Science.gov

Sample records for 1-acid glycoprotein agp

  1. Increased α1-3 fucosylation of α-1-acid glycoprotein (AGP) in pancreatic cancer.

    PubMed

    Balmaña, Meritxell; Giménez, Estela; Puerta, Angel; Llop, Esther; Figueras, Joan; Fort, Esther; Sanz-Nebot, Victoria; de Bolós, Carme; Rizzi, Andreas; Barrabés, Sílvia; de Frutos, Mercedes; Peracaula, Rosa

    2016-01-30

    Pancreatic cancer (PDAC) lacks reliable diagnostic biomarkers and the search for new biomarkers represents an important challenge. Previous results looking at a small cohort of patients showed an increase in α-1-acid glycoprotein (AGP) fucosylation in advanced PDAC using N-glycan sequencing. Here, we have analysed AGP glycoforms in a larger cohort using several analytical techniques including mass spectrometry (MS), capillary zone electrophoresis (CZE) and enzyme-linked lectin assays (ELLAs) for determining AGP glycoforms which could be PDAC associated. AGP from 31 serum samples, including healthy controls (HC), chronic pancreatitis (ChrP) and PDAC patients, was purified by immunoaffinity chromatography. Stable isotope labelling of AGP released N-glycans and their analysis by zwitterionic hydrophilic interaction capillary liquid chromatography electrospray MS (μZIC-HILIC-ESI-MS) showed an increase in AGP fucosylated glycoforms in PDAC compared to ChrP and HC. By CZE-UV analysis, relative concentrations of some of the AGP isoforms were found significantly different compared to those in PDAC and HC. Finally, ELLAs using Aleuria aurantia lectin displayed a significant increase in AGP fucosylation, before and after AGP neuraminidase treatment, in advanced PDAC compared to ChrP and HC, respectively. Altogether, these results indicate that α1-3 fucosylated glycoforms of AGP are increased in PDAC and could be potentially regarded as a PDAC biomarker. PMID:26563517

  2. Identification of alpha-1 acid glycoprotein (AGP) as a potential marker of impaired growth in the newborn piglet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two studies were conducted to investigate the relationship between the circulating levels of the acute phase proteins haptoglobin (HP) and alpha 1 acid glycoprotein (AGP) and growth potential in neonatal pigs. In runts, the circulating level of AGP, but not HP in serum of newborn piglets was higher...

  3. Transcriptional induction of the alpha-1 acid glycoprotein (AGP) gene by synergistic interaction of two alternative activator forms of AGP/enhancer-binding protein (C/EBP beta) and NF-kappaB or Nopp140.

    PubMed Central

    Lee, Y M; Miau, L H; Chang, C J; Lee, S C

    1996-01-01

    Alpha-1 acid glycoprotein/enhancer-binding protein (AGP/EBP) (C/EBPbeta), a member of the C/EBP family, is one of the key transcription factors responsible for the induction of a wide array of genes, some of which are expressed during the acute-phase response. Both activator and repressor were shown to be encoded by the intronless agp/ebp or its rat and human homologs, which contain a common bZIP domain at their C-terminal regions. Expression of the AGP gene (agp) is regulated by AGP/EBP in liver during the acute-phase response. However, the molecular mechanism for this regulation is poorly understood. The experiments reported here demonstrate that two activator forms of AGP/EBP, one of which has an additional 21 amino acids at its N-terminal region, are expressed in liver as well as in a number of cell lines. We have also demonstrated that NF-kappaB and a phosphoprotein of 140 kDa, Nopp140, interact with different AGP/EBP activators synergistically, which results in induction of the agp gene in an AGP/EBP-binding-motif-dependent manner. Furthermore, extracellular stimuli that are known to be NF-kappaB inducers can selectively activate the agp gene by cooperating with one of the two activator forms of AGP/EBP. The physiological significance of differential regulation for the function of two activator forms of AGP/EBP through selective interaction with different transcription factors is discussed. PMID:8754826

  4. Transcriptional induction of the alpha-1 acid glycoprotein (AGP) gene by synergistic interaction of two alternative activator forms of AGP/enhancer-binding protein (C/EBP beta) and NF-kappaB or Nopp140.

    PubMed

    Lee, Y M; Miau, L H; Chang, C J; Lee, S C

    1996-08-01

    Alpha-1 acid glycoprotein/enhancer-binding protein (AGP/EBP) (C/EBPbeta), a member of the C/EBP family, is one of the key transcription factors responsible for the induction of a wide array of genes, some of which are expressed during the acute-phase response. Both activator and repressor were shown to be encoded by the intronless agp/ebp or its rat and human homologs, which contain a common bZIP domain at their C-terminal regions. Expression of the AGP gene (agp) is regulated by AGP/EBP in liver during the acute-phase response. However, the molecular mechanism for this regulation is poorly understood. The experiments reported here demonstrate that two activator forms of AGP/EBP, one of which has an additional 21 amino acids at its N-terminal region, are expressed in liver as well as in a number of cell lines. We have also demonstrated that NF-kappaB and a phosphoprotein of 140 kDa, Nopp140, interact with different AGP/EBP activators synergistically, which results in induction of the agp gene in an AGP/EBP-binding-motif-dependent manner. Furthermore, extracellular stimuli that are known to be NF-kappaB inducers can selectively activate the agp gene by cooperating with one of the two activator forms of AGP/EBP. The physiological significance of differential regulation for the function of two activator forms of AGP/EBP through selective interaction with different transcription factors is discussed.

  5. 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...

  6. 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 ...

  7. 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...

  8. 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

  9. 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

  10. Activated human PMN synthesize and release a strongly fucosylated glycoform of alpha1-acid glycoprotein, which is transiently deposited in human myocardial infarction.

    PubMed

    Poland, Dennis C W; García Vallejo, Juan-Jesús; Niessen, Hans W M; Nijmeyer, Remco; Calafat, Jero; Hack, C Erik; Van het Hof, Bert; Van Dijk, Willem

    2005-08-01

    Alpha1-acid glycoprotein (AGP) is a major acute-phase protein present in human plasma as well as in polymorphonuclear leukocytes (PMN). In this report, we show that PMN synthesize a specific glycoform of AGP, which is stored in the specific and azurophilic granules. Activation of PMN results in the rapid release of soluble AGP. PMN AGP exhibits a substantially higher apparent molecular weight than plasma AGP (50-60 kD vs. 40-43 kD), owing to the presence of strongly fucosylated and sialylated polylactosamine units on its five N-linked glycans. PMN AGP is also released in vivo from activated PMN, as appeared from studies using well-characterized myocard slices of patients that had died within 2 weeks after an acute myocardial infarction. AGP was found deposited transiently on damaged cardiomyocytes in areas with infiltrating PMN only. It is interesting that this was inversely related to the deposition of activated complement C3. Strongly fucosylated and sialylated AGP glycoforms have the ability to bind to E-selectin and to inhibit complement activation. We suggest that AGP glycoforms in PMN provide an endogenous feedback-inhibitory response to excessive inflammation.

  11. Drug-binding energetics of human α-1-acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

    PubMed Central

    Huang, Johnny X.; Cooper, Matthew A.; Baker, Mark A.; Azad, Mohammad A.K.; Nation, Roger L.; Li, Jian; Velkov, Tony

    2012-01-01

    This study utilizes sensitive, modern isothermal titration calorimetric (ITC) methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α-1-acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug-AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug-binding thermodynamics were characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed enthalpy-entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (ΔH°) and favorable (positive) entropic (ΔS°) contributions to the Gibbs free energy (ΔG°). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug-serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side-chain sub-domains within the multi-lobed AGP ligand binding cavity. PMID:23192962

  12. Expression of α1-acid glycoprotein and lipopolysaccharide binding protein in visceral and subcutaneous adipose tissue of dairy cattle.

    PubMed

    Rahman, Mizanur M; Lecchi, Cristina; Sauerwein, Helga; Mielenz, Manfred; Häußler, Susanne; Restelli, Laura; Giudice, Chiara; Ceciliani, Fabrizio

    2015-02-01

    Adipose tissue is an endocrine compartment that plays an important role in immune defence by producing and releasing a wide range of proteins, including acute phase proteins (APPs). The liver is the main organ of APP synthesis, although extrahepatic production has also been reported. In the present study, expression of two APPs in dairy cattle, lipopolysaccharide binding protein (LBP) and α1-acid glycoprotein (AGP), was determined in four visceral (pericardial, mesenteric, omental and retroperitoneal) and three subcutaneous (withers, tail head and sternum) adipose tissue depots. mRNA expression was evaluated using qualitative and quantitative PCR, protein profiles were assessed by Western blot analysis and cellular localisation was determined by immunohistochemistry. The presence of LBP and AGP was demonstrated at mRNA and protein levels in all seven adipose tissue depots. Expression of AGP and LBP suggests that they may have roles as local and systemic inflammatory adipokines. PMID:25542063

  13. Serum α-1 acid glycoprotein and serum amyloid A concentrations in cats receiving antineoplastic treatment for lymphoma.

    PubMed

    Winkel, Valter M; Pavan, Tatiana L R; Wirthl, Vera A B F; Alves, Ana L N; Lucas, Silvia R R

    2015-11-01

    OBJECTIVE To characterize serum α-1 acid glycoprotein (AGP) and serum amyloid A (SAA) concentrations at diagnosis and during treatment in cats with lymphoma. ANIMALS 16 cats with various anatomic forms of lymphoma and 25 healthy cats. PROCEDURES Blood samples were collected from healthy cats once and from cats with lymphoma at diagnosis and 2-week intervals until the 12th week of antineoplastic treatment. Serum harvested from blood samples was assessed for AGP and SAA concentrations. Differences in serum AGP and SAA values were investigated between healthy cats and cats with lymphoma (at diagnosis) and, for cats with lymphoma, between diagnosis and various points during treatment. RESULTS Serum AGP and SAA concentrations were higher in cats with lymphoma at diagnosis (median, 832.60 and 1.03 μg/mL, respectively), compared with those in healthy cats (median, 269.85 and 0.10 μg/mL). Treatment resulted in a gradual decrease in serum AGP concentration after 4 weeks and in SAA concentration after 8 weeks of treatment, and these concentrations returned to values comparable with those of healthy cats by 12 weeks of treatment, by which point all cats had achieved complete remission of the disease. CONCLUSIONS AND CLINICAL RELEVANCE Serum AGP and SAA concentrations in cats with lymphoma were higher at diagnosis than after antineoplastic treatment. Decreases to values established for healthy cats corresponded with achievement of complete disease remission. Serum AGP and SAA may be useful protein markers for monitoring of antineoplastic treatment in cats with lymphoma.

  14. 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

  15. Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

    PubMed

    Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

    2016-05-15

    A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

  16. Serum albumin and α-1 acid glycoprotein impede the killing of Schistosoma mansoni by the tyrosine kinase inhibitor Imatinib

    PubMed Central

    Beckmann, Svenja; Long, Thavy; Scheld, Christina; Geyer, Rudolf; Caffrey, Conor R.; Grevelding, Christoph G.

    2014-01-01

    In the search for new drugs and drug targets to treat the flatworm disease schistosomiasis, protein kinases (PKs) have come under particular scrutiny because of their essential roles in developmental and physiological processes in schistosome parasites. In this context the application of the anti-cancer Abl tyrosine kinase (TK) inhibitor Imatinib (Gleevec/Glivec; STI-571) to adult Schistosoma mansoni in vitro has indicated negative effects on diverse physiological processes including survival. Motivated by these in vitro findings, we performed in vivo experiments in rodent models of S. mansoni infection. Unexpectedly, Imatinib had no effect on worm burden or egg-production. We found that the blood components serum albumin (SA) and alpha-1 acid glycoprotein (AGP or orosomucoid) negated Imatinib’s deleterious effects on adult S. mansoni and schistosomula (post-infective larvae) in vitro. This negative effect was partially reversed by erythromycin. AGP synthesis can increase as a consequence of inflammatory processes or infection; in addition upon infection AGP levels are 6–8 times higher in mice compared to humans. Therefore, mice and probably other rodents are poor infection models for measuring the effects of Imatinib in vivo. Accordingly, we suggest the routine evaluation of the ability of AGP and SA to block in vitro anti-schistosomal effects of small molecules like Imatinib prior to laborious and expensive animal experiments. PMID:25516839

  17. Analysis of free drug fractions in serum by ultrafast affinity extraction and two-dimensional affinity chromatography using α1-acid glycoprotein microcolumns.

    PubMed

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

    2016-02-01

    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 110-830ms during sample passage through the AGP microcolumns and allowed free drug fractions to be determined within 10-20min when using only 3-10μL 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

  18. 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.

  19. 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. PMID:27289464

  20. Amyloid fibril formation by bovine α1-acid glycoprotein in a reducing environment: The role of disulfide bridges on the observed aggregation kinetics.

    PubMed

    Baldassarre, Maurizio; Maggiore, Beatrice; Scirè, Andrea; Tanfani, Fabio

    2015-11-01

    Bovine α1-acid glycoprotein (bAGP), a thermostable counterpart of its human homologue, is a positive acute phase protein involved in binding and transportation of a large number of bio-active molecules and drugs across the body. We have investigated the effect of low pH and reducing conditions on the structure of the protein and found that it aggregates at high temperatures. The aggregates show a fibrillar structure when observed with electron microscopy. Aggregation assays using the amyloid-specific dye Thioflavin T show the presence of a lag phase which was neither abolished nor shortened when seeds were added. A priori reduction of the two disulfide bridges of bAGP, on the other hand, abolished the lag phase and reveals a connection between the kinetics of reduction and aggregation. We provide a kinetic interpretation and the corresponding rate laws allowing to model the process of fibril formation by bAGP under reducing conditions. Our interpretation allows to assess the role of disulfide bridges on the fibrillation kinetics of bAGP and can provide a more accurate interpretation of the fibrillation kinetics of other amyloidogenic proteins containing disulfide bridges.

  1. Coactivator TIF1β Interacts with Transcription Factor C/EBPβ and Glucocorticoid Receptor To Induce α1-Acid Glycoprotein Gene Expression

    PubMed Central

    Chang, Ching-Jin; Chen, Ya-Ling; Lee, Sheng-Chung

    1998-01-01

    The transcription of the α1-acid glycoprotein gene is induced by inflammatory cytokines and glucocorticoids. C/EBPβ is a major transcription factor involved in the induction of the agp gene by some cytokines. In this report, we have identified a novel transcriptional intermediary factor, TIF1β, which could enhance the transcription of the agp gene by the glucocorticoid receptor (GR) and C/EBPβ. TIF1β belongs to a subgroup of RING (really interesting new gene) finger proteins that contain a RING finger preceding two B box-type fingers and a putative coiled-coil domain (RBCC domain). Immunoprecipitation experiments showed that the interaction between GR and TIF1β is ligand independent. The overexpression of the TIF1β gene enhances GR-regulated expression in a ligand- and glucocorticoid-responsive element (GRE)-dependent manner. TIF1β can also augment C/EBPβ-mediated activity on wild-type and GRE-mutated agp genes, but this augmentation is diminished when all three C/EBPβ-binding elements are mutated. Functional and biochemical characterizations indicated that the bZIP domain of C/EBPβ and the RBCC domain, plant homeodomain finger, and bromodomain of TIF1β are crucial for the interactions of these proteins. Taken together, these results suggest that TIF1β serves as a converging mediator of signal transduction pathways of glucocorticoids and some inflammatory cytokines. PMID:9742105

  2. The effect of age and teat order on alpha1-acid glycoprotein, neutrophil-to-lymphocyte ratio, cortisol, and average daily gain in commercial growing pigs.

    PubMed

    Stull, C L; Kachulis, C J; Farley, J L; Koenig, G J

    1999-01-01

    The objectives of the study were to evaluate age and teat order on a performance trait, average daily gain, and on physiological stress indicators, alpha1-acid glycoprotein (AGP), neutrophil-to-lymphocyte ratio (N:L), and cortisol in commercial growing pigs from weaning to market age. Pigs (n = 129) from five commercial California farms were weighed and blood-sampled at 28-d intervals from 28 to 168 d of age. Laboratory assays were performed from blood samples to quantify cortisol, AGP, and N:L. Age and facility effects (P<.001), but not teat order effects (P>.05), were found for all three physiological traits and ADG. Pigs that routinely suckled from teats 1, 4, or 6 (numbered from anterior to posterior on the upper teat bank) had similar (P>.05) ADG and BW throughout the production cycle. No correlation (P> .05) was found between cortisol, AGP, and N:L. The use of these physiological and production traits as stress and health indices of growing pigs in commercial facilities has limitations in comparing data between facilities or different ages of pigs. PMID:10064029

  3. 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.

  4. Urinary monocyte chemoattractant protein 1 and alpha 1 acid glycoprotein as biomarkers of renal disease activity in juvenile-onset systemic lupus erythematosus.

    PubMed

    Watson, L; Midgley, A; Pilkington, C; Tullus, K; Marks, Sd; Holt, Rcl; Jones, Ca; Beresford, Mw

    2012-04-01

    A higher proportion of patients with juvenile-onset systemic lupus erythematosus (JSLE) will have renal involvement compared with adult-onset disease, some progressing to renal failure in adulthood. Histological examination is the gold standard for diagnosing lupus nephritis (LN), but its invasive nature limits routine use. Using cross-sectional cohort analysis, we aimed to determine whether urinary concentrations of monocyte chemoattractant protein-1 (MCP1), alpha-1-acid glycoprotein (AGP) and interferon-inducible protein 10 (IP10) are biomarkers of active LN. Sixty JSLE patients recruited to the UK JSLE Cohort Study were categorized according to the British Isles Lupus Assessment Group (BILAG) activity index. Patients with active renal JSLE (n = 8; renal BILAG score A, B) had significantly higher urinary MCP1 concentrations than patients with inactive renal disease (n = 52; renal BILAG score C, D, E; 582 pg/mg creatinine [Cr], 207 pg/mg Cr; p = 0.018) or healthy controls (n = 23; 117 pg/mg Cr; p = 0.005). Urinary AGP concentration was significantly elevated in patients with active renal disease compared with inactive renal disease (1517 ng/mg Cr, 485 ng/mg Cr; p = 0.027) or healthy controls (313 ng/mg Cr; p = 0.013). Urinary IP10 concentration was not significantly different between groups, but did strongly correlate with uMCP and uAGP levels (rho = 0.38, p = 0.009; rho = 0.33, p = 0.021). Urinary MCP1 and AGP are biomarkers of LN, providing insight into its pathophysiology. Longitudinal studies are warranted.

  5. 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.

  6. 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

  7. Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

    PubMed

    Gross, V; Tran-Thi, T A; Schwarz, R T; Elbein, A D; Decker, K; Heinrich, P C

    1986-06-15

    The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted. PMID:2947571

  8. Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

    PubMed Central

    Gross, V; Tran-Thi, T A; Schwarz, R T; Elbein, A D; Decker, K; Heinrich, P C

    1986-01-01

    The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted. Images Fig. 1. Fig. 2. Fig. 5. PMID:2947571

  9. Α₁-acid glycoprotein modulates phagocytosis and killing of Escherichia coli by bovine polymorphonuclear leucocytes and monocytes.

