fetuin, α(1)-acid glycoprotein, immunoglobulin, and thyroglobulin. All N-glycans associated with these model glycoproteins were detected using the online PNGase F reactor setup.

  2. The Design, Synthesis, and Biological Evaluation of New Paclitaxel Analogs With the Ability to Evade Efflux by P-Glycoprotein

    DTIC Science & Technology

    2005-05-01

    Seelig observed that compounds which carry a negative charge at physiological pH, such as those that contain a carboxylic acid , sulphonate , or nitro...observed during the conversion of acid fluorides to amides.5,6 These properties make shelf-stable acid fluorides possible and allow for their isolation...glycoprotein (Pgp). In accord with Seelig’s model and our previous reports, all carboxylic acid analogs had no apparent interactions with Pgp

  3. 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.

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

    PubMed

    Tian, Yuan; Zhang, Hui

    2013-02-01

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

  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. Selective extraction and enrichment of glycoproteins based on boronate affinity SPME and determination by CIEF-WCID.

    PubMed

    Li, Lixian; Xia, Zhining; Pawliszyn, Janusz

    2015-07-30

    In this study, a new thin-film boronic acid coating was developed for solid-phase microextraction (SPME) followed by capillary isoelectric focusing with whole-column imaging detection (CIEF-WCID). Boronate functionalized particles of phenylboronic acid (PBA) and 3-aminophenylboronic acid (3-aPBA) were utilized as boronate affinity solid phase coating on thin-film stainless steel blades for selective extraction and enrichment of glycoproteins. The process of extraction and elution could be easily controlled by adjusting pH. To test specificity, asialofetuin and lactoferrin were selected as glycoproteins test molecules, while BSA and myoglobin were used as control non-glycoproteins in this study. The boronate affinity coating was characterized. The effect of buffer, pH, extraction profiles and elution profiles were investigated. The developed method was successfully applied to extract glycoproteins from standard buffer, PBS, human plasma and 10-fold diluted human blood using two kinds of boronate affinity blades. Boronate affinity SPME could be a promising tool for selective extraction and enrichment of low-abundance glycoproteins in real biological samples.

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

    PubMed Central

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

    1985-01-01

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

  8. Antiviral and anti-proliferative glycoproteins from the rhizome of Smilax glabra Roxb (Liliaceae).

    PubMed

    Ooi, Linda S M; Wong, Elaine Y L; Chiu, Lawrence C M; Sun, Samuel S M; Ooi, Vincent E C

    2008-01-01

    The glycoproteins possessing antiviral and anti-proliferative activities were isolated from the Chinese medicinal herb Smilax glabra (known as tufuling), by extraction with 0.2 M NaCl, ammonium sulfate precipitation, fetuin-agarose affinity chromatography and gel filtration. The molecular mass of the fetuin-binding glycoprotein (designated SGPF2) was estimated to be about 58 kDa, with a major protein subunit of 26 kDa. The non-fetuin binding glycoproteins (in the unadsorbed fraction) were further separated into 5 different subfractions (SGPF1a-SGPF1e) with anion-exchange chromatography, all of which also contained the major band at 26 kDa. All the isolated proteins of 26 kDa had similar N-terminal amino acid sequences, implying that they were probably the isoforms originated putatively from a multigene family with different binding affinity and ionic strength. The glycoprotein SGPF2 exhibited antiviral activity against respiratory syncytial virus (RSV) with a median inhibitory concentration (IC(50)) of 62.5 microg/ml and Herpes simplex virus type 1 (HSV-1) had an IC(50) of 31.3 microg/ml. The glycoprotein potencies for antiviral activity appeared to depend on the molecules' binding affinity for fetuin, that is, the fetuin-binding protein was more potent than the non-fetuin binding proteins. Further examination revealed that these glycoproteins also had the ability to suppress the proliferation of MCF-7 cells. The possible mechanism of anti-proliferative action as analyzed by DNA flow cytometry indicated that they could induce apoptosis mediated via sub-G(1) phase of the MCF-7 cell cycle. For example, there was an increase by 75.8% of the control level of apoptosis after incubation with SGPF1a.

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

    PubMed

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

    2015-09-01

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

  10. A Tyrosine Motif in the Cytoplasmic Domain of Mason-Pfizer Monkey Virus Is Essential for the Incorporation of Glycoprotein into Virions

    PubMed Central

    Song, Chisu; Dubay, Susan R.; Hunter, Eric

    2003-01-01

    Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site. PMID:12692221

  11. Characterization of an equine herpesvirus type 1 gene encoding a glycoprotein (gp13) with homology to herpes simplex virus glycoprotein C.

    PubMed

    Allen, G P; Coogle, L D

    1988-08-01

    The molecular structure of the equine herpesvirus type 1 (EHV-1) gene encoding glycoprotein 13 (gp13) was analyzed. The gene is contained within a 1.8-kilobase AccI-EcoRI restriction fragment mapping at map coordinates 0.136 to 0.148 in the UL region of the EHV-1 genome and is transcribed from right to left. Determination of the nucleotide sequence of the DNA fragment revealed a complete transcriptional unit composed of typical regulatory promoter elements upstream to a long open reading frame (1,404 base pairs) that encoded a 468-amino-acid primary translation product of 51 kilodaltons. The predicted protein has the characteristic features of a membrane-spanning protein: an N-terminal signal sequence, a hydrophobic membrane anchor region, a charged C-terminal cytoplasmic tail, and an exterior domain with nine potential N-glycosylation sites. The EHV-1 DNA sequences expressed in lambda gt11 as gp13 epitopes were present in the open reading frame. Amino acid sequences composing a major antigenic site, recognized by 35% of a panel of 42 anti-gp13 monoclonal antibodies, were identified in the N-terminal surface domain of the deduced gp13 molecule. Comparison of the EHV-1 gp13 DNA sequence with that encoding glycoproteins of other alphaherpesviruses revealed no detectable homology. However, a search for homology at the amino acid level showed regions of significant sequence similarity between the amino acids of the carboxy half of EHV-1 gp13 and those of the same region of gC-like glycoproteins of herpes simplex virus (gC-1 and gC-2), pseudorabies herpesvirus (gIII), and varicella-zoster virus (gp66). The sequences of the N-terminal portion of gp13, by contrast, were much less conserved. The results of these studies indicate that EHV-1 gp13 is the structural homolog of herpes simplex virus glycoprotein C and further suggest that the epitope-containing N-terminal amino acid sequences of the herpesvirus gC-like glycoproteins have undergone more extensive evolutionary

  12. Glycoprotein patterns in normal and malignant cervical tissue.

    PubMed

    O'Brien, M E; Souberbielle, B E; Cowan, M E; Allen, C A; Luesley, D M; Mould, J J; Blackledge, G R; Skinner, G R

    1991-07-04

    Glycoproteins from normal and malignant human cervix were studied using an organ culture system and compared by gel electrophoresis and autoradiography. Five glycoproteins of 178 kDa, 95 kDa, 93 kDa, 82 kDa and 38 kDa and 1 glycolipid (46 kDa) were detected more frequently in squamous carcinomas. Certain glycoproteins were shown to be oncofoetal and some had affinity for Concanavalin A (Con A). The 82 kDa glycoprotein was present in 16/17 squamous carcinomas but in only 1/13 normal cervices. This band represented a glycoprotein containing glucosamine, mannose, small quantities of methionine and no fucose. These preliminary results suggest that these glycoproteins and in particular the 82-kDa glycoprotein are worthy of further investigation and characterisation.

  13. Cyclic phosphatidic acid inhibits alkyl-glycerophosphate-induced downregulation of histone deacetylase 2 expression and suppresses the inflammatory response in human coronary artery endothelial cells.

    PubMed

    Tsukahara, Tamotsu; Haniu, Hisao; Matsuda, Yoshikazu

    2014-01-01

    Activation of the endothelium by alkyl-glycerophosphate (AGP) has been implicated in the development of atherosclerosis. Our previous study suggested that cyclic phosphatidic acid (cPA) inhibits arterial wall remodeling in a rat model in vivo. However, the mechanisms through which specific target genes are regulated during this process remain unclear. Here, we examined whether cPA inhibited AGP-induced expression of class I histone deacetylases (HDACs, namely HDAC1, HDAC2, HDAC3, and HDAC8), which may affect subsequent transcriptional activity of target genes. Our experimental results showed that human coronary artery endothelial cells (HCAECs) expressed high levels of HDAC2 and low levels HDAC1, HDAC3, and HDAC8. Moreover, AGP treatment induced downregulation of HDAC2 expression in HCAECs. However, cotreatment with cPA inhibited this downregulation of HDAC2 expression. Interestingly, treatment with AGP increased the expression and secretion of endogenous interleukin (IL)-6 and IL-8; however, this effect was inhibited when HCAECs were cotreated with cPA or the synthetic peroxisome proliferator-activator receptor gamma (PPARγ) antagonist T0070907. Thus, our data suggested that cPA may have beneficial effects in inflammation-related cardiovascular disease by controlling HDAC2 regulation.

  14. On-plate glycoproteins/glycopeptides selective enrichment and purification based on surface pattern for direct MALDI MS analysis.

    PubMed

    Zeng, Zhoufang; Wang, Yandong; Guo, Xinhua; Wang, Ling; Lu, Nan

    2013-05-21

    In this paper, a novel method has been proposed to achieve selective enrichment and purification of glycoproteins/glycopeptides on a surface patterned sample support, which consists of a hydrophobic outer layer (F-SAM) and an internal boronic acid-modified gold microspot (900 μm). Upon deposition, the sample solution is firstly concentrated in a small area by repulsion of the hydrophobic outer layer, and then the glycoproteins/glycopeptides are selectively captured through boronic acid covalently binding in the inner layer. However, the non-glycosylated proteins/peptides or high concentration salts are removed after rinsing with alkaline solution. As a result, the detection sensitivity is improved by an order of magnitude greater than when using a stainless steel MALDI plate. With surface patterned sample support, the glycoproteins/glycopeptides can be detected even under interference from the excessive existing non-glycosylated proteins/peptides (10 times more than glycoproteins/glycopeptides). Simultaneously, high-quality mass spectra can be obtained even in the presence of urea (1 M), NaCl (1 M), or NH4HCO3 (200 mM). Therefore, this novel technique may be applied to high-throughput analysis of low-abundance glycoproteins/glycopeptides in complicated proteome research.

  15. Drastic differences in glycosylation of related S-layer glycoproteins from moderate and extreme halophiles.

    PubMed

    Mengele, R; Sumper, M

    1992-04-25

    The outer surface of the moderate halophilic archaebacterium Haloferax volcanii (formerly named Halobacterium volcanii) is covered with a hexagonally packed surface (S) layer glycoprotein. The polypeptide (794 amino acid residues) contains 7 N-glycosylation sites. Four of these sites were isolated as glycopeptides and the structure of one of the corresponding saccharides was determined. Oligosaccharides consisting of beta-1,4-linked glucose residues are attached to the protein via the linkage unit asparaginyl-glucose. In the related glycoprotein from the extreme halophile Halobacterium halobium, the glucose residues are replaced by sulfated glucuronic acid residues, causing a drastic increase in surface charge density. This is discussed in terms of a recent model explaining the stability of halophilic proteins.

  16. 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

  17. Serum glycoprotein-derived N- and O-linked glycans as cancer biomarkers

    PubMed Central

    Lan, Ying; Hao, Cui; Zeng, Xuan; He, Yanli; Zeng, Pengjiao; Guo, Zhihua; Zhang, Lijuan

    2016-01-01

    Early detection of cancer is the key to improving survival. Since most clinically used serum cancer biomarkers are either glycoproteins or glycan structures that can be recognized by specific monoclonal antibodies, developing glycan structure-based biomarkers from human serum/plasma glycoproteins through mass spectrometry (MS) analysis are active research field during the past decades. Numerous studies have shown that changes in serum/plasma glycan structures occur during cancer initiation, progression, and treatment. This review describes N- and O-linked glycan structures identified from serum/plasma glycoprotein (s) by MS analysis with focus on alterations associated with different types of human cancers. The global changes in serum N- and O-linked glycan structures, especially the glycans that are not made by cancer cells such as B lymphocyte-derived IgG and liver-synthesized haptoglobin and α1 acid glycoprotein, suggest that glycans might be the long sought diagnostic biomarkers associated with system malfunction in the blood circulation of cancer patients. Therefore, N- and O-linked glycan structures have great potential to serve as cancer diagnosis, prognosis, and treatment monitoring biomarkers to facilitate personalized medicine. PMID:27904760

  18. Post-Synthetic Defucosylation of AGP by Aspergillus nidulans α-1,2-Fucosidase Expressed in Arabidopsis Apoplast Induces Compensatory Upregulation of α-1,2-Fucosyltransferases

    PubMed Central

    Pogorelko, Gennady V.; Reem, Nathan T.; Young, Zachary T.; Chambers, Lauran; Zabotina, Olga A.

    2016-01-01

    Cell walls are essential components of plant cells which perform a variety of important functions for the different cell types, tissues and organs of a plant. Besides mechanical function providing cell shape, cell walls participate in intercellular communication, defense during plant-microbe interactions, and plant growth. The plant cell wall consists predominantly of polysaccharides with the addition of structural glycoproteins, phenolic esters, minerals, lignin, and associated enzymes. Alterations in the cell wall composition created through either changes in biosynthesis of specific constituents or their post-synthetic modifications in the apoplast compromise cell wall integrity and frequently induce plant compensatory responses as a result of these alterations. Here we report that post-synthetic removal of fucose residues specifically from arabinogalactan proteins in the Arabidopsis plant cell wall induces differential expression of fucosyltransferases and leads to the root and hypocotyl elongation changes. These results demonstrate that the post-synthetic modification of cell wall components presents a valuable approach to investigate the potential signaling pathways induced during plant responses to such modifications that usually occur during plant development and stress responses. PMID:27448235

  19. Expression of small regions of equine herpesvirus 1 glycoprotein C in Escherichia coli.

    PubMed

    Crabb, B S; Studdert, M J

    1995-09-01

    A series of truncated equine herpesvirus 1 (EHV1) glycoprotein C (gC) molecules was examined for use as serodiagnostic antigens for EHV1 and EHV4. Small regions of EHV1 glycoprotein C, an immunodominant EHV1 glycoprotein, were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins using the bacterial expression vector pGEX-2T. Sera obtained from horses, including sera from specific-pathogen-free (SPF) foals, following exposure to either EHV1, EHV4 or both viruses were used. Several of the fusion proteins were shown to encompass EHV1 specific epitopes while others encompassed strong, cross-reactive epitopes. One clone, termed pEC-3, produced a soluble and stable fusion protein which encompassed amino acids 107-275 of EHV1 gC. Strong cross-reactive epitopes on pEC-3 were localised to a region encompassed by amino acids 137 to approximately 152 while EHV1 specific epitope(s) were identified downstream of this region, i.e., approximately amino acids 152 to 275. E. coli expressed EHV1 gC polypeptides showed clear potential for use as diagnostic reagents for the detection of cross-reactive and type-specific EHV1 and EHV4 antibodies present in convalescent equine sera.

  20. An update on post-translational modifications of hydroxyproline-rich glycoproteins: toward a model highlighting their contribution to plant cell wall architecture

    PubMed Central

    Hijazi, May; Velasquez, Silvia M.; Jamet, Elisabeth; Estevez, José M.; Albenne, Cécile

    2014-01-01

    Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs) is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp)-rich O-glycoproteins (HRGPs) were classified into three categories: (i) moderately glycosylated extensins (EXTs) able to form covalent scaffolds; (ii) hyperglycosylated arabinogalactan proteins (AGPs); and (iii) Hyp/proline (Pro)-Rich proteins (H/PRPs) that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs), biosynthesis, structure, and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture. PMID:25177325

  1. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

    USGS Publications Warehouse

    Bjorklund, H.V.; Higman, K.H.; Kurath, G.

    1996-01-01

    The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2-33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

  2. The glycoprotein genes and gene junctions of the fish rhabdoviruses spring viremia of carp virus and hirame rhabdovirus: Analysis of relationships with other rhabdoviruses

    USGS Publications Warehouse

    Bjorklund, H.V.; Higman, K.H.; Kurath, G.

    1996-01-01

    The nucleotide sequences of the glycoprotein genes and all of the internal gene junctions of the fish pathogenic rhabdoviruses spring viremia of carp virus (SVCV) and hirame rhabdovirus (HIRRV) have been determined from cDNA clones generated from viral genomic RNA. The SVCV glycoprotein gene sequence is 1588 nucleotides (nt) long and encodes a 509 amino acid (aa) protein. The HIRRV glycoprotein gene sequence comprises 1612 nt, coding for a 508 aa protein. In sequence comparisons of 15 rhabdovirus glycoproteins, the SVCV glycoprotein gene showed the highest amino acid sequence identity (31.2–33.2%) with vesicular stomatitis New Jersey virus (VSNJV), Chandipura virus (CHPV) and vesicular stomatitis Indiana virus (VSIV). The HIRRV glycoprotein gene showed a very high amino acid sequence identity (74.3%) with the glycoprotein gene of another fish pathogenic rhabdovirus, infectious hematopoietic necrosis virus (IHNV), but no significant similarity with glycoproteins of VSIV or rabies virus (RABV). In phylogenetic analyses SVCV was grouped consistently with VSIV, VSNJV and CHPV in the Vesiculovirus genus of Rhabdoviridae. The fish rhabdoviruses HIRRV, IHNV and viral hemorrhagic septicemia virus (VHSV) showed close relationships with each other, but only very distant relationships with mammalian rhabdoviruses. The gene junctions are highly conserved between SVCV and VSIV, well conserved between IHNV and HIRRV, but not conserved between HIRRV/IHNV and RABV. Based on the combined results we suggest that the fish lyssa-type rhabdoviruses HIRRV, IHNV and VHSV may be grouped in their own genus within the family Rhabdoviridae. Aquarhabdovirus has been proposed for the name of this new genus.

  3. 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

  4. Developmental regulation of neuraminidase-sensitive lectin-binding glycoproteins during myogenesis of rat L6 myoblasts.

    PubMed Central

    Holland, P C; Pena, S D; Guerin, C W

    1984-01-01

    Intact monolayers of L6 myoblasts were treated with neuraminidase, with the aim of selectively removing sialic acid residues of cell-surface glycoproteins. Neuraminidase treatment unmasked binding sites for Ricinus communis agglutinin I and peanut agglutinin, thus allowing the identification of the major binding proteins for these lectins. For Ricinus communis agglutinin I these neuraminidase-sensitive glycoproteins had apparent Mr values of 136000, 115000, 87000, 83000 and 49000. For peanut agglutinin the major neuraminidase-sensitive glycoproteins had apparent Mr values of 200000, 136000, 87000 and 83000. We found highly reproducible, developmentally regulated, changes in the lectin-binding capacity of certain of these glycoproteins as L6 myoblasts differentiated into myotubes. Coincident with myoblast fusion there was a co-ordinate decrease in Ricinus communis agglutinin I binding by glycoproteins of apparent Mr of 136000 and 49000. There was also a co-ordinate shift in mobility of the broad band of glycoprotein, centred at an apparent Mr of 115000 in myoblasts, to a new average apparent Mr of 107000 in mid-fusion cultures and myotube cultures. Peanut agglutinin binding by the major protein of apparent Mr 136000 also decreased at the mid-fusion stage of myogenesis, and was barely detectable in 7-day-old fused cultures. These developmentally regulated changes in neuraminidase-sensitive glycoproteins were all inhibited by growth of myoblasts in 6.4 microM-5-bromo-2'-deoxyuridine, indicating that they are associated with myoblast differentiation. In contrast, an increase in fibronectin was seen in mid-fusion cultures, which was not inhibited by growth of myoblasts in 5-bromo-2'-deoxyuridine. This initial increase in fibronectin is, therefore, unlikely to be directly related to myoblast fusion or differentiation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6712625

  5. Modularity of the oncoprotein-like properties of platelet glycoprotein Ibalpha.

    PubMed

    Li, Youjun; Lu, Jie; Prochownik, Edward V

    2009-01-16

    Glycoprotein Ib alpha (GpIbalpha), a trans-membrane glycoprotein, is expressed on the surface of megakaryocytes and platelets, where, in association with glycoprotein Ib beta, glycoprotein V, and glycoprotein IX, it normally forms the von Willebrand factor receptor (vWFR). A fully functional vWFR is necessary for platelet attachment, aggregation, and activation and has also been shown to regulate megakaryocyte ploidy. We have recently shown that the gene encoding GpIbalpha is a transcriptional target for the c-Myc oncoprotein and is more widely expressed than previously thought, with particularly high levels occurring in transformed cells. Indeed, GpIbalpha can substitute for c-Myc in promoting growth, transformation, and genomic instability. In the current work, we have demonstrated that, despite the promiscuous expression of GpIbalpha, other vWFR subunits remain largely restricted to megakaryocytes. We have characterized a panel of GpIbalpha mutants and shown that some regions of the protein essential for vWFR activity are not necessary for c-Myc-like functions. Specifically, the six C-terminal amino acids of the cytoplasmic domain, which mediate vWFR signaling, are entirely dispensible for the c-Myc-like functions of GpIbalpha. Instead, these require a more membrane-proximal filamin-binding domain. Also important is the GpIbalpha signal peptide, which, in the absence of other vWFR subunits, directs GpIbalpha to the endoplasmic reticulum rather than the membrane. Together, these results provide strong evidence that the domains of GpIbalpha mediating c-Myc-like functions are modular, genetically distinct, and independent of those involved in vWFR signaling.