    PubMed

    Lecchi, Cristina; Scarafoni, Alessio; Bronzo, Valerio; Martino, Piera Anna; Cavallini, Alice; Sartorelli, Paola; Ceciliani, Fabrizio

    2013-04-01

    α1-Acid glycoprotein (AGP) is an acute phase protein that modulates innate immunity and increases in response to infection or injury. The effects of native (phosphorylated) and partially dephosphorylated AGP on the antimicrobial activities of bovine polymorphonuclear leucocytes (PMNs) and monocytes were evaluated. Native AGP inhibited phagocytosis and killing of Escherichia coli by PMNs and monocytes. Engulfment and killing of E. coli were reduced at the acute phase concentration of AGP (0.9 mg/mL) compared with a physiological concentration (0.3mg/mL). The ability of AGP to inhibit phagocytosis by monocytes and the killing of E. coli by PMNs was reduced following dephosphorylation. The findings indicate that the functions of PMNs and monocytes are differentially regulated by varying concentrations of AGP and its phosphorylation state.

  10. Bovine α₁-acid glycoprotein, a thermostable version of its human counterpart: insights from Fourier transform infrared spectroscopy and in silico modelling.

    PubMed

    Baldassarre, Maurizio; Galeazzi, Roberta; Maggiore, Beatrice; Tanfani, Fabio; Scirè, Andrea

    2014-07-01

    α1-Acid glycoprotein (AGP) is a plasma protein and a member of the acute phase response. AGP is known to bind and carry several biologically active compounds, as well as to down-modulate the immune system activities. In this work, the structure of bovine AGP has been investigated by Fourier-Transform infrared spectroscopy. A model structure has been obtained on the basis of human AGP and refined by molecular dynamics. In spite of the similar structure, bovine AGP shows an unexpectedly higher (∼20 °C) thermostability than its human counterpart. Inspection of the model structure has pointed out the presence of 12 ionic bridges and 2 sulphur-aromatic interactions, whereas only 6 ionic bridges were detected in human AGP. The high number (9) of glutamic acid residues involved in the ionic interactions might explain the significantly decreased thermostability measured at pH 5.5 (Tm ∼ 71 °C) with respect to pH 7.4 (Tm ∼ 81 °C), whereas thermostability of human AGP was only slightly affected by lowering the pH. As in human AGP and several other lipocalins, a temperature-induced molten globule state has been observed in the denaturation pathway of bovine AGP. PMID:24530968

  11. Protective effects of alpha1-acid glycoprotein and serum amyloid A on concanavalin A-induced liver failure via interleukin-6 induction by ME3738.

    PubMed

    Kuzuhara, Hiroyuki; Nakano, Yoshihisa; Yamashita, Nobuyuki; Imai, Masako; Kawamura, Yuji; Kurosawa, Tohru; Nishiyama, Shoji

    2006-07-17

    We examined whether the 22beta-methoxyolean-12-ene-3beta,24(4beta)-diol (ME3738)-mediated selective induction of interleukin-6 increased alpha1-acid glycoprotein and serum amyloid A expression, and whether these proteins protected against liver injury in vitro and in vivo. ME3738 treatment in male mice increased gene expression of alpha1-acid glycoprotein subtypes and serum amyloid A 2 genes, and plasma concentration of serum amyloid A. Treatment with alpha1-acid glycoprotein at 5 mg/animal or serum amyloid A at 0.03 and 0.1 mg/animal prior to concanavalin A administration reduced multifocal necrosis in the liver. Treatment with alpha1-acid glycoprotein and serum amyloid A, but not alpha1-antitrypsin, protected Hep G2 cells against cell injury. These results suggest that alpha1-acid glycoprotein and serum amyloid A, increased by ME3738-induced interleukin-6, might protect against concanavalin A-induced liver injury. PMID:16765939

  12. Binding of oxprenolol and propranolol to serum, albumin and alpha 1-acid glycoprotein in man and other species.

    PubMed

    Belpaire, F M; Braeckman, R A; Bogaert, M G

    1984-07-01

    Species differences in binding of basic drugs have only occasionally been studied and we have therefore measured the binding of the beta-adrenergic blockers oxprenolol and propranolol in (1) serum of healthy humans, dogs, rats and rabbits and of rabbits with experimental arthritis, (2) a solution of albumin of these species and (3) a solution of human alpha 1-AGP. In humans, dogs, rats and arthritic rabbits, binding of oxprenolol and propranolol was much higher in serum than in albumin solution; in healthy rabbits serum binding was very low and not different from albumin binding. For both drugs, concentration-dependency was seen in serum of dogs, humans and rats and of arthritic rabbits; a similar concentration-dependency was found for human alpha 1-AGP solution, but not for human albumin and for serum of healthy rabbits. Tris (2-butoxyethyl)-phosphate (TBEP), a known displacer of drugs from alpha 1-AGP in humans, decreased binding in serum of all species except the rabbit. For both beta-blockers, species differences in capacity constants were found; species differences in affinity constants were present only for propranolol. These results suggest that in humans, dog and rat, but much less in rabbits, oxprenolol and propranolol bind mainly to alpha 1-AGP and that binding to alpha 1-AGP is more important for oxprenolol than for propranolol. PMID:6743355

  13. AtAGP18 is localized at the plasma membrane and functions in plant growth and development.

    PubMed

    Zhang, Yizhu; Yang, Jie; Showalter, Allan M

    2011-04-01

    Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins (HRGPs). AtAGP17, 18 and 19 comprise the lysine-rich classical AGP subfamily in Arabidopsis. Overexpression of GFP-AtAGP17/18/19 fusion proteins in Arabidopsis revealed localization of the fusion proteins on the plant cell surface of different organs. Subcellular localization of the fusion proteins at the plasma membrane was further determined by plasmolysis of leaf trichome cells. To elucidate AtAGP17/18/19 function(s), these AGPs were expressed without the green fluorescent protein (GFP) tag under the control of 35S cauliflower mosaic virus promoter. In contrast to AtAGP17/AtAGP19 overexpressors which showed phenotypes identical to wild-type plants, AtAGP18 overexpressors displayed several phenotypes distinct from wild-type plants. Specifically, these overexpressors had smaller rosettes and shorter stems and roots, produced more branches and had less viable seeds. Moreover, these AtAGP18 overexpressors exhibited similar phenotypes to tomato LeAGP-1 overexpressors, suggesting these two AGP genes may have similar function(s) in Arabidopsis and tomato. PMID:21165646

  14. Protein binding of fentanyl and its metabolite nor-fentanyl in human plasma, albumin and α-1 acid glycoprotein.

    PubMed

    Bista, Sudeep Raj; Haywood, Alison; Hardy, Janet; Lobb, Michael; Tapuni, Angela; Norris, Ross

    2015-03-01

    1.Fentanyl is a highly lipophilic opioid commonly used to treat cancer pain. Plasma protein binding (PPB) of fentanyl in human plasma is reported as 80-85%, however it is unclear whether fentanyl binds primarily to albumin (ALB) or α-1 acid glycoprotein (AAG) and no studies have been conducted on the metabolite, nor-fentanyl. Fentanyl is also known to bind to plasticware and ultrafiltration (UF) devices which impacts adversely on binding experiments. 2.PPB of fentanyl and nor-fentanyl to ALB and AAG in isotonic phosphate buffer solution and seeded human plasma was quantified. PPB was also performed in plasma samples obtained from cancer patients receiving transdermal fentanyl. The adsorption of fentanyl and nor-fentanyl to UF devices and plasticware commonly used in PPB studies was also assessed. 3.Fentanyl was shown to bind primarily to ALB as opposed to AAG, with nor-fentanyl exhibiting negligible binding to plasma proteins. Total PPB of fentanyl was 86-89% in seeded human plasma. PPB in 56 cancer patient samples was 95.1 ± 3.52% for fentanyl and 32.4 ± 21.9% for nor-fentanyl. 4.UF was shown to be a reliable and convenient method for PPB studies, thereby removing the need for complex testing for adsorption of the drug to plasticware during UF.

  15. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    PubMed

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  16. Studying the Impact of Presence of Alpha Acid Glycoprotein and Protein Glycoprotein in Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate in the State of Qatar

    PubMed Central

    Al-Dewik, Nader I.; Jewell, Andrew P.; Yassin, Mohammed A.; Morsi, Hisham M.

    2015-01-01

    Despite the efficacy of imatinib mesylate (IM) in treating chronic myeloid leukemia (CML), there is a high degree of resistance. Alpha- 1-acid glycoprotein may reduce drug efficacy through its ability to interact with IM and blocks it from reaching its target, while protein glycoprotein (PGP) may reduce the intracellular concentration of the drug via an active pump mechanism. We thus investigated the correlation between AGP and PGP levels and the resistance/response to treatment. A total of 26 CML patients were investigated for AGP and PGP levels at diagnosis and during treatment. There was no significant difference or correlation between AGP levels and the different groups of patients. There was also no significant difference in the fluorescence intensities of PGP levels among the different patient groups. The resistance observed in our CML patient population could not be correlated with AGP and PGP levels. There was no significant pattern of AGP and PGP expression, irrespective of the response or resistance to treatment. PMID:26640393

  17. 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.

  18. 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

  19. 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.

  20. Secretion of high-mannose-type alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein by primary cultures of rat hepatocytes in the presence of the mannosidase I inhibitor 1-deoxymannojirimycin.

    PubMed

    Gross, V; Steube, K; Tran-Thi, T A; McDowell, W; Schwarz, R T; Decker, K; Gerok, W; Heinrich, P C

    1985-07-01

    Two different forms of alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein were found in primary cultures of rat hepatocytes. After a 2.5-h labeling period with [35S]methionine the high-mannose-type precursor of alpha 1-proteinase inhibitor (Mr 49000) and alpha 1-acid glycoprotein (Mr 39 000) and the mature-complex-type alpha 1-proteinase inhibitor (Mr 54 000) and alpha 1-acid glycoprotein (Mr 43 000-60 000) could be immunoprecipitated from the cells, but only the complex-type forms of the two glycoproteins were secreted into the hepatocyte media. When hepatocytes were incubated with the mannosidase I inhibitor 1-deoxymannojirimycin at a concentration of 4 mM, the 49 000-Mr form of alpha 1-proteinase inhibitor and the 39 000-Mr form of alpha 1-acid glycoprotein could be detected in the cells as well as in their media. Neither the secretion of alpha 1-proteinase inhibitor nor that of alpha 1-acid glycoprotein was impaired by 1-deoxymannojirimycin. While alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by control cells, were resistant to endoglucosaminidase H, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by hepatocytes treated with 4 mM 1-deoxymannojirimycin, could be deglycosylated by endoglucosaminidase H. When the [3H]mannose-labeled oligosaccharides of alpha 1-proteinase inhibitor, secreted by 1-deoxymannojirimycin-treated hepatocytes, were cleaved off by endoglucosaminidase H and analyzed by Bio-Gel P-4 chromatography, they eluted at the position of Man9GlcNAc, indicating that mannosidase I had been efficiently inhibited. 1-Deoxymannojirimycin did not inhibit the synthesis or the cotranslational N-glycosylation of alpha 1-proteinase inhibitor or alpha 1-acid glycoprotein. PMID:3160588

  1. Glycosylation of alpha1-acid glycoprotein in inflammatory disease: analysis by high-pH anion-exchange chromatography and concanavalin A crossed affinity immunoelectrophoresis.

    PubMed

    Rydén, I; Skude, G; Lundblad, A; Påhlsson, P

    1997-06-01

    High-pH anion-exchange chromatography with pulsed amperometric detection is a highly sensitive technique that can be used for detecting changes in sialylation and fucosylation, as well as different branching patterns of N-linked oligosaccharides in glycoproteins. We examined the N-glycans of alpha1-acid glycoprotein obtained from twelve patients with various inflammatory conditions with this technique, as well as traditional concanavalin A crossed affinity immunoelectrophoresis. We found the chromatographic profiles of N-glycans in all patients with rheumatoid arthritis to be very similar, but significantly different from normal controls. N-glycans from patients with ulcerative colitis also showed specific alterations in their chromatographic profiles. However, some heterogeneity was found between these patients, perhaps reflecting changes in glycosylation secondary to certain states of the disease, or to medical treatment. We conclude that this technique is useful for detailed mapping of glycosylation changes in alpha1-acid glycoprotein in clinical samples, and that it may be used to further increase our knowledge about glycosylation changes in response to inflammatory disease.

  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. 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

  4. Temperature Effects on Agrobacterium Phytochrome Agp1

    PubMed Central

    Njimona, Ibrahim; Lamparter, Tilman

    2011-01-01

    Phytochromes are widely distributed biliprotein photoreceptors with a conserved N-terminal chromophore-binding domain. Most phytochromes bear a light-regulated C-terminal His kinase or His kinase-like region. We investigated the effects of light and temperature on the His kinase activity of the phytochrome Agp1 from Agrobacterium tumefaciens. As in earlier studies, the phosphorylation activity of the holoprotein after far-red irradiation (where the red-light absorbing Pr form dominates) was stronger than that of the holoprotein after red irradiation (where the far red-absorbing Pfr form dominates). Phosphorylation activities of the apoprotein, far red-irradiated holoprotein, and red-irradiated holoprotein decreased when the temperature increased from 25°C to 35°C; at 40°C, almost no kinase activity was detected. The activity of a holoprotein sample incubated at 40°C was nearly completely restored when the temperature returned to 25°C. UV/visible spectroscopy indicated that the protein was not denatured up to 45°C. At 50°C, however, Pfr denatured faster than the dark-adapted sample containing the Pr form of Agp1. The Pr visible spectrum was unaffected by temperatures of 20–45°C, whereas irradiated samples exhibited a clear temperature effect in the 30–40°C range in which prolonged irradiation resulted in the photoconversion of Pfr into a new spectral species termed Prx. Pfr to Prx photoconversion was dependent on the His-kinase module of Agp1; normal photoconversion occurred at 40°C in the mutant Agp1-M15, which lacks the C-terminal His-kinase module, and in a domain-swap mutant in which the His-kinase module of Agp1 is replaced by the His-kinase/response regulator module of the other A. tumefaciens phytochrome, Agp2. The temperature-dependent kinase activity and spectral properties in the physiological temperature range suggest that Agp1 serves as an integrated light and temperature sensor in A. tumefaciens. PMID:22043299

  5. 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.

  6. 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. PMID:26851488

  7. 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.

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

    PubMed

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

    2012-03-01

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

  9. 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.

  10. Demonstration of alpha 1-acid glycoprotein (orosomucoid) by double one-dimensional slab gel electrophoresis: evidence for intra- and interindividual variability of the microheterogeneity pattern in health and disease.

    PubMed

    Altland, K; Roeder, T; Jakin, H M; Zimmer, H G; Neuhoff, V

    1982-04-01

    This double one-dimensional slab gel electrophoresis technique, in the sequence polyacrylamide gel electrophoresis followed by isoelectric focusing in polyacrylamide gels, permits the selective demonstration and comparison of the micro-heterogeneity pattern of alpha 1-acid glycoprotein (orosomucoid) in as many as 96 human plasma or serum samples on one gel. The electrophoretic analysis is performed on 8 microL of serum or plasma without prior purification. Several hundred samples can be analyzed by one investigator during a working day. Densitometric evaluation of the patterns revealed two new findings: 1) The microheterogeneity pattern can be described by a parameter that is independent of the concentration of orosomucoid in plasma: the center of density of the pattern, with the peak number as coordinate. 2) There is a considerable average shift of the center of density towards more basic components among the patterns obtained for samples from hospital patients. The results suggest that followup of the intra-individual variation of the orosomucoid pattern in health and disease might help in studying the still-uncertain function of this protein as well as the diseases affecting the pattern.

  11. 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

  12. Arabinogalactan protein 31 (AGP31), a putative network-forming protein in Arabidopsis thaliana cell walls?

    PubMed Central

    Hijazi, May; Roujol, David; Nguyen-Kim, Huan; del Rocio Cisneros Castillo, Liliana; Saland, Estelle; Jamet, Elisabeth; Albenne, Cécile

    2014-01-01

    Background and Aims Arabinogalactan protein 31 (AGP31) is a remarkable plant cell-wall protein displaying a multi-domain organization unique in Arabidopsis thaliana: it comprises a predicted signal peptide (SP), a short AGP domain of seven amino acids, a His-stretch, a Pro-rich domain and a PAC (PRP-AGP containing Cys) domain. AGP31 displays different O-glycosylation patterns with arabinogalactans on the AGP domain and Hyp-O-Gal/Ara-rich motifs on the Pro-rich domain. AGP31 has been identified as an abundant protein in cell walls of etiolated hypocotyls, but its function has not been investigated thus far. Literature data suggest that AGP31 may interact with cell-wall components. The purpose of the present study was to identify AGP31 partners to gain new insight into its function in cell walls. Methods Nitrocellulose membranes were prepared by spotting different polysaccharides, which were either obtained commercially or extracted from cell walls of Arabidopsis thaliana and Brachypodium distachyon. After validation of the arrays, in vitro interaction assays were carried out by probing the membranes with purified native AGP31 or recombinant PAC-V5-6xHis. In addition, dynamic light scattering (DLS) analyses were carried out on an AGP31 purified fraction. Key Results It was demonstrated that AGP31 interacts through its PAC domain with galactans that are branches of rhamnogalacturonan I. This is the first experimental evidence that a PAC domain, also found as an entire protein or a domain of AGP31 homologues, can bind carbohydrates. AGP31 was also found to bind methylesterified polygalacturonic acid, possibly through its His-stretch. Finally, AGP31 was able to interact with itself in vitro through its PAC domain. DLS data showed that AGP31 forms aggregates in solution, corroborating the hypothesis of an auto-assembly. Conclusions These results allow the proposal of a model of interactions of AGP31 with different cell-wall components, in which AGP31 participates in

  13. Transcriptional induction of the agp/ebp (c/ebp beta) gene by hepatocyte growth factor.

    PubMed

    Shen, B J; Chang, C J; Lee, H S; Tsai, W H; Miau, L H; Lee, S C

    1997-06-01

    Hepatocyte growth factor (HGF) is a pleiotropic factor with mitogenic, morphogenic, motogenic, cytotoxic, or growth inhibitory activity. Although the signaling of HGF is mediated through the cell membrane receptor c-Met, the molecular mechanism of downstream signal transduction remains obscure. In this report, we present evidence that shows HGF can stimulate the expression of AGP/EBP (C/EBP beta) and NF-kappaB, which are both key transcription factors responsible for the regulation of many genes under stress conditions or during the acute-phase response. Biochemical and functional analysis indicates that the HGF-responsive element is located in the region -376 to -352 (URE1) of the 5'-upstream regulatory sequence of agp/ebp. Activation of NF-kappaB by HGF was observed to precede the induction of agp/ebp. Further studies indicate that NF-kappaB can cooperate with AGP/EBP or other members of the C/EBP family to activate the agp/ebp gene in both URE1 and URE2-dependent manner. These results suggest that the induction of the agp/ebp gene by HGF is mediated at least in part by its activation of NF-kappaB. The activated NF-kappaB then interacts with AGP/EBP, resulting in the induction of agp/ebp.

  14. AGP: a multimethods web server for alignment-free genome phylogeny.

    PubMed

    Cheng, Jinkui; Cao, Fuliang; Liu, Zhihua

    2013-05-01

    Phylogenetic analysis based on alignment method meets huge challenges when dealing with whole-genome sequences, for example, recombination, shuffling, and rearrangement of sequences. Thus, various alignment-free methods for phylogeny construction have been proposed. However, most of these methods have not been implemented as tools or web servers. Researchers cannot use these methods easily with their data sets. To facilitate the usage of various alignment-free methods, we implemented most of the popular alignment-free methods and constructed a user-friendly web server for alignment-free genome phylogeny (AGP). AGP integrated the phylogenetic tree construction, visualization, and comparison functions together. Both AGP and all source code of the methods are available at http://www.herbbol.org:8000/agp (last accessed February 26, 2013). AGP will facilitate research in the field of whole-genome phylogeny and comparison.

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

    PubMed Central

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

    2015-01-01

    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-effective pharmacological tools for inhibiting AGPS, which may provide chemical scaffolds for future AGPS inhibitor development for cancer therapy. PMID:26322624

  16. 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.

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

    PubMed

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

    2014-01-01

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

  18. 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

  19. 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.

  20. CsAGP1, a Gibberellin-Responsive Gene from Cucumber Hypocotyls, Encodes a Classical Arabinogalactan Protein and Is Involved in Stem Elongation

    PubMed Central

    Park, Me Hea; Suzuki, Yoshihito; Chono, Makiko; Knox, J. Paul; Yamaguchi, Isomaro

    2003-01-01

    Fluorescence differential display was used to isolate the gibberellin (GA)-responsive gene, CsAGP1, from cucumber (Cucumis sativus) hypocotyls. A sequence analysis of CsAGP1 indicated that the gene putatively encodes a “classical” arabinogalactan protein (AGP) in cucumber. Transgenic tobacco (Nicotiana tabacum) plants overexpressing CsAGP1 under the control of the cauliflower mosaic virus 35S promoter produced a Y(βGlc)3-reactive proteoglycan in addition to AGPs present in wild-type tobacco plants. Immuno-dot blotting of the product, using anti-AGP antibodies, showed that the CsAGP1 protein had the AGP epitopes common to AGP families. The transcription level of CsAGP1 in cucumber hypocotyls increased in response not only to GA but also to indole-3-acetic acid. Although CsAGP1 is expressed in most vegetative tissues of cucumber, including the shoot apices and roots, the GA treatment resulted in an increase in the mRNA level of CsAGP1 only in the upper part of the hypocotyls. Y(βGlc)3, which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls. Transgenic plants ectopically expressing CsAGP1 showed a taller stature and earlier flowering than the wild-type plants. These observations suggest that CsAGP1 is involved in stem elongation. PMID:12644694

  1. CsAGP1, a gibberellin-responsive gene from cucumber hypocotyls, encodes a classical arabinogalactan protein and is involved in stem elongation.

    PubMed

    Park, Me Hea; Suzuki, Yoshihito; Chono, Makiko; Knox, J Paul; Yamaguchi, Isomaro

    2003-03-01

    Fluorescence differential display was used to isolate the gibberellin (GA)-responsive gene, CsAGP1, from cucumber (Cucumis sativus) hypocotyls. A sequence analysis of CsAGP1 indicated that the gene putatively encodes a "classical" arabinogalactan protein (AGP) in cucumber. Transgenic tobacco (Nicotiana tabacum) plants overexpressing CsAGP1 under the control of the cauliflower mosaic virus 35S promoter produced a Y(betaGlc)(3)-reactive proteoglycan in addition to AGPs present in wild-type tobacco plants. Immuno-dot blotting of the product, using anti-AGP antibodies, showed that the CsAGP1 protein had the AGP epitopes common to AGP families. The transcription level of CsAGP1 in cucumber hypocotyls increased in response not only to GA but also to indole-3-acetic acid. Although CsAGP1 is expressed in most vegetative tissues of cucumber, including the shoot apices and roots, the GA treatment resulted in an increase in the mRNA level of CsAGP1 only in the upper part of the hypocotyls. Y(betaGlc)(3), which selectively binds AGPs, inhibited the hormone-promoted elongation of cucumber seedling hypocotyls. Transgenic plants ectopically expressing CsAGP1 showed a taller stature and earlier flowering than the wild-type plants. These observations suggest that CsAGP1 is involved in stem elongation.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  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

    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.

  8. 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.

  9. 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.

  10. Glycoprotein synthesis

    DOEpatents

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

    2009-07-14

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

  11. 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.

  12. Redox regulation of glycogen biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803: analysis of the AGP and glycogen synthases.

    PubMed

    Díaz-Troya, Sandra; López-Maury, Luis; Sánchez-Riego, Ana María; Roldán, Miguel; Florencio, Francisco J

    2014-01-01

    Glycogen constitutes the major carbon storage source in cyanobacteria, as starch in algae and higher plants. Glycogen and starch synthesis is linked to active photosynthesis and both of them are degraded to glucose in the dark to maintain cell metabolism. Control of glycogen biosynthesis in cyanobacteria could be mediated by the regulation of the enzymes involved in this process, ADP-glucose pyrophosphorylase (AGP) and glycogen synthase, which were identified as putative thioredoxin targets. We have analyzed whether both enzymes were subjected to redox modification using purified recombinant enzymes or cell extracts in the model cyanobacterium Synechocystis sp. PCC 6803. Our results indicate that both AGP and glycogen synthases are sensitive to copper oxidation. However, only AGP exhibits a decrease in its enzymatic activity, which is recovered after reduction by DTT or reduced thioredoxin (TrxA), suggesting a redox control of AGP. In order to elucidate the role in redox control of the cysteine residues present on the AGP sequence (C45, C185, C320, and C337), they were replaced with serine. All AGP mutant proteins remained active when expressed in Synechocystis, although they showed different electrophoretic mobility profiles after copper oxidation, reflecting a complex pattern of cysteines interaction.

  13. Ether lipid generating enzyme AGPS alters the balance of structural and signaling lipids to fuel cancer pathogenicity.

    PubMed

    Benjamin, Daniel I; Cozzo, Alyssa; Ji, Xiaodan; Roberts, Lindsay S; Louie, Sharon M; Mulvihill, Melinda M; Luo, Kunxin; Nomura, Daniel K

    2013-09-10

    Aberrant lipid metabolism is an established hallmark of cancer cells. In particular, ether lipid levels have been shown to be elevated in tumors, but their specific function in cancer remains elusive. We show here that the metabolic enzyme alkylglyceronephosphate synthase (AGPS), a critical step in the synthesis of ether lipids, is up-regulated across multiple types of aggressive human cancer cells and primary tumors. We demonstrate that ablation of AGPS in cancer cells results in reduced cell survival, cancer aggressiveness, and tumor growth through altering the balance of ether lipid, fatty acid, eicosanoid, and fatty acid-derived glycerophospholipid metabolism, resulting in an overall reduction in the levels of several oncogenic signaling lipids. Taken together, our results reveal that AGPS, in addition to maintaining ether lipids, also controls cellular utilization of fatty acids, favoring the generation of signaling lipids necessary for promoting the aggressive features of cancer. PMID:23980144

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

    PubMed

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

    2014-06-01

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

  15. 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.

  16. Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion.

    PubMed

    Mancera-Arteu, Montserrat; Giménez, Estela; Barbosa, José; Sanz-Nebot, Victòria

    2016-10-12

    In this study, a ZIC-HILIC-MS methodology for the analysis of N-glycan isomers was optimized to obtain greater detection sensitivity and thus identify more glycan structures in hAGP. In a second step, this method was combined with glycan reductive isotope labelling (GRIL) through [(12)C6]/[(13)C6]-aniline and exoglycosidase digestion to characterize the different glycan isomers. The GRIL method allows the peak areas resulting from two different labelled samples to be compared, since neither retention time shifts nor variations in the ionization of glycans between these samples are obtained. First, sialic acid linkage assignations were performed for most hAGP glycan isomers with α2-3 sialidase digestion. Bi-, tri- and tetraantennary glycan isomers with different terminal sialic acid linkages to galactose (α2-3 or α2-6) were assigned, and the potential of this technique for the structural characterization of isobaric isomers was therefore demonstrated. Furthermore, fucose linkage isomers of hAGP glycans were also characterized using this isotope-labelling approach in combination with α1-3,4 fucosidase and β1-4 galactosidase digestion. α1-3 antennary fucoses and α1-6 core fucosylation were detected in hAGP fucosylated glycans. These established methodologies can be extremely useful for patho-glycomic studies to characterize glycoproteins of biomedical interest and find novel glycan isomers that could be used as biomarkers in cancer research. PMID:27662763

  17. The SAGA histone acetyltransferase complex regulates leucine uptake through the Agp3 permease in fission yeast.

    PubMed

    Takahashi, Hidekazu; Sun, Xiaoying; Hamamoto, Makiko; Yashiroda, Yoko; Yoshida, Minoru

    2012-11-01

    Metabolic responses of unicellular organisms are mostly acute, transient, and cell-autonomous. Regulation of nutrient uptake in yeast is one such rapid response. High quality nitrogen sources such as NH(4)(+) inhibit uptake of poor nitrogen sources, such as amino acids. Both transcriptional and posttranscriptional mechanisms operate in nutrient uptake regulation; however, many components of this system remain uncharacterized in the fission yeast, Schizosaccharomyces pombe. Here, we demonstrate that the Spt-Ada-Gcn acetyltransferase (SAGA) complex modulates leucine uptake. Initially, we noticed that a branched-chain amino acid auxotroph exhibits a peculiar adaptive growth phenotype on solid minimal media containing certain nitrogen sources. In fact, the growth of many auxotrophic strains is inhibited by excess NH(4)Cl, possibly through nitrogen-mediated uptake inhibition of the corresponding nutrients. Surprisingly, DNA microarray analysis revealed that the transcriptional reprogramming during the adaptation of the branched-chain amino acid auxotroph was highly correlated with reprogramming observed in deletions of the SAGA histone acetyltransferase module genes. Deletion of gcn5(+) increased leucine uptake in the prototrophic background and rendered the leucine auxotroph resistant to NH(4)Cl. Deletion of tra1(+) caused the opposite phenotypes. The increase in leucine uptake in the gcn5Δ mutant was dependent on an amino acid permease gene, SPCC965.11c(+). The closest budding yeast homolog of this permease is a relatively nonspecific amino acid permease AGP3, which functions in poor nutrient conditions. Our analysis identified the regulation of nutrient uptake as a physiological function for the SAGA complex, providing a potential link between cellular metabolism and chromatin regulation.

  18. 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.

  19. 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. PMID:19819408

  20. 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. PMID:27291677

  1. 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.

  2. dbAARD & AGP: A computational pipeline for the prediction of genes associated with age related disorders.

    PubMed

    Srivastava, Isha; Gahlot, Lokesh Kumar; Khurana, Pooja; Hasija, Yasha

    2016-04-01

    The atrocious behavioral and physiological shift with aging accelerate occurrence of deleterious disorders. Contemporary research is focused at uncovering the role of genetic associations in age-related disorders (ARDs). While the completion of the Human Genome Project and the HapMap project has generated huge amount of data on genetic variations; Genome-Wide Association Studies (GWAS) have identified genetic variations, essentially SNPs associated with several disorders including ARDs. However, a repository that houses all such ARD associations is lacking. The present work is aimed at filling this void. A database, dbAARD (database of Aging and Age Related Disorders) has been developed which hosts information on more than 3000 genetic variations significantly (p-value <0.05) associated with 51 ARDs. Furthermore, a machine learning based gene prediction tool AGP (Age Related Disorders Gene Prediction) has been constructed by employing rotation forest algorithm, to prioritize genes associated with ARDs. The tool achieved an overall accuracy in terms of precision 75%, recall 76%, F-measure 76% and AUC 0.85. Both the web resources have been made available online at http://genomeinformatics.dce.edu/dbAARD/ and http://genomeinformatics.dce.edu/AGP/ respectively for easy retrieval and usage by the scientific community. We believe that this work may facilitate the analysis of plethora of variants associated with ARDs and provide cues for deciphering the biology of aging. PMID:26836976

  3. 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

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Recommendations for standardizing glucose reporting and analysis to optimize clinical decision making in diabetes: the Ambulatory Glucose Profile (AGP).

    PubMed

    Bergenstal, Richard M; Ahmann, Andrew J; Bailey, Timothy; Beck, Roy W; Bissen, Joan; Buckingham, Bruce; Deeb, Larry; Dolin, Robert H; Garg, Satish K; Goland, Robin; Hirsch, Irl B; Klonoff, David C; Kruger, Davida F; Matfin, Glenn; Mazze, Roger S; Olson, Beth A; Parkin, Christopher; Peters, Anne; Powers, Margaret A; Rodriguez, Henry; Southerland, Phil; Strock, Ellie S; Tamborlane, William; Wesley, David M

    2013-03-01

    Abstract Underutilization of glucose data and lack of easy and standardized glucose data collection, analysis, visualization, and guided clinical decision making are key contributors to poor glycemic control among individuals with type 1 diabetes. An expert panel of diabetes specialists, facilitated by the International Diabetes Center and sponsored by the Helmsley Charitable Trust, met in 2012 to discuss recommendations for standardization of analysis and presentation of glucose monitoring data, with the initial focus on data derived from CGM systems. The panel members were introduced to a universal software report, the Ambulatory Glucose Profile (AGP), and asked to provide feedback on its content and functionality, both as a research tool and in clinical settings. This paper provides a summary of the topics and issues discussed during the meeting and presents recommendations from the expert panel regarding the need to standardize glucose profile summary metrics and the value of a uniform glucose report to aid clinicians, researchers, and patients.

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

    PubMed Central

    Bezkorovainy, Anatoly; Grohlich, Dietmar

    1969-01-01

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

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

    PubMed

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

    2015-11-01

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

  8. 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.

  9. Characterization of TRIF Selectivity in the AGP Class of Lipid A Mimetics: Role of Secondary Lipid Chains

    PubMed Central

    Khalaf, Juhienah K.; Bowen, William S.; Bazin, Hélène G.; Ryter, Kendal T.; Livesay, Mark T.; Ward, Jon R.; Evans, Jay T.; Johnson, David A.

    2014-01-01

    TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and D-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the D-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4–MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction. PMID:25553892

  10. Analysis of the five glycosylation sites of human alpha 1-acid glycoprotein.

    PubMed Central

    Treuheit, M J; Costello, C E; Halsall, H B

    1992-01-01

    Orosomucoid (OMD) contains complex bi-, tri- and tetra-antennary glycan chains. Subfractionation of OMD into three molecular variants using concanavalin A lectin chromatography is based on variations in these complex structures. Standard h.p.l.c. profiles have been developed to analyse the percentage and distribution of the glycoforms present at each glycosylation site in OMD and its molecular variants. The ability to quantify the glycoforms present at each site allows us to extend the earlier results of others and resolve the remaining questions concerning the glycan structures of these variants. Most significantly, the proportions of bi-, tri- and tetra-antennary chains differ at each site for the three molecular variants. The most strongly retained variant from concanavalin A is uniquely capable of possessing biantennary chains at all five sites, whereas the unretained variant is completely devoid of biantennary chains. Only glycosylation site II of the five present is 100% biantennary in the retained and weakly retained variants. In addition, the two gene products of OMD were differentially glycosylated. Molecular masses of the glycoforms were verified by matrix-assisted u.v. laser desorption mass spectrometry. On the basis of the site distribution of oligosaccharides in the variants, efforts were made to understand the factors that control the processing of the carbohydrate chains in OMD. The results indicate that the 'site-directed' model of processing offers the most consistent explanation for the structures seen at the individual glycosylation sites of OMD. PMID:1567356

  11. The orosomucoid 1 protein (α1 acid glycoprotein) is overexpressed in odontogenic myxoma

    PubMed Central

    2012-01-01

    Background Odontogenic myxoma (OM) is a benign, but locally invasive, neoplasm occurring in the jaws. However, the molecules implicated in its development are unknown. OM as well as Dental Follicle (DF), an odontogenic tissue surrounding the enamel organ, is derived from ectomesenchymal/mesencyhmal elements. To identify some protein that could participate in the development of this neoplasm, total proteins from OM were separated by two-dimensional electrophoresis and the profiles were compared with those obtained from DF, used as a control. Results We identified eight proteins with differential expression; two of them were downregulated and six upregulated in OM. A spot consistently overexpressed in odontogenic myxoma, with a molecular weight of 44-kDa and a pI of 3.5 was identified as the orosomucoid 1 protein. Western blot experiments confirmed the overexpression of this protein in odontogenic myxoma and immunohistochemical assays showed that this protein was mainly located in the cytoplasm of stellate and spindle-shaped cells of this neoplasm. Conclusion Orosomucoid 1, which belongs to a group of acute-phase proteins, may play a role in the modulation of the immune system and possibly it influences the development of OM. PMID:22888844

  12. Identification and characterization of a nucleolar phosphoprotein, Nopp140, as a transcription factor.

    PubMed

    Miau, L H; Chang, C J; Tsai, W H; Lee, S C

    1997-01-01

    Expression of the alpha-1 acid glycoprotein (AGP) gene (agp) is activated by a key transcription factor, AGP/enhancer-binding protein (AGP/EBP, commonly called C/EBP beta), in the liver during the acute-phase response. In addition to this positive regulation, agp is negatively regulated by nucleolin (T. H. Yang et al., Mol. Cell. Biol. 14:6068-6074, 1994). Other factors involve in positive regulation of the agp gene are poorly characterized. In a systematic search for factors that may interact with AGP/EBP, we have identified Nopp 140, a phosphoprotein of 140 kDa, by immunoaffinity chromatography. Nopp 140 not only functions as a transcriptional activator per se but also interacts with AGP/EBP to synergistically activate the agp gene in an AGP/EBP-binding motif-dependent manner. In addition to interacting with AGP/EBP, Nopp140 interacts specifically with TFIIB. Distinct regions of Nopp140 that interact with AGP/EBP and TFIIB have been characterized. The sequence of Nopp140 contains several stretches of serine- and acidic amino acid-rich sequences which are also found in ICP4 of herpes simplex virus type 1, a known transcription factor that interacts with TFIIB. The physical interaction between TFIIB and wild-type Nopp140 or several deletion mutants of Nopp140 correlates with the ability of Nopp140 to activate the agp gene synergistically with AGP/EBP. Thus, the molecular mechanism for agp gene activation may involve the interaction of AGP/EBP and TFIIB mediated by coactivator Nopp140.