  6. Cell-wall polysaccharides and glycoproteins of parenchymatous tissues of runner bean (Phaseolus coccineus).

    PubMed

    Ryden, P; Selvendran, R R

    1990-07-15

    1. Polymers were solubilized from the cell walls of parenchyma from mature runner-bean pods with minimum degradation by successive extractions with cyclohexane-trans-1,2-diamine-NNN'N'-tetra-acetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue, which contained cross-linked pectic polysaccharides and Hyp-rich glycoproteins. These were solubilized with chlorite/acetic acid and cellulase. The polymers were fractionated by anion-exchange chromatography, and fractions were subjected to methylation analysis. 2. The pectic polysaccharides differed in their ease of extraction, and a small proportion were highly cross-linked. The bulk of the pectic polysaccharides solubilized by CDTA and Na2CO3 were less branched than those solubilized by KOH. There was good evidence that most of the pectic polysaccharides were not degraded during extraction. 3. The protein-containing fractions included Hyp-rich and Hyp-poor glycoproteins associated with easily extractable pectic polysaccharides, Hyp-rich glycoproteins solubilized with 4M-KOH+borate, the bulk of which were not associated with pectic polysaccharides, and highly cross-linked Hyp-rich glycoproteins. 4. Isodityrosine was not detected, suggesting that it does not have a (major) cross-linking role in these walls. Instead, it is suggested that phenolics, presumably linked to C-5 of 3,5-linked Araf residues of Hyp-rich glycoproteins, serve to cross-link some of the polymers. 5. There were two main types of xyloglucan, with different degrees of branching. The bulk of the less branched xyloglucans were solubilized by more-concentrated alkali. The anomeric configurations of the sugars in one of the highly branched xyloglucans were determined by 13C-n.m.r. spectroscopy. 6. The structural features of the cell-wall polymers and complexes are discussed in relation to the structure of the cell walls of parenchyma tissues.

  7. HNK-1 Carrier Glycoproteins Are Decreased in the Alzheimer's Disease Brain.

    PubMed

    García-Ayllón, María-Salud; Botella-López, Arancha; Cuchillo-Ibañez, Inmaculada; Rábano, Alberto; Andreasen, Niels; Blennow, Kaj; Ávila, Jesús; Sáez-Valero, Javier

    2017-01-01

    The human natural killer-1 (HNK-1), 3-sulfonated glucuronic acid, is a glycoepitope marker of cell adhesion that participates in cell-cell and cell-extracellular matrix interactions and in neurite growth. Very little is known about the regulation of the HNK-1 glycan in neurodegenerative disease, particularly in Alzheimer's disease (AD). In this study, we investigate changes in the levels of HNK-1 carrier glycoproteins in AD. We demonstrate an overall decrease in HNK-1 immunoreactivity in glycoproteins extracted from the frontal cortex of AD subjects, compared with levels from non-demented controls (NDC). Immunoblotting of ventricular post-mortem and lumbar ante-mortem cerebrospinal fluid with HNK-1 antibodies indicate similar levels of carrier glycoproteins in AD and NDC samples. Decrease in HNK-1 carrier glycoproteins were not paralleled by changes in messenger RNA (mRNA) levels of the enzymes involved in the synthesis of the glycoepitope, β-1,4-galactosyltransferase (β4GalT), glucuronyltransferases GlcAT-P and GlcAT-S, or sulfotransferase HNK-1ST. Over-expression of amyloid precursor protein in Tg2576 transgenic mice and in vitro treatment of SH-SY5Y neuroblastoma cells with the amyloidogenic Aβ42 peptide resulted in a decrease in HNK-1 immunoreactivity levels in brain and cellular extracts, whereas the levels of soluble HNK-1 glycoproteins detected in culture media were not affected by Aβ treatment. HNK-1 levels remain unaffected in the brain extracts of Tg-VLW mice, a model of mutant hyperphosphorylated tau, and in SH-SY5Y cells over-expressing hyperphosphorylated wild-type tau. These results provide evidence that cellular levels of HNK-1 carrier glycoforms are decreased in the brain of AD subjects, probably influenced by the β-amyloid protein.

  8. Identification of structural and secretory lectin-binding glycoproteins of normal and cancerous human prostate.

    PubMed

    Lad, P M; Cooper, J F; Learn, D B; Olson, C V

    1984-12-07

    We have utilized the technique of lectin-loading of SDS gels with iodinated concanavalin A and wheat germ agglutinin to identify glycoproteins in prostatic and seminal fluids as well as in prostate tissue fractions. The following subunits which bound both lectins were detected: (a) 50, 43 and 38 kDa subunits common to prostatic and seminal fluids, and an additional 55 kDa subunit which predominates only in prostatic fluid; (b) 78, 55, 50 and 43 kDa subunits in prostatic tissue cytosol and (c) 195, 170, 135, 116 and 95 kDa subunits present in the particulate fractions of prostatic tissue. Immunoblotting using specific rabbit antibodies revealed the 50 kDa band to be prostatic acid phosphatase and the 38 kDa band to be prostate-specific antigen. Interestingly, antibodies directed toward prostatic acid phosphatase were found to cross-react with the 43 kDa band. Fractionation on sucrose gradients showed that several of these particulate glycoproteins were associated with a vesicle fraction enriched in adenylate cyclase activity, implying that they are plasma membrane glycoproteins. Comparison of soluble and particulate fractions of normal and cancerous tissue homogenates was made by densitometric scanning of autoradiograms of lectin-loaded gels. Similar relative intensities of lectin-binding were obtained for corresponding proteins in normal and cancerous tissue fractions. Also, immunoblotting showed no differences in prostatic acid phosphatase or prostate-specific antigen between normal and cancerous soluble homogenate fractions. Our results suggest that major lectin-binding proteins are conserved in the transition from normal to cancerous tissue. These results may be useful in developing a multiple-marker profile of metastatic prostate cancer and for the design of imaging agents, such as monoclonal antibodies, to prominent soluble and particulate prostate glycoproteins.

  9. Markers of Ovarian Cancer Using a Glycoprotein/Antibody Array

    DTIC Science & Technology

    2014-05-01

    204. 4. Zhao J, Patwa TH, Qiu W, Shedden K, Hinderer R, Misek DE, Anderson MA, Simeone DM, and Lubman DM. Glycoprotein microarrays with multi -lectin...quantitative strategy, clusterin (CLUS), leucine-rich alpha-2-glycoprotein (LRG1), hemopexin (HEMO), vitamin D-binding protein (VDB), and complement...Part of complement system P02750 LRG1 Leucine-rich alpha-2- glycoprotein 0.019 It is expressed during granulocyte differentiation P02774 VDB Vitamin

  10. Maturation of murine bone marrow dendritic cells induced by acidic Ginseng polysaccharides.

    PubMed

    Wang, Zuozhou; Meng, Jingjuan; Xia, Yanjie; Meng, Yiming; Du, Lin; Zhang, Zhenjie; Wang, Enhua; Shan, Fengping

    2013-02-01

    In this study, we report that a acidic polysaccharide (AGP) isolated from a Chinese medicinal herb, named Ginseng (Panax giseng C.A. Meyer), induces maturation of bone marrow dendritic cells (BMDCs) via concrete changes both inside and outside BMDCs. The impacts of AGP on BMDCs were assessed with use of conventional scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) for morphology, flow cytometry (FCM) for key surface molecules, cytochemistry assay, FITC-dextran, bio-assay for phagocytosis and enzyme linked immunosorbent assay (ELISA) for production of cytokines. Our results elucidated that PPS promoted maturation of BMDCs via changes as reflected by the down-regulation of acid phosphatase (ACP) activity inside the BMDCs, which occurs when phagocytosis of BMDCs to antigen decreased, while antigen presentation increased upon maturation, higher expression of key surface molecules of MHC II, CD80, CD86, CD83, and CD40, and releasing higher level of cytokines IL-12 and low level of TNF-α. Our study suggest that AGP play marked immunostimulating role on the maturation of murine BMDCs through precise regulation of phagocytosis and enzyme activities inside the BMDCs.

  11. A monoclonal antibody to a synthetic fragment of rabies virus glycoprotein binds ligands of the nicotinic cholinergic receptor.

    PubMed

    Rustici, M; Santucci, A; Lozzi, L; Petreni, S; Spreafico, A; Neri, P; Bracci, L; Soldani, P

    1989-09-01

    Rabies virus glycoprotein and snake venom curaremimetic neurotoxins share a region of high homology (30-45 for neurotoxins and 190-203 for the glycoprotein) in the regions that are believed to be responsible for binding the nicotinic acetylcholine receptor. Monoclonal antibodies raised to the 190-203 synthetic fragment of rabies virus glycoprotein were immobilized on a high performance affinity chromatography column and were able to bind neurotoxins. Toxins were displaced from the affinity column by elution at acidic pH and by affinity competition with acetylcholine at neutral pH. Furthermore, the affinity column proved to be useful for the purification of cholinergic ligands. Overall, these results indicate that the paratope of our monoclonal antibodies could behave as an 'internal image' of the nicotinic cholinergic receptor acetylcholine binding site.

  12. Preparation and evaluation of a phenylboronate affinity monolith for selective capture of glycoproteins by capillary liquid chromatography.

    PubMed

    Lin, Zi An; Pang, Ji Lei; Lin, Yao; Huang, Hui; Cai, Zong Wei; Zhang, Lan; Chen, Guo Nan

    2011-08-21

    A phenylboronate affinity monolith was prepared and applied to the selective capture of glycoproteins from unfractionated protein mixtures. The monolith was synthesized in a 100 μm i.d capillary by an in situ polymerization procedure using a pre-polymerization mixture consisting of 4-vinylphenylboronic acid (VPBA) as functional monomer, ethylene dimethacrylate (EDMA) as crosslinker, diethylene glycol and ethylene glycol as binary porogenic solvents, and azobisisobutyronitrile (AIBN) as initiator. The prepared monolith was characterized in terms of the morphology, pore property, and recognition property. The selectivity and dynamic binding capacity were evaluated by using standard glycoproteins and nonglycoproteins as model proteins. The chromatographic results demonstrated that the phenylboronate affinity monolith had higher selectivity and binding capacity for glycoprotein than nonglycoprotein. The resulting phenylboronate affinity monolith was used as the sorbent for in-tube solid phase microextraction (in-tube SPME), and the extraction performance of the monolith was assessed by capture of ovalbumin from egg white sample.

  13. Studies on the protein composition of human serum very low density lipoproteins: demonstration of the beta 2-glycoprotein-I.

    PubMed

    Polz, E; Kostner, G M; Holasek, A

    1979-08-01

    Human serum VLDL isolated by polyanion precipitation and ultracentrifugation have been delipidated with ethanal/diethyl ether. By electrophoresis in 10% polyacrylamide gels containing 8M urea, we found a protein which comigrated with apolipoprotein E. This protein was purified by column chromatography and turned out to be identical with beta 2-glycoprotein-I, the serum factor which is necessary for the precipitation of triglyceride-rich lipoproteins with sodium decyl sulfate or sodium dodecyl sulfate. Upon analytical isoelectric focusing, beta 2-glycoprotein-I gave four major bands in the pH region 5.7--6.6. All four bands gave an immunochemical reaction of identity with a monospecific antiserum. From its unique amino acid composition we conclude that beta 2-glycoprotein-I is distinct from all apolipoproteins described previously in the literature.

  14. Pachytene spermatocyte protein(s) stimulate Sertoli cells grown in bicameral chambers: dose-dependent secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin.

    PubMed

    Onoda, M; Djakiew, D

    1991-03-01

    Interactions between pachytene spermatocytes and Sertoli cells were investigated using the bicameral culture chamber system. Pachytene spermatocytes were isolated from adult rats with a purity in excess of 90% by centrifugal elutriation. The pachytene spermatocytes were cultured in a defined media and pachytene spermatocyte protein prepared from the conditioned media by dialysis and lyophilization. This pachytene spermatocyte protein was reconstituted at various concentrations and incubated with confluent epithelial sheets of immature Sertoli cells cultured in bicameral chambers. Pachytene spermatocyte protein stimulated secretion of total [35S]methionine-labeled protein from Sertoli cells in a dose-dependent manner predominantly in an apical direction. This stimulatory effect of pachytene spermatocyte protein was domain specific from the apical surface of Sertoli cells, and seemed specific for secretion because total intracellular protein did not increase under the influence of pachytene spermatocyte protein. Pachytene spermatocyte protein and follicle-stimulating hormone additively stimulated Sertoli cell secretion. The physicochemical characteristics of the stimulatory pachytene spermatocyte protein are indicative of heat stability, whereas the stimulatory pachytene spermatocyte protein exhibit acid, dithiothreitol and trypsin sensitivity, and partial urea sensitivity. Furthermore, Sertoli cell secretion of ceruloplasmin, sulfated glycoprotein-1, sulfated glycoprotein-2, and transferrin in response to various concentrations of pachytene spermatocyte protein were determined by immunoprecipitate of these [35S]methionine-labeled proteins with polyclonal antibodies. Maximal stimulation of ceruloplasmin and sulfated glycoprotein-1 secretion from Sertoli cells was observed at a dose of 50 micrograms/ml pachytene spermatocyte protein, whereas maximal stimulation of sulfated glycoprotein-2 and transferrin secretion from Sertoli cells was observed at a dose of 100

  15. 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.

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

    PubMed

    Böcker, Sophia; Laaf, Dominic; Elling, Lothar

    2015-07-24

    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.

  17. Laurus nobilis L. Seed Extract Reveals Collateral Sensitivity in Multidrug-Resistant P-Glycoprotein-Expressing Tumor Cells.

    PubMed

    Saab, Antoine M; Guerrini, Alessandra; Zeino, Maen; Wiench, Benjamin; Rossi, Damiano; Gambari, Roberto; Sacchetti, Gianni; Greten, Henry Johannes; Efferth, Thomas

    2015-01-01

    The frequent failure of standard cancer chemotherapy requires the development of novel drugs capable of killing otherwise drug-resistant tumors. Here, we have investigated a chloroform extract of Laurus nobilis seeds. Fatty acids and 23 constituents of the volatile fraction were identified by gas chromotography/flame ionization detection (GC/FID) and gas chromatography/mass spectrometry (GC/MS), in good agreement with (1)H NMR (nuclear magnetic resonance) spectrum. Multidrug-resistant P-glycoprotein-expressing CEM/ADR5000 leukemia cells were hypersensitive (collaterally sensitive) toward this extract compared to drug-sensitive CCRF-CEM cells, whereas CEM/ADR5000 cells were 2586-fold resistant to doxorubicin as control drug. Collateral sensitivity was verified by measurement of apoptotic cells by flow cytometry. The log10IC50 values of 3 compounds in the extract (limonene, eucalyptol, oleic acid) did not correlate with mRNA expression of the P-glycoprotein-coding ABCB1/MDR1 gene and accumulation of the P-glycoprotein substrate rhodamine in the NCI panel of tumor cell lines. A microarray-based profile of 20 genes predicted resistance to doxorubicin and 7 other anticancer drugs involved in the multidrug resistance phenotype but not to limonene, eucalyptol and oleic acid. In conclusion, our results show that Laurus nobilis seed extract is suitable to kill multidrug-resistant P-glycoprotein expressing tumor cells.

  18. Beta-hexosaminidase activity of the oral pathogen Tannerella forsythia influences biofilm formation on glycoprotein substrates.

    PubMed

    Roy, Sumita; Phansopa, Chatchawal; Stafford, Prachi; Honma, Kiyonobu; Douglas, C W Ian; Sharma, Ashu; Stafford, Graham P

    2012-06-01

    Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes β-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as β-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that β-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that β-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.

  19. Automated sample preparation facilitated by PhyNexus MEA purification system for oligosaccharide mapping of glycoproteins.

    PubMed

    Prater, Bradley D; Anumula, Kalyan R; Hutchins, Jeff T

    2007-10-15

    A reproducible high-throughput sample cleanup method for fluorescent oligosaccharide mapping of glycoproteins is described. Oligosaccharides are released from glycoproteins using PNGase F and labeled with 2-aminobenzoic acid (anthranilic acid, AA). A PhyNexus MEA system was adapted for automated isolation of the fluorescently labeled oligosaccharides from the reaction mixture prior to mapping by HPLC. The oligosaccharide purification uses a normal-phase polyamide resin (DPA-6S) in custom-made pipette tips. The resin volume, wash, and elution steps involved were optimized to obtain high recovery of oligosaccharides with the least amount of contaminating free fluorescent dye in the shortest amount of time. The automated protocol for sample cleanup eliminated all manual manipulations with a recycle time of 23 min. We have reduced the amount of excess AA by 150-fold, allowing quantitative oligosaccharide mapping from as little as 500 ng digested recombinant immunoglobulin G (rIgG). This low sample requirement allows early selection of a cell line with desired characteristics (e.g., oligosaccharide profile and high specific productivity) for the production of glycoprotein drugs. In addition, the use of Tecan or another robotic platform in conjunction with this method should allow the cleanup of 96 samples in 23 min, a significant decrease in the amount of time currently required to process such a large number of samples.

  20. Transient translocation of the cytoplasmic (endo) domain of a type I membrane glycoprotein into cellular membranes

    PubMed Central

    1993-01-01

    The E2 glycoprotein of the alphavirus Sindbis is a typical type I membrane protein with a single membrane spanning domain and a cytoplasmic tail (endo domain) containing 33 amino acids. The carboxyl terminal domain of the tail has been implicated as (a) attachment site for nucleocapsid protein, and (b) signal sequence for integration of the other alpha-virus membrane proteins 6K and E1. These two functions require that the carboxyl terminus be exposed in the cell cytoplasm (a) and exposed in the lumen of the endoplasmic reticulum (b). We have investigated the orientation of this glycoprotein domain with respect to cell membranes by substituting a tyrosine for the normally occurring serine, four amino acids upstream of the carboxyl terminus. Using radioiodination of this tyrosine as an indication of the exposure of the glycoprotein tail, we have provided evidence that this domain is initially translocated into a membrane and is returned to the cytoplasm after export from the ER. This is the first demonstration of such a transient translocation of a single domain of an integral membrane protein and this rearrangement explains some important aspects of alphavirus assembly. PMID:8432728

  1. Cell surface glycoproteins from Thermoplasma acidophilum are modified with an N-linked glycan containing 6-C-sulfofucose.

    PubMed

    Vinogradov, Evgeny; Deschatelets, Lise; Lamoureux, Marc; Patel, Girishchandra B; Tremblay, Tammy-Lynn; Robotham, Anna; Goneau, Marie-France; Cummings-Lorbetskie, Cathy; Watson, David C; Brisson, Jean-Robert; Kelly, John F; Gilbert, Michel

    2012-09-01

    Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at pH 2 and 59°C. This extremophile is remarkable by the absence of a cell wall or an S-layer. Treating the cells with Triton X-100 at pH 3 allowed the extraction of all of the cell surface glycoproteins while keeping cells intact. The extracted glycoproteins were partially purified by cation-exchange chromatography, and we identified five glycoproteins by N-terminal sequencing and mass spectrometry of in-gel tryptic digests. These glycoproteins are positive for periodic acid-Schiff staining, have a high content of Asn including a large number in the Asn-X-Ser/Thr sequon and have apparent masses that are 34-48% larger than the masses deduced from their amino acid sequences. The pooled glycoproteins were digested with proteinase K and the purified glycopeptides were analyzed by NMR. Structural determination showed that the carbohydrate part was represented by two structures in nearly equal amounts, differing by the presence of one terminal mannose residue. The larger glycan chain consists of eight residues: six hexoses, one heptose and one sugar with an unusual residue mass of 226 Da which was identified as 6-deoxy-6-C-sulfo-D-galactose (6-C-sulfo-D-fucose). Mass spectrometry analyses of the peptides obtained by trypsin and chymotrypsin digestion confirmed the principal structures to be those determined by NMR and identified 14 glycopeptides derived from the main glycoprotein, Ta0280, all containing the Asn-X-Ser/Thr sequons. Thermoplasma acidophilum appears to have a "general" protein N-glycosylation system that targets a number of cell surface proteins.