  13. Identification and characterization of a nucleolar phosphoprotein, Nopp140, as a transcription factor.

    PubMed Central

    Miau, L H; Chang, C J; Tsai, W H; Lee, S C

    1997-01-01

    Expression of the alpha-1 acid glycoprotein (AGP) gene (agp) is activated by a key transcription factor, AGP/enhancer-binding protein (AGP/EBP, commonly called C/EBP beta), in the liver during the acute-phase response. In addition to this positive regulation, agp is negatively regulated by nucleolin (T. H. Yang et al., Mol. Cell. Biol. 14:6068-6074, 1994). Other factors involve in positive regulation of the agp gene are poorly characterized. In a systematic search for factors that may interact with AGP/EBP, we have identified Nopp 140, a phosphoprotein of 140 kDa, by immunoaffinity chromatography. Nopp 140 not only functions as a transcriptional activator per se but also interacts with AGP/EBP to synergistically activate the agp gene in an AGP/EBP-binding motif-dependent manner. In addition to interacting with AGP/EBP, Nopp140 interacts specifically with TFIIB. Distinct regions of Nopp140 that interact with AGP/EBP and TFIIB have been characterized. The sequence of Nopp140 contains several stretches of serine- and acidic amino acid-rich sequences which are also found in ICP4 of herpes simplex virus type 1, a known transcription factor that interacts with TFIIB. The physical interaction between TFIIB and wild-type Nopp140 or several deletion mutants of Nopp140 correlates with the ability of Nopp140 to activate the agp gene synergistically with AGP/EBP. Thus, the molecular mechanism for agp gene activation may involve the interaction of AGP/EBP and TFIIB mediated by coactivator Nopp140. PMID:8972203

  14. Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Chi, Lisha; Ruan, Dandan; Xuan, Yuanhu; Cong, Weitao; Jin, Litai

    2014-06-01

    A fluorescent detection method for glycoproteins in SDS-PAGE by using 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH) was developed in this study. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be specifically detected by the BH staining method, which is twofold more sensitive than that of the most commonly used Pro-Q Emerald 488 glycoprotein stain. Furthermore, the specificity of the newly developed stain for glycoproteins was demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that BH stain may provide new choices for convenient, sensitive, specific and economic visualization of gel-separated glycoproteins. PMID:24712021

  15. Fluorescent staining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Chi, Lisha; Ruan, Dandan; Xuan, Yuanhu; Cong, Weitao; Jin, Litai

    2014-06-01

    A fluorescent detection method for glycoproteins in SDS-PAGE by using 4H-[1]-benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH) was developed in this study. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be specifically detected by the BH staining method, which is twofold more sensitive than that of the most commonly used Pro-Q Emerald 488 glycoprotein stain. Furthermore, the specificity of the newly developed stain for glycoproteins was demonstrated by 1-D and 2-D SDS-PAGE, deglycosylation, glycoprotein affinity enrichment and LC-MS/MS, respectively. According to the results, it is concluded that BH stain may provide new choices for convenient, sensitive, specific and economic visualization of gel-separated glycoproteins.

  16. Analysis of Glycoproteins for Biomarker Discovery

    PubMed Central

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

    2012-01-01

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

  17. Lubrication by glycoprotein brushes.

    NASA Astrophysics Data System (ADS)

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

    2006-03-01

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

  18. Prestaining of glycoproteins in SDS-PAGE via 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide with weak influence on protein mobility.

    PubMed

    Zhu, Zhongxin; Zhou, Xuan; Wang, Yang; Yu, Qing; Zhu, Xinliang; Niu, Chao; Cong, Weitao; Jin, Litai

    2014-12-01

    A new fluorescent prestaining method for gel-separated glycoproteins in 1D and 2D SDS-PAGE was developed by using 4H-[1]-Benzopyrano[4,3-b]thiophene-2-carboxylic acid hydrazide (BH). The prestained gels were readily imaged after electrophoresis without any time-consuming steps needed for poststain. As low as 4-8 ng glycoproteins (transferrin, α1-acid glycoprotein) could be selectively detected, which is comparable to the most commonly used Pro-Q Emerald 488 glycoprotein stain. In addition, subsequent study of deglycosylation, glycoprotein affinity chromatography, and LC-MS/MS analysis were performed to confirm the specificity of the newly developed method. As a result, BH prestain provides a new choice for quick, sensitive, specific, economical, and MS compatible visualization of gel-separated glycoproteins. PMID:25229714

  19. 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.

  20. Spectroscopic Investigation on the Primary Photoreaction of Bathy Phytochrome Agp2-Pr of Agrobacterium fabrum: Isomerization in a pH-dependent H-bond Network.

    PubMed

    Singer, Patrick; Wörner, Sybille; Lamparter, Tilman; Diller, Rolf

    2016-05-01

    Bathy phytochrome Agp2 from Agrobacterium fabrum exhibits an unusually low pKa =7.6 in the Pr state in contrast to a pKa >11 in the Pfr state, indicating a pH-dependent charge distribution and H-bond network in the Pr chromophore binding pocket around neutral pH. Here, we report on ultrafast UV/Vis absorption spectroscopy of the primary Pr photoisomerization of Agp2 at pH 6 and pH 9 and upon H2 O/D2 O buffer exchange. The triexponential Pr kinetics slows down at increased pH and pronounced pH-dependent kinetic isotope effects are observed. The results on the Pr photoreaction suggest: 1) component-wise hindered dynamics on the chromophore excited-state potential energy surface at high pH and 2) proton translocation processes either via single-proton transfer or via significant reorganization of H-bond networks. Both effects reflect the interplay between the pH-dependent charge distribution in the Pr chromophore binding pocket on the one hand and chromophore excitation and its Z→E isomerization on the other hand. PMID:27075723

  1. 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.

  2. 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

  3. The Functional Property Changes of Muscular Na(v)1.4 and Cardiac Na(v)1.5 Induced by Scorpion Toxin BmK AGP-SYPU1 Mutants Y42F and Y5F.

    PubMed

    Meng, Xiangxue; Xu, Yijia; Zhao, Mingyi; Wang, Fangyang; Ma, Yuanyuan; Jin, Yao; Liu, Yanfeng; Song, Yongbo; Zhang, Jinghai

    2015-05-19

    Scorpion toxins are invaluable therapeutic leads and pharmacological tools which influence the voltage-gated sodium channels. However, the details were still unclear about the structure-function relationship of scorpion toxins on VGSC subtypes. In the previous study, we reported one α-type scorpion toxin Bmk AGP-SYPU1 and its two mutants (Y5F and Y42F) which had been demonstrated to ease pain in mice acetic acid writhing test. However, the function of Bmk AGP-SYPU1 on VGSCs is still unknown. In this study, we examined the effects of BmK AGP-SYPU1 and its two mutants (Y5F and Y42F) on hNa(v)1.4 and hNa(v)1.5 heterologously expressed CHO cell lines by using Na⁺-specialized fluorescent dye and whole-cell patch clamp. The data showed that BmK AGP-SYPU1 displayed as an activator of hNa(v)1.4 and hNa(v)1.5, which might indeed contribute to its biotoxicity to muscular and cardiac system and exhibited the functional properties of both the α-type and β-type scorpion toxin. Notably, Y5F mutant exhibited lower activatory effects on hNa(v)1.4 and hNa(v)1.5 compared with BmK AGP-SYPU1. Y42F was an enhanced activator and confirmed that the conserved Tyr42 was the key amino acid involved in bioactivity or biotoxicity. These data provided a deep insight into the structure-function relationship of BmK AGP-SYPU1, which may be the guidance for engineering α-toxin with high selectivity on VGSC subtypes. PMID:25919575

  4. 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

  5. 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

  6. 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.

  7. 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. PMID:27466363

  8. 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.

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

    PubMed

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

    2014-10-01

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

  10. Phosphorylation of the multidrug resistance associated glycoprotein

    SciTech Connect

    Mellado, W.; Horwitz, S.B.

    1987-11-03

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

  11. Structure and Function of RSV Surface Glycoproteins

    PubMed Central

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

    2014-01-01

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

  12. 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.

  13. 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.

  14. Functional roles of membrane glycoprotein CD36.

    PubMed

    Daviet, L; McGregor, J L

    1996-01-01

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

  15. 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...

  16. Differential effects of alpha 1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During bacterial-mediated diseases of dairy cows, such as mastitis, neutrophils (PMN’s) play a critical role in defending the host against invading pathogens. To carry out this role, PMN’s travel from the blood to the mammary gland in response to a variety of inflammatory mediators, including cytok...

  17. 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.

  18. Clinical applications of bacterial glycoproteins.

    PubMed

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

    2016-01-01

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

  19. [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.

  20. 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.

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

    PubMed

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

    2012-05-01

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

  2. Purification and characterization of nucleolin and its identification as a transcription repressor.

    PubMed Central

    Yang, T H; Tsai, W H; Lee, Y M; Lei, H Y; Lai, M Y; Chen, D S; Yeh, N H; Lee, S C

    1994-01-01

    Expression of the acute-phase response genes, such as that for alpha-1 acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor. Images PMID:8065340

  3. 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.

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

    PubMed Central

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

    2015-01-01

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

  5. 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.

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

    PubMed Central

    Song, Ehwang; Mechref, Yehia

    2016-01-01

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

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

    PubMed

    Song, Ehwang; Mechref, Yehia

    2015-01-01

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

  8. 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.

  9. 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.

  10. Characterization of disease-associated N-linked glycoproteins

    PubMed Central

    Tian, Yuan; Zhang, Hui

    2013-01-01

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

  11. 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.

  12. 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

  13. 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

    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

  14. 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

  15. Sweet new world: glycoproteins in bacterial pathogens.

    PubMed

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

    2003-12-01

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

  16. 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 Central

    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. PMID:25133777

  17. 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

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

    PubMed

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

    2009-08-01

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

  19. Native functionality and therapeutic targeting of arenaviral glycoproteins.

    PubMed

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

    2016-06-01

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

  20. Swainsonine: an inhibitor of glycoprotein processing.

    PubMed Central

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

    1981-01-01

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

  1. Swainsonine: an inhibitor of glycoprotein processing.

    PubMed

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

    1981-12-01

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

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

    PubMed Central

    Wojtaszek, P; Bolwell, G P

    1995-01-01

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

  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. PMID:27442696

  4. 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)

  5. Enhanced detection of glycoproteins in polyacrylamide gels.

    PubMed

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

    1988-05-01

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

  6. 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.

  7. 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.

  8. Podoplanin - a small glycoprotein with many faces

    PubMed Central

    Ugorski, Maciej; Dziegiel, Piotr; Suchanski, Jaroslaw

    2016-01-01

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

  9. Podoplanin - a small glycoprotein with many faces.

    PubMed

    Ugorski, Maciej; Dziegiel, Piotr; Suchanski, Jaroslaw

    2016-01-01

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

  10. Evidence for glycoprotein transport into complex plastids.

    PubMed

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

    2013-06-25

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

  11. Ammonia transport in the kidney by Rhesus glycoproteins

    PubMed Central

    Verlander, Jill W.

    2014-01-01

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

  12. 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.

  13. Role of envelope glycoproteins in intracellular virus maturation

    SciTech Connect

    Matsuoka, Y.

    1988-01-01

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

  14. 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.

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

    PubMed

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

    2008-01-01

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

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

    PubMed

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

    1987-05-01

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

  17. Solubilization of glycoproteins of envelope viruses by detergents

    SciTech Connect

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

    1986-11-20

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

  18. 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.

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

    PubMed Central

    Grant, Anne M. S.; Neuberger, Albert

    1973-01-01

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

  20. 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.

  1. Binding of PAI-1 to endothelial cells stimulated by thymosin beta4 and modulation of their fibrinolytic potential.

    PubMed

    Boncela, Joanna; Smolarczyk, Katarzyna; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2006-01-13

    Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.

  2. EBV glycoproteins: where are we now?

    PubMed Central

    Hutt-Fletcher, Lindsey M

    2015-01-01

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

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

    PubMed

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

    2014-01-15

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

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

    PubMed

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

    1986-01-01

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

  5. 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...

  6. 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...

  7. 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...

  8. 21 CFR 866.5440 - Beta-2-glycoprotein III immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Beta-2-glycoprotein III immunological test system....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...

  9. 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...

  10. Arabinogalactan proteins 6 and 11 are required for stamen and pollen function in Arabidopsis.

    PubMed

    Levitin, Bella; Richter, Dganit; Markovich, Inbal; Zik, Moriyah

    2008-11-01

    Successful male reproductive function in plants is dependent on the correct development and functioning of stamens and pollen. AGP6 and AGP11 are two homologous Arabidopsis genes encoding cell wall-associated arabinogalactan glycoproteins (AGPs). Both genes were found to be specifically expressed in stamens, pollen grains and pollen tubes, suggesting that these genes may play a role in male organ development and function. RNAi lines with reduced AGP6 and AGP11 expression were generated. These, together with lines harboring point mutations in the coding region of AGP6, were used to show that loss of function in AGP6 and AGP11 led to reduced fertility, at least partly as a result of inhibition of pollen tube growth. Our results also suggest that AGP6 and AGP11 play an additional role in the release of pollen grains from the mature anther. Thus, our study demonstrates the involvement of specific AGPs in pollen tube growth and stamen function.

  11. 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.

  12. 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.

  13. 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.

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

    PubMed

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

    2015-01-01

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

  15. 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.

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

    SciTech Connect

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

    1987-11-01

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

  17. KDN-containing glycoprotein from loach skin mucus.

    PubMed

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

    2001-01-01

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

  18. Concanavalin A binding glycoprotein in human stratum corneum

    SciTech Connect

    Brysk, M.M.; Miller, J.

    1984-03-01

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

  19. 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.

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

    PubMed Central

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

    1976-01-01

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

  1. Homology modelling of human P-glycoprotein.

    PubMed

    Domicevica, Laura; Biggin, Philip C

    2015-10-01

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

  2. Antifreeze glycoprotein agents: structural requirements for activity.

    PubMed

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

    2011-11-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  5. Environmental temperature and stocking density effects on acute phase proteins, heat shock protein 70, circulating corticosterone and performance in broiler chickens

    NASA Astrophysics Data System (ADS)

    Najafi, Pardis; Zulkifli, Idrus; Amat Jajuli, Nurfarahin; Farjam, Abdoreza Soleimani; Ramiah, Suriya Kumari; Amir, Anna Aryani; O'Reily, Emily; Eckersall, David

    2015-11-01

    An experiment was conducted to determine the effect of different stocking densities on serum corticosterone (CORT), ovotransferrin (OVT), α1-acid glycoprotein (AGP) and ceruloplasmin (CP) concentrations, brain heat shock protein (HSP) 70 expression and performance in broiler chickens exposed to unheated and heated conditions. Day-old chicks were stocked at 0.100 m2/bird (low density (LD)) or 0.063 m2/bird (high density (HD)), in battery cages and housed in environmentally controlled rooms. From 21 to 35 days of age, birds from each stocking density group were exposed to either 24 or 32 °C. Growth performance was recorded during the heat treatment period, and blood and brain samples were collected to determine CORT, OVT, AGP, CP and HSP 70 levels on day 35. Heat treatment but not stocking density was detrimental to growth performance. There were significant temperature × density interactions for CORT, CP and OVT on day 35. Although HD elevated CORT, CP and OVT when compared to LD, the effects of the former were more obvious under heated condition. Both temperature and density had significant effect on AGP and HSP 70. In conclusion, irrespective of temperature, high stocking density was physiologically stressful to broiler chickens, as indicated by CORT, AGP, CP, OVT and HSP 70, but not detrimental to growth performance and survivability. As it was shown in the present study, AGP, CP and OVT could be useful biomarkers to determine the effect of overcrowding and high temperature on the welfare of broiler chickens.

  6. Acute phase proteins as biomarkers of urinary tract infection in dairy cows: diagnostic and prognostic accuracy.

    PubMed

    El-Deeb, Wael M; Elmoslemany, Ahmed M

    2016-02-01

    The aims of this study were to investigate the level of acute phase proteins in dairy cows with urinary tract infection (UTI) and to evaluate their diagnostic and prognostic value. Eighty-four lactating cows with clinical and laboratory evidence of UTI and 15 healthy controls were included in this study. Serum samples were evaluated for the levels of Haptoglobin (Hp), serum amyloid A (SAA), fibrinogen (Fb), α1-Acid glycoprotein (AGP), total protein, and globulin. The diagnostic and prognostic performance of each parameter was evaluated by estimating the area under receiver operating characteristics curve (AUROC). Escherichia coli and Corynebacterium spp. were the primary bacteria associated with UTI. The levels of serum Hp, SAA, Fb, AGP, total protein, and globulin were significantly higher in UTI cows. Successfully treated cows (n = 51) had lower levels of Hp, SAA, AGP, total protein, and globulin than non-responsive cows. Overall, Hp, SAA, Fb, and AGP showed comparable diagnostic accuracy (AUROC ranged from 0.93 to 0.98). Both Hp and SAA showed high accuracy in predicting treatment response (AUROC > 0.95), whereas Fb level was of no prognostic value (AUROC = 0.48). From this study, acute phase proteins levels can be used as markers for UTI in cows and higher levels of Hp, SAA and AGP are related to poor treatment response. PMID:27348889

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

    PubMed Central

    Lafuma, C; Moczar, M; Robert, L

    1982-01-01

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

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

    PubMed

    Gupta, Satish Kumar

    2015-04-01

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

  9. Glycoproteins from the cell wall of Phaseolus coccineus.

    PubMed

    O'Neill, M A; Selvendran, R R

    1980-04-01

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

  10. P-glycoprotein activity and biological response

    SciTech Connect

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

    2005-09-01

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

  11. 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.

  12. 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

  13. 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.

  14. 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.

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

    PubMed

    Schwarz, P M; Elbein, A D

    1985-11-25

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

  16. 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.

  17. 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.

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

    SciTech Connect

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

    1985-05-01

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

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

    PubMed Central

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

    2014-01-01

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

  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. Magnetic enzyme reactors for isolation and study of heterogeneous glycoproteins

    NASA Astrophysics Data System (ADS)

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

    2005-05-01

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

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

    PubMed Central

    Clark, David; Mao, Li

    2012-01-01

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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

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

    PubMed

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

    1996-02-01

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

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

    PubMed

    Miyake, A; Beyer, J

    1974-08-16

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

  10. 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.

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

    PubMed

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

    1979-04-25

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. 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.

  14. 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.

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

    Mirković, S

    1991-01-01

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

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

    PubMed

    Gardiner, Elizabeth E; Andrews, Robert K

    2014-04-01

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

  18. [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.

  19. 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

  20. Marine Natural Products with P-Glycoprotein Inhibitor Properties

    PubMed Central

    Lopez, Dioxelis; Martinez-Luis, Sergio

    2014-01-01

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

  1. 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

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

    PubMed

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

    2007-02-01

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

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

    PubMed

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

    1989-04-01

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

  4. 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.

  5. 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

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

    PubMed

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

    1991-01-01

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

  7. 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.

  8. 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

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

    PubMed Central

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

    2008-01-01

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

  10. Identification of glycoproteins from mouse skin tumors and plasma

    PubMed Central

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

    2010-01-01

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

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

    PubMed

    Trombetta, E S; Helenius, A

    2000-03-20

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2005-11-01

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

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

    PubMed

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

    2016-11-15

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

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

    PubMed Central

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

    1989-01-01

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

  16. Glycoproteins That Exhibit Extensive Size Polymorphisms in Dictyostelium Discoideum

    PubMed Central

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

    1989-01-01

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

  17. 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.

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

    PubMed

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

    2015-10-01

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

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

    PubMed

    Lindenthal, C; Elsinghorst, E A

    1999-08-01

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

  20. Identification of a Glycoprotein Produced by Enterotoxigenic Escherichia coli

    PubMed Central

    Lindenthal, Christoph; Elsinghorst, Eric A.