  2. Double Recognition and Selective Extraction of Glycoprotein Based on the Molecular Imprinted Graphene Oxide and Boronate Affinity.

    PubMed

    Luo, Jing; Huang, Jing; Cong, Jiaojiao; Wei, Wei; Liu, Xiaoya

    2017-03-01

    Specific recognition and separation of glycoproteins from complex biological solutions is very important in clinical diagnostics considering the close relationship between glycoproteins with the occurrence of diverse diseases, but the lack of materials with high selectivity and superior capture capacity still makes it a challenge. In this work, graphene oxide (GO) based molecularly imprinted polymers (MIPs) possessing double recognition abilities have been synthesized and applied as highly efficient adsorbents for glycoprotein recognition and separation. Boronic acid functionalized graphene oxide (GO-APBA) was first prepared and a template glycoprotein (ovalbumin, OVA) was then immobilized onto the surface of GO-APBA through boronate affinity. An imprinting layer was subsequently deposited onto GO-APBA surface by a sol-gel polymerization of organic silanes in aqueous solution. After the removal of the template glycoprotein, 3D cavities with double recognition abilities toward OVA were obtained in the as-prepared imprinted materials (GO-APBA/MIPs) because of the combination of boronate affinity and molecularly imprinted spatial matched cavities. The obtained GO-APBA/MIPs exhibited superior specific recognition toward OVA with imprinted factor (α) as high as 9.5, significantly higher than the corresponding value (4.0) of GO/MIPs without the introduction of boronic acid groups. Meanwhile, because of the synergetic effect of large surface area of graphene and surface imprinting, high binding capacity and fast adsorption/elution rate of GO-APBA/MIPs toward OVA were demonstrated and the saturation binding capacity of GO-APBA/MIPs could reach 278 mg/g within 40 min. The outstanding recognizing behavior (high adsorption capacity, highly specific recognition, and rapid binding rate) coupled to the facile and environmentally friendly preparation procedure makes GO-APBA/MIPs promising in the recognition, separation, and analysis of glycoproteins in clinics in the future.

  3. [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.

  4. Characterization of Murine Gammaherpesvirus 68 Glycoprotein B

    PubMed Central

    Lopes, Filipa B.; Colaco, Susanna; May, Janet S.; Stevenson, Philip G.

    2004-01-01

    Murine gammaherpesvirus 68 (MHV-68) glycoprotein B (gB) was identified in purified virions by immunoblotting, immunoprecipitation, and immunoelectron microscopy. It was synthesized as a 120-kDa precursor in infected cells and cleaved into 65-kDa and 55-kDa disulfide-linked subunits close to the time of virion release. The N-linked glycans on the cleaved, virion gB remained partially endoglycosidase H sensitive. The processing of MHV-68 gB therefore appears similar to that of Kaposi's sarcoma-associated herpesvirus gB and human cytomegalovirus gB. PMID:15542690

  5. 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)

  6. Effect of collecting duct-specific deletion of both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on renal response to metabolic acidosis.

    PubMed

    Lee, Hyun-Wook; Verlander, Jill W; Handlogten, Mary E; Han, Ki-Hwan; Weiner, I David

    2014-02-15

    The Rhesus (Rh) glycoproteins, Rh B and Rh C Glycoprotein (Rhbg and Rhcg, respectively), are ammonia-specific transporters expressed in renal distal nephron and collecting duct sites that are necessary for normal rates of ammonia excretion. The purpose of the current studies was to determine the effect of their combined deletion from the renal collecting duct (CD-Rhbg/Rhcg-KO) on basal and acidosis-stimulated acid-base homeostasis. Under basal conditions, urine pH and ammonia excretion and serum HCO3(-) were similar in control (C) and CD-Rhbg/Rhcg-KO mice. After acid-loading for 7 days, CD-Rhbg/Rhcg-KO mice developed significantly more severe metabolic acidosis than did C mice. Acid loading increased ammonia excretion, but ammonia excretion increased more slowly in CD-Rhbg/Rhcg-KO and it was significantly less than in C mice on days 1-5. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice on days 1, 3, and 5 of acid loading. Metabolic acidosis increased phosphenolpyruvate carboxykinase (PEPCK) and Na(+)/H(+) exchanger NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that 1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis; 2) the significantly greater changes in PEPCK, NHE-3, and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared with acid-loaded C mice cause the role of Rhbg and Rhcg to be underestimated quantitatively; and 3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3, and GS expression despite the presence of persistent metabolic acidosis.

  7. Early and Delayed Effects of Naturally Occurring Asbestos on Serum Biomarkers of Inflammation and Metabolism

    EPA Science Inventory

    Studies recently showed that intratracheal (IT) instillation of Libby amphibole (LA) increases circulating acute-phase proteins (APP; a-2 macroglobulin, A2M; and a-1 acid glycoprotein, AGP) and inflammatory biomarkers (osteopontin and lipocalin) in rats. In this study, objectives...

  8. Tamm-Horsfall glycoprotein and calcium nephrolithiasis.

    PubMed

    Hess, B

    1994-01-01

    Available data on the effects of Tamm-Horsfall glycoprotein (THP) on calcium oxalate crystallization processes are apparently conflicting. With the main emphasis on calcium oxalate crystal aggregation, this review demonstrates that THP has a dual role as a modifier of crystal aggregation: in solutions with high pH, low ionic strength (IS) and low concentrations of calcium and THP itself, the glycoprotein acts as a powerful inhibitor of calcium oxalate crystal aggregation. Conversely, low pH, high IS and high concentrations of calcium and THP all favor self-aggregation of THP molecules which lowers their inhibitory activity against calcium oxalate crystal aggregation. Some patients with severely recurrent Ca stone disease excrete abnormal THPs which self-aggregate at levels of pH, IS and concentrations of Ca and THP at which normal THPs remain in monomeric form. With high Ca concentrations, such abnormal THPs become strong promoters of crystal aggregation, since conformational changes in crystal-bound THP molecules induce strong viscous binding forces which overcome repulsive electrostatic surface charges. By chelating free Ca ions, citrate reduces self-aggregation of THP molecules and turns promoting THPs into inhibitors of calcium oxalate crystal aggregation.

  9. 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.

  10. 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.

  11. Human immune responses to major human cytomegalovirus glycoprotein complexes.

    PubMed Central

    Liu, Y N; Kari, B; Gehrz, R C

    1988-01-01

    Sera from both human cytomegalovirus (HCMV)-seropositive adults and infants with congenital HCMV infection recognized two major HCMV glycoprotein complexes. However, proliferative responses of peripheral blood mononuclear cells to these complexes varied among seropositive adults and were not detected in any of the infants. Thus, these glycoproteins alone may not be sufficient to develop a subviral HCMV vaccine. Images PMID:2828655

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

    PubMed

    Okamoto, Ryo; Izumi, Masayuki; Kajihara, Yasuhiro

    2014-10-01

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

  13. Cloning and sequencing of the gene encoding the cell surface glycoprotein of Haloarcula japonica strain TR-1.

    PubMed

    Wakai, H; Nakamura, S; Kawasaki, H; Takada, K; Mizutani, S; Aono, R; Horikoshi, K

    1997-02-01

    The triangular disk-shaped halophilic archaeon Haloarcula japonica strain TR-1 has a glycoprotein on its cell surface. The complete gene encoding the cell surface glycoprotein (CSG) was cloned and sequenced. The gene has an open reading frame of 2586 bp, and a potential archaeal promoter sequence approximately 150 bp upstream of the ATG initiation codon. The mature CSG is composed of 828 amino acids and is preceded by a signal sequence of 34 amino acid residues. A hydropathy analysis showed a hydrophobic stretch at the C-terminus, that probably serves as a transmembrane domain. The amino acid sequence of the Ha. japonica CSG showed 52.1% and 43.2% identities to those from the Halobacterium halobium and Haloferax volcanii CSGs, respectively. Five potential N-glycosylation sites were found in the mature Ha. japonica CSG, sites that were distinctly different from those in Hb. halobium and Hf. volcanii. The Ha. japonica CSG gene was expressed in Escherichia coli.

  14. Blue eye disease porcine rubulavirus (PoRv) infects pig neurons and glial cells using sialo-glycoprotein as receptor.

    PubMed

    Mendoza-Magaña, Maria L; Godoy-Martinez, Diana V; Guerrero-Cazares, Hugo; Rodriguez-Peredo, Alejandra; Dueñas-Jimenez, Judith M; Dueñas-Jiménez, Sergio H; Ramírez-Herrera, Mario A

    2007-03-01

    Pig neural cells express glycoproteins with sialylated N-linked oligosaccharide chains (SNOC) which are used by the porcine rubulavirus (PoRv) as receptors. Pig neuronal or glial cell cultures were employed to investigate (a) whether PoRv infects such cells using a molecule expressing SNOC, and (b) the role of viral envelope glycoproteins in establishing the infection. Enriched neuronal or glial cell cultures were exposed to PoRv and infection was detected immunocytochemically. Neuronal cultures prepared from neonatal pigs were treated enzymatically to eliminate sialic acid or N-linked oligosaccharide chains. Primary neural cultures were exposed to anti-HN or anti-F preincubated with PoRv to study the role of the viral glycoproteins. In enriched cultures, PoRv infected neurons and glial cells, and sialic acid expressed in N-linked oligosaccharide chains appeared to play a central role in infection. It was concluded that HN and F viral glycoproteins are required to infect neurons and glial cells.

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

    PubMed

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

    2010-07-01

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

  16. 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.

  17. Concanavalin A is synthesized as a glycoprotein precursor.

    PubMed

    Herman, E M; Shannon, L M; Chrispeels, M J

    1985-07-01

    Concanavalin A (Con A) is a tetrameric lectin which is synthesized in the cotyledons of developing jack-bean (Canavalia ensiformis (L.) D.C.) seeds and accumulates in the protein bodies of storage-parenchyma cells. The polypeptides of Con A have a molecular weight of 27000 and a relative molecular mass (Mr) of 30000 when analyzed by gel electrophoresis on denaturing polyacrylamide gels. In-vitro translation of RNA isolated from immature jack-bean cotyledons shows that Con A is synthesized as a polypeptide with Mr 34000. In-vivo pulse labeling of cotyledons with radioactive amino acids or glucosamine also resulted in the formation of a 34000-Mr polypeptide. In-vivo labeling with radioactive amino acids in the presence of tunicamycin yielded an additional polypeptide of 32000 Mr. Together these results indicate that Con A is cotranslationally processed by the removal of a signal sequence and the addition of an oligosaccharide side chain of corresponding size. Analysis of the structure of the oligogosaccharide side chain was accomplished through glycosidase digestion of glycopeptides isolated from [(3)H]glucosamine-labeled Con A. Incubation of the labeled glycopeptides with endoglycosidase H, α-mannosidase or β-N-acetylglucosaminidase, followed by gel filtration, allowed us to deduce that the oligosaccharide side chain of pro-Con A is a high-mannose oligosaccharide. Pulse-chase experiments with labeled amino acids are consistent with the interpretation that the glycosylated precursor of Con A is processed to mature Con A (Mr=30000). The 4000 decrease in Mr is interpreted to result from the removal of a small glycopeptide. The implications of the conversion of a glycoprotein pro-Con A to mature Con A are discussed in the context of the unique circular permutation of the primary structure of Con A.

  18. 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.

  19. Inhibitory effect of glycoprotein isolated from Opuntia ficus-indica var. saboten MAKINO on activities of allergy-mediators in compound 48/80-stimulated mast cells.

    PubMed

    Lim, Kye-Taek

    2010-01-01

    The present study was performed to investigate the anti-allergy potentials of glycoprotein (90kDa) isolated from Opuntia ficus-indica var. saboten MAKINO (OFI glycoprotein) in vivo (ICR mice) and in vitro (RBL-2H3 cells). At first, to know whether the OFI glycoprotein has an inhibitory ability for allergy in vivo, we evaluated the activities of allergy-related factors such as histamine and beta-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in compound 48/80 (8 ml/kg BW)-treated ICR mice. After that, we studied to found the effect for anti-allergy in vitro such as nuclear factor kappa B (NF-kappaB) and inducible nitric oxide synthase (iNOS), extracellular signal-regulated kinase (ERK) 1/2, arachidonic acid, and cyclooxygenase-2 (COX-2) in compound 48/80 (5 microg/ml)-treated RBL-2H3 cells. Our results showed that the OFI glycoprotein (5 mg/kg) inhibited histamine and beta-hexosaminidase release, lactate dehydrogenase (LDH), and interleukin 4 (IL-4) in mice serum. Also OFI glycoprotein (25 microg/ml) has suppressive effects on the expression of MAPK (ERK1/2), and on protein expression of anti-allergic proteins (iNOS and COX-2). Thus, we speculate that the OFI glycoprotein is an example of natural compound that blocks anti-allergic signal transduction pathways.

  20. Use of influenza C virus glycoprotein HEF for generation of vesicular stomatitis virus pseudotypes.

    PubMed

    Hanika, Andrea; Larisch, Birthe; Steinmann, Eike; Schwegmann-Wessels, Christel; Herrler, Georg; Zimmer, Gert

    2005-05-01

    Influenza C virus contains two envelope glycoproteins: CM2, a putative ion channel protein; and HEF, a unique multifunctional protein that performs receptor-binding, receptor-destroying and fusion activities. Here, it is demonstrated that expression of HEF is sufficient to pseudotype replication-incompetent vesicular stomatitis virus (VSV) that lacks the VSV glycoprotein (G) gene. The pseudotyped virus showed characteristic features of influenza C virus with respect to proteolytic activation, receptor usage and cell tropism. Chimeric glycoproteins composed of HEF ectodomain and VSV-G C-terminal domains were efficiently incorporated into VSV particles and showed receptor-binding and receptor-destroying activities but, unlike authentic HEF, did not mediate efficient infection, probably because of impaired fusion activity. HEF-pseudotyped VSV efficiently infected polarized Madin-Darby canine kidney cells via the apical plasma membrane, whereas entry of VSV-G-complemented virus was restricted to the basolateral membrane. These findings suggest that pseudotyping of viral vectors with HEF might be useful for efficient apical gene transfer into polarized epithelial cells and for targeting cells that express 9-O-acetylated sialic acids.

  1. Potent glycan inhibitors of myelin-associated glycoprotein enhance axon outgrowth in vitro.

    PubMed

    Vyas, Alka A; Blixt, Ola; Paulson, James C; Schnaar, Ronald L

    2005-04-22

    Myelin-associated glycoprotein (MAG, Siglec-4) is one of several endogenous axon regeneration inhibitors that limit recovery from central nervous system injury and disease. Molecules that block such inhibitors may enhance axon regeneration and functional recovery. MAG, a member of the Siglec family of sialic acid-binding lectins, binds to sialoglycoconjugates on axons and particularly to gangliosides GD1a and GT1b, which may mediate some of the inhibitory effects of MAG. In a prior study, we identified potent monovalent sialoside inhibitors of MAG using a novel screening platform. In the current study, the most potent of these were tested for their ability to reverse MAG-mediated inhibition of axon outgrowth from rat cerebellar granule neurons in vitro. Monovalent sialoglycans enhanced axon regeneration in proportion to their MAG binding affinities. The most potent glycoside was disialyl T antigen (NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc-R), followed by 3-sialyl T antigen (NeuAcalpha2-3Galbeta1-3GalNAc-R), structures expressed on O-linked glycoproteins as well as on gangliosides. Prior studies indicated that blocking gangliosides reversed MAG inhibition. In the current study, blocking O-linked glycoprotein sialylation with benzyl-alpha-GalNAc had no effect. The ability to reverse MAG inhibition with monovalent glycosides encourages further exploration of glycans and glycan mimetics as blockers of MAG-mediated axon outgrowth inhibition.

  2. Natural cell-mediated cytotoxicity: possible role of N-linked glycoproteins

    SciTech Connect

    Oeltmann, T.N.; Chambers, W.H.

    1986-05-01

    The authors have examined the role of N-linked glycoproteins in natural cell-mediated cytotoxicity (NCMC) by treating effector cells or target cells with swainsonine, a specific inhibitor of golgi mannosidase II which is critical for N-linked glycoprotein processing. They have also examined the effects of alpha-mannosidase, an exoglycosidase specific for alpha-linked mannose residues, on both target and effector cells. Pretreatment of nonadherent mononuclear cells with swainsonine (18 hr) resulted in an inhibition of lysis of K-562 target cells as measured by LDH release. Protein synthesis was not inhibited as measured by incorporation of /sup 14/C-amino acids. However, oligosaccharide processing was altered as measured by incorporation of 2(/sup 3/H)-mannose. Similar treatment did not inhibit target cell-effector cell conjugation. Pretreatment of nonadherent mononuclear cells with alpha-mannosidase (1 hr) did not result in a reduction in NK cell function. However, alpha-mannosidase did cause a release of mannose from treated cells. These results suggest that N-linked glycoproteins may play a role in NCMC, but not at the level of recognition and binding.

  3. Natural cell-mediated cytotoxicity: possible role of N-linked glycoproteins

    SciTech Connect

    Chambers, W.H.; Oeltmann, T.N.

    1986-03-01

    The authors have examined the role of N-linked glycoproteins in natural cell-mediated cytotoxicity (NCMC) by treating effector cells or target cells with swainsonine, a specific inhibitor of golgi mannosidase II which is critical for N-linked glycoprotein processing. They have also examined the effects of alpha-mannosidase, an exoglycosidase specific for alpha-linked mannose residues, on both target and effector cells. Pretreatment of nonadherent mononuclear cells with swainsonine (18 hr) resulted in an inhibition of lysis of K-562 target cells as measured by LDH release. Protein synthesis was not inhibited as measured by incorporation of /sup 14/C-amino acids. However, oligosaccharide processing was altered as measured by incorporation of 2(/sup 3/H)-mannose. Similar treatment did not inhibit target cell-effector cell conjugation. Pretreatment of nonadherent mononuclear cells with alpha-mannosidase (1 hr) did not result in a reduction in NK cell function. However, alpha-mannosidase did cause a release of mannose from treated cells. These results suggest that N-linked glycoproteins may play a role in NCMC, but not at the level of recognition and binding.

  4. Glycosylation of VSV glycoprotein is similar in cystic fibrosis, heterozygous carrier, and normal human fibroblasts.

    PubMed

    Hunt, L A; Summers, D F

    1977-01-01

    The single envelope glycoprotein of vesicular stomatitis virus was used as a specific probe of glycosyltransferase activities in fibroblasts from two cystic fibrosis patients, an obligate heterozygous carrier and a normal individual. Gel filtration of pronase-digested glycopeptides from both purified virions and infected cell-associated VSV glycoprotein which had been labeled with[3H] glucosamine did not reveal any significant differences in the glycosylation patterns between the different cell cultures. All 4 cell lines were apparently able to synthesize the mannose- and glucosamine- containing core structure and branch chains terminating in sialic acid which are characteristic of asparagine-linked carbohydrate side chains in cellular glycoproteins. Analysis of tryptic glycopeptides by anion-exchange chromotography indicated that the same 2 major sites on the virus polypeptide were recognized and glycosylated in all 4 VSV-infected cell cultures. These studies suggest that the basic biochemical defect(s) in cystic fibrosis is not an absence or deficiency in enzymes responsible for the biosynthesis of complex carbohydrate side chains.

  5. The Use of Chimeric Virus-like Particles Harbouring a Segment of Hantavirus Gc Glycoprotein to Generate a Broadly-Reactive Hantavirus-Specific Monoclonal Antibody

    PubMed Central

    Zvirbliene, Aurelija; Kucinskaite-Kodze, Indre; Razanskiene, Ausra; Petraityte-Burneikiene, Rasa; Klempa, Boris; Ulrich, Rainer G.; Gedvilaite, Alma

    2014-01-01

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80–89) or site #4 (aa 280–289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications. PMID:24513568

  6. The use of chimeric virus-like particles harbouring a segment of hantavirus Gc glycoprotein to generate a broadly-reactive hantavirus-specific monoclonal antibody.