    1999-01-01

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

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

    PubMed Central

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

    1986-01-01

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

  2. The effect of transport stress on turkey (Meleagris gallopavo) liver acute phase proteins gene expression.

    PubMed

    Marques, Andreia Tomás; Lecchi, Cristina; Grilli, Guido; Giudice, Chiara; Nodari, Sara Rota; Vinco, Leonardo J; Ceciliani, Fabrizio

    2016-02-01

    The aim of this study was to investigate the effects of transport-related stress on the liver gene expression of four acute phase proteins (APP), namely α1-acid glycoprotein (AGP), C-Reactive Protein (CRP), Serum Amyloid A (SAA) and PIT54, in turkeys (Meleagris gallopavo). A group of seven BUT BIG 6 commercial hens was subjected to a two-hour long road transportation and the quantitative gene expression of APP in the liver was compared to that of a non transported control group. The expression of AGP and CRP mRNA was found to be increased in animals slaughtered after road transport. The presence of AGP protein was also confirmed by immunohistochemistry and Western blotting. The results of this study showed that road-transport may induce the mRNA expression of immune related proteins. The finding that AGP and CRP can be upregulated during transport could suggest their use as for the assessment of turkey welfare during transport.

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

    SciTech Connect

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

    1990-01-01

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

  4. Stereoselective binding of chiral drugs to plasma proteins

    PubMed Central

    Shen, Qi; Wang, Lu; Zhou, Hui; Jiang, Hui-di; Yu, Lu-shan; Zeng, Su

    2013-01-01

    Chiral drugs show distinct biochemical and pharmacological behaviors in the human body. The binding of chiral drugs to plasma proteins usually exhibits stereoselectivity, which has a far-reaching influence on their pharmacological activities and pharmacokinetic profiles. In this review, the stereoselective binding of chiral drugs to human serum albumin (HSA), α1-acid glycoprotein (AGP) and lipoprotein, three most important proteins in human plasma, are detailed. Furthermore, the application of AGP variants and recombinant fragments of HSA for studying enantiomer binding properties is also discussed. Apart from the stereoselectivity of enantiomer-protein binding, enantiomer-enantiomer interactions that may induce allosteric effects are also described. Additionally, the techniques and methods used to determine drug-protein binding parameters are briefly reviewed. PMID:23852086

  5. 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.

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

    PubMed

    Kamiya, Yukiko; Satoh, Tadashi; Kato, Koichi

    2014-06-01

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

  7. Mannostatin A, a new glycoprotein-processing inhibitor

    SciTech Connect

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

    1990-10-01

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

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

    SciTech Connect

    Varner, J.E.

    1990-01-01

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

  9. HIV Entry and Envelope Glycoprotein-mediated Fusion*

    PubMed Central

    Blumenthal, Robert; Durell, Stewart; Viard, Mathias

    2012-01-01

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

  10. Ebolavirus Glycoprotein Directs Fusion through NPC1+ Endolysosomes

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Peschke, Madeleine; Hempel, Franziska

    2013-10-01

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

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

    PubMed

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

    2016-09-01

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

  13. 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-06-19

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

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

    PubMed

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

    2013-06-01

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

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

    PubMed

    Lu, Y; Ye, J; Wold, F

    1993-02-15

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

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

    PubMed

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

    2015-01-14

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

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    1995-01-01

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

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

    PubMed

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

    2016-11-01

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

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

    PubMed Central

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

    1984-01-01

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

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed

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

    2012-09-01

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

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

    PubMed

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

    2012-09-01

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

  6. Glycoprotein Gene Sequence Variation in Rhesus Monkey Rhadinovirus

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2013-11-01

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

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

    ERIC Educational Resources Information Center

    Smith, Christopher A.; And Others

    1991-01-01

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

  9. 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.

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    1987-09-01

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

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

    SciTech Connect

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

    1989-08-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-28

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

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

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

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

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

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

    PubMed

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

    1985-07-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2015-12-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1989-12-01

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

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

    PubMed

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

    2015-01-01

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

  4. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    PubMed Central

    Liu, Bo

    2013-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  7. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    SciTech Connect

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

    2014-08-26

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

  8. Crystal Structure of the Human Cytomegalovirus Glycoprotein B

    PubMed Central

    Burke, Heidi G.; Heldwein, Ekaterina E.

    2015-01-01

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

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

    PubMed

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

    2010-06-01

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

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

    PubMed

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

    2016-03-15

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

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

    PubMed

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

    2011-12-01

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

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

    PubMed

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

    2015-07-01

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

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

    PubMed

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

    2015-10-01

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

  14. 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.

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

    PubMed Central

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

    1987-01-01

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

  16. 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.

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

    PubMed

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

    2015-06-01

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

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

    PubMed Central

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

    1992-01-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    SciTech Connect

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

    1984-06-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Chuh, Kelly N; Pratt, Matthew R

    2015-10-01

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

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

    PubMed

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

    1987-11-01

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

  4. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  5. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  6. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  7. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  8. 21 CFR 866.5425 - Alpha-2-glycoproteins immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

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

    PubMed

    Feng, Daishen; Jia, Renyong

    2016-01-01

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

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

    SciTech Connect

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

    2007-09-21

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

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2015-01-01

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

  13. 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

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

    PubMed

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

    2007-01-01

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

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

    PubMed

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

    2004-01-16

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

  16. 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.

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

    PubMed

    Lim, Kye-Taek

    2005-01-01

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

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

    PubMed Central

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

    1991-01-01

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

  19. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    SciTech Connect

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

    2014-02-01

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

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

    PubMed Central

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

    2016-01-01

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

  1. 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.

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

    PubMed

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

    2012-10-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-08-01

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

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

    SciTech Connect

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

    1981-12-01

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

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

    PubMed

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

    1994-04-01

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

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

    PubMed

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

    1997-02-01

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

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

    PubMed

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

    2014-11-01

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

  11. 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.

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

    PubMed

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

    2013-01-01

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

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

    SciTech Connect

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

    2011-09-16

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

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

    Orlow, S.J.

    1986-01-01

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

  16. Effects of hydrocortisone on pulsatile pituitary glycoprotein secretion.

    PubMed

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

    1994-01-01

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

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

    PubMed

    Morla, Sudhir; Makhija, Aditi; Kumar, Sachin

    2016-06-10

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    1994-01-01

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

  1. Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-01

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

  3. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein

    PubMed Central

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

    2015-01-01

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

  4. 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.

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed

    Mecham, Robert P; Gibson, Mark A

    2015-09-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2006-09-30

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

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

    PubMed

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

    2014-02-21

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

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

    PubMed

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

    2004-08-01

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

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

    PubMed

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

    1984-04-01

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

  12. 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

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

    NASA Astrophysics Data System (ADS)

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

    2003-03-01

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

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

    PubMed

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

    1977-12-01

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

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

    PubMed

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

    1996-09-01

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

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

    PubMed

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

    1994-01-01

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

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

    PubMed

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

    2014-11-10

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

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

    PubMed

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

    2012-06-01

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

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

    PubMed Central

    1975-01-01

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

  20. The onset of the progression of acute phase response mechanisms induced by extreme impacts can be followed by the decrease in blood levels of positive acute phase proteins.

    NASA Astrophysics Data System (ADS)

    Larina, Olga; Bekker, Anna

    Studies performed at space flights and earth-based simulation models detected the plasma indices of acute phase reaction (APR), i.e. the increase of APR cytokine mediators and alterations in the production of blood acute phase proteins (APP) at the initial stages of adaptation to altered gravity conditions. Acute phase response is the principal constituent of the functional activity of innate immunity system. Changes in plasma APPs contents are considered to serve the restoration of homeostasis state. According to trends of their concentration shifts at the evolving of acute phase reaction APPs are denoted as positive, neutral, or negative. Plasma concentrations of positive acute phase proteins α1-acid glycoprotein (α1-AGP), α1-antitrypsin (α1-AT), and neutral α2-macroglobulin (α2-M) were measured in human study at 12-hour antiorthostatic position (AOP) with 15° head down tilt and hypoxia experiments at 14% oxygen in pressure chamber. Both of these impacts were shown to produce alterations in the APP levels indicative for acute phase response. Nevertheless, in AOP experiment noticeable decrease in α1-AGP concentration occurred by hour 12, and even more pronounced decline of α1-AGP and α1-AT were found on hypoxia hours 12 and 36. Acute phase proteins α1-AGP and α2-M possess the features of proteinase inhibitors. This function is implemented by the formation of complexes with the molecules of proteolytic enzymes which subsequently are removed from the blood flow. Transient decrease in plasma concentrations of protease inhibitors on early phases of APR development was reported to result from the growth of plasma protease activity due to cathepsin release from activated leukocytes, which had not yet been compensated by enhanced APP synthesis. Being a carrier protein for positively charged and neutral substances, α1-AGP shows pronounced elevation in its blood content during APR development. As assumed, it is required for the transportation of the increased

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

    PubMed Central

    1993-01-01

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

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

    SciTech Connect

    Ickes, D.E.

    1989-01-01

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

  3. 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.

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

    SciTech Connect

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

    1988-05-03

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Zhou, Guoying; Roizman, Bernard

    2001-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    1993-01-01

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

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

    PubMed

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

    1988-05-01

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

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

    PubMed

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

    2015-05-21

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

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

    PubMed

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

    2015-07-21

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

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

    SciTech Connect

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

    2013-12-14

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

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

    PubMed

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

    2015-05-21

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

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

    PubMed

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

    2016-07-01

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

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2014-02-01

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

  20. Pneumocystis carinii glycoprotein A binds macrophage mannose receptors.

    PubMed Central

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

    1995-01-01

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

  1. 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.

  2. Alterations of intestinal glycoprotein hydrolases in congenital diabetes

    SciTech Connect

    Najjar, S.M.

    1989-01-01

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

  3. Thyroid Hormone and P-Glycoprotein in Tumor Cells

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2011-07-01

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

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

    PubMed

    Gilljam, G

    1993-05-01

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

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

    SciTech Connect

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

    1991-03-26

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

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

    Kandiba, Lina; Eichler, Jerry

    2014-01-01

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

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

    PubMed

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

    1992-06-21

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

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

    PubMed

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

    1991-11-01

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

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

    PubMed Central

    Yang, Weiming; Jackson, Brooks; Zhang, Hui

    2016-01-01

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

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

    PubMed

    Su, Yuting; Xu, Yongjian

    2015-07-01

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

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

    PubMed

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

    1999-07-01

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

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    1995-09-26

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

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

    PubMed

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

    2007-07-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

    Wei, Xin; Dulberger, Charles; Li, Lingjun

    2010-01-01

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

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

    PubMed

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

    2010-03-01

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

  1. 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

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

    PubMed

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

    2001-11-10

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

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

    SciTech Connect

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

    1987-05-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    1986-01-01

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

  6. Acute phase proteins response to feed deprivation in broiler chickens.

    PubMed

    Najafi, P; Zulkifli, I; Soleimani, A F; Goh, Y M

    2016-04-01

    Feed deprivation in poultry farming imposes some degree of stress to the birds, and adversely affects their well -being. Serum levels of acute phase proteins (APP) are potential physiological indicators of stress attributed to feed deprivation. However, it has not been determined how long it takes for a measurable APP response to stressors to occur in avian species. An experiment was designed to delineate the APP and circulating levels of corticosterone responses in commercial broiler chickens to feed deprivation for 30 h. It was hypothesized that feed deprivation would elicit both APP and corticosterone (CORT) reactions within 30 h that is probably associated with stress of hunger. Twenty-one day old birds were subjected to one of 5 feed deprivation periods: 0 (ad libitum, AL), 6, 12, 18, 24, and 30 h. Upon completion of the deprivation period, blood samples were collected to determine serum CORT, ovotransferrin (OVT), α1-acid glycoprotein (AGP), and ceruloplasmin (CP) concentrations. Results showed that feed deprivation for 24 h or more caused a marked elevation in CORT (P=0.002 and P<0.0001, respectively) when compared to AL. However, increases in AGP (P=0.0005), CP (P=0.0002), and OVT (P=0.0003) were only noted following 30 h of feed deprivation. It is concluded that elicitation of AGP, CP, and OVT response may represent a more chronic stressful condition than CORT response in assessing the well-being of broiler chickens.

  7. Matrix-assisted laser desorption/ionization mass spectrometry of neutral and acidic oligosaccharides with collision-induced dissociation.

    PubMed

    Mechref, Y; Baker, A G; Novotny, M V

    1998-12-15

    Using ribonuclease B and human alpha 1-acid glycoprotein (AGP) as model glycoproteins, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with collision-induced dissociation (CID) is validated here as an effective tool for oligosaccharide sequencing. The spectra acquired for high-mannose and complex oligosaccharide structures show characteristic fragments resulting from cleavages of the glycosidic bonds and a few cross-ring cleavages. Esterification of the sialic acid residues is essential in stabilizing the acidic N-linked oligosaccharides. An important analytical feature observed in all acquired spectra is the occurrence of cleavages on the same antenna up to the branching point, as deduced from the absence of fragmentation due to the simultaneous cleavages on two or more antennas.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2007-11-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

    Strenge, K; Schauer, R; Kelm, S

    1999-02-01

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

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

    PubMed

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

    2012-12-01

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

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

    SciTech Connect

    Grotjan, H.E. Jr.

    1982-04-01

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

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

    PubMed Central

    Schochetman, G; Kortright, K; Schlom, J

    1975-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    1990-01-01

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

  17. The effect of castanospermine on the synthesis of synaptic glycoproteins by rat brain slices.

    PubMed

    Howes, S; Bissoon, N; Ito, M; Beesley, P W; Gurd, J W

    1990-03-01

    Slices were prepared from rat forebrains and the incorporation of [3H]mannose and [35S]methionine into proteins and glycoproteins determined. The incorporation of methionine continued to increase for up to 8 hours whereas mannose incorporation was maximal between 2 and 4 hours and declined thereafter. Glycopeptides prepared by pronase digestion of [3H]mannose-labeled glycoproteins were digested with endoglucosaminidase H (endo H) and analysed by gel filtration. The major endo H-sensitive oligosaccharide eluted in a position similar to standard Man8GlcNAc. In the presence of castanospermine, which inhibits glucosidase I, the first enzymatic step in the processing of N-linked oligosaccharides, a new endo H-sensitive glycan similar in size to standard Glc3Man9GlcNAc2 accumulated. Synaptic membranes (SMs) were isolated from slices which had been incubated with either [3H]mannose or [35S]methionine in the presence and absence of castanospermine. In the presence of inhibitor the relative incorporation of [3H]mannose into high-mannose glycans of synaptic glycoproteins was increased. The incorporation of newly synthesized, [35S] methionine-labeled, Con A-binding glycoproteins into SMs was not affected by the addition of inhibitor. Many of the glycoproteins synthesized in the presence of castanospermine exhibited a decreased electrophoretic mobility indicative of the presence of altered oligosaccharide chains. The results indicate that changes in oligosaccharide composition produced by castanospermine had little effect on the subsequent transport and incorporation of glycoproteins into synaptic membranes. PMID:2195374

  18. The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins.

    PubMed

    Elbein, A D; Legler, G; Tlusty, A; McDowell, W; Schwarz, R

    1984-12-01

    Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species. PMID:6240228

  19. Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

    PubMed

    Li, Y; Drone, C; Sat, E; Ghosh, H P

    1993-07-01

    The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.

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

    PubMed

    Freitag, Tanja C; Maisner, Andrea

    2016-03-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  1. Nephrotoxic potency of antisera to three rat glomerular basement membrane glycoproteins.

    PubMed Central

    Devulder, B; Bardos, P; Plouvier, B; Martin, J C; Muh, J P; Tacquet, A

    1978-01-01

    In a previous article, we cited studies which have allowed us to isolate diverse glycoproteins of the rat glomerular basement membrane (GMB) and to study their biochemical structures and antigenicity. This present study attempts to examine, using the heterologous nephrotoxic nephritis model (Masugi's nephritis) the nephrotoxicity of immune sera prepared from three of these glycoproteins: one fairly rich in collagen-like structures (A3), another lacking collagen-like structures (A1), and a third of intermediate composition (A2). The results obtained are discussed in relation to those already published concerning the nature of the GBM antigen(s) responsible for the nephrotoxicity of the sera. PMID:357054

  2. Isolation and partial chemical characterization of a 64,000-dalton glycoprotein of human cytomegalovirus

    SciTech Connect

    Clark, B.R.; Zaia, J.A.; Balce-Directo, L.; Ting, Y.P.

    1984-01-01

    A guanidinium chloride extract of (/sup 3/H)glucosamine- and (/sup 35/S)methionine-labeled virions plus dense bodies of human cytomegalovirus (Towne) was separated by reverse-phase high-pressure liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eluate revealed the major peak to be a glycoprotein with a relative mass of 64,000. This glycoprotein (HCMVgp64) was characterized by amino acid analysis and a high-pressure liquid chromatographic map of its tryptic peptides.

  3. Quantitative characterization of glycoproteins in neurodegenerative disorders using iTRAQ.

    PubMed

    Shi, Min; Hwang, Hyejin; Zhang, Jing

    2013-01-01

    Aberrant protein glycosylation has been recognized to be associated with many neurodegenerative disorders, including Alzheimer and Parkinson disease. Using mass spectrometry-based technologies to catalog and quantify glycoproteins in these diseases is expected to provide insight into not only the biochemical pathogenesis of neurodegeneration but also the biomarker discovery. This chapter describes a multidisciplinary approach to accomplish the goal of glycoprotein identification and quantification in human brain tissue and cerebrospinal fluid, which includes sample preparation, isobaric tag labeling of digested peptides, glycopeptide enrichment using hydrazide chemistry, protein/peptide identification and quantification by liquid chromatography-based high-resolution tandem mass spectrometry, as well as bioinformatic data processing. PMID:23296538

  4. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

    PubMed

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain.

  5. The B-cell lymphoma 2 (BCL2)-inhibitors, ABT-737 and ABT-263, are substrates for P-glycoprotein

    SciTech Connect

    Vogler, Meike; Dickens, David; Dyer, Martin J.S.; Owen, Andrew; Pirmohamed, Munir; Cohen, Gerald M.

    2011-05-06

    Highlights: {yields} The BCL2-inhibitor ABT-263 is a substrate for P-glycoprotein. {yields} Apoptosis is inhibited by P-glycoprotein expression. {yields} Overexpression of P-glycoprotein may contribute to resistance to ABT-263 or ABT-737. -- Abstract: Inhibition of BCL2 proteins is one of the most promising new approaches to targeted cancer therapy resulting in the induction of apoptosis. Amongst the most specific BCL2-inhibitors identified are ABT-737 and ABT-263. However, targeted therapy is often only effective for a limited amount of time because of the occurrence of drug resistance. In this study, the interaction of BCL2-inhibitors with the drug efflux transporter P-glycoprotein was investigated. Using {sup 3}H labelled ABT-263, we found that cells with high P-glycoprotein activity accumulated less drug. In addition, cells with increased P-glycoprotein expression were more resistant to apoptosis induced by either ABT-737 or ABT-263. Addition of tariquidar or verapamil sensitized the cells to BCL2-inhibitor treatment, resulting in higher apoptosis. Our data suggest that the BCL2-inhibitors ABT-737 and ABT-263 are substrates for P-glycoprotein. Over-expression of P-glycoprotein may be, at least partly, responsible for resistance to these BCL2-inhibitors.

  6. Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry

    SciTech Connect

    Follis, Kathryn E.; York, Joanne; Nunberg, Jack H. . E-mail: jack.nunberg@umontana.edu

    2006-07-05

    The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctional region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.

  7. Regulation of P-glycoprotein efflux activity by Z-guggulsterone of Commiphora mukul at the blood-brain barrier.

    PubMed

    Xu, Hong-Bin; Yu, Jing; Xu, Lu-Zhong; Fu, Jun

    2016-04-15

    The present study was to investigate whether Z-guggulsterone had the regulatory effect on the activity and expression of P-glycoprotein in rat brain microvessel endothelial cells (rBMECs) and in rat brain. Inorganic phosphate liberation assay, high performance liquid chromatography, and western blot analysis were performed to assess the P-glycoprotein ATPase activity, the accumulation of NaF and rhodamine 123, and P-glycoprotein and MRP1 expression. The results showed that Z-guggulsterone (0-100 μM) significantly enhanced basal P-glycoprotein ATPase activity in a concentration-dependent manner. Tetrandrine (0.1, 0.3, 1 μM) or cyclosporine A (0.1, 0.3, 1 μM) had non-competitively inhibitory manner on Z-guggulsterone-stimulated P-glycoprotein ATPase activity, suggesting that Z-guggulsterone might have unique binding site or regulating site on P-glycoprotein. However, Z-guggulsterone (30, 100 μM) had almost no influence on MRP1 expression in rBMECs. Further results revealed that Z-guggulsterone (50mg/kg) significantly increased the accumulation of rhodamine 123 by down-regulating P-glycoprotein expression in rat brain, as compared with control (P<0.05). Our studies suggested that Z-guggulsterone potentially inhibited the activity and expression of P-glycoprotein in rBMECs and in rat brain. PMID:27000241

  8. Determination of N-linked glycosylation in viral glycoproteins by negative ion mass spectrometry and ion mobility

    PubMed Central

    Bitto, David; Harvey, David J.; Halldorsson, Steinar; Doores, Katie J.; Pritchard, Laura K.; Huiskonen, Juha T.; Bowden, Thomas A.; Crispin, Max

    2016-01-01

    Summary Glycan analysis of virion-derived glycoproteins is challenging due to the difficulties in glycoprotein isolation and low sample abundance. Here, we describe how ion mobility mass spectrometry can be used to obtain spectra from virion samples. We also describe how negative ion fragmentation of glycans can be used to probe structural features of virion glycans. PMID:26169737

  9. Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

    PubMed

    McClements, W L; Armstrong, M E; Keys, R D; Liu, M A

    1996-10-15

    DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

  10. Evaluation of relative concentration of bacteria in 1% acid red 52- stainable and non-stainable dentin.

    PubMed

    Shirol, D D; Deshpande, S D; Shigli, A

    2004-01-01

    Modern management of caries stresses on non-invasive techniques wherever possible with maximum protection of sound tooth structure. Various dyes provide a highly contrasting visual clue by staining only infected dentin, thus clearly demarcating it from the affected normal dentin. Utilizing this property of preferential staining of the "Caries Detector" the current study was carried out to evaluate the relative concentration of bacteria in 1% Acid Red 52-stainable and non-stainable dentin. Sample of ten carious permanent molars or premolars in individual patient's mouth and carious teeth extracted due to periodontal diseases were examined for the two parts of the clinical study. Each clinical aspect was later subjected to laboratory procedure. Cariously infected dentin delineated by the solution, "Caries Detector" (1% Acid Red 52 solution in propylene glycol) contained six times more CFU/ml than dentin that did not take up dye.

  11. Use of lambdagt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    SciTech Connect

    Petrovskis, E.A.; Timmins, J.G.; Post, L.E.

    1986-10-01

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector lambdagt11. The library was screened with antisera which reacted with mixtures of PRV proteins to isolate recombinant bacteriophages expressing PRV proteins. By the nature of the lambdagt11 vector, the cloned proteins were expressed in Escherichia coli as ..beta..-galactosidase fusion proteins. The fusion proteins from 35 of these phages were purified and injected into mice to raise antisera. The antisera were screened by several different assays, including immunoprecipitation of (/sup 14/C)glucosamine-labeled PRV proteins. This method identified phages expressing three different PRV glycoproteins: the secreted glycoprotein, gX; gI; and a glycoprotein that had not been previously identified, which we designate gp63. The gp63 and gI genes map adjacent to each other in the small unique region of the PRV genome. The DNA sequence was determined for the region of the genome encoding gp63 and gI. It was found that gp63 has a region of homology with a herpes simplex virus type 1 (HSV-1) protein, encoded by US7, and also with varicella-zoster virus (VZV) gpIV. The gI protein sequence has a region of homology with HSV-1 gE and VZV gpI. It is concluded that PRV, HSV, and VZV all have a cluster of homologous glycoprotein genes in the small unique components of their genomes and that the organization of these genes is conserved.

  12. Progesterone regulates the expression and activity of two mouse isoforms of the glycoprotein folding sensor UDP-Glc: glycoprotein glucosyltransferase (UGGT).

    PubMed

    Prados, María B; Caramelo, Julio J; Miranda, Silvia E

    2013-12-01

    UDP-Glucose:glycoprotein glucosyltransferase (UGGT) is a central component of the endoplasmic reticulum (ER) glycoprotein-folding quality control system, which prevents the exit of partially folded species. UGGT activity can be regulated by the accumulation of misfolded proteins in the ER, a stimulus that triggers a complex signaling pathway known as unfolded protein response (UPR) which is closely associated with inflammation and disease. In this work, we investigated the effect of progesterone (P4) on the expression and activity of UGGT in a mouse hybridoma. We detected the expression of two UGGT isoforms, UGGT1 and UGGT2, and demonstrated that both isoforms are active in these cells. Interestingly, the expression of each isoform is regulated by high physiological P4 concentrations. This work provides the first evidence of a hormonal regulation of UGGT isoform expression and activity, which might influence the glycoprotein quality control mechanism. These findings could contribute to the study of pathologies triggered by the accumulation of misfolded proteins.

  13. Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane.

    PubMed

    Hwangbo, S; Azuma, N; Kurisaki, J; Kanno, C

    1997-09-01

    A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.

  14. Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

    PubMed Central

    Rauh, I; Weiland, F; Fehler, F; Keil, G M; Mettenleiter, T C

    1991-01-01

    The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is

  15. Identification of key structural characteristics of Schisandra chinensis lignans involved in P-glycoprotein inhibition.

    PubMed

    Slanina, Jiří; Páchniková, Gabriela; Carnecká, Martina; Porubová Koubíková, Ludmila; Adámková, Lenka; Humpa, Otakar; Smejkal, Karel; Slaninová, Iva

    2014-10-24

    The aim of the present study was to determine the structural requirements for dibenzocyclooctadiene lignans essential for P-glycoprotein inhibition. Altogether 15 structurally related lignans isolated from Schisandra chinensis or prepared by modification of their backbone were investigated, including three pairs of enantiomers. P-Glycoprotein inhibition was quantified using a doxorubicin accumulation assay in human promyelotic leukemia HL60/MDR cells overexpressing P-glycoprotein. A preliminary quantitative structure-activity relationship analysis revealed three main structural features involved in P-glycoprotein inhibition: a 1,2,3-trimethoxy moiety, a 6-acyloxy group, and the absence of a 7-hydroxy group. The most effective inhibitors, (-)-gomisin N (1) and (+)-deoxyschizandrin [(+)-2], were selected for further evaluation of their effects. Both these lignans restored the cytotoxic effect of doxorubicin in HL60/MDR cells and when combined with a subtoxic concentration of this compound increased the proportion of G2/M cells significantly, which is a usual response to treatment with this anticancer drug.

  16. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  17. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  18. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  19. Variations in Spike Glycoprotein Gene of MERS-CoV, South Korea, 2015.

    PubMed

    Kim, Dae-Won; Kim, You-Jin; Park, Sung Han; Yun, Mi-Ran; Yang, Jeong-Sun; Kang, Hae Ji; Han, Young Woo; Lee, Han Saem; Kim, Heui Man; Kim, Hak; Kim, A-Reum; Heo, Deok Rim; Kim, Su Jin; Jeon, Jun Ho; Park, Deokbum; Kim, Joo Ae; Cheong, Hyang-Min; Nam, Jeong-Gu; Kim, Kisoon; Kim, Sung Soon

    2016-01-01

    An outbreak of nosocomial infections with Middle East respiratory syndrome coronavirus occurred in South Korea in May 2015. Spike glycoprotein genes of virus strains from South Korea were closely related to those of strains from Riyadh, Saudi Arabia. However, virus strains from South Korea showed strain-specific variations.

  20. Identification of a glycoprotein ligand for E-selectin on mouse myeloid cells

    PubMed Central

    1993-01-01

    E-selectin is an inducible endothelial cell adhesion molecule for neutrophils which functions as a Ca(2+)-dependent lectin. Using a recombinant, antibody-like form of mouse E-selectin, we have searched for glycoprotein ligands on mouse neutrophils and the neutrophil progenitor cell line 32D cl 3. We have identified a 150-kD glycoprotein as the only protein which could be affinity-isolated with soluble E- selectin from [35S]methionine/[35S]cysteine-labeled 32D cl 3 cells. Binding of this protein was strictly Ca(2+)-dependent, was blocked by a cell adhesion-blocking mAb against mouse E-selectin, and required the presence of sialic acid on the 150-kD ligand. This glycoprotein was also affinity-isolated from mature neutrophils, in addition to a minor component at 250 kD, but could not be isolated from several other non- myeloid cell lines. The 150-kD glycoprotein was the only protein from 32D cl 3 cells, which was detectable by silver-staining after a one- step affinity-isolation. PMID:7682218

  1. Localization and Characterization of Flavivirus Envelope Glycoprotein Cross-Reactive Epitopes

    PubMed Central

    Crill, Wayne D.; Chang, Gwong-Jen J.

    2004-01-01

    The flavivirus E glycoprotein, the primary antigen that induces protective immunity, is essential for membrane fusion and mediates binding to cellular receptors. Human flavivirus infections stimulate virus species-specific as well as flavivirus cross-reactive immune responses. Flavivirus cross-reactive antibodies in human sera create a serious problem for serodiagnosis, especially for secondary flavivirus infections, due to the difficulty of differentiating primary from secondary cross-reactive serum antibodies. The presence of subneutralizing levels of flavivirus cross-reactive serum antibodies may result in a dramatic increase in the severity of secondary flavivirus infections via antibody-dependent enhancement. An understanding of flavivirus E-glycoprotein cross-reactive epitopes is therefore critical for improving public health responses to these serious diseases. We identified six E-glycoprotein residues that are incorporated into three distinct flavivirus cross-reactive epitopes. Two of these epitopes which are recognized by distinct monoclonal antibodies contain overlapping continuous residues located within the highly conserved fusion peptide. The third epitope consists of discontinuous residues that are structurally related to the strictly conserved tryptophan at dengue virus serotype 2 E-glycoprotein position 231. PMID:15564505

  2. Averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using Jsubtomo.

    PubMed

    Huiskonen, Juha T; Parsy, Marie-Laure; Li, Sai; Bitto, David; Renner, Max; Bowden, Thomas A

    2014-01-01

    Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein 'spikes' is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health. PMID:25350719

  3. Support studies for installing the phosphodiester residues of the Thy-1 glycoprotein membrane anchor.

    PubMed

    Campbell, A S; Fraser-Reid, B

    1994-11-01

    Support studies for late-stage installation of the three different types of phosphodiesters found in the rat brain Thy-1 glycoprotein membrane anchor are described. The strategy is geared towards optimizing convergency and the development of chemoselective procedures including deprotection, phosphorylation, esterification and cysteinylation has been investigated. Some of these procedures are being designed for oligosaccharides containing several unprotected hydroxy groups.

  4. Structure of a trimeric variant of the Epstein-Barr virus glycoprotein B

    SciTech Connect

    Backovic, Marija; Longnecker, Richard; Jardetzky, Theodore S

    2009-03-16

    Epstein-Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular membranes. The fusion apparatus is comprised of envelope glycoprotein B (gB) and a heterodimeric complex made of glycoproteins H and L. Glycoprotein B is the most conserved envelope glycoprotein in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is available. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has been solved in its pre and postfusion states, shed light on gB residues that may be involved in conformational changes and membrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distributions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.

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

    PubMed

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

    2015-03-01

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

  6. Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins.

    PubMed

    Cova, Marta; Oliveira-Silva, Rui; Ferreira, José Alexandre; Ferreira, Rita; Amado, Francisco; Daniel-da-Silva, Ana Luísa; Vitorino, Rui

    2015-01-01

    Protein post-translational modifications (PTMs) have increasingly become a research field of incredible importance to fully understand the regulation of biological processes in health and disease. Among PTMs, glycosylation is one of the most studied for which contributed the development and improvement of enrichment techniques. Nowadays, glycoprotein enrichment methods are based on lectin affinity, covalent interactions, and hydrophilic interaction liquid chromatography (HILIC). Nonetheless, the nanotechnology era has fetched new methods to enrich glycoproteins from complex samples as human biological fluids. For instance, magnetic nanoparticles (MNPs) are being used as an interesting enrichment approach allowing a better characterization of glycoproteins and glycopeptides.In this chapter, we describe an enrichment method based on MNPs functionalized with lectins (Concavalin A, wheat germ agglutinin, and Maackia amurensis lectin) to enrich specific sets of glycoproteins from biological fluids. Moreover, it is proposed a bioinformatic strategy to deal with data retrieved from mass spectrometry analysis of enriched samples aiming the identification of relevant biological processes modulated by a given stimuli and, ultimately, of new biomarkers for disease screening/management.

  7. Intracellular transport and stability of varicella-zoster virus glycoprotein K

    SciTech Connect

    Hall, Susan L.; Govero, Jennifer L.; Heineman, Thomas C. . E-mail: heinemtc@slu.edu

    2007-02-20

    VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established that gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.

  8. Paramyxovirus Glycoprotein Incorporation, Assembly and Budding: A Three Way Dance for Infectious Particle Production

    PubMed Central

    El Najjar, Farah; Schmitt, Anthony P.; Dutch, Rebecca Ellis

    2014-01-01

    Paramyxoviruses are a family of negative sense RNA viruses whose members cause serious diseases in humans, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus and rinderpest virus. Paramyxovirus particles form by assembly of the viral matrix protein, the ribonucleoprotein complex and the surface glycoproteins at the plasma membrane of infected cells and subsequent viral budding. Two major glycoproteins expressed on the viral envelope, the attachment protein and the fusion protein, promote attachment of the virus to host cells and subsequent virus-cell membrane fusion. Incorporation of the surface glycoproteins into infectious progeny particles requires coordinated interplay between the three viral structural components, driven primarily by the matrix protein. In this review, we discuss recent progress in understanding the contributions of the matrix protein and glycoproteins in driving paramyxovirus assembly and budding while focusing on the viral protein interactions underlying this process and the intracellular trafficking pathways for targeting viral components to assembly sites. Differences in the mechanisms of particle production among the different family members will be highlighted throughout. PMID:25105277

  9. Altering Entry Site Preference of Lentiviral Vectors into Neuronal Cells by Pseudotyping with Envelope Glycoproteins.

    PubMed

    Kobayashi, Kenta; Kato, Shigeki; Inoue, Ken-Ichi; Takada, Masahiko; Kobayashi, Kazuto

    2016-01-01

    A lentiviral vector system provides a powerful strategy for gene therapy trials against a variety of neurological and neurodegenerative disorders. Pseudotyping of lentiviral vectors with different envelope glycoproteins not only confers the neurotropism to the vectors, but also alters the preference of sites of vector entry into neuronal cells. One major group of lentiviral vectors is a pseudotype with vesicular stomatitis virus glycoprotein (VSV-G) that enters preferentially cell body areas (somata/dendrites) of neurons and transduces them. Another group contains lentiviral vectors pseudotyped with fusion envelope glycoproteins composed of different sets of rabies virus glycoprotein and VSV-G segments that enter predominantly axon terminals of neurons and are transported through axons retrogradely to their cell bodies, resulting in enhanced retrograde gene transfer. This retrograde gene transfer takes a considerable advantage of delivering the transgene into neuronal cell bodies situated in regions distant from the injection site of the vectors. The rational use of these two vector groups characterized by different entry mechanisms will further extend the strategy for gene therapy of neurological and neurodegenerative disorders.

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

    PubMed Central

    Garcia, Natalie K.; Lee, Kelly K.

    2016-01-01

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system. PMID:26761026

  11. Correction of defective protein kinesis of human P-glycoprotein mutants by substrates and modulators.

    PubMed

    Loo, T W; Clarke, D M

    1997-01-10

    There is growing evidence that abnormal protein folding or trafficking (protein kinesis) leads to diseases. We have used P-glycoprotein as a model protein to develop strategies to overcome defects in protein kinesis. Misprocessed mutants of the human P-glycoprotein are retained in the endoplasmic reticulum as core-glycosylated biosynthetic intermediates and rapidly degraded. Synthesis of the mutant proteins in the presence of drug substrates or modulators such as capsaicin, cyclosporin, vinblastine, or verapamil, however, resulted in the appearance of a fully glycosylated and functional protein at the cell surface. These effects were dose-dependent and occurred within a few hours after the addition of substrate. The ability to facilitate processing of the misfolded mutants appeared to be independent of the cell lines used and location of the mutation. P-glycoproteins with mutations in transmembrane segments, extracellular or cytoplasmic loops, the nucleotide-binding domains, or the linker region were processed to the fully mature form in the presence of these substrates. These drug substrates or modulators acted as specific chemical chaperones for P-glycoprotein because they were ineffective on the deltaF508 mutant of cystic fibrosis transmembrane conductance regulator. Therefore, one possible strategy to prevent protein misfolding is to carry out synthesis in the presence of specific substrates or modulators of the protein.

  12. Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these i...

  13. Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

    PubMed Central

    Huiskonen, Juha T.; Parsy, Marie-Laure; Li, Sai; Bitto, David; Renner, Max; Bowden, Thomas A.