    PubMed

    Zvirbliene, Aurelija; Kucinskaite-Kodze, Indre; Razanskiene, Ausra; Petraityte-Burneikiene, Rasa; Klempa, Boris; Ulrich, Rainer G; Gedvilaite, Alma

    2014-02-07

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have used initially a recombinant yeast-expressed full-length Puumala virus (PUUV) Gc protein. However, this approach was unsuccessful. As an alternative recombinant antigen, chimeric virus-like particles (VLPs) harboring a segment of PUUV Gc glycoprotein were generated in yeast Saccharomyces cerevisiae. A 99 amino acid (aa)-long segment of Gc protein was inserted into the major capsid protein VP1 of hamster polyomavirus at previously defined positions: either site #1 (aa 80-89) or site #4 (aa 280-289). The chimeric proteins were found to self-assemble to VLPs as evidenced by electron microscopy. Chimeric VLPs induced an efficient insert-specific antibody response in immunized mice. Monoclonal antibody (clone #10B8) of IgG isotype specific to hantavirus Gc glycoprotein was generated. It recognized recombinant full-length PUUV Gc glycoprotein both in ELISA and Western blot assay and reacted specifically with hantavirus-infected cells in immunofluorescence assay. Epitope mapping studies revealed the N-terminally located epitope highly conserved among different hantavirus strains. In conclusion, our approach to use chimeric VLPs was proven useful for the generation of virus-reactive MAb against hantavirus Gc glycoprotein. The generated broadly-reactive MAb #10B8 might be useful for various diagnostic applications.

  7. Glycoproteins and glycoproteomics in pancreatic cancer

    PubMed Central

    Pan, Sheng; Brentnall, Teresa A; Chen, Ru

    2016-01-01

    Aberrations in protein glycosylation and polysaccharides play a pivotal role in pancreatic tumorigenesis, influencing cancer progression, metastasis, immuno-response and chemoresistance. Abnormal expression in sugar moieties can impact the function of various glycoproteins, including mucins, surface receptors, adhesive proteins, proteoglycans, as well as their effectors and binding ligands, resulting in an increase in pancreatic cancer invasiveness and a cancer-favored microenvironment. Recent advance in glycoproteomics, glycomics and other chemical biology techniques have been employed to better understand the complex mechanism of glycosylation events and how they orchestrate molecular activities in genomics, proteomics and metabolomics implicated in pancreatic adenocarcinoma. A variety of strategies have been demonstrated targeting protein glycosylation and polysaccharides for diagnostic and therapeutic development. PMID:27895417

  8. Conformational Changes of the Flavivirus E Glycoprotein

    PubMed Central

    Zhang, Ying; Zhang, Wei; Ogata, Steven; Clements, David; Strauss, James H.; Baker, Timothy S.; Kuhn, Richard J.; Rossmann, Michael G.

    2014-01-01

    Summary Dengue virus, a member of the Flaviviridae family, has a surface composed of 180 copies each of the envelope (E) glycoprotein and the membrane (M) protein. The crystal structure of an N-terminal fragment of E has been determined and compared with a previously described structure. The primary difference between these structures is a 10° rotation about a hinge relating the fusion domain DII to domains DI and DIII. These two rigid body components were used for independent fitting of E into the cryo-electron microscopy maps of both immature and mature dengue viruses. The fitted E structures in these two particles showed a difference of 27° between the two components. Comparison of the E structure in its postfusion state with that in the immature and mature virions shows a rotation approximately around the same hinge. Flexibility of E is apparently a functional requirement for assembly and infection of flaviviruses. PMID:15341726

  9. Glycoprotein expression in human milk during lactation.

    PubMed

    Froehlich, John W; Dodds, Eric D; Barboza, Mariana; McJimpsey, Erica L; Seipert, Richard R; Francis, Jimi; An, Hyun Joo; Freeman, Samara; German, J Bruce; Lebrilla, Carlito B

    2010-05-26

    While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.

  10. Immunological aspects of pregnancy-associated glycoproteins.

    PubMed

    Dosogne, H; Massart-Leën, A M; Burvenich, C

    2000-01-01

    The incidence of severe cases of acute E. coli mastitis in dairy cows is highest during early lactation. This phenomenon has been associated with a decreased function and decreased numbers of circulating polymorphonuclear neutrophil leukocytes (PMN). The cause of this impaired function and decreased number is poorly understood. Stress, hormonal and metabolic alterations around parturition and the onset of lactation may play a role in this phenomenon. Several molecules, such as cortisol and beta-hydroxybutyrate have been found to alter the oxidative burst activity of circulating PMN around parturition. Pregnancy-Associated Glycoprotein (bPAG) could also be involved. The theory of immunosuppression by bPAG was investigated because analogous glycoproteins produced by the placenta of other species exert local immunosuppression in order to maintain the histoincompatible feto-maternal unit. The production and subsequent release into the maternal circulation of bPAG is ensured by the binucleate cells from the trophoblast and starts already at implantation. However, peak levels are only reached 1 week before parturition. Due to the long half-life time of this molecule, high levels are found in plasma until 2 weeks after calving. The co-occurrence of the impairment of PMN oxidative burst activity in the early postpartum period and a peak in plasma bPAG concentrations might support the hypothesis of an immunosuppressive effect of PAG. Moreover, an inhibitory effect of bPAG on the proliferation of bovine bone marrow progenitor cells has been found recently in our laboratory. bPAG occurs in colostrum, but its effect on milk cells has not been clarified. It is concluded that interaction between the physiology of reproduction and lactation on the one side and immune function on the other side in dairy cattle requires further research.

  11. 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.

  12. 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.

  13. Ubiquitination of the Prototype Foamy Virus Envelope Glycoprotein Leader Peptide Regulates Subviral Particle Release

    PubMed Central

    Stanke, Nicole; Stange, Annett; Lüftenegger, Daniel; Zentgraf, Hanswalter; Lindemann, Dirk

    2005-01-01

    Foamy virus (FV) particle egress is unique among retroviruses because of its essential requirement for Gag and Env coexpression for budding and particle release. The FV glycoprotein undergoes a highly unusual biosynthesis resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM), derived from a precursor protein by posttranslational proteolysis mediated by furin or furinlike proteases. Previously at least three LP products of different molecular weights were detected in purified FV particles. Here we demonstrate that the higher-molecular-weight forms gp28LP and gp38LP are ubiquitinated variants of the major gp18LP cleavage product, which has a type II membrane topology. Furthermore, we show that all five lysine residues located within the N-terminal 60-amino-acid cytoplasmic domain of gp18LP can potentially be ubiquitinated, however, there seems to be a preference for using the first three. Inactivation of ubiquitination sites individually resulted in no obvious phenotype. However, simultaneous inactivation of the first three or all five ubiquitination sites in gp18LP led to a massive increase in subviral particles released by these mutant glycoproteins that were readily detectable by electron microscopy analysis upon expression of the ubiquitination-deficient glycoprotein by itself or in a proviral context. Surprisingly, only the quintuple ubiquitination mutant showed a two- to threefold increase in single-cycle infectivity assays, whereas all other mutants displayed infectivities similar to that of the wild type. Taken together, these data suggest that the balance between viral and subviral particle release of FVs is regulated by ubiquitination of the glycoprotein LP. PMID:16306578

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

    PubMed

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

    2015-04-15

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

  15. A multigene family encoding surface glycoproteins in Trypanosoma congolense

    PubMed Central

    Thonnus, Magali; Guérin, Amandine; Rivière, Loïc

    2017-01-01

    Trypanosoma congolense, the causative agent of the most important livestock disease in Africa, expresses specific surface proteins involved in its parasitic lifestyle. Unfortunately, the complete repertoire of such molecules is far from being deciphered. As these membrane components are exposed to the host environment, they could be used as therapeutic or diagnostic targets. By mining the T. congolense genome database, we identified a novel family of lectin-like glycoproteins (TcoClecs). These molecules are predicted to have a transmembrane domain, a tandem repeat amino acid motif, a signal peptide and a C-type lectin-like domain (CTLD). This paper depicts several experimental arguments in favor of a surface localization in bloodstream forms of T. congolense. A TcoClec gene was heterologously expressed in U-2 OS cells and the product could be partially found at the plasma membrane. TcoClecs were also localized at the surface of T. congolense bloodstream forms. The signal was suppressed when the cells were treated with a detergent to remove the plasma membrane or with trypsin to « shave » the parasites and remove their external proteins. This suggests that TcoClecs could be potential diagnostic or therapeutic antigens of African animal trypanosomiasis. The potential role of these proteins in T. congolense as well as in other trypanosomatids is discussed. PMID:28357394

  16. [Glycoprotein hexoses in feces of infants with lactose intolerance].

    PubMed

    Filippvskiĭ, G K; Klimov, L Ia

    1995-01-01

    A modified method for estimation of total glycoprotein hexoses in feces, based on their measurements in the blood serum, is presented. Sixty-six nursing children with lactose intolerance, breastfed or formula fed, were examined; formula fed babies were kept on mixtures with high and low lactose content. Glycoprotein hexose parameters were as follows (X +/- m): 13.51 +/- 1.93, 12.05 +/- 2.20, and 3.69 +/- 0.47 g/l feces. In control children without lactose intolerance (n = 33) this value was 3.6 +/- 0.79 g/l. Increased glycoprotein excretion is connected with glycocalix and small intestinal enterocyte alteration.

  17. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  18. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  19. 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....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...

  20. 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....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...

  1. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  3. 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...

  4. Peptide mimotopes of rabies virus glycoprotein with immunogenic activity.

    PubMed

    Houimel, Mehdi; Dellagi, Koussay

    2009-07-23

    A random constrained hexapeptide phage display library (Cys-6aa-Cys) was screened with purified neutralizing human anti-rabies virus IgG antibodies (hRABVIgG) to identify peptides that correspond to or mimic natural epitopes on rabies virus glycoprotein (RABVG) and to investigate their immunogenicities in vivo. After four rounds of biopanning, 20 phage clones randomly selected for their specificity to hRABVIgG, effectively blocked the binding of the inactive rabies virus (RABV) to hRABVIgG. The phage clones were sequenced and the deduced amino acid sequences were derived (C-KRDSTW-C; C-KYLWSK-C; C-KYWLSR-C; C-KYWWSK-C; C-KYAWSR-C; C-KYSMSK-C). Alignments to the amino acid sequence of RABVG showed good match with the antigenic site III (at 330-338 aa), indicating that the hRABVIgG antibodies most likely recognize preferentially this antigenic site. The selected mimotopes were able to inhibit the interactions of the hRABVIgG antibodies with RABV in a dose-dependent manner. Subcutaneous administration of phageKRDSTW expressing the RABVG site III mimotope induced an RABVG-specific IgG response in BALB/c mice. The results indicated that peptide mimotopes when displayed on phages, are accessible to the mice immune system to trigger a humoral response and to induce IgG production. The RABVG site III mimotope (C-KRDSTW-C) would provide a new and promising concept for the development of rabies vaccine.

  5. Metabolism of proteins and glycoproteins in tumour bearing mice treated with Aeromonas L-asparaginase.

    PubMed

    Benny, P J; Muraleedhara, K G; Sreejith, K; Jayashree, G

    1996-12-01

    L-asparaginase, isolated in our laboratory, from Aeromonas had been found to be antileukaemic. In the present study, changes in the levels of proteins and glycoproteins in leukaemic mice and under treatment with Aeromonas L-asparaginase have been compared. Levels of protein bound hexose, fucose and sialic acid which were increased during leukaemia attained normal levels when treated with L-asparaginase. The increased blood urea level declined significantly during enzyme therapy. Effects of L-asparaginase are compared with 'Leunase', a commercially available drug used in the treatment of leukaemia.

  6. Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Emmenegger, Eveline J.; Huang, C.; LaPatra, S.; Winton, James R.

    1995-01-01

    Summary ― Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

  7. The surface glycoproteins of human skin fibroblasts detected after electrophoresis by the binding of peanut (Arachis hypogaea) agglutinin and Ricinus communis (castor-bean) agglutinin I.

    PubMed

    Gordon, B B; Pena, S D

    1982-11-15

    A new methodology was developed to study the cell-surface glycoproteins of cultured human skin fibroblasts. This was based on the binding of a variety of biotinyl-lectins to nitrocellulose electrophoretic transfers of total fibroblast lysates after separation in sodium dodecyl sulphate/polyacrylamide gels, followed by reaction with avidin-biotinyl-peroxidase complexes and detection with 3,3'-diaminobenzidine. The technique proved to be very sensitive and a large number of glycoproteins were detected by binding of concanavalin A and wheat-germ agglutinin. Binding of peanut agglutinin and to a lesser extent of Ricinus communis agglutinin I were found to be dependent on prior removal of sialic acid residues from the glycoproteins. Since by treatment of intact viable cells with neuraminidase only external sialic acid residues were removed, peanut agglutinin and Ricinus communis agglutinin I could thus be utilized for selective detection of cell-surface glycoproteins. Also, because peanut agglutinin was known to bind preferentially to oligosaccharides of the O-glycosidic type, and Ricinus communis agglutinin I to those of the N-glycosidic type, the two lectins were complementary in displaying the surface glycoproteins and in providing information about their oligosaccharide composition.

  8. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice.

    PubMed

    Wang, Xueqian; Cabrera, Robert M; Li, Yue; Miller, David S; Finnell, Richard H

    2013-03-01

    Folate deficiency has been associated with many adverse clinical manifestations. The blood-brain barrier (BBB), formed by brain capillary endothelial cells, protects the brain from exposure to neurotoxicants. The function of BBB is modulated by multiple ABC transporters, particularly P-glycoprotein. A proton-coupled folate transporter (PCFT)-deficient mouse has been previously described as a model for systemic folate deficiency. Herein, we demonstrate that exposing mouse brain capillaries to the antiepileptic drug, valproic acid (VPA; 5 μM), significantly increased P-glycoprotein transport function in the wild-type animals. A ligand to the aryl hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produced a similar induction of P-glycoprotein, which tightened the BBB, thereby increasing the neuroprotection. However, VPA- or TCDD-induced P-glycoprotein transport was blocked in the PCFT-nullizygous mice, indicating that multiple neuroprotective mechanisms are compromised under folate-deficient conditions. Brain capillaries from S-folinic acid (SFA; 40 mg/kg)-treated PCFT-nullizygous mice exhibited increased P-glycoprotein transport following VPA exposure. This suggests that SFA supplementation restored the normal BBB function. In addition, we show that tight-junction proteins are disintegrated in the PCFT mutant mice. Taken together, these findings strongly suggest that folate deficiency disrupts the BBB function by targeting the transporter and tight junctions, which may contribute to the development of neurological disorders.

  9. Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR

    PubMed Central

    Barb, Adam W.; Glushka, John N.; Prestegard, James H.

    2011-01-01

    The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into the cell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virally-produced neuraminidase. Here we present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real time nuclear magnetic resonance (NMR) spectroscopy to monitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A time-resolved two dimensional data processing technique, statistical total correlation spectroscopy (STOCSY), enhances the resolution of the complicated 1D glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through a wide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology and data processing techniques. PMID:22058570

  10. Kinetics of Neuraminidase Action on Glycoproteins by 1D and 2D NMR.

    PubMed

    Barb, Adam W; Glushka, John N; Prestegard, James H

    2011-01-01

    The surfaces of mammalian cells are coated with complex carbohydrates, many terminated with a negatively charged N-acetylneuraminic acid residue. This motif is specifically targeted by pathogens, including influenza viruses and many pathogenic bacteria, to gain entry into the cell. A necessary step in the influenza virus life cycle is the release of viral particles from the cell surface; this is achieved by cleaving N-acetylneuraminic acid from cell surface glycans with a virally-produced neuraminidase. Here we present a laboratory exercise to model this process using a glycoprotein as a glycan carrier and using real time nuclear magnetic resonance (NMR) spectroscopy to monitor N-acetylneuraminic acid release as catalyzed by neuraminidase. A time-resolved two dimensional data processing technique, statistical total correlation spectroscopy (STOCSY), enhances the resolution of the complicated 1D glycoprotein spectrum and isolates characteristic peaks corresponding to substrates and products. This exercise is relatively straightforward and leads students through a wide range of biologically and chemically relevant procedures, including use of NMR spectroscopy, enzymology and data processing techniques.

  11. Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis.

    PubMed

    Oyama, Takehiro; Yodohsi, Masahiro; Yamane, Ayako; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

    2011-10-01

    Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N(4)-(N-acetyl-β-D-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α(1)-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100fmol and 4fmol, respectively.

  12. Identification of the S-layer glycoproteins and their covalently linked glycans in the halophilic archaeon Haloarcula hispanica.

    PubMed

    Lu, Hua; Lü, Yang; Ren, Jinwei; Wang, Zhongfu; Wang, Qian; Luo, Yuanming; Han, Jing; Xiang, Hua; Du, Yuguo; Jin, Cheng

    2015-11-01

    Haloarcula hispanica is one of members of the Halobacteriaceae, which displays particularly low restriction activity and is therefore important as one of the most tractable haloarchaea for archaeal genetic research. Although the Har. hispanica S-layer protein has been reported glycosylated, the S-layer glycoprotein and its glycosylation have not been investigated yet. In this study, the S-layer proteins of Har. hispanica were extracted and characterized. The S-layer was found containing two different glycoproteins which shared highly similar amino acid sequences. The genes coding for these two S-layer glycoproteins were found next to each other in the genome. Moreover, the N- and O-linked glycans were released from these two S-layer glycoproteins for structural determination. Based on the mass spectrometry and nuclear magnetic resonance, the N-glycan was determined as a branched trisaccharide containing a 225 Da residue corresponded to a 2-amino-6-sulfo-2, 6-dideoxy-quinovose, which was the first time that a naturally occurring form of sulfoquinovosamine was identified. Besides, the O-glycan was characterized as a Glcα-1,4-Gal disaccharide by mass spectrometry combined with monosaccharide composition analysis and glycosidase treatment. The determination of the N- and O-glycan structure will be helpful for studying the diverse protein glycosylation pathways in archaea utilizing H. hispanica as a new model.

  13. 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.

  14. Herpesvirus Glycoproteins Undergo Multiple Antigenic Changes before Membrane Fusion

    PubMed Central

    Glauser, Daniel L.; Kratz, Anne-Sophie; Stevenson, Philip G.

    2012-01-01

    Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4) entry machinery—gB, gH/gL and gp150—changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion. PMID:22253913

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

    PubMed

    Sethi, Manveen K; Fanayan, Susan

    2015-08-03

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

  16. Regenerated bacterial cellulose microfluidic column for glycoproteins separation.

    PubMed

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

    2016-02-10

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

  17. Association of dystrophin and an integral membrane glycoprotein.

    PubMed

    Campbell, K P; Kahl, S D

    1989-03-16

    Duchenne muscular dystrophy (DMD) is caused by a defective gene found on the X-chromosome. Dystrophin is encoded by the DMD gene and represents about 0.002% of total muscle protein. Immunochemical studies have shown that dystrophin is localized to the sarcolemma in normal muscle but is absent in muscle from DMD patients. Many features of the predicted primary structure of dystrophin are shared with membrane cytoskeletal proteins, but the precise function of dystrophin in muscle is unknown. Here we report the first isolation of dystrophin from digitonin-solubilized skeletal muscle membranes using wheat germ agglutinin (WGA)-Sepharose. We find that dystrophin is not a glycoprotein but binds to WGA-Sepharose because of its tight association with a WGA-binding glycoprotein. The association of dystrophin with this glycoprotein is disrupted by agents that dissociate cytoskeletal proteins from membranes. We conclude that dystrophin is linked to an integral membrane glycoprotein in the sarcolemma. Our results indicate that the function of dystrophin could be to link this glycoprotein to the underlying cytoskeleton and thus help either to preserve membrane stability or to keep the glycoprotein non-uniformly distributed in the sarcolemma.

  18. Removal of a N-linked Glycosylation Site on the Classical Swine Fever Virus Strain Brescia E(rns) Glycoprotein Affects Virulence in Swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Classical Swine Fever Virus (CSFV) E(rns) glycoprotein is involved in several functions; including virus attachment and entry to target cells, production of antibodies, and virulence. Here, we describe the role of CSFV strain Brescia E(rns) glycosylation on virulence in swine. Amino acid residue N...

  19. Glucosylation of glycoproteins in Crithidia fasciculata.

    PubMed

    Gotz, G; Gañán, S; Parodi, A J

    1991-04-01

    High mannose-type, N-linked oligosaccharides devoid of glucose units may be glucosylated directly from UDP-Glc in mammalian, plant, fungal and protozoan cells. The glucosylated compounds thus formed (protein-linked Glc1Man5-9GlcNAc2, depending on the organisms) are immediately deglucosylated by glucosidase II, an enzyme located, the same as the glucosylating activity, in the endoplasmic reticulum. In order to evaluate the molar proportion of N-linked oligosaccharides that are glucosylated in the trypanosomatid Crithidia fasciculata (a microorganism transferring Man7GlcNAc2 in protein N-glycosylation) cells of the parasite were grown in the presence of [14C]glucose and concentrations of the glucosidase II inhibitors deoxynojirimycin and/or castanospermine that were several hundred-fold higher than those required to inhibit 50% of the activity of the protozoan enzyme. The inhibitors did not affect the cell growth rate and, although glucose analogs, did not interfere with the entry of glucose into the cells. About 40-43% of total N-linked oligosaccharides appeared to be glucosylated. As on the average there are several N-linked oligosaccharides per glycoprotein, more than 40-43% (but probably not all of them) are transiently glucosylated in the endoplasmic reticulum.