    2014-01-01

    Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein ‘spikes’ is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 Å and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health. PMID:25350719

  14. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    PubMed Central

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

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

    PubMed

    Garcia, Natalie K; Lee, Kelly K

    2016-01-11

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

  16. P-glycoprotein regulates trafficking of CD8(+) T cells to the brain parenchyma.

    PubMed

    Kooij, Gijs; Kroon, Jeffrey; Paul, Debayon; Reijerkerk, Arie; Geerts, Dirk; van der Pol, Susanne M A; van Het Hof, Bert; Drexhage, Joost A; van Vliet, Sandra J; Hekking, Liesbeth H P; van Buul, Jaap D; Pachter, Joel S; de Vries, Helga E

    2014-05-01

    The trafficking of cytotoxic CD8(+) T lymphocytes across the lining of the cerebral vasculature is key to the onset of the chronic neuro-inflammatory disorder multiple sclerosis. However, the mechanisms controlling their final transmigration across the brain endothelium remain unknown. Here, we describe that CD8(+) T lymphocyte trafficking into the brain is dependent on the activity of the brain endothelial adenosine triphosphate-binding cassette transporter P-glycoprotein. Silencing P-glycoprotein activity selectively reduced the trafficking of CD8(+) T cells across the brain endothelium in vitro as well as in vivo. In response to formation of the T cell-endothelial synapse, P-glycoprotein was found to regulate secretion of endothelial (C-C motif) ligand 2 (CCL2), a chemokine that mediates CD8(+) T cell migration in vitro. Notably, CCL2 levels were significantly enhanced in microvessels isolated from human multiple sclerosis lesions in comparison with non-neurological controls. Endothelial cell-specific elimination of CCL2 in mice subjected to experimental autoimmune encephalomyelitis also significantly diminished the accumulation of CD8(+) T cells compared to wild-type animals. Collectively, these results highlight a novel (patho)physiological role for P-glycoprotein in CD8(+) T cell trafficking into the central nervous system during neuro-inflammation and illustrate CCL2 secretion as a potential link in this mechanism.

  17. Structure–Function Relationships of Glycoprotein Hormones and Their Subunits’ Ancestors

    PubMed Central

    Cahoreau, Claire; Klett, Danièle; Combarnous, Yves

    2015-01-01

    Glycoprotein hormones (GPHs) are the most complex molecules with hormonal activity. They exist only in vertebrates but the genes encoding their subunits’ ancestors are found in most vertebrate and invertebrate species although their roles are still unknown. In the present report, we review the available structural and functional data concerning GPHs and their subunits’ ancestors. PMID:25767463

  18. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits.

    PubMed

    Robinson, James E; Hastie, Kathryn M; Cross, Robert W; Yenni, Rachael E; Elliott, Deborah H; Rouelle, Julie A; Kannadka, Chandrika B; Smira, Ashley A; Garry, Courtney E; Bradley, Benjamin T; Yu, Haini; Shaffer, Jeffrey G; Boisen, Matt L; Hartnett, Jessica N; Zandonatti, Michelle A; Rowland, Megan M; Heinrich, Megan L; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C; Andersen, Kristian G; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J; Fonnie, Richard; Jalloh, Simbirie C; Kargbo, Brima; Vandi, Mohamed A; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A; Okokhere, Peter O; Follarin, Onikepe A; Schieffelin, John S; Pitts, Kelly R; Geisbert, Joan B; Kulakoski, Peter C; Wilson, Russell B; Happi, Christian T; Sabeti, Pardis C; Gevao, Sahr M; Khan, S Humarr; Grant, Donald S; Geisbert, Thomas W; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  19. Glycoprotein Biochemistry (Biosynthesis)--A Vehicle for Teaching Many Aspects of Biochemistry and Molecular Biology.

    ERIC Educational Resources Information Center

    Cole, Clair R.; Smith, Christopher A.

    1990-01-01

    Information about the biosynthesis of the carbohydrate portions or glycans of glycoproteins is presented. The teaching of glycosylation can be used to develop and emphasize many general aspects of biosynthesis, in addition to explaining specific biochemical and molecular biological features associated with producing the oligosaccharide portions of…

  20. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  1. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Beta-2-glycoprotein III immunological test system. 866.5440 Section 866.5440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  4. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  5. 21 CFR 866.5430 - Beta-2-glycoprotein I immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Beta-2-glycoprotein I immunological test system. 866.5430 Section 866.5430 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  6. Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway.

    PubMed

    Gardner, Thomas J; Tortorella, Domenico

    2016-09-01

    The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

  7. Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus.

    PubMed

    Coleman, Stewart; Hornig, Julia; Maddux, Sarah; Choi, K Yeon; McGregor, Alistair

    2015-01-01

    Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene

  8. Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus

    PubMed Central

    Maddux, Sarah; Choi, K. Yeon; McGregor, Alistair

    2015-01-01

    Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene

  9. Blood-brain barrier P-glycoprotein function in Alzheimer's disease.

    PubMed

    van Assema, Daniëlle M E; Lubberink, Mark; Bauer, Martin; van der Flier, Wiesje M; Schuit, Robert C; Windhorst, Albert D; Comans, Emile F I; Hoetjes, Nikie J; Tolboom, Nelleke; Langer, Oliver; Müller, Markus; Scheltens, Philip; Lammertsma, Adriaan A; van Berckel, Bart N M

    2012-01-01

    A major pathological hallmark of Alzheimer's disease is accumulation of amyloid-β in senile plaques in the brain. Evidence is accumulating that decreased clearance of amyloid-β from the brain may lead to these elevated amyloid-β levels. One of the clearance pathways of amyloid-β is transport across the blood-brain barrier via efflux transporters. P-glycoprotein, an efflux pump highly expressed at the endothelial cells of the blood-brain barrier, has been shown to transport amyloid-β. P-glycoprotein function can be assessed in vivo using (R)-[(11)C]verapamil and positron emission tomography. The aim of this study was to assess blood-brain barrier P-glycoprotein function in patients with Alzheimer's disease compared with age-matched healthy controls using (R)-[(11)C]verapamil and positron emission tomography. In 13 patients with Alzheimer's disease (age 65 ± 7 years, Mini-Mental State Examination 23 ± 3), global (R)-[(11)C]verapamil binding potential values were increased significantly (P = 0.001) compared with 14 healthy controls (aged 62 ± 4 years, Mini-Mental State Examination 30 ± 1). Global (R)-[(11)C]verapamil binding potential values were 2.18 ± 0.25 for patients with Alzheimer's disease and 1.77 ± 0.41 for healthy controls. In patients with Alzheimer's disease, higher (R)-[(11)C]verapamil binding potential values were found for frontal, parietal, temporal and occipital cortices, and posterior and anterior cingulate. No significant differences between groups were found for medial temporal lobe and cerebellum. These data show altered kinetics of (R)-[(11)C]verapamil in Alzheimer's disease, similar to alterations seen in studies where P-glycoprotein is blocked by a pharmacological agent. As such, these data indicate that P-glycoprotein function is decreased in patients with Alzheimer's disease. This is the first direct evidence that the P-glycoprotein transporter at the blood-brain barrier is compromised in sporadic

  10. Inhibition of Arenavirus Infection by a Glycoprotein-Derived Peptide with a Novel Mechanism

    PubMed Central

    Spence, Jennifer S.; Melnik, Lilia I.; Badani, Hussain; Wimley, William C.

    2014-01-01

    ABSTRACT The family Arenaviridae includes a number of viruses of public health importance, such as the category A hemorrhagic fever viruses Lassa virus, Junin virus, Machupo virus, Guanarito virus, and Sabia virus. Current chemotherapy for arenavirus infection is limited to the nucleoside analogue ribavirin, which is characterized by considerable toxicity and treatment failure. Using Pichinde virus as a model arenavirus, we attempted to design glycoprotein-derived fusion inhibitors similar to the FDA-approved anti-HIV peptide enfuvirtide. We have identified a GP2-derived peptide, AVP-p, with antiviral activity and no acute cytotoxicity. The 50% inhibitory dose (IC50) for the peptide is 7 μM, with complete inhibition of viral plaque formation at approximately 20 μM, and its antiviral activity is largely sequence dependent. AVP-p demonstrates activity against viruses with the Old and New World arenavirus viral glycoprotein complex but not against enveloped viruses of other families. Unexpectedly, fusion assays reveal that the peptide induces virus-liposome fusion at neutral pH and that the process is strictly glycoprotein mediated. As observed in cryo-electron micrographs, AVP-p treatment causes morphological changes consistent with fusion protein activation in virions, including the disappearance of prefusion glycoprotein spikes and increased particle diameters, and fluorescence microscopy shows reduced binding by peptide-treated virus. Steady-state fluorescence anisotropy measurements suggest that glycoproteins are destabilized by peptide-induced alterations in viral membrane order. We conclude that untimely deployment of fusion machinery by the peptide could render virions less able to engage in on-pathway receptor binding or endosomal fusion. AVP-p may represent a potent, highly specific, novel therapeutic strategy for arenavirus infection. IMPORTANCE Because the only drug available to combat infection by Lassa virus, a highly pathogenic arenavirus, is toxic

  11. Interactions of HIV-1 and HIV-2 envelope glycoproteins with sulphated polysaccharides and mannose-6-phosphate.

    PubMed

    Mbemba, E; Gluckman, J C; Gattegno, L

    1994-02-01

    Envelope glycoproteins of human immunodeficiency viruses (HIV-1 and HIV-2) can interact with high-mannose glycans and with the mannosyl or N-acetylglucosaminyl core of complex-type oligosaccharidic structures. HIV-1 glycoproteins also specifically bind sulphated polysaccharides such as dextran sulphate (DS) and heparin. Here, we show that the latter property is shared by HIV-2 recombinant gp140 (rgp140) precursor glycoprotein. Binding of rgp140 and of corresponding rgp160 of HIV-1 to heparin- and DS-substituted (sulphated dextran beads; SDB) affinity matrices was inhibited by the soluble specific ligand and also by fetuin, asialofetuin or the anionic simple carbohydrate derivative mannose-6-phosphate (M6P). Interaction of HIV-1 rgp120 subunit with the two affinity matrices was also inhibited by M6P, but only rgp120 binding to heparin-agarose, and not that to SDB, was affected by fetuin and asialofetuin. These results suggest that HIV-1 and HIV-2 envelope glycoproteins presumably display different sulphated polysaccharide and carbohydrate recognition sites. Some of these may be common or in close proximity: with respect to rgp160, for example, the sites may be common on the gp41 moiety and/or in a region of gp120 which would be more accessible when expressed on rgp160 than on processed gp120, while they may be distinct on the cleaved gp120 subunit. Finally, because M6P is a marker of lysosomal enzymes, we verified that HIV-1 and HIV-2 envelope glycoproteins could specifically bind in a M6P-inhibitable manner to a representative lysosomal enzyme, bovine liver beta-glucuronidase coupled to agarose, suggesting that they may possibly interfere with lysosomal enzyme sorting in HIV-infected cells.

  12. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    NASA Astrophysics Data System (ADS)

    Ney, Yap Wei; Rahman, Badarulhisam Abdul; Aziz, Azila Abdul

    Baculovirus Expression Vector System (BEVS) is widely used for the production of recombinant glycoproteins, but it is not ideal for pharmaceutical glycoprotein production due to incomplete glycosylation. The factors that ensure successful glycosylation are the presence of sufficient amount of glycosyltransferases, sugar nucleotides as the substrate donor and the recombinant protein as the substrate acceptor. In this study, we analyzed the galactosylation process by the introduction of ß-1,4galactosyltransferase (ß-1,4GalT) as the glycosyltransferase of interest and uridine-5`-diphosphogalactose (UDP-Gal) as the substrate donor. Recombinant human transferrin (rhTf) as a model protein was used as the substrate acceptor. Insect cell lines have been reported to produce a small amount of ß-1,4GalT and thus insufficient for effective galactosylation. In this study, we developed a method to produce galactosylated rhTf and optimized the expression of rhTf with better N-glycan quality. Recombinant ß-1,4GalT was introduced during protein expression by the coinfection of the BEVS with baculovirus carrying bovine ß-1,4GalT. To evaluate the extent of galactosylation by the coinfection strategy, a binding assay was established. In this binding assay, glycoprotein acceptor was absorbed onto ELISA plate surface. A lectin known as Ricinus communis agglutinin-I (RCA-I) labeled with peroxidase, was added and allowed to recognize Gal ß1>4GlcNAc group on the N-glycan of the glycoprotein, followed by appropriate color reaction measurements. Coexpression between rhTf and ß-1,4GalT did not show encouraging results due to the reduction of UDP-Gal upon baculovirus infection. This interesting finding suggested that the introduction of ß-1,4GalT alone was not sufficient for successful galactosylation. Alternatively, post harvest glycosylation method strategy seems to be a promising technique in the improvement of glycoprotein quality.

  13. Emergence of an Ancestral Glycoprotein Hormone in the Pituitary of the Sea Lamprey, a Basal Vertebrate.

    PubMed

    Sower, Stacia A; Decatur, Wayne A; Hausken, Krist N; Marquis, Timothy J; Barton, Shannon L; Gargan, James; Freamat, Mihael; Wilmot, Michael; Hollander, Lian; Hall, Jeffrey A; Nozaki, Masumi; Shpilman, Michal; Levavi-Sivan, Berta

    2015-08-01

    The gnathostome (jawed vertebrates) classical pituitary glycoprotein hormones, FSH, LH, and TSH, consist of a common α-subunit (GpA1) and unique β-subunits (Gpβ1, -2, and -3), whereas a recently identified pituitary glycoprotein hormone, thyrostimulin, consists of GpA2 and GpB5. This paper reports the identification, expression, and function of an ancestral, nonclassical, pituitary heterodimeric glycoprotein hormone (GpH) consisting of the thyrostimulin A2 subunit with the classical β-subunit in the sea lamprey, Petromyzon marinus, a jawless basal vertebrate. Lamprey (l) GpA2, and lGpHβ were shown to form a heterodimer by coimmunoprecipitation of lGpA2 with FLAG-tagged lGpHβ after the overexpression in transiently transfected COS7 cells using a bipromoter vector. Dual-label fluorescent in situ hybridization and immunohistochemistry showed the coexpression of individual subunits in the proximal pars distalis of the pituitary. GnRH-III (1μΜ) significantly increased the expression of lGpHβ and lGpA2 in in vitro pituitary culture. Recombinant lamprey GpH was constructed by tethering the N terminal of lGpA2 to the C terminal of lGpHβ with a linker region composed of six histidine residues followed by three glycine-serine repeats. This recombinant lamprey GpH activated the lamprey glycoprotein hormone receptor I as measured by increased cAMP/luciferase activity. These data are the first to demonstrate a functional, unique glycoprotein heterodimer that is not found in any other vertebrate. These data suggest an intermediate stage of the structure-function of the gonadotropin/thyroid-stimulating hormone in a basal vertebrate, leading to the emergence of the highly specialized gonadotropin hormones and thyroid stimulating hormones in gnathostomes.

  14. Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein

    SciTech Connect

    Lee, Jeffrey E.; Fusco, Marnie L.; Abelson, Dafna M.; Hessell, Ann J.; Burton, Dennis R.; Saphire, Erica Ollmann

    2009-11-01

    Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing of the trimeric ebolavirus glycoprotein are described. The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008 ▶), Nature (London), 454, 177–182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the

  15. Analysis of the apparent biphasic axonal transport kinetics of fucosylated glycoproteins

    SciTech Connect

    Goodrum, J.F.; Morell, P.

    1984-07-01

    Following intraocular injection of (/sup 3/H)fucose, the accumulation of transported radioactivity arriving at the superior colliculus peaks within a few hours and decays with a time course of hours. Then, over a period of several days, radioactivity again accumulates at the superior colliculus and then decays with a half-life of days. Such data have been interpreted as evidence for both a group of rapidly released, rapidly transported glycoproteins (first peak) and a group of slowly released but rapidly transported glycoproteins (second peak). This supposition was investigated by studying in more detail the metabolism of some individual fucosylated proteins in both the retina and superior colliculus. It was noted that much of the radioactivity incorporated in fucosylated glycoproteins at the retina was rapidly metabolized, while the remainder of the fucosylated moieties had a metabolic half-life on the order of days. In other experiments (/sup 35/S)methionine was injected intraocularly, the metabolism in the retina was examined and a study was made of the kinetics of transport to the superior colliculus of the peptide backbone of these same individual proteins. In contrast to the two waves of accumulation of radioactivity from (/sup 3/H)fucose, accumulation of radioactivity of the peptide backbone of the same glycoproteins was monophasic. The author's explanation of these data involves the presence of two types of fucose moieties on the peptides. One group of fucose moieties is labile and is lost from the peptide backbone over a period of hours. Other fucose moieties are approximately as metabolically stable as the peptide backbones to which they are attached. The actual peptide backbones of the glycoproteins are committed to rapid transport over a period of several days.

  16. Feasibility and challenges in the development of immunocontraceptive vaccine based on zona pellucida glycoproteins.

    PubMed

    Choudhury, Sangeeta; Srivastava, Neelu; Narwal, P S; Rath, Archana; Jaiswal, Sonika; Gupta, Satish K

    2007-01-01

    The zona pellucida (ZP) glycoproteins play a crucial role during fertilization and thus are considered as important target antigens for the development of immunocontraceptive vaccines aiming to inhibit fertility at a pre-fertilization stage. In order to evaluate the immunocontraceptive potential of ZP glycoproteins, bonnet monkey (Macaca radiata) ZP2, ZP3 and ZP4 have been cloned and expressed using either E. coli or baculovirus expression systems. Active immunization studies with the recombinant ZP glycoproteins in female baboons (Papio anubis) and bonnet monkeys revealed curtailment of fertility. In order to minimize the ovarian pathology, synthetic peptides corresponding to B cell epitopes that are devoid of 'oophoritogenic' T cell epitopes were designed and their in vitro immunocontraceptive potential explored. There are several issues that need to be addressed before ZP glycoproteins based immunocontraceptive vaccines become feasible for use in humans. Nonetheless, the utility of such a vaccine is imminent for controlling wild life population. In this direction, active immunization of female non-descript dogs with recombinant canine ZP3 conjugated to diphtheria toxoid led to curtailment of fertility. Further, canine ZP3 has also been expressed in insect cells as a fusion protein with rabies virus glycoprotein G (RV-G), an antigen that is involved in providing protection against rabies. The immunogenicity of such a recombinant protein and its potential to curtail fertility was explored both in female mice and dogs. Simultaneously, DNA vaccine encoding canine ZP3 and RV-G have been made and evaluated for their immunogenicity. The results obtained so far, current shortcomings and the possible ways to circumvent these have been discussed in the present manuscript. PMID:17566293

  17. Characterization of the oligosaccharide units of the bovine erythrocyte membrane glycoprotein.

    PubMed

    Emerson, W A; Kornfeld, S

    1976-04-20

    The major glycoprotein of the bovine erythrocyte membrane was purified by extraction of the ghosts with lithium 3,5-diiodosalicylate followed by phenol-water extraction and acidification. The glycoprotein contains 20% protein and 80% carbohydrate by weight and gives a single band on sodium dodecyl sulfate-polyacrylamide gels with an estimated molecular weight of 230000 daltons. The carbohydrate composition of the glycoprotein was determined to be (in residues relative to sialic acid): sialic acid, 1.0; fucose, less than 0.01; mannose, 0.1; galactose, 3.3; N-acetylgalactosamine, 0.9; and N-acetylglucosamine, 2.4. Pronase digestion of the isolated glycoprotein followed by Sephadex G-75 gel filtration resulted in the separation of a small pool of glycopeptides (pool III), which included all of the mannose-containing glycopeptides, from the bulk of the glycopeptide material which was in the void fractions of the column (pool I). Alkaline borohydride treatment released over 95% of the oligosaccharide units in pool I and approximately 30% of the oligosaccharide units in pool III. These oligosaccharides were isolated by gel filtration and ion-exchange chromatography. The oligosaccharides released from pool I had molecular weights of 1100-1400 daltons and contained sialic acid, galactose, and N-acetylglucosamine in molar ratios of 0.5-1:3:2 as well as a partial residue of N-acetylgalactosaminitol. The oligosaccharides released from pool III by alkali had molecular weights of 1300-1600 daltons and contained sialic acid, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-ACETYLgalactosaminitol in molar ratios of 1-2:2:1:1:1. These data indicate that the majority of the oligosaccharide units of the bovine erythrocyte glycoprotein are linked O-glycosidically to the peptide backbone of the molecule. PMID:1268191

  18. Carbohydrate Structure of Sindbis Virus Glycoprotein E2 from Virus Grown in Hamster and Chicken Cells

    PubMed Central

    Burke, David; Keegstra, Kenneth

    1979-01-01

    Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways. PMID:430605

  19. P-glycoprotein alters blood–brain barrier penetration of antiepileptic drugs in rats with medically intractable epilepsy

    PubMed Central

    Ma, Aimei; Wang, Cuicui; Chen, Yinghui; Yuan, Weien

    2013-01-01

    P-glycoprotein is one of the earliest known multidrug transporters and plays an important role in resistance to chemotherapeutic drugs. In this study, we detected levels of P-glycoprotein and its mRNA expression in a rat brain model of medically intractable epilepsy established by amygdala kindling and drug selection. We investigated whether inhibition of P-glycoprotein affects the concentration of antiepileptic drugs in cortical extracellular fluid. We found that levels of P-glycoprotein and its mRNA expression were upregulated in epileptic cerebral tissue compared with cerebral tissue from normal rats. The concentrations of two antiepileptic drugs, carbamazepine and phenytoin, were very low in the cortical extracellular fluid of rats with medically intractable epilepsy, and were restored after blockade of P-glycoprotein by verapamil. These results show that increased P-glycoprotein levels alter the ability of carbamazepine and phenytoin to penetrate the blood–brain barrier and reduce the concentrations of these agents in extracellular cortical fluid. High P-glycoprotein levels may be involved in resistance to antiepileptic drugs in medically intractable epilepsy. PMID:24348021

  20. Glucose persistence on high-mannose oligosaccharides selectively inhibits the macroautophagic sequestration of N-linked glycoproteins.