  20. Cell surface expression of biologically active influenza C virus HEF glycoprotein expressed from cDNA.

    PubMed

    Pekosz, A; Lamb, R A

    1999-10-01

    The hemagglutinin, esterase, and fusion (HEF) glycoprotein of influenza C virus possesses receptor binding, receptor destroying, and membrane fusion activities. The HEF cDNAs from influenza C/Ann Arbor/1/50 (HEF-AA) and influenza C/Taylor/1223/47 (HEF-Tay) viruses were cloned and expressed, and transport of HEF to the cell surface was monitored by susceptibility to cleavage by exogenous trypsin, indirect immunofluorescence microscopy, and flow cytometry. Previously it has been found in studies with the C/Johannesburg/1/66 strain of influenza C virus (HEF-JHB) that transport of HEF to the cell surface is severely inhibited, and it is thought that the short cytoplasmic tail, Arg-Thr-Lys, is involved in blocking HEF cell surface expression (F. Oeffner, H.-D. Klenk, and G. Herrler, J. Gen. Virol. 80:363-369, 1999). As the cytoplasmic tail amino acid sequences of HEF-AA and HEF-Tay are identical to that of HEF-JHB, the data indicate that cell surface expression of HEF-AA and HEF-Tay is not inhibited by this amino acid sequence. Furthermore, the abundant cell surface transport of HEF-AA and HEF-Tay indicates that their cell surface expression does not require coexpression of another viral protein. The HEF-AA and HEF-Tay HEF glycoproteins bound human erythrocytes, promoted membrane fusion in a low-pH and trypsin-dependent manner, and displayed esterase activity, indicating that the HEF glycoprotein alone mediates all three known functions at the cell surface.

  1. Molecular characterization of glycoprotein genes and phylogenetic analysis of two swine paramyxoviruses isolated from United States.

    PubMed

    Qiao, Dan; Janke, Bruce H; Elankumaran, Subbiah

    2009-08-01

    Two swine paramyxoviruses (SPMV)-(81-19252 (Texas-81) and 92-7783 (ISU-92)-were isolated from encephalitic pigs in the United States in 1981 and 1992. Antigenic, morphologic, and biological characteristics of these two viruses were essentially similar to members of the family Paramyxoviridae. Antigenic analysis by indirect fluorescent antibody, immunoblot, and one-way cross-neutralization tests placed these viruses along with bovine parainfluenza 3 (BPIV3) viruses. Purified virions were 50-300 nm in size and morphologically indistinguishable from other paramyxoviruses. These two viruses hemagglutinated red blood cells and had neuraminidase activity. The gene junctions of fusion (F) and hemagglutinin (HN) glycoprotein genes of these viruses contained highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions similar to other Paramyxoviridae. The F gene of ISU-92 was longer than Texas-81 due to insertion of a 24-nucleotide "U"-rich 3' untranslated region. Structure-based sequence alignment of glycoproteins of these two SPMVs indicated that they are essentially similar in structure and function to parainfluenzaviruses. The Texas-81 strain was closely related to BPIV3 Shipping Fever (SF) strain at nucleotide and amino acid level, while the ISU-92 strain was more closely related to BPIV3 910N strain. The envelope glycoproteins of ISU-92 had only approximately 92 and approximately 96% identity at nucleotide and amino acid levels with BPIV3-SF strain, respectively. The high sequence identities to BPIV3 indicated cross-species infection in pigs. Phylogenetic analyses based on both F protein and HN protein suggested the classification of these viruses into the subfamily Paramyxovirinae, genus Respirovirus, and genotype A of BPIV3.

  2. Glycoprotein isolated from Styrax japonica Siebold et al. Zuccarini inhibits oxidative and pro-inflammatory responses in HCT116 colonic epithelial cells and dextran sulfate sodium-treated ICR mice.

    PubMed

    Lee, Sei-Jung; Lee, Jin; Song, Sooyeon; Lim, Kye-Taek

    2016-01-01

    This study was carried out to investigate the anti-inflammatory potentials of a 38 kDa glycoprotein isolated from Styrax japonica Siebold et al Zuccarini (SJSZ glycoprotein). We found that SJSZ glycoprotein has concentration-dependent scavenging activity against DPPH and hydroxyl radicals in the cell-free systems. In colonic epithelial cells (HCT116 cells), the results showed that SJSZ glycoprotein inhibits the production of reactive oxygen species (ROS) induced by glucose/glucose oxidase (G/GO) in a concentration-dependent manner. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (w/v) for 7 days. We figured out that administration of SJSZ glycoprotein (10 mg/kg) lowers the levels of disease activity index, myeloperoxidase activity, and histological inflammation in DSS-treated mice. In addition, SJSZ glycoprotein inhibited plasmic thiobarbituric acid reactive substances (TBARS) formation, nitric oxide (NO) production, and lactate dehydrogenase (LDH) release, accompanying the inhibition of colonic inflammatory signal proteins (NF-κB, iNOS, and COX-2) and inflammation-related cytokines (IL-1β, IL-6, and TNF-α). These results indicate that SJSZ glycoprotein inhibits oxidative and pro-inflammatory responses in mouse colitis.

  3. Inhibitory effect of SJSZ glycoprotein (38 kDa) on expression of heat shock protein 27 and 70 in chromium (VI)-treated hepatocytes.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2012-01-01

    Chromium (VI) is as an extremely toxic chemical substance, and is also an internationally recognized human carcinogen. The principal objective of this study was to determine whether or not Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents hepatocarcinogenesis in chromium-treated BNL CL.2 cells and ICR mice. Firstly, it was evaluated that SJSZ glycoprotein has strong antioxidant character and scavenges radicals. In an effort to assess the chemopreventive effects of SJSZ glycoprotein on hepatocarcinogenesis, ICR mice were intraperitoneally injected with chromium (10 mg/kg, BW) for 8 weeks. After sacrifice, we evaluated indicators of liver tissue damage [the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS)], antioxidative enzymes [activities of superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (GPx)], and initiating hepatocarcinogenic indicator [heat shock protein (HSP) 27 and 70] and protein kinase C (PKC), p38 MAPK and PCNA via biochemical methods and immunoblot analysis. The results obtained from this study demonstrated that the SJSZ glycoprotein (50 μg/ml) inhibited the production of intracellular ROS in BNL CL.2 cells. In addition, the SJSZ glycoprotein (10 mg/kg, BW) attenuated the levels of LDH, ALT, and TBARS, whereas it increased antioxidative enzymes in mouse serum. SJSZ glycoprotein attenuated the activity of HSP27, HSP70, PKC, MAPKs, and PCNA in BNL CL.2 cells and liver tissue. Taken together, our results indicate that SJSZ glycoprotein might be have a potent preventive effect against hepatocarcinogenesis induced by oxidative stress.

  4. 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

    2014-11-07

    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.

  5. Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins.

    PubMed

    Elovson, J

    1980-06-25

    As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.

  6. Glycoproteins E2 of the Venezuelan and eastern equine encephalomyelitis viruses contain multiple cross-reactive epitopes.

    PubMed

    Pereboev, A V; Razumov, I A; Svyatchenko, V A; Loktev, V B

    1996-01-01

    Enzyme immunoassay (EIA) with sixty types of monoclonal antibodies (MAbs) was used to study cross-reactive epitopes on the attenuated and virulent strains of the Eastern equine encephalomyelitis (EEE) and Venezuelan equine encephalomyelitis (VEE) viruses. All three structural proteins of the EEE and VEE viruses were demonstrated to have both cross-reactive and specific antigenic determinants. The glycoprotein E1 of EEE and VEE viruses possesses three cross-reactive epitopes for binding to MAbs. The glycoprotein E2 has a cluster of epitopes for 20 cross-reacting MAbs produced to EEE and VEE viruses. Cross-reactive epitopes were localised within five different sites of glycoprotein E2 of VEE virus and within four sites of that of the EEE virus. There are no cross-neutralising MAbs to the VEE and EEE viruses. Only one type of the protective Mabs was able to cross-protect mice against lethal infection by the virulent strains of the VEE and EEE viruses. Eight MAbs blocked the hemagglutination activity (HA) of both viruses. Antigenic alterations of neutralising and protective sites were revealed for all attenuated strains of the VEE and EEE viruses. Comparative studies of the E2 proteins amino acid sequences show that the antigenic modifications observed with the attenuated strains of the VEE virus may be caused by multiple amino acid changes in positions 7, 62, 120, 192 and 209-213. The escape-variants of the VEE virus obtained with cross-reactive MAbs 7D1, 2D4 and 7A6 have mutations of the E2 protein at positions 59, 212-213 and 232, respectively. Amino acid sequences in these regions of the VEE and EEE viruses are not homologous. These observations indicate that cross-reactive MAbs are capable of recognising discontinuous epitopes on the E2 glycoprotein.

  7. Asparagine-linked sugar chains of glycoproteins in calf thymocyte plasma membrane. Isolation and fractionation of oligosaccharides liberated by hydrazinolysis.

    PubMed

    Yoshima, H; Takasaki, S; Kobata, A

    1980-07-01

    The plasma membrane glycoproteins of calf thymocytes were converted to glycopeptides by exhaustive pronase digestion. Glycopeptides with asparagine-linked sugar chains were separated from those with mucine-type sugar chains by Bio-Gel P-10 column chromatography. The asparagine-linked sugar chains were released as oligosaccharides from the peptide moiety by hydrazinolysis and labeled by reduction with NaB[3H]4. The radioactive oligosaccharides were fractionated into fifteen acidic components and ten neutral components by combination of paper electrophoresis and Bio-Gel P-4 column chromatography. The acidic nature of all fifteen acidic components can be ascribed to their N-acetylneuraminic acid residues. The Bio-Gel P-4 column chromatographic patterns of the neutral oligosaccharide fraction and of the neutral fraction obtained on sialidase treatment of the pooled acidic oligosaccharide fraction were totally different, indicating that the acidic oligosaccharides are not simple sialyl derivatives of the neutral oligosaccharides.

  8. A carbohydrate-binding affinity ligand for the specific enrichment of glycoproteins.

    PubMed

    Chen, Chen; El Khoury, Graziella; Zhang, Peiqing; Rudd, Pauline M; Lowe, Christopher R

    2016-04-29

    One challenge facing the production of glycoprotein therapeutics is the lack of stable and selective affinity ligands for their enrichment. Synthetic affinity ligands based on the solid phase multi-component Ugi reaction represent a desirable option, particularly those incorporating benzoboroxole and its derivatives, which have been shown to enrich glycoproteins under physiological conditions. Thus, an Ugi ligand, A21C11I8, comprising 5-amino-2-hydroxymethylphenylboronic acid was synthesised on aldehyde-functionalised Sepharose™. Immobilised A21C11I8 displayed affinity for the glycosylated protein, glucose oxidase (GOx), which bound primarily through its glycan moiety. The ligand had a preference for sugar alcohols and the furanose form of the monosaccharides tested. Compared with immobilised 3-aminophenylboronic acid and Concanavalin A, the Ugi ligand was able to purify GOx from spiked Escherichia coli supernatants with retention of its maximum enzymatic activity and protein recovery. Glycan profiles of human immunoglobulin G tested on A21C11I8 columns suggested that the adsorbent possesses the potential to resolve sialylated and neutral glycoforms. The benzoboroxole-functionalised Ugi ligand may find application in selective glycoform separation.

  9. Structural features affecting variant surface glycoprotein expression in Trypanosoma brucei.

    PubMed

    Wang, Jun; Böhme, Ulrike; Cross, George A M

    2003-05-01

    The glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) of Trypanosoma brucei is the most abundant GPI-anchored protein expressed on any cell, and is an essential virulence factor. To determine what structural features affect efficient expression of VSG, we made a series of mutations in two VSGs. Inserting 18 amino acids, between the amino- and carboxy-terminal domains, reduced the expression of VSG 221 to about 3% of the wild-type level. When this insertion was combined with deletion of the single carboxy-terminal subdomain, expression was reduced a further three-fold. In VSG 117, which contains two carboxy-terminal subdomains, point mutation of the intervening N-glycosylation site reduced expression about 15-fold. Deleting the most carboxy-terminal subdomain and intervening region, including the N-glycosylation site, reduced expression to 15-20% of wild type VSG, and deletion of both subdomains reduced expression to <1%. Despite their low abundance, all VSG mutants were GPI anchored on the cell surface. Our results suggest that, for a protein to be efficiently displayed on the surface of bloodstream-form T. brucei, it is essential that it contains the conserved structural motifs of a T. brucei VSG. Serum resistance-associated protein (SRA), which confers human infectivity on T. brucei, strongly resembles a VSG deletion mutant. Expression of three epitope-tagged versions of SRA in T. brucei conferred total resistance to human serum. SRA possesses a canonical GPI signal sequence, but we were unable to obtain unequivocal evidence for the presence of a GPI anchor. SRA was not released during osmotic lysis, indicating that it is not GPI anchored on the cell surface.

  10. The catalytic triad of the influenza C virus glycoprotein HEF esterase: characterization by site-directed mutagenesis and functional analysis.

    PubMed

    Pleschka, S; Klenk, H D; Herrler, G

    1995-10-01

    Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-KB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex.

  11. Genetic variability of platelet glycoprotein Ibalpha gene.

    PubMed

    Ozelo, Margareth C; Costa, Devanira S P; Siqueira, Lucia H; Machado, Tania M F; Castro, Vagner; Gonçalves, Marilda S; Menezes, Raimundo C; Soares, Manoel; Annichino-Bizzacchi, Joyce M; Costa, Fernando F; Arruda, Valder R

    2004-10-01

    Platelet membrane glycoprotein (GP) Ibalpha is a critical component of platelet adhesion complex to subendothelium structures following tissue injury or pathological surfaces, such as atherosclerotic plaques. Polymorphisms of the GPIbalpha gene have been associated with a high risk for occlusive vascular disease, and its distribution varies considerably among distinct populations. These polymorphisms comprise the human platelet antigen (HPA)-2 system, the -5C/T dimorphism of the Kozak sequence, and the variable number of tandem 39-bp repeats (VNTR). Here we report the prevalence of the GPIbalpha gene polymorphisms among Brazilians, a highly ethnically diverse population. We analyzed 492 subjects of European, African, or Indigenous origin. It was possible to determine ten distinct haplotypes. The most common ( reverse similar 40%) haplotype was the Kozak-TT/HPA-2aa/VNTR-CC for both Caucasian and African descent. However, among Indigenous, Kozak-TT/HPA-2aa/VNTR-CC and Kozak-TC/HPA-2aa/VNTR-CC were equally present. Although a strong linkage disequilibrium between VNTR and HPA-2 polymorphism had also been observed, here we determined incomplete linkage disequilibrium in 10% of subjects from all ethnic groups. VNTR-E, a rare variant lacking the 39-bp repeat, was identified in two unrelated subjects, and functional platelet studies revealed no abnormalities. The VNTR-A allele, the largest variant containing four copies of the repeats, was not identified in this population. However, homozygosity for the VNTR-A allele (Kozak-TT/HPA-2aa/VNTR-AA) was determined in two distinct species of nonhuman primates. These results suggest a greater complex evolutionary mechanism in the macroglycoprotein region of the GPIbalpha gene and may be useful in the design of gene-disease association studies for vascular disease.

  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. Recognition and invasion of human erythrocytes by malarial parasites: contribution of sialoglycoproteins to attachment and host specificity

    SciTech Connect

    Friedman, M.J.; Blankenberg, T.; Sensabaugh, G.; Tenforde, T.S.

    1984-05-01

    The receptivity of human erythrocytes to invasion by Plasmodium falciparum merozoites can be decreased by neuraminidase or trypsin treatment, an observation that supports a role for the erythrocyte sialoglycoproteins (glycophorins) in invasion. We have found that ..cap alpha../sub 1/-acid glycoprotein (AGP), added to in vitro cultures, can restore invasion of enzyme-treated human erythrocytes. AGP is structurally different from the glycophorins although it does carry 12% sialic acid. Its ability to restore receptivity to desialylated cells is dependent on its sialic acid complement, its concentration, and its binding to the erythrocyte surface. We present evidence that AGP forms a bridge between the merozoite and the enzyme-treated erythrocyte that allows the stronger and more complex interactions of invasion to proceed. We suggest that the glycophorins play the same role on the surface of the intact erythrocyte. 31 references, 3 figures, 6 tables.

  14. Recognition and invasion of human erythrocytes by malarial parasites: contribution of sialoglycoproteins to attachment and host specificity

    PubMed Central

    1984-01-01

    The receptivity of human erythrocytes to invasion by Plasmodium falciparum merozoites can be decreased by neuraminidase or trypsin treatment, an observation that supports a role for the erythrocyte sialoglycoproteins (glycophorins) in invasion. We have found that alpha 1-acid glycoprotein (AGP), added to in vitro cultures, can restore invasion of enzyme-treated human erythrocytes. AGP is structurally different from the glycophorins although it does carry 12% sialic acid. Its ability to restore receptivity to desialylated cells is dependent on its sialic acid complement, its concentration, and its binding to the erythrocyte surface. We present evidence that AGP forms a bridge between the merozoite and the enzyme-treated erythrocyte that allows the stronger and more complex interactions of invasion to proceed. We suggest that the glycophorins play the same role on the surface of the intact erythrocyte. PMID:6373782

  15. Characterization of a human glycoprotein with a potential role in sperm-egg fusion: cDNA cloning, immunohistochemical localization, and chromosomal assignment of the gene (AEGL1)

    SciTech Connect

    Hayashi, Masaru; Fujimoto, Seiichiro; Takano, Hiroko

    1996-03-05

    Acidic epididymal glycoprotein (AEG), thus far identified only in rodents, is one of the sperm surface proteins involved in the fusion of the sperm and egg plasma membranes. In the present study, we describe the isolation and characterization of cDNA encoding a human glycoprotein related to AEG. Although this protein, designated ARP (AEG-related protein), is not the ortholog of rodent AEG, it resembles AEG in that it is an epididymal secretory glycoprotein that binds to the postacrosomal region of the sperm head. The fact that no AEG mRNA can be detected in the human epididymis suggests that ARP might be the functional counterpart of rodent AEG. The gene encoding ARP (AEGL1) was mapped by fluorescence in situ hybridization to 6p21.1-p21.2. This result indicates that AEGL1 and the mouse gene for AEG are located in the chromosomal segments with conserved syntenies. 43 refs., 6 figs.

  16. PCR cloning and characterization of multiple ADP-glucose pyrophosphorylase cDNAs from tomato

    NASA Technical Reports Server (NTRS)

    Chen, B. Y.; Janes, H. W.; Gianfagna, T.

    1998-01-01

    Four ADP-glucose pyrophosphorylase (AGP) cDNAs were cloned from tomato fruit and leaves by the PCR techniques. Three of them (agp S1, agp S2, and agp S3) encode the large subunit of AGP, the fourth one (agp B) encodes the small subunit. The deduced amino acid sequences of the cDNAs show very high identities (96-98%) to the corresponding potato AGP isoforms, although there are major differences in tissue expression profiles. All four tomato AGP transcripts were detected in fruit and leaves; the predominant ones in fruit are agp B and agp S1, whereas in leaves they are agp B and agp S3. Genomic southern analysis suggests that the four AGP transcripts are encoded by distinct genes.

  17. Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

    PubMed Central

    Edson, C M; Hosler, B A; Respess, R A; Waters, D J; Thorley-Lawson, D A

    1985-01-01

    Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection. Images PMID:2993665

  18. Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Positive (ER+) and Negative (ER-) Human Breast Cancer Tissues by LC-LTQ/FT-ICR Mass Spectrometry

    PubMed Central

    Semaan, Suzan M.; Wang, Xu; Marshall, Alan G.; Sang, Qing-Xiang Amy

    2012-01-01

    Breast cancer is the second most fatal cancer in American women. To increase the life expectancy of patients with breast cancer new diagnostic and prognostic biomarkers and drug targets must be identified. A change in the glycosylation on a glycoprotein often causes a change in the function of that glycoprotein; such a phenomenon is correlated with cancerous transformation. Thus, glycoproteins in human breast cancer estrogen receptor positive (ER+) tissues and those in the more advanced stage of breast cancer, estrogen receptor negative (ER-) tissues, were compared. Glycoproteins showing differences in glycosylation were examined by 2-dimensional gel electrophoresis with double staining (glyco- and total protein staining) and identified by reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/ Fourier transform ion cyclotron resonance mass spectrometer. Among the identified glycosylated proteins are alpha 1 acid glycoprotein, alpha-1-antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor that were further verified by Western blotting for both ER+ and ER- human breast tissues. Results show the presence of a possible glycosylation difference in alpha-1-antitrypsin, a potential tumor-derived biomarker for breast cancer progression, which was expressed highest in the ER- samples. PMID:22773931

  19. Towards a reliable molecular mass determination of intact glycoproteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    PubMed

    Giménez, Estela; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

    2007-01-01

    Different matrices and sample-matrix preparation procedures have been tested in order to study their influence on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra of intact glycoproteins, which present different degrees of glycosylation (human transferrin; bovine fetuin; bovine alpha(1)-acid-glycoprotein; recombinant human erythropoietin; and the novel erythropoiesis stimulating protein). Using sinapinic acid (SA) and the fast evaporation method, the studied glycoproteins became susceptible to fragmentation at any laser intensity, suggesting that this 'hot' matrix is unsuitable for a reliable molecular mass determination of glycosylated compounds. In contrast, 2,5-dihydroxybenzoic acid (DHB) and 6-aza-2-thiothymine (ATT), with an adequate sample-matrix preparation, provided improved results. Samples containing DHB after crystallization by vacuum drying demonstrated the best performance because the labile functional groups from the glycoforms were apparently fragmented to a lower extent. The average molecular masses obtained using this methodology were in all cases a better estimation than those values reported in the literature. The results were reproducible, and sensitivity was similar to that obtained with SA and the fast evaporation method. These excellent results suggest that this MALDI-TOF-MS methodology could be useful for an improved determination of the average molecular mass values of microheterogeneous compounds such as glycoproteins, glycosylated compounds or, in general, molecular mass values of molecules with similar labile functional groups.

  20. A single point mutation of the influenza C virus glycoprotein (HEF) changes the viral receptor-binding activity.

    PubMed

    Szepanski, S; Gross, H J; Brossmer, R; Klenk, H D; Herrler, G

    1992-05-01

    From strain JHB/1/66 of influenza C virus a mutant was derived with a change in the cell tropism. The mutant was able to grow in a subline of Madin-Darby canine kidney cells (MDCK II) which is resistant to infection by the parent virus due to a lack of receptors. Inactivation of cellular receptors by either neuraminidase or acetylesterase and generation of receptors by resialylation of cells with N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) indicated that 9-O-acetylated sialic acid is a receptor determinant for both parent and mutant virus. However, the mutant required less Neu5,9Ac2 on the cell surface for virus attachment than the parent virus. The increased binding efficiency enabled the mutant to infect cells with a low content of 9-O-acetylated sialic acid which were resistant to the parent virus. By comparing the nucleotide sequences of the glycoprotein (HEF) genes of the parent and the mutant virus only a single point mutation could be identified on the mutant gene. This mutation at nucleotide position 872 causes an amino acid exchange from threonine to isoleucine at position 284 on the amino acid sequence. Sequence similarity with a stretch of amino acids involved in the receptor-binding pocket of the influenza A hemagglutinin suggests that the mutation site on the influenza C glycoprotein (HEF) is part of the receptor-binding site.

  1. 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

  2. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice

    PubMed Central

    Wang, Xueqian; Cabrera, Robert M.; Li, Yue; Miller, David S.; Finnell, Richard H.

    2013-01-01

    Folate deficiency has been associated with many adverse clinical manifestations. The blood-brain barrier (BBB), formed by brain capillary endothelial cells, protects the brain from exposure to neurotoxicants. The function of BBB is modulated by multiple ABC transporters, particularly P-glycoprotein. A proton-coupled folate transporter (PCFT)-deficient mouse has been previously described as a model for systemic folate deficiency. Herein, we demonstrate that exposing mouse brain capillaries to the antiepileptic drug, valproic acid (VPA; 5 μM), significantly increased P-glycoprotein transport function in the wild-type animals. A ligand to the aryl hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produced a similar induction of P-glycoprotein, which tightened the BBB, thereby increasing the neuroprotection. However, VPA- or TCDD-induced P-glycoprotein transport was blocked in the PCFT-nullizygous mice, indicating that multiple neuroprotective mechanisms are compromised under folate-deficient conditions. Brain capillaries from S-folinic acid (SFA; 40 mg/kg)-treated PCFT-nullizygous mice exhibited increased P-glycoprotein transport following VPA exposure. This suggests that SFA supplementation restored the normal BBB function. In addition, we show that tight-junction proteins are disintegrated in the PCFT mutant mice. Taken together, these findings strongly suggest that folate deficiency disrupts the BBB function by targeting the transporter and tight junctions, which may contribute to the development of neurological disorders.—Wang, X., Cabrera, R. M., Li, Y., Miller, D. S., Finnell, R. H. Functional regulation of P-glycoprotein at the blood-brain barrier in proton-coupled folate transporter (PCFT) mutant mice. PMID:23212123

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

    PubMed

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

    2015-08-14

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

  4. 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.

  5. Structure and heterologous expression of the gene encoding the cell surface glycoprotein from Haloarcula japonica strain TR-1.

    PubMed

    Wakai, H; Takada, K; Nakamura, S; Horikoshi, K

    1995-01-01

    The gene encoding the cell surface glycoprotein (CSG) of Haloarcula japonica strain TR-1 was cloned and sequenced. The structural gene consisted from an open reading frame of 2,586 bp. A potential promoter sequence was found about 150 bp upstream of the ATG initiation codon. N-terminal amino acid sequence of the Ha. japonica CSG revealed that the mature CSG consisted of 828 amino acids. Five potential N-glycosylation sites were found in the mature sequence. The cloned CSG gene of Ha. japonica was expressed in closely-related halophilic archaea.

  6. Characterization of Trypanosoma brucei gambiense variant surface glycoprotein LiTat 1.5.

    PubMed

    Van Nieuwenhove, L; Rogé, S; Lejon, V; Guisez, Y; Büscher, P

    2012-05-09

    At present, all available diagnostic antibody detection tests for Trypanosoma brucei gambiense human African trypanosomiasis are based on predominant variant surface glycoproteins (VSGs), such as VSG LiTat 1.5. During investigations aiming at replacement of the native VSGs by recombinant proteins or synthetic peptides, the sequence of VSG LiTat 1.5 was derived from cDNA and direct N-terminal amino acid sequencing. Characterization of the VSG based on cysteine distribution in the amino acid sequence revealed an unusual cysteine pattern identical to that of VSG Kinu 1 of T. b. brucei. Even though both VSGs lack the third of four conserved cysteines typical for type A N-terminal domains, they can be classified as type A.

  7. Localization and synthesis of an antigenic determinant of herpes simplex virus glycoprotein D that stimulates the production of neutralizing antibody

    SciTech Connect

    Cohen, G.H.; Dietzschold, B.; Ponce de Leon, M.; Long, D.; Golub, E.; Varrichio, A.; Pereira, L.; Eisenberg, R.J.

    1984-01-01

    An antigenic determinant capable of inducing type-common herpes simplex virus (HSV)-neutralizing antibodies has been located on glycoprotein D (gD) of HSV type 1 (HSV-1). A peptide of 16 amino acids corresponding to residues 8 to 23 of the mature glycoprotein (residues 33 to 48 of the predicted gD-1 sequence) was synthesized. This peptide reacted with an anti-gD monoclonal antibody (group VII) previously shown to neutralize the infectivity of HSV-1 and HSV-2. The peptide was also recognized by polyclonal antibodies prepared against purified gD-1 but was less reactive with anti-gD2 sera. Sera from animals immunized with the synthetic peptide reacted with native gD and neutralized both HSV-1 and HSV-2.

  8. A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation.

    PubMed

    Potorac, Iulia; Rivero-Müller, Adolfo; Trehan, Ashutosh; Kiełbus, Michał; Jozwiak, Krzysztof; Pralong, Francois; Hafidi, Aicha; Thiry, Albert; Ménagé, Jean-Jacques; Huhtaniemi, Ilpo; Beckers, Albert; Daly, Adrian F

    2016-12-01

    Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone (LH) is essential for the postnatal development and maturation of the male gonad. Inactivating Luteinizing hormone beta (LHB) gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe in males. We describe a family with selective LH deficiency and hypogonadism in two brothers. DNA sequencing of LHB was performed and the effects of genetic variants on hormone function and secretion were characterized by mutagenesis studies, confocal microscopy and functional assays. A 20-year-old male from a consanguineous family had pubertal delay, hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was identified in the patient and his brother, who subsequently had the same phenotype. Treatment with hCG led to pubertal development, increased circulating testosterone and spermatogenesis. Experiments in HeLa cells revealed that the mutant LH is retained intracellularly and showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common alpha-subunit and can activate its receptor. Deletion of flanking glutamic acid residues at positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is functionally important across all heterodimeric glycoprotein hormones, because deletion of the corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in the protein that is crucial for normal secretion of all human glycoprotein hormones.

  9. Palmitoylation of the Rous Sarcoma Virus Transmembrane Glycoprotein Is Required for Protein Stability and Virus Infectivity

    PubMed Central

    Ochsenbauer-Jambor, Christina; Miller, David C.; Roberts, Charles R.; Rhee, Sung S.; Hunter, Eric

    2001-01-01

    The Rous sarcoma virus (RSV) transmembrane (TM) glycoprotein is modified by the addition of palmitic acid. To identify whether conserved cysteines within the hydrophobic anchor region are the site(s) of palmitoylation, and to determine the role of acylation in glycoprotein function, cysteines at residues 164 and 167 of the TM protein were mutated to glycine (C164G, C167G, and C164G/C167G). In CV-1 cells, palmitate was added to env gene products containing single mutations but was absent in the double-mutant Env. Although mutant Pr95 Env precursors were synthesized with wild-type kinetics, the phenotypes of the mutants differed markedly. Env-C164G had properties similar to those of the wild type, while Env-C167G was degraded faster, and Env containing the double mutant C164G/C167G was very rapidly degraded. Degradation occurred after transient plasma membrane expression. The decrease in steady-state surface expression and increased rate of internalization into endosomes and lysosomes paralleled the decrease in palmitoylation observed for the mutants. The phenotypes of mutant viruses were assessed in avian cells in the context of the pATV8R proviral genome. Virus containing the C164G mutation replicated with wild-type kinetics but exhibited reduced peak reverse transcriptase levels. In contrast, viruses containing either the C167G or the C164G/C167G mutation were poorly infectious or noninfectious, respectively. These phenotypes correlated with different degrees of glycoprotein incorporation into virions. Infectious revertants of the double mutant demonstrated the importance of cysteine-167 for efficient plasma membrane expression and Env incorporation. The observation that both cysteines within the membrane-spanning domain are accessible for acylation has implications for the topology of this region, and a model is proposed. PMID:11689636

  10. Core structure of S2 from the human coronavirus NL63 spike glycoprotein.

    PubMed

    Zheng, Qi; Deng, Yiqun; Liu, Jie; van der Hoek, Lia; Berkhout, Ben; Lu, Min

    2006-12-26

    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 degrees 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.

  11. 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.

  12. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica.

    PubMed

    Roehrig, J T; Bolin, R A; Kelly, R G

    1998-07-05

    Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.

  13. P-Glycoprotein Transport of Neurotoxic Pesticides.

    PubMed

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

    2015-10-01

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

  14. Emerging Technologies for Making Glycan-Defined Glycoproteins

    PubMed Central

    Wang, Lai-Xi; Lomino, Joseph V.

    2011-01-01

    Protein glycosylation is a common and complex posttranslational modification of proteins, which expands functional diversity while boosting structural heterogeneity. Glycoproteins, the end products of such a modification, are typically produced as mixtures of glycoforms possessing the same polypeptide backbone but differ in the site of glycosylation and/or in the structures of pendant glycans, from which single glycoforms are difficult to isolate. The urgent need for glycan-defined glycoproteins in both detailed structure-function relationship studies and therapeutic applications has stimulated an extensive interest in developing various methods for manipulating protein glycosylation. This review highlights emerging technologies that hold great promise in making a variety of glycan-defined glycoproteins, with a particular emphasis in the following three areas: specific glycoengineering of host biosynthetic pathways, in vitro chemoenzymatic glycosylation remodeling, and chemo-selective and site-specific glycosylation of proteins. PMID:22141574

  15. Retroviral env glycoprotein trafficking and incorporation into virions.

    PubMed

    Murakami, Tsutomu

    2012-01-01

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

  16. Interactions of retinoids with the ABC transporters P-glycoprotein and Breast Cancer Resistance Protein

    PubMed Central

    Tarapcsák, Szabolcs; Szalóki, Gábor; Telbisz, Ágnes; Gyöngy, Zsuzsanna; Matúz, Krisztina; Csősz, Éva; Nagy, Péter; Holb, Imre J.; Rühl, Ralph; Nagy, László; Szabó, Gábor; Goda, Katalin

    2017-01-01

    Retinoids – derivatives of vitamin A – are important cell permeant signaling molecules that regulate gene expression through activation of nuclear receptors. P-glycoprotein (Pgp) and ABCG2 are plasma membrane efflux transporters affecting the tissue distribution of numerous structurally unrelated lipophilic compounds. In the present work we aimed to study the interaction of the above ABC transporters with retinoid derivatives. We have found that 13-cis-retinoic acid, retinol and retinyl-acetate inhibited the Pgp and ABCG2 mediated substrate transport as well as the substrate stimulated ATPase activity of these transporters. Interestingly, 9-cis-retinoic acid and ATRA (all-trans retinoic acid), both are stereoisomers of 13-cis-retinoic acid, did not have any effect on the transporters’ activity. Our fluorescence anisotropy measurements revealed that 13-cis-retinoic acid, retinol and retinyl-acetate selectively increase the viscosity and packing density of the membrane. Thus, the mixed-type inhibition of both transporters by retinol and ABCG2 by 13-cis-retinoic acid may be the collective result of direct interactions of these retinoids with the substrate binding site(s) and of indirect interactions mediated by their membrane rigidifying effects. PMID:28145501

  17. Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide

    SciTech Connect

    Plassmeyer, Matthew L.; Soldan, Samantha S.; Stachelek, Karen M.; Roth, Susan M.; Martin-Garcia, Julio; Gonzalez-Scarano, Francisco . E-mail: scarano@mail.med.upenn.edu

    2007-02-20

    The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.

  18. A model of the rabies virus glycoprotein active site.

    PubMed

    Rustici, M; Bracci, L; Lozzi, L; Neri, P; Santucci, A; Soldani, P; Spreafico, A; Niccolai, N

    1993-06-01

    The glycoprotein from the neurotropic rabies virus shows a significant homology with the alpha neurotoxin that binds to the nicotinic acetylcholine receptor. The crystal structure of the alpha neurotoxins suggests that the Arg 37 guanidinium group and the Asp 31 side-chain carboxylate of the erabutoxin have stereochemical features resembling those of acetylcholine. Conformational studies on the Asn194-Ser195-Arg196-Gly197 tetrapeptide, an essential part of the binding site of the rabies virus glycoprotein, indicate that the side chains of Asn and Arg could also mimic the acetylcholine structure. This observation is consistent with the recently proposed mechanism of the viral infection.

  19. Reversers of the multidrug resistance transporter P-glycoprotein.

    PubMed

    Stein, Wilfred D

    2002-05-01

    Multidrug resistance can arise from the presence of the membrane-bound pump, P-glycoprotein, in a tumor. Major efforts have been made to develop inhibitors of this pump, and a number of promising blockers have reached late stages of clinical trials. The kinetics of the inhibition of P-glycoprotein is complex, with binding sites that can interact synergistically. Reversers of increased affinity and specificity could, in principle, be developed on the basis of these synergies, and offer some promise in cancer therapeutics.

  20. 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...

  1. Protein glycosylation as an adaptive response in Archaea: growth at different salt concentrations leads to alterations in Haloferax volcanii S-layer glycoprotein N-glycosylation.

    PubMed

    Guan, Ziqiang; Naparstek, Shai; Calo, Doron; Eichler, Jerry

    2012-03-01

    To cope with life in hypersaline environments, halophilic archaeal proteins are enriched in acidic amino acids. This strategy does not, however, offer a response to transient changes in salinity, as would post-translational modifications. To test this hypothesis, N-glycosylation of the Haloferax volcanii S-layer glycoprotein was compared in cells grown in high (3.4 M NaCl) and low (1.75 M NaCl) salt, as was the glycan bound to dolichol phosphate, the lipid upon which the N-linked glycan is assembled. In high salt, S-layer glycoprotein Asn-13 and Asn-83 are modified by a pentasaccharide, while dolichol phosphate is modified by a tetrasaccharide comprising the first four pentasaccharide residues. When the same targets were considered from cells grown in low salt, substantially less pentasaccharide was detected. At the same time, cells grown at low salinity contain dolichol phosphate modified by a distinct tetrasaccharide absent in cells grown at high salinity. The same tetrasaccharide modified S-layer glycoprotein Asn-498 in cells grown in low salt, whereas no glycan decorated this residue in cells grown in the high-salt medium. Thus, in response to changes in environmental salinity, Hfx. volcanii not only modulates the N-linked glycans decorating the S-layer glycoprotein but also the sites of such post-translational modification.

  2. The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

    PubMed

    Voigt, Jürgen; Frank, Ronald; Wöstemeyer, Johannes

    2009-02-01

    Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.

  3. Characterization of the glycoprotein of infectious hematopoietic necrosis virus using neutralizing monoclonal antibodies

    USGS Publications Warehouse

    Huang, Chienjin; Chien, Maw-Sheng; Landolt, Marsha; Winton, James

    1994-01-01

    To study the antigenic nature of the glycoprotein (G protein) of infectious hematopoietic necrosis virus (IHNV), 31 neutralizing monoclonal antibodies (MAbs) were produced against a reference isolate of the virus. The MAbs were compared using a neutralization assay, an enzyme-linked immunosorbent assay (ELISA), and by immunoblotting of the G protein in the native, reduced, and deglycosylated forms. Hybridoma culture fluids of the various MAbs could be diluted from 1:2 to 1:512 and still completely neutralize 1 X 104 plaque-forming units of IHNV. Similarly, the end point dilutions that produced optical density readings of 0.1 or greater in the ELISA were 1:40 to 1:10240. Western blotting showed that all of the MAbs reacted with the G protein in the unreduced (i.e. native) conformation; however, only 9 nine of the MAbs were able to react with the G protein following reduction by 2-mercaptoethanol. Deglycosylation of the protein did not influence the binding ability of any of the MAbs. These data indicate that all the MAbs recognized amino acid sequences on the protein itself and that the IHNV glycoprotein contains linear as well as conformation-dependent neutralizing epitopes. When rainbow trout Oncorhynchus mykiss fingerlings were passively immunized with MAbs against either a linear or a conformation-dependent epitope, the fish were protected against challenge with wild-type IHNV.

  4. Persistent influenza C virus possesses distinct functional properties due to a modified HEF glycoprotein.

    PubMed

    Marschall, M; Herrler, G; Böswald, C; Foerst, G; Meier-Ewert, H

    1994-09-01

    A model of long term viral persistence has been established by selecting a spontaneous mutant strain of influenza C/Ann Arbor/1/50 virus in a permanent carrier culture of MDCK cells. Infectivity and cell tropism are mainly determined by the multifunctional viral membrane glycoprotein (HEF). HEF analysis was aimed at identifying a putative correlation between sequence and function, i.e. receptor binding, enzymatic activity, antigenicity and rate of infection. The current experimental picture is summarized by the following findings: (i) C/Ann Arbor/1/50 persistent virus carries a modified receptor-binding sequence, (ii) receptor-binding activity is altered, as indicated by a higher efficiency in recognizing low amounts of the receptor determinant N-acetyl-9-O-acetylneuraminic acid, (iii) direct attachment to cell surfaces differs from that of wild-type virus, as measured by slower kinetics of viral elution, (iv) receptor-destroying enzymatic activity is diminished, (v) characteristic features of virion surface morphology are altered or unstable, (vi) persistent-type HEF epitopes are distinguishable by monoclonal antibodies from wild-type and (vii) viral infectivity is intensified for cells bearing a low number of receptors. The sum of these changes highlights a structurally and functionally modified HEF glycoprotein that allows long term viral persistence. In order to clarify which of the described points are required for the persistent viral phenotype, a working concept is presented.

  5. Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

    SciTech Connect

    Raub, T.J.; Denny, J.B.; Roberts, R.M.