    PubMed Central

    Ogier-Denis, E; Bauvy, C; Cluzeaud, F; Vandewalle, A; Codogno, P

    2000-01-01

    The macroautophagic-lysosomal pathway is a bulk degradative process for cytosolic proteins and organelles including the endoplasmic reticulum (ER). We have previously shown that the human colonic carcinoma HT-29 cell population is characterized by a high rate of autophagic degradation of N-linked glycoproteins substituted with ER-type glycans. In the present work we demonstrate that glucosidase inhibitors [castanospermine (CST) and deoxynojirimycin] have a stabilizing effect on newly synthesized glucosylated N-linked glycoproteins and impaired their lysosomal delivery as shown by subcellular fractionation on Percoll gradients. The inhibition of macroautophagy was restricted to N-linked glycoproteins because macroautophagic parameters such as the rate of sequestration of cytosolic markers and the fractional volume occupied by autophagic vacuoles were not affected in CST-treated cells. The protection of glucosylated glycoproteins from autophagic sequestration was also observed in inhibitor-treated Chinese hamster ovary (CHO) cells and in Lec23 cells (a CHO mutant deficient in glucosidase I activity). The interaction of glucosylated glycoproteins with the ER chaperone binding protein (BiP) was prolonged in inhibitor-treated cells in comparison with untreated CHO cells. These results show that the removal of glucose from N-glycans of glycoproteins is a key event for their delivery to the autophagic pathway and that interaction with BiP could prevent or delay newly synthesized glucosylated N-linked glycoproteins from being sequestered by the autophagic pathway. PMID:10642502

  1. 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.

  2. Benzoboroxole-functionalized magnetic core/shell microspheres for highly specific enrichment of glycoproteins under physiological conditions.

    PubMed

    Zhang, Yuting; Ma, Wanfu; Li, Dian; Yu, Meng; Guo, Jia; Wang, Changchun

    2014-04-01

    Efficient enrichment of specific glycoproteins from complex biological samples is of great importance towards the discovery of disease biomarkers in biological systems. Recently, phenylboronic acid-based functional materials have been widely used for enrichment of glycoproteins. However, such enrichment was mainly carried out under alkaline conditions, which is different to the status of glycoproteins in neutral physiological conditions and may cause some unpredictable degradation. In this study, on-demand neutral enrichment of glycoproteins from crude biological samples is accomplished by utilizing the reversible interaction between the cis-diols of glycoproteins and benzoboroxole-functionalized magnetic composite microspheres (Fe3O4/PAA-AOPB). The Fe3O4/PAA-AOPB composite microspheres are deliberately designed and constructed with a high-magnetic-response magnetic supraparticle (MSP) core and a crosslinked poly(acrylic acid) (PAA) shell anchoring abundant benzoboroxole functional groups on the surface. These nanocomposites possessed many merits, such as large enrichment capacity (93.9 mg/g, protein/beads), low non-specific adsorption, quick enrichment process (10 min) and magnetic separation speed (20 s), and high recovery efficiency. Furthermore, the as-prepared Fe3O4/PAA-AOPB microspheres display high selectivity to glycoproteins even in the E. coli lysate or fetal bovine serum, showing great potential in the identify of low-abundance glycoproteins as biomarkers in real complex biological systems for clinical diagnoses.

  3. Platelet interaction with von Willebrand factor is enhanced by shear-induced clustering of glycoprotein Ibα

    PubMed Central

    Gitz, Eelo; Koopman, Charlotte D.; Giannas, Alèkos; Koekman, Cornelis A; van den Heuvel, Dave J.; Deckmyn, Hans; Akkerman, Jan-Willem N.; Gerritsen, Hans C.; Urbanus, Rolf T.

    2013-01-01

    Initial platelet arrest at the exposed arterial vessel wall is mediated through glycoprotein Ibα binding to the A1 domain of von Willebrand factor. This interaction occurs at sites of elevated shear force, and strengthens upon increasing hydrodynamic drag. The increased interaction requires shear-dependent exposure of the von Willebrand factor A1 domain, but the contribution of glycoprotein Ibα remains ill defined. We have previously found that glycoprotein Ibα forms clusters upon platelet cooling and hypothesized that such a property enhances the interaction with von Willebrand factor under physiological conditions. We analyzed the distribution of glycoprotein Ibα with Förster resonance energy transfer using time-gated fluorescence lifetime imaging microscopy. Perfusion at a shear rate of 1,600 s−1 induced glycoprotein Ibα clusters on platelets adhered to von Willebrand factor, while clustering did not require von Willebrand factor contact at 10,000 s−1. Shear-induced clustering was reversible, not accompanied by granule release or αIIbβ3 activation and improved glycoprotein Ibα-dependent platelet interaction with von Willebrand factor. Clustering required glycoprotein Ibα translocation to lipid rafts and critically depended on arachidonic acid-mediated binding of 14-3-3ζ to its cytoplasmic tail. This newly identified mechanism emphasizes the ability of platelets to respond to mechanical force and provides new insights into how changes in hemodynamics influence arterial thrombus formation. PMID:23753027

  4. Total synthesis of the α-subunit of human glycoprotein hormones: toward fully synthetic homogeneous human follicle-stimulating hormone.

    PubMed

    Aussedat, Baptiste; Fasching, Bernhard; Johnston, Eric; Sane, Neeraj; Nagorny, Pavel; Danishefsky, Samuel J

    2012-02-22

    Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.

  5. Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

    PubMed

    Zeinyeh, Waël; Mahiout, Zahia; Radix, Sylvie; Lomberget, Thierry; Dumoulin, Axel; Barret, Roland; Grenot, Catherine; Rocheblave, Luc; Matera, Eva-Laure; Dumontet, Charles; Walchshofer, Nadia

    2012-10-01

    Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

  6. In vitro, in silico and integrated strategies for the estimation of plasma protein binding. A review.

    PubMed

    Lambrinidis, George; Vallianatou, Theodosia; Tsantili-Kakoulidou, Anna

    2015-06-23

    Plasma protein binding (PPB) strongly affects drug distribution and pharmacokinetic behavior with consequences in overall pharmacological action. Extended plasma protein binding may be associated with drug safety issues and several adverse effects, like low clearance, low brain penetration, drug-drug interactions, loss of efficacy, while influencing the fate of enantiomers and diastereoisomers by stereoselective binding within the body. Therefore in holistic drug design approaches, where ADME(T) properties are considered in parallel with target affinity, considerable efforts are focused in early estimation of PPB mainly in regard to human serum albumin (HSA), which is the most abundant and most important plasma protein. The second critical serum protein α1-acid glycoprotein (AGP), although often underscored, plays also an important and complicated role in clinical therapy and thus the last years it has been studied thoroughly too. In the present review, after an overview of the principles of HSA and AGP binding as well as the structure topology of the proteins, the current trends and perspectives in the field of PPB predictions are presented and discussed considering both HSA and AGP binding. Since however for the latter protein systematic studies have started only the last years, the review focuses mainly to HSA. One part of the review highlights the challenge to develop rapid techniques for HSA and AGP binding simulation and their performance in assessment of PPB. The second part focuses on in silico approaches to predict HSA and AGP binding, analyzing and evaluating structure-based and ligand-based methods, as well as combination of both methods in the aim to exploit the different information and overcome the limitations of each individual approach. Ligand-based methods use the Quantitative Structure-Activity Relationships (QSAR) methodology to establish quantitate models for the prediction of binding constants from molecular descriptors, while they provide

  7. Human platelet glycoprotein V: characterization of the polypeptide and the related Ib-V-IX receptor system of adhesive, leucine-rich glycoproteins.

    PubMed Central

    Hickey, M J; Hagen, F S; Yagi, M; Roth, G J

    1993-01-01

    Human platelet glycoprotein (GP) V (M(r) 83,300), whose primary structure is reported here, is a part of the Ib-V-IX system of surface glycoproteins (GPs Ib alpha, Ib beta, V, IX) that constitute the receptor for von Willebrand factor (vWf) and mediate the adhesion of platelets to injured vascular surfaces in the arterial circulation, a critical initiating event in hemostasis. System members share physical associations, leucine-rich glycoprotein (LRG) structures, and a congenital deficiency state, Bernard-Soulier syndrome. With PCR techniques and platelet cDNA templates, 1.4 kb of GP V cDNA sequence was obtained that encodes 469 GP V amino acids. A genomic 3.5-kb BamHI fragment was then isolated that includes 3.46 kb of GP V cDNA sequence: the 1.7-kb open reading frame plus 2 bases of the 5' and 1.8 kb of the 3' untranslated regions. Northern blot analysis reveals three GP V platelet transcripts of 3.8, 4.2, and 5.2 kb. A 16-amino acid signal peptide is present. Mature GP V is a 544-amino acid transmembrane protein with a 504-amino acid extracellular domain that encompasses a set of 15 tandem LRG repeats in a "flank-LRG center-flank" array [Roth, G. J. (1991) Blood 77, 5-19] along with eight putative N-linked glycosylation sites and cleavage sites for thrombin and calpain. GP V is a transmembrane, adhesive LRG protein that plays an undefined, but potentially critical, role in the expression and/or function of the Ib-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries. Images Fig. 2 PMID:7690959

  8. 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.

  9. Properties of potato lectin and the nature of its glycoprotein linkages

    PubMed Central

    Allen, Anthony K.; Desai, Nila N.; Neuberger, Albert; Creeth, J. Michael

    1978-01-01

    1. Potato lectin is a glycoprotein that contains about 47% (by weight) l-arabinose, 3% d-galactose and 11% hydroxyproline. It has a monomeric molecular weight of about 50000 and probably exists as a monomer–dimer system in aqueous solution, with the monomer predominating. It has a very high viscosity, which would indicate either that the molecule is very expanded or that it is an elongated ellipsoid. 2. After prolonged proteolytic digestion of a reduced and carboxymethylated derivative of the lectin, a glycopeptide was isolated (of mol.wt. 32000–34000) that included all the carbohydrate and hydroxyproline of the original glycoprotein but less than 30% of the total original amino acid residues. 3. The arabinose of the glycoprotein is present exclusively as the β-arabinofuranoside and this includes those residues that are directly linked to the hydroxyproline residues of the polypeptide chain. All the arabinose of the glycoprotein is linked to the polypeptide chain through the hydroxyproline residues; the ratio of arabinose to hydroxyproline is 3.4:1. Although α-arabinofuranosides are known to be present in arabinans and arabinogalactans, the natural occurrence of β-arabinofuranosides has not previously been reported. 4. Nine or ten serine residues of the polypeptide chain are substituted with single α-galactopyranoside residues that can be removed by the action of α-galactosidase from coffee beans but not by a β-galactosidase. This is the first report of an α-galactoside linkage to serine. The effect of α-galactosidase is much greater on a glycopeptide from which the arabinose has been already removed, which indicates a steric hindrance of the galactosidase action by adjacent chains of arabinosides. 5. In 0.5m-NaOH (pH13.7), galactose residues were removed from the serine residues of the glycopeptide by a process of β-elimination. This reaction took place very slowly in the intact glycopeptide but much more rapidly when the arabinofuranoside residues

  10. Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

    PubMed

    Zhou, Xuan; Hong, Guo-Ying; Huang, Bin-Bin; Duan, Yuan-Meng; Shen, Jia-Yi; Ni, Mao-Wei; Cong, Wei-Tao; Jin, Li-Tai

    2014-05-01

    An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins. PMID:24591039

  11. Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum.

    PubMed Central

    Trombetta, E S; Helenius, A

    1999-01-01

    UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle. We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP. This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences. It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER. PMID:10369669

  12. On-line high-performance liquid chromatography/mass spectrometric characterization of native oligosaccharides from glycoproteins.

    PubMed

    Barroso, Begona; Dijkstra, Rene; Geerts, Marlieke; Lagerwerf, Fija; van Veelen, Peter; de Ru, Arnoud

    2002-01-01

    An on-line high-performance liquid chromatography/mass spectrometry (HPLC/MS) method is described for the rapid characterization of any type of oligosaccharide released from glycoproteins. The procedure can be applied without further manipulation to fractions collected from a high-performance anion-exchange chromatography-pulse amperometric detection (HPAEC-PAD) system commonly used for glycosylation mapping of glycoproteins, or to a pool of oligosaccharides directly released from glycoproteins. The system consists of a porous graphitized high-performance chromatography column (Hypercarb) coupled to a quadrupole time-of-flight (TOF) mass spectrometer. Oligosaccharides are eluted from the column with a gradient of ammonium acetate/acetonitrile and directly identified following in-source fragmentation. Some applications of the method are presented, as well as information about the spectra and fragmentation behavior observed for N- and O-linked oligosaccharides released from some recombinant glycoproteins. Low femtomole limits of detection are achieved using proper miniaturization. PMID:12112260

  13. A selective method for sequential splitting of O- and N-linked glycans from N,O-glycoproteins.

    PubMed

    Likhosherstov, L M; Novikova, O S; Derevitskaya, V A; Kochetkov, N K

    1990-05-15

    O-Linked oligosaccharides from N,O-glycoproteins were selectively split off by treatment with alkaline sodium borohydride in the presence of cadmium salt. The side reaction of reductive cleavage of N-glycosylamide and peptide bonds, observed under standard conditions of splitting of O-linked chains (M NaBH4 and 50mM NaOH, 16 h, 50 degrees), was inhibited by addition of 50-10 mM cadium acetate and 5-10mM EDTA.Na4, as shown by treatment of model compounds and several glycoproteins (ovomucoid, group-specific glycoproteins H and B, fetuin, and asialofetuin). This treatment, in combination with the previously developed procedure for the release of the N-linked oligosaccharide chains by lithium borohydride, allows a sequential, selective cleavage of O-, and then N-linked oligosaccharides from N,O-glycoproteins by chemical methods.

  14. Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells

    SciTech Connect

    Owens, R.J.; Compans, R.W.

    1989-02-01

    Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. The authors have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, /sup 125/I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.

  15. Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses.

    PubMed

    Zimmer, Gert; Locher, Samira; Berger Rentsch, Marianne; Halbherr, Stefan J

    2014-08-01

    Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment.

  16. Effects of carbohydrate precursors of glycoproteins on retention performance of a brightness discrimination task in rats.

    PubMed

    Popov, N; Hecker, C; Riechert, U; Matthies, H

    1985-01-01

    The influence of intraventricularly applied carbohydrate precursors of glycoproteins on acquisition and retention of a brightness discrimination task in rats was tested. The injection of 0.8 mumoles L-fucose/animal, N-acetylneuraminic acid or D-galactosamine as well as 2.4 mumoles/animal D-galactose or D-glucosamine 30 min before starting the behavioural experiments significantly improved the retention performance of the acquired behaviour. The intraventricular application of 2.4 mumoles D-mannose/animal had no influence on the behavioural parameters tested. The results are discussed in the light of an activation of glycoprotein formation in brain tissue mainly by carbohydrates which occupy terminal positions in the oligosaccharide chains.

  17. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology.

    PubMed

    Chua, Chong Long; Sam, I-Ching; Chan, Yoke Fun

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection. PMID:27233260

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

    PubMed

    Wenzl, S; Thym, D; Sumper, M

    1984-04-01

    We report the chemical characterization of the highly sulphated glycoprotein SSG 185 from Volvox carteri. SSG 185 is a hydroxyproline-containing, extracellular glycoprotein. The sulphate residues are clustered within the parent saccharide structure of SSG 185, since on mercaptolysis all the sulphate residues are recovered in a small saccharide fragment containing mannose, arabinose and sulphate (in a molar ratio of 112). SSG 185 is a short-lived molecule, serving as a precursor for a high mol. wt. component of the extracellular matrix. Synthesis of SSG 185 is developmentally controlled. Different SSG 185 variants, with unknown modifications in the sulphated saccharide fragment, are synthesized at different developmental stages or under the influence of the sexual inducer. These modifications remain conserved in the aggregated state of SSG 185, indicating the development-dependent modification of the extracellular matrix. PMID:16453512

  19. A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms.

    PubMed

    Machiah, Deepa K; Girish, K S; Gowda, T Veerabasappa

    2006-06-01

    Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis.

  20. Evidence for activity-regulated hormone-binding cooperativity across glycoprotein hormone receptor homomers.

    PubMed

    Zoenen, Maxime; Urizar, Eneko; Swillens, Stéphane; Vassart, Gilbert; Costagliola, Sabine

    2012-01-01

    Glycoprotein hormone receptors show strong negative cooperativity. As a consequence, at physiological hormone concentrations, a single agonist binds to a receptor dimer. Here we present evidence that constitutively active receptors lose cooperative allosteric regulation in direct relation with their basal activity. The most constitutive mutants lost nearly all cooperativity and showed an increase of initial tracer binding, reflecting the ability of each protomer to bind with equal affinity. Allosteric interaction between the protomers takes place at the transmembrane domain. The allosteric message resulting from hormone binding to the ectodomain of one protomer travels 'downward' to its transmembrane domain, before affecting the transmembrane domain of the other protomer. This results in transmission of an 'upward' message lowering the binding affinity of the ectodomain of the second protomer. Our results demonstrate a direct relation between the conformational changes associated with activation of the transmembrane domain and the allosteric behaviour of glycoprotein hormone receptors dimers.