    1986-01-01

    The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4/sup 0/C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with (/sup 3/H)D-glucosamine or (/sup 3/H)L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of (/sup 125/I)anti-DNP antibodies. Within 15 min at 37/sup 0/C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ approx. 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

  6. A major stress-inducible Mr-42000 wall glycoprotein of French bean (Phaseolus vulgaris L.).

    PubMed

    Millar, D J; Slabas, A R; Sidebottom, C; Smith, C G; Allen, A K; Bolwell, G P

    1992-05-01

    A major wall protein of suspension-cultured cells of French bean has been isolated and characterised. It can be prepared from walls or the culture filtrate and in composition it is particularly rich in proline, valine and glutamic acid/glutamine and contains appreciable amounts of hydroxyproline. The N-terminus shows some glycosylation, while following chemical deglycosylation the first 38 residues were found to be identical to those of proline-rich proteins from soybean. However, the composition of the highly purified Mr-42000 bean protein differs considerably from the soybean proteins and must contain its own specific domains. An antibody was raised and used to demonstrate the inducibility of the Mr-42000 bean protein in response to elicitor action. The protein was found to be mainly localised in the intercellular spaces of the cortical cells of bean hypocotyls and at the wall-plasmalemma interface of xylem vessels, another potentially accessible compartment for pathogens. Following wounding, the protein was found to be generally distributed in the wall of epidermal and cortical cells of the hypocotyls. The Mr-42000 protein is cross reactive with antibodies raised to glycoproteins of the Rhizobium infection thread and the chitin-binding hydroxyproline-rich glycoprotein, potato lectin. These common epitopes together with the previously demonstrated chitin-binding properties of the bean protein indicate a role in host-microbial interactions. Furthermore, the Mr-42000 protein itself bound to the growing hyphal tips of the bean pathogen, Colletotrichum lindemuthianum.

  7. Antioxidant activity of glycoprotein purified from Undaria pinnatifida measured by an in vitro digestion model.

    PubMed

    Rafiquzzaman, S M; Kim, Eun-Young; Kim, Yu-Ri; Nam, Taek-Jeong; Kong, In-Soo

    2013-11-01

    The present study was performed to investigate the chemical composition and antioxidant activity of glycoprotein purified from Undaria pinnatifida Harvey (UPGP). On SDS-PAGE, UPGP migrated as a single band with a molecular weight of approximately 10 kDa and confirmed by staining with Schiff's reagent as glycoprotein. It consists of a carbohydrate component (42.53%) and protein component (57.47%). Amino acid profile, FT-IR spectrum and enzymatic glycosylation analysis suggested that protein is linked with carbohydrate by O-glycosylation. UPGP showed dose-dependent antioxidant activities as detected by different assays before and after in vitro digestion. The IC50 values of undigested UPGP were 0.25 ± 0.03, 0.08 ± 0.005, 0.69 ± 0.12, and 0.25 ± 0.08 mg/mL for DPPH, ABTS, FRAP, and NO, respectively. Following in vitro digestion, the antioxidant activities of UPGP were decreased during the gastric phase compared to those of undigested UPGP, with an increase occurring during the duodenal phase in all assays. However, the reducing power was unchanged after in vitro digestion. Furthermore, UPGP showed protective activity against oxidative DNA damage both undigested, after saliva and duodenal phase of digestion. These results indicate that the antioxidant and DNA protection activities of UPGP may be pH-dependent and assay specific.

  8. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    NASA Technical Reports Server (NTRS)

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  9. Functional characterization of the Sindbis virus E2 glycoprotein by transposon linker-insertion mutagenesis

    SciTech Connect

    Navaratnarajah, Chanakha K.; Kuhn, Richard J. . E-mail: kuhnr@purdue.edu

    2007-06-20

    The glycoprotein envelope of alphaviruses consists of two proteins, E1 and E2. E1 is responsible for fusion and E2 is responsible for receptor binding. An atomic structure is available for E1, but one for E2 has not been reported. In this study, transposon linker-insertion mutagenesis was used to probe the function of different domains of E2. A library of mutants, containing 19 amino acid insertions in the E2 glycoprotein sequence of the prototype alphavirus, Sindbis virus (SINV), was generated. Fifty-seven independent E2 insertions were characterized, of which more than half (67%) gave rise to viable virus. The wild-type-like mutants identify regions that accommodate insertions without perturbing virus production and can be used to insert targeting moieties to direct SINV to specific receptors. The defective and lethal mutants give insight into regions of E2 important for protein stability, transport to the cell membrane, E1-E2 contacts, and receptor binding.

  10. Foreign Glycoproteins Can Be Actively Recruited to Virus Assembly Sites during Pseudotyping▿

    PubMed Central

    Jorgenson, Rebecca L.; Vogt, Volker M.; Johnson, Marc C.

    2009-01-01

    Retroviruses like human immunodeficiency virus type 1 (HIV-1), as well as many other enveloped viruses, can efficiently produce infectious virus in the absence of their own surface glycoprotein if a suitable glycoprotein from a foreign virus is expressed in the same cell. This process of complementation, known as pseudotyping, often can occur even when the glycoprotein is from an unrelated virus. Although pseudotyping is widely used for engineering chimeric viruses, it has remained unknown whether a virus can actively recruit foreign glycoproteins to budding sites or, alternatively, if a virus obtains the glycoproteins through a passive mechanism. We have studied the specificity of glycoprotein recruitment by immunogold labeling viral glycoproteins and imaging their distribution on the host plasma membrane using scanning electron microscopy. Expressed alone, all tested viral glycoproteins were relatively randomly distributed on the plasma membrane. However, in the presence of budding HIV-1 or Rous sarcoma virus (RSV) particles, some glycoproteins, such as those encoded by murine leukemia virus and vesicular stomatitis virus, were dramatically redistributed to viral budding sites. In contrast, the RSV Env glycoprotein was robustly recruited only to the homologous RSV budding sites. These data demonstrate that viral glycoproteins are not in preformed membrane patches prior to viral assembly but rather that glycoproteins are actively recruited to certain viral assembly sites. PMID:19224995

  11. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

    SciTech Connect

    Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G. . E-mail: vtgusk@lsu.edu

    2007-04-10

    The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

  12. Evaluation of the presence of arabinogalactan proteins and pectins during Quercus suber male gametogenesis

    PubMed Central

    Costa, Mário Luís; Sobral, Rómulo; Costa, Maria Manuela Ribeiro; Amorim, Maria Isabel; Coimbra, Sílvia

    2015-01-01

    Background and Aims Quercus suber (cork oak) is a dominant tree of the Fagaceae in forests of the south-west Iberian Peninsula. It is monoecious with a long progamic phase that provides a comprehensive system for comparative studies in development and sexual reproduction. In this study the distribution of arabinogalactan protein (AGPs) and pectin epitopes in anthers of Q. suber was assessed to map these hydroxyproline-rich glycoproteins and the galacturonate-rich acidic polysaccharides during pollen development. Methods Immunolocalization in male flowers was performed with a set of monoclonal antibodies directed against the carbohydrate moiety that recognizes AGPs and pectins. To identify AGP genes involved in cork oak male flower development, a search was conducted for annotated AGP genes in the available transcriptome data of the Cork Oak EST Consortium database (www.corkoakdb.org). Key Results Ubiquitous labelling in all cell types was obtained with anti-homogalacturan antibodies for methyl-esterified pectins. In contrast, the antibody that labelled non-methyl-esterified homogalacturans had a preferential presence in microsporocyte cells walls at the beginning of pollen development. Intense labelling was obtained with anti-AGP antibodies both in the tapetum and in the intine wall near the pollen apertures and later in the generative cell wall and vegetative cell. Evaluation of the putative AGPs highly expressed in the male gametophyte was achieved by quantitative RT-PCR analysis in male and female cork oak flowers. Conclusions Four putative AGP genes were identified that are preferentially expressed in the male flower compared with the female flower. The putative Arabidopsis thaliana orthologues of these genes are associated with preferential expression in pollen, suggesting that the AGPs probably play a significant role in cork oak reproduction. PMID:25452249

  13. Boronate affinity monolith with a gold nanoparticle-modified hydrophilic polymer as a matrix for the highly specific capture of glycoproteins.

    PubMed

    Wu, Ci; Liang, Yu; Zhao, Qun; Qu, Yanyan; Zhang, Shen; Wu, Qi; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2014-07-07

    As low abundance is the great obstacle for glycoprotein analysis, the development of materials with high efficiency and selectivity for glycoprotein enrichment is a prerequisite in glycoproteome research. Herein, we report a new kind of hydrophilic boronate affinity monolith by attaching 4-mercaptophenylboronic acid (MPBA) with 2-mercaptoethylamine (MPA) on the gold nanoparticle-modified poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate)) monolith for glycoprotein enrichment. With poly(ethylene glycol) diacrylate as the cross-linker and the further modification of gold nanoparticles, the matrix has advantages of good hydrophilicity and enhanced surface area, which are beneficial to improve the enrichment selectivity and efficiency for glycoproteins. The attachment of MPBA and MPA provide intramolecular BN coordination, which could further enhance the specificity of glycoprotein capture. Such a boronate affinity monolith was applied to enrich horseradish peroxidase (HRP) from the mixture of HRP and bovine serum albumin (BSA), and high selectivity was obtained even at a mass ratio of 1:1000. In addition, the binding capacity of ovalbumin on such monolith reached 390 μg g(-1) . Furthermore, the average recovery of HRP on the prepared affinity monoliths was (84.8±1.9) %, obtained in three times enrichment with the same column. Finally, the boronate affinity monolith was successfully applied for the human-plasma glycoproteome analysis. As a result, 160 glycoproteins were credibly identified from 9 μg of human plasma, demonstrating the great potential of such a monolith for large-scale glycoproteome research.

  14. SJSZ glycoprotein (38 kDa) inhibits cell cycle and oxidative stress in N-methyl-N'-nitro-N-nitrosoguanidine-induced ICR mice.

    PubMed

    Lee, Jin; Lim, Kye-Taek

    2013-05-01

    The initiation stage of liver cancer is closely related to abnormal cell proliferation as observed for other types of carcinogenesis. Recently, we isolated a glycoprotein from Styrax japonica Siebold et al Zuccarini (SJSZ glycoprotein), which consists of a carbohydrate moiety (52.64%) and a protein moiety (47.36%). In this study, the antitumoric mechanism of SJSZ glycoprotein during the initiation stage in N-Methyl-N`-nitro-N-nitrosoguanidine (MNNG; 40 mg/kg, BW)-induced ICR was investigated. First, we evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), thiobarbituric acid-reactive substances (TBARS), and activities of antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)] in mouse liver tissue and serum. The alpha-fetoprotein (AFP), cell cycle-related factors [cyclin D1/ cyclin dependent kinase (CDK) 4], cell cycle inhibitors (CKIs; p53, p21, and p27), and proliferating cell nuclear antigen (PCNA) were then assessed using Western Blot analysis. The results of this analysis showed that the SJSZ glycoprotein (10 mg/kg, BW) decreased the levels of LDH, ALT, TBARS, and the expression of AFP but it increased the activity of hepatic anti-oxidant enzymes (SOD, GPx and CAT). In addition, the SJSZ glycoprotein (10 mg/kg, BW)was shown to decrease the expression of cyclin D1/CDK4 and PCNA and increase the expression of CKIs (p53, p21, and p27). The results in this study indicate that the SJSZ glycoprotein displays anti-oxidative stress and anti-cell proliferation activity in MNNG induced ICR.

  15. 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.

  16. 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

  17. Interaction of forskolin with the P-glycoprotein multidrug transporter

    SciTech Connect

    Ming s, D.I.; Seamon, K.B. ); Speicher, L.A.; Tew, K.D. ); Ruoho, A.E. )

    1991-08-27

    Forskolin and 1,9-dideoxyforskolin, an analogue that does not activate adenylyl cyclase, were tested for their ability to enhance the cytotoxic effects of adriamycin in human ovarian carcinoma cells, SKOV3, which are sensitive to adriamycin and express low levels of P-glycoprotein, and a variant cell line, SKVLB, which overexpresses the P-glycoprotein and has the multidrug reing ance (MDR) phenotype. Forskolin and 1,9-dideoxyforskolin both increased the cytotoxic effects of adriamycin in SKVLB cells, yet had no effect on SKOV3 cells. Two photoactive derivatives of forskolin have been synthesized, 7-O-((2-(3-(4-azido-3-({sup 125}I)iodophenyl)propionamido)ethyl)carbamyl)forskolin, {sup 125}I-6-AIPP-Fsk, and 6-O-((2-(3-(4-azido-3-({sup 125}I)iodophenyl)propionamido)ethyl)carbamyl)forskolin, {sup 125}I-6-AIPP-Fsk, which exhibit specificity for labeling the glucose transporter and aing lyl cyclase, respectively. Both photolabels identified a 140-kDa protein in membranes from SKVLB cells whose labeling was inhibited by forskolin and 1,9-dideoxyforskolin. The data are consistent with forskolin binding to the P-glycoprotein analogous to that of other chemosensitizing drugs that have been shown to partially reverse MDR. The ability of forskolin photolabels to specifically label the transporter, the adenylyl cyclase, and the P-glycoprotein suggests that these proteins may share a common biing g domain for forskolin analogues.

  18. 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.

  19. NEW CLINICALLY SIGNIFICANT METHOD OF DETERMINING GLYCOPROTEINS IN BLOOD SERUM

    DTIC Science & Technology

    The reaction with ammonium molybdate is a simple and at the same time a very sensitive method for determining the level of glycoproteins in the blood ... serum , which has a number of advantages over the diphehylamine reaction. The reaction with ammonium molybdate is a valuable supplementary test for

  20. Glycoproteins identified from heart failure and treatment models.

    PubMed

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

    2015-01-01

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

  1. 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.

  2. Synthesis of cell envelope glycoproteins of Cryptococcus laurentii

    PubMed Central

    Schutzbach, John; Ankel, Helmut; Brockhausen, Inka

    2007-01-01

    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 C. laurentii is an excellent model for studies of polysaccharide and glycoprotein synthesis in the 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

  3. 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.

  4. Glycoprotein (90 kDa) isolated from Opuntia ficus-indica var. saboten MAKINO lowers plasma lipid level through scavenging of intracellular radicals in Triton WR-1339-induced mice.

    PubMed

    Oh, Phil-Sun; Lim, Kye-Taek

    2006-07-01

    The Opuntia ficus-indica var. saboten MAKINO (OFI) has been traditionally used as health food and herbal agent in folk medicine in Korea. In this study, we investigated whether the OFI glycoprotein has antioxidative activity and hypolipidemic effect on Triton WR-1339-induced A/J mice. The OFI glycoprotein inhibits the production of reactive oxygen species (ROS) generated by glucose/glucose oxidase (G/GO) in BNL CL.2 cells. With its antioxidative property, the mice were orally administered in the OFI glycoprotein [50 mg/kg body weight (BW)] for two weeks. Our finding resulted in a significant decrease of plasma lipid levels in Triton WR-1339-treated mice such as total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL). Indeed, mice which induced by Triton WR-1339 were significantly increased the levels of TC, TG and LDL, whereas the high-density lipoprotein (HDL) level obviously decreased. However, the values were reversed at pretreatment with OFI glycoprotein in Triton WR-1339-treated mice. The data also showed that pretreatment with OFI glycoprotein resulted in decrease of thiobarbituric acid-reactive substances (TBARS) level and in increase of nitric oxide (NO) amount in presence of Triton WR-1339-treated mice, while the activities of antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] were augmented. Therefore, we speculate that the OFI glycoprotein would be effective in lowering of plasma lipid levels.

  5. Interaction of digitalis-like compounds with p-glycoprotein.

    PubMed

    Gozalpour, Elnaz; Wittgen, Hanneke G M; van den Heuvel, Jeroen J M W; Greupink, Rick; Russel, Frans G M; Koenderink, Jan B

    2013-02-01

    Digitalis-like compounds (DLCs), or cardiac glycosides, are produced and sequestered by certain plants and animals as a protective mechanism against herbivores or predators. Currently, the DLCs digoxin and digitoxin are used in the treatment of cardiac congestion and some types of cardiac arrhythmia, despite a very narrow therapeutic index. P-glycoprotein (P-gp; ABCB1) is the only known ATP-dependent efflux transporter that handles digoxin as a substrate. Ten alanine mutants of human P-gp drug-binding amino acids-Leu(65), Ile(306), Phe(336), Ile(340), Phe(343), Phe(728), Phe(942), Thr(945), Leu(975), and Val(982)-were generated and expressed in HEK293 cells with a mammalian baculovirus system. The uptake of [(3)H]-N-methyl-quinidine (NMQ), the P-gp substrate in vesicular transport assays, was determined. The mutations I306A, F343A, F728A, T945A, and L975A abolished NMQ transport activity of P-gp. For the other mutants, the apparent affinities for six DLCs (cymarin, digitoxin, digoxin, peruvoside, proscillaridin A, and strophanthidol) were determined. The affinities of digoxin, proscillaridin A, peruvoside, and cymarin for mutants F336A and I340A were decreased two- to fourfold compared with wild type, whereas that of digitoxin and strophanthidol did not change. In addition, the presence of a hydroxyl group at position 12β seems to reduce the apparent affinity when the side chain of Phe(336) and Phe(942) is absent. Our results showed that a δ-lactone ring and a sugar moiety at 3β of the steroid body are favorable for DLC binding to P-gp. Moreover, DLC inhibition is increased by hydroxyl groups at positions 5β and 19, whereas inhibition is decreased by those at positions 1β, 11α, 12β, and 16β. The understanding of the P-gp-DLC interaction improves our insight into DLCs toxicity and might enhance the replacement of digoxin with other DLCs that have less adverse drug effects.

  6. Surface-exposed glycoproteins of hyperthermophilic Sulfolobus solfataricus P2 show a common N-glycosylation profile.

    PubMed

    Palmieri, Gianna; Balestrieri, Marco; Peter-Katalinić, Jasna; Pohlentz, Gottfried; Rossi, Mosè; Fiume, Immacolata; Pocsfalvi, Gabriella

    2013-06-07

    Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the N-glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of S. solfataricus, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex4(GlcNAc)2 plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of S. acidocaldarius, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of S. solfataricus are N-glycosylated by the same sulfated oligosaccharide and we identified 56 N-glycosylation sites in this subproteome.

  7. The cholesterol-binding motif of the HIV-1 glycoprotein gp41 regulates lateral sorting and oligomerization.

    PubMed

    Schwarzer, Roland; Levental, Ilya; Gramatica, Andrea; Scolari, Silvia; Buschmann, Volker; Veit, Michael; Herrmann, Andreas

    2014-10-01

    Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.

  8. One-step post-imprint modification achieve dual-function of glycoprotein fluorescent sensor by "Click Chemistry".

    PubMed

    Zhao, Tao; Wang, Junping; He, Jianli; Deng, Qiliang; Wang, Shuo

    2017-05-15

    A novel molecular imprinting fluorescent sensor synthesized avoiding complicated design and synthesis of a fluorescent functional monomer, and without the extra cost of additional fluorescence materials (quantum dots, carbon dots and fluorescein), only by a click reaction making the common protein imprinting polymers upgraded to a fluorescent sensing material which can be applied to the fast detection of glycoproteins. In this study, a common protein imprinted polymer (protein-IP) which containing a disulfide bond in the imprinted cavities was first synthesized, then the disulfide linkage was cleaved by reduction, the exposed thiol groups were used for the click reaction to introduce 4-vinyl phenylboronic acid to improve the imprinted recognition for glycoproteins and convert recognition behavior into fluorescent signal simultaneously in one-step site-specific post-imprinting modifications (PIMs). Good linear relationship was obtained in the range of 0.1-10mgmL(-1) by fluorescence assay, comparison of the adsorption capacity of the MIPs before and after modification proved that the method has no negative impact on the imprinting effect and exhibit good imprinting effect for glycoproteins of different molecular size and weight.

  9. The first biantennary bacterial secondary cell wall polymer and its influence on S-layer glycoprotein assembly.

    PubMed Central

    Steindl, Christian; Schäffer, Christina; Wugeditsch, Thomas; Graninger, Michael; Matecko, Irena; Müller, Norbert; Messner, Paul

    2002-01-01

    The cell surface of Aneurinibacillus thermoaerophilus DSM 10155 is covered with a square surface (S)-layer glycoprotein lattice. This S-layer glycoprotein, which was extracted with aqueous buffers after a freeze-thaw cycle of the bacterial cells, is the only completely water-soluble S-layer glycoprotein to be reported to date. The purified S-layer glycoprotein preparation had an overall carbohydrate content of 19%. Detailed chemical investigations indicated that the S-layer O-glycans of previously established structure accounted for 13% of total glycosylation. The remainder could be attributed to a peptidoglycan-associated secondary cell wall polymer. Structure analysis was performed using purified secondary cell wall polymer-peptidoglycan complexes. NMR spectroscopy revealed the first biantennary secondary cell wall polymer from the domain Bacteria, with the structure alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->4)-[alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->4)-beta-L-Gal p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)]-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-beta-L-Man p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->3)-alpha-L-Glc p NAc-(1-->O)-PO(2)(-)-O-PO(2)(-)-(O-->6)-MurNAc- (where MurNAc is N -acetylmuramic acid). The neutral polysaccharide is linked via a pyrophosphate bond to the C-6 atom of every fourth N -acetylmuramic acid residue, in average, of the A1gamma-type peptidoglycan. In vivo, the biantennary polymer anchored the S-layer glycoprotein very effectively to the cell wall, probably due to the doubling of motifs for a proposed lectin-like binding between the polymer and the N-terminus of the S-layer protein. When the cellular support was removed during S-layer glycoprotein isolation, the co-purified polymer mediated the solubility of the S

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

    PubMed

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

    2011-06-01

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

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

    PubMed

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

    2011-07-01

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

  12. Glycoprotein Synthesis in Maize Endosperm Cells

    PubMed Central

    Riedell, Walter E.; Miernyk, Jan A.

    1988-01-01

    Microsomal membrane preparations from maize (Zea mays L., inbred A636) endosperm cultures contained enzymes that transferred sugar moieties from uridine diphosphate-N-acetylglucosamine, guanosine diphosphate-mannose, and uridine diphosphate-glucose to dolichol-phosphate. These enzyme activities were characterized with respect to detergent and pH optima, substrate kinetic constants, and product and antibiotic inhibition constants. It was demonstrated by mild acid hydrolysis and high performance liquid chromatography that the products of the N-acetylglucosamine transferases were N-acetylglucosamine-pyrophosphoryl-dolichol and N,N′-diacetyl-chitobiosyl-pyrophosphoryl-dolichol and that the product of the mannose transferase was mannosyl-phosphoryl-dolichol. A large proportion of the products of the glucose transferase activity was stable to mild acid hydrolysis. However, the proportion that was labile was identified as glucosyl-phosphoryl-dolichol. Rate zonal sedimentation and isopycnic banding in linear sucrose density gradients in the presence of 1 millimolar ethylenediaminetetraacetic acid indicated that the glycosyltransferase activities were located in the endoplasmic reticulum. The glycosyltransferases were not solubilized by 500 millimolar KCl or by sequential washes with tris-(hydroxymethyl)aminomethane and water, treatments that release peripheral membrane proteins. Solubilization was achieved with low concentrations of Triton X-100. When sealed microsomal vesicles were incubated with trypsin for 30 minutes in absence of detergent, the activity of N-acetylglucosaminyl-transferase was substantially reduced, while the activity of the glucosyl-transferase was somewhat reduced. Activity of the mannosyl-transferase was resistant to inactivation by incubation with trypsin unless Triton was present. PMID:16666157

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

    PubMed

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

    2013-09-01

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

  14. Constituents of Carpobrotus edulis inhibit P-glycoprotein of MDR1-transfected mouse lymphoma cells.

    PubMed

    Martins, A; Vasas, A; Schelz, Zs; Viveiros, M; Molnár, J; Hohmann, J; Amaral, L

    2010-03-01

    A bioassay-guided separation protocol, including the testing of the extracts, fractions and pure compounds for their ability to inhibit P-glycoprotein (the efflux pump responsible for the multidrug resistance of the used cell line) of mouse lymphoma cells containing the human efflux pump gene MDR1, led to the isolation of seven compounds from the chloroform and ethyl acetate soluble fractions of the methanolic extract of Carpobrotus edulis. The compounds were identified by 1D, 2D NMR and MS investigations as triterpens (beta-amyrin, uvaol and oleanolic acid), monogalactosyldiacylglycerol, catechin, epicatechin and procyanidin B5. Uvaol was the most effective and promising compound in the reversal of multidrug resistance in MDR mouse lymphoma cell line.

  15. From the ganglioside GQ1balpha to glycomimetic antagonists of the myelin-associated glycoprotein (MAG).

    PubMed

    Ernst, Beat; Schwardt, Oliver; Mesch, Stefanie; Wittwer, Matthias; Rossato, Gianluca; Vedani, Angelo

    2010-01-01

    The tetrasaccharide 4, a substructure of ganglioside GQ1balpha, shows a remarkable affinity for the myelin-associated glycoprotein (MAG) and was therefore selected as starting point for a lead optimization program. In our search for structurally simplified and pharmacokinetically improved mimics of 4, antagonists with modifications of the core disaccharide Galbeta(1-3)GalNAc, as well as the terminal alpha(2-3)- and the internal alpha(2-6)-linked neuraminic acid were synthesized and tested in target-based binding assays. Compared to the reference tetrasaccharide 4, the most potent antagonist 17 exhibits a 360-fold improved affinity. Furthermore, pharmacokinetic parameters such as stability in the cerebrospinal fluid, logD and permeation through the BBB indicate the drug-like properties of antagonist 17.

  16. Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

    PubMed Central

    Geisler, Christoph; Jarvis, Donald L.

    2012-01-01

    The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2′-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems. PMID:23022569

  17. Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2

    SciTech Connect

    Eisenberg, R.J.; Long, D.; Hogue-Angeletti, R.; Cohen, G.H.

    1984-01-01

    Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. The authors caried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared out sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells.

  18. The selective expression of distinct fucosylated glycoproteins on murine T and B lymphocyte subsets.

    PubMed

    Abdul-Salam, Fatma; Mansour, Mohamed H; Al-Shemary, Tahany

    2005-01-01

    The putative expression of distinct terminally fucosylated glycoconjugates among murine lymphocyte subpopulations was sought using Ulex europaeus agglutinin-I (UEA-I) and Anguilla anguilla agglutinin (AAA), each with a distinctive primary binding preference to type II and type I blood group H oligosaccharide determinants, respectively. In newly born and adult mice, direct labeling of isolated lymphocyte subsets in suspension, as well as immunohistochemical assays were indicative of the age-regulated co-expression of the UEA-I-reactive ligand among thymic epithelial cells and a subset of the mature (PNA-), medullary thymocytes. In the spleen, UEA-I-ligand expression was selectively confined to a subset of the CD4+ T lymphocytes scattered around red pulp sinuses in newly born mice, but distinctively localized within the T cell-dependent periarteriolar lymphoid sheath compartment in adult mice. Among thymocytes of adult mice, two-dimensional Western blots demonstrated the expression of the UEA-I-reactive ligand among multiple isoforms of three major 50, 114 and 180kDa acidic glycoproteins, of which, heterogeneous weight and charge variants of the 114kDa component were also evident among splenocytes. The expression of the AAA-reactive ligand was, on the other hand, restricted to a single major 120 kDa acidic glycoprotein, in addition to a minor molecular weight variant of 115kDa, associated with a subset of immature IgM+ B lymphocytes localized within the red pulp, in both newly born and adult mice. The significance of these findings is discussed in relation to mechanisms that govern lymphocyte maturation, selection and migration.

  19. Synthesis of Glc1Man9-Glycoprotein Probes by a Misfolding/Enzymatic Glucosylation/Misfolding Sequence.

    PubMed

    Izumi, Masayuki; Oka, Yukiho; Okamoto, Ryo; Seko, Akira; Takeda, Yoichi; Ito, Yukishige; Kajihara, Yasuhiro

    2016-03-14

    Glycoproteins in non-native conformations are often toxic to cells and may cause diseases, thus the quality control (QC) system eliminates these unwanted species. Lectin chaperone calreticulin and glucosidase II, both of which recognize the Glc1 Man9 oligosaccharide on glycoproteins, are important components of the glycoprotein QC system. Reported herein is the preparation of Glc1 Man9 -glycoproteins in both native and non-native conformations by using the following sequence: misfolding of chemically synthesized Man9 -glycoprotein, enzymatic glucosylation, and another misfolding step. By using synthetic glycoprotein probes, calreticulin was found to bind preferentially to a hydrophobic non-native glycoprotein whereas glucosidase II activity was not affected by glycoprotein conformation. The results demonstrate the ability of chemical synthesis to deliver homogeneous glycoproteins in several non-native conformations for probing the glycoprotein QC system.

  20. Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence in Trypanosoma brucei.

    PubMed

    Böhme, Ulrike; Cross, George A M

    2002-02-15

    The variant surface glycoproteins (VSG) of Trypanosoma brucei are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. All GPI-anchored proteins are synthesized with a C-terminal signal sequence, which is replaced by a GPI-anchor in a rapid post-translational transamidation reaction. VSG GPI signal sequences are extraordinarily conserved. They contain either 23 or 17 amino acids, a difference that distinguishes the two major VSG classes, and consist of a spacer sequence followed by a more hydrophobic region. The omega amino acid, to which GPI is transferred, is either Ser, Asp or Asn, the omega+2 amino acid is always Ser, and the omega+7 amino acid is almost always Lys. In order to determine whether this high conservation is necessary for GPI anchoring, we introduced several mutations into the signal peptide. Surprisingly, changing the most conserved amino acids, at positions omega+1, omega+2 and omega+7, had no detectable effect on the efficiency of GPI-anchoring or on protein abundance. Several more extensive changes also had no discernable impact on GPI-anchoring. Deleting the entire 23 amino-acid signal sequence or the 15 amino-acid hydrophobic region generated proteins that were not anchored. Instead of being secreted, these truncated proteins accumulated in the endoplasmic reticulum prior to lysosomal degradation. Replacing the GPI signal sequence with a proven cell-surface membrane-spanning domain reduced expression by about 99% and resulted not in cell surface expression but in accumulation close to the flagellar pocket and in non-lysosomal compartments. These results indicate that the high conservation of the VSG GPI signal sequence is not necessary for efficient expression and GPI attachment. Instead, the GPI anchor is essential for surface expression of VSG. However, because the VSG is a major virulence factor, it is possible that small changes in the efficiency of GPI anchoring, undetectable in our experiments, might have

  1. Homogeneous human complex-type oligosaccharides in correctly folded intact glycoproteins: evaluation of oligosaccharide influence on protein folding, stability, and conformational properties.

    PubMed

    Kajihara, Yasuhiro; Tanabe, Yasutaka; Sasaoka, Shun; Okamoto, Ryo

    2012-05-07

    The N-glycosylation of proteins is generated at the consensus sequence NXS/T (where X is any amino acid except proline) by the biosynthetic process, and occurs in the endoplasmic reticulum and Golgi apparatus. In order to investigate the influence of human complex-type oligosaccharides on counterpart protein conformation, crambin and ovomucoide, which consist of 46 and 56 amino acid residues, respectively, were selected for synthesis of model glycoproteins. These small glycoproteins were intentionally designed to be glycosylated at the α-helix (crambin: 8 position), β-sheet (crambin: 2 position) and loop position between the antiparallel β-sheets (ovomucoide: 28 position), and were synthesized by using a peptide-segment coupling strategy. After preparation of these glycosylated polypeptide chains, protein folding experiments were performed under redox conditions by using cysteine-cystine. Although the small glycoproteins bearing intentional glycosylation at the α-helix and β-sheet exhibited a suitable folding process, glycosylation at the loop position between the antiparallel β-strands caused multiple products. The conformational differences in the isolated homogeneous glycoproteins compared with non-glycosylated counterparts were evaluated by circular dichroism (CD) and NMR spectroscopy. These analyses suggested that this intentional N-glycosylation did not result in large conformational changes in the purified protein structures, including the case of glycosylation at the loop position between the antiparallel β-strands. In addition to these experiments, the conformational properties of three glycoproteins were evaluated by CD spectroscopy under different temperatures. The oligosaccharides on the protein surface fluctuated considerably; this was dependent on the increase in the solution temperature and was thought to disrupt the protein tertiary structure. Based on the measurement of the CD spectra, however, the glycoproteins bearing three disulfide

  2. The Presence of Cysteine in the Cytoplasmic Domain of the Vesicular Stomatitis Virus Glycoprotein is Required for Palmitate Addition

    NASA Astrophysics Data System (ADS)

    Rose, John K.; Adams, Gregg A.; Gallione, Carol J.

    1984-04-01

    The transmembrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is known to contain 1-2 mol of covalently linked fatty acid (palmitate) per mol of protein. G protein is oriented in cellular membranes such that the carboxyl-terminal 29 amino acids protrude into the cytoplasm. We have obtained expression in eukaryotic cells of mutagenized cDNA clones that encode VSV G proteins lacking portions of this cytoplasmic domain. Labeling of these truncated proteins with [3H]palmitate indicated that the palmitate might be linked to an amino acid residue within the first 14 residues on the carboxyl-terminal side of the transmembrane domain. Using oligonucleotide directed mutagenesis, we changed the single codon specifying cysteine in this domain to a codon specifying serine. Expression of this mutant gene results in synthesis of a G protein lacking palmitate. We suggest that linkage of palmitate to G protein is through the cysteine in the cytoplasmic domain and that such a linkage may occur in many viral and cellular glycoproteins. The G protein lacking palmitate is glycosylated and is transported normally to the cell surface.

  3. Developmental expression of a sialyltransferase responsible for sialylation of cortical alveolus glycoprotein during oogenesis in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Asahina, Shinji; Sato, Chihiro; Kitajima, Ken

    2004-08-01

    Polysialoglycoprotein (PSGP) is a major cortical alveolus glycoprotein of rainbow trout eggs that is characterized by the attachment of a polysialic acid structure to its O-glycan chains. It has been demonstrated that the polysialic acid structure is synthesized by at least three sialyltransferases mostly localized in cortical alveoli. Here we have cloned a cDNA encoding the sialyltransferase, designated rtST6GalNAc, responsible for the transfer of the first sialic acid residue onto the O-glycan chain of PSGP. This enzyme belongs to the vertebrate ST6GalNAc II family, and is strongly expressed in ovaries. Of those O-glycoproteins tested as substrates, asialo-PSGP is the best substrate. These results indicate that rtST6GalNAc is the enzyme responsible for the sialylation of PSGP during oogenesis. Furthermore, the rtST6GalNAc mRNA is expressed throughout oogenesis, is down-regulated at the late yolk vesicle stage (May), and then up-regulated during vitellogenesis (until August). This developmental profile is highly similar to that of STL2, a cortical alveolus lectin, while it is quite different from that of PSGP, which is extensively expressed at the yolk vesicle stage and down-regulated at later stages. Thus, not all cortical alveolus components are transcribed concomitantly. This is the first description of a developmental change in the transcription of a glycosyltransferase during oogenesis.

  4. Haloferax volcanii AglB and AglD are involved in N-glycosylation of the S-layer glycoprotein and proper assembly of the surface layer.

    PubMed

    Abu-Qarn, Mehtap; Yurist-Doutsch, Sophie; Giordano, Assunta; Trauner, Andrej; Morris, Howard R; Hitchen, Paul; Medalia, Ohad; Dell, Anne; Eichler, Jerry

    2007-12-14

    In this study, the effects of deleting two genes previously implicated in Haloferax volcanii N-glycosylation on the assembly and attachment of a novel Asn-linked pentasaccharide decorating the H. volcanii S-layer glycoprotein were considered. Mass spectrometry revealed the pentasaccharide to comprise two hexoses, two hexuronic acids and an additional 190 Da saccharide. The absence of AglD prevented addition of the final hexose to the pentasaccharide, while cells lacking AglB were unable to N-glycosylate the S-layer glycoprotein. In AglD-lacking cells, the S-layer glycoprotein-based surface layer presented both an architecture and protease susceptibility different from the background strain. By contrast, the absence of AglB resulted in enhanced release of the S-layer glycoprotein. H. volcanii cells lacking these N-glycosylation genes, moreover, grew significantly less well at elevated salt levels than did cells of the background strain. Thus, these results offer experimental evidence showing that N-glycosylation endows H. volcanii with an ability to maintain an intact and stable cell envelope in hypersaline surroundings, ensuring survival in this extreme environment.

  5. Pepscan mapping of viral hemorrhagic septicemia virus glycoprotein G major lineal determinants implicated in triggering host cell antiviral responses mediated by type I interferon.

    PubMed

    Chico, V; Martinez-Lopez, A; Ortega-Villaizan, M; Falco, A; Perez, L; Coll, J M; Estepa, A

    2010-07-01

    Surface glycoproteins of enveloped virus are potent elicitors of type I interferon (IFN)-mediated antiviral responses in a way that may be independent of the well-studied genome-mediated route. However, the viral glycoprotein determinants responsible for initiating the IFN response remain unidentified. In this study, we have used a collection of 60 synthetic 20-mer overlapping peptides (pepscan) spanning the full length of glycoprotein G (gpG) of viral hemorrhagic septicemia virus (VHSV) to investigate what regions of this protein are implicated in triggering the type I IFN-associated immune responses. Briefly, two regions with ability to increase severalfold the basal expression level of the IFN-stimulated mx gene and to restrict the spread of virus among responder cells were mapped to amino acid residues 280 to 310 and 340 to 370 of the gpG protein of VHSV. In addition, the results obtained suggest that an interaction between VHSV gpG and integrins might trigger the host IFN-mediated antiviral response after VHSV infection. Since it is known that type I IFN plays an important role in determining/modulating the protective-antigen-specific immune responses, the identification of viral glycoprotein determinants directly implicated in the type I IFN induction might be of special interest for designing new adjuvants and/or more-efficient and cost-effective viral vaccines as well as for improving our knowledge on how to stimulate the innate immune system.

  6. Pepscan Mapping of Viral Hemorrhagic Septicemia Virus Glycoprotein G Major Lineal Determinants Implicated in Triggering Host Cell Antiviral Responses Mediated by Type I Interferon▿

    PubMed Central

    Chico, V.; Martinez-Lopez, A.; Ortega-Villaizan, M.; Falco, A.; Perez, L.; Coll, J. M.; Estepa, A.

    2010-01-01

    Surface glycoproteins of enveloped virus are potent elicitors of type I interferon (IFN)-mediated antiviral responses in a way that may be independent of the well-studied genome-mediated route. However, the viral glycoprotein determinants responsible for initiating the IFN response remain unidentified. In this study, we have used a collection of 60 synthetic 20-mer overlapping peptides (pepscan) spanning the full length of glycoprotein G (gpG) of viral hemorrhagic septicemia virus (VHSV) to investigate what regions of this protein are implicated in triggering the type I IFN-associated immune responses. Briefly, two regions with ability to increase severalfold the basal expression level of the IFN-stimulated mx gene and to restrict the spread of virus among responder cells were mapped to amino acid residues 280 to 310 and 340 to 370 of the gpG protein of VHSV. In addition, the results obtained suggest that an interaction between VHSV gpG and integrins might trigger the host IFN-mediated antiviral response after VHSV infection. Since it is known that type I IFN plays an important role in determining/modulating the protective-antigen-specific immune responses, the identification of viral glycoprotein determinants directly implicated in the type I IFN induction might be of special interest for designing new adjuvants and/or more-efficient and cost-effective viral vaccines as well as for improving our knowledge on how to stimulate the innate immune system. PMID:20463070

  7. 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

  8. 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

  9. Ice growth in supercooled solutions of antifreeze glycoproteins

    NASA Technical Reports Server (NTRS)

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

    1987-01-01

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

  10. Structural insights into the human metapneumovirus glycoprotein ectodomain.

    PubMed

    Leyrat, Cedric; Paesen, Guido C; Charleston, James; Renner, Max; Grimes, Jonathan M

    2014-10-01

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

  11. Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

    PubMed Central

    Maturo, Joseph M.; Hollenberg, Morley D.

    1978-01-01

    In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 Å and 38 Å. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 Å) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 Å to 72 Å. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin. PMID:277909

  12. Structural Insights into the Human Metapneumovirus Glycoprotein Ectodomain

    PubMed Central

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

    2014-01-01

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

  13. Goblet Cell Glycoprotein and Tracheal Gland Hypertrophy in Rat Airways: The Effect of Tobacco Smoke with or without the Anti-Inflammatory Agent Phenylmethyloxadiazole

    PubMed Central

    Jones, R.; Bolduc, P.; Reid, L.

    1973-01-01

    A quantitative analysis has been made of tracheal gland size and of histochemical changes occurring in goblet cells of the respiratory epithelium, in rats exposed either to tobacco smoke alone or to tobacco smoke with phenylmethyloxadiazole (PMO). Exposure to tobacco smoke alone causes an increase in goblet cell number with a shift from the production of neutral to acid glycoprotein, mainly sialidase resistant sialomucin but some sialidase sensitive sialomucin and sulphomucin. Acid glycoprotein, and each of its types, appears first at the cell apex. The addition of PMO to the tobacco protects against the increase in goblet cell number but gives no protection against the shift from neutral to acid glycoprotein and causes a larger secretory mass within the goblet cell. In the tracheal gland exposure to smoke from either tobacco alone or tobacco with PMO causes a significant increase in cell size and acinar diameter and a lesser increase in lumen diameter. There is also an increase in the thickness of the gland and its depth. Each of these gland changes is more pronounced in those animals receiving PMO with the tobacco. PMID:4121724

  14. 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

  15. 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

  16. 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.

  17. Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins