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Sample records for 5t4 oncofetal glycoprotein

  1. Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

    Mulryan, Kate; Ryan, Matthew G; Myers, Kevin A; Shaw, David; Wang, Who; Kingsman, Susan M; Stern, Peter L; Carroll, Miles W

    2002-10-01

    The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy. PMID:12481437

  2. Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells--A Key Role for Heat-Shock Protein 70 and Receptor CD91.

    Salimu, Josephine; Spary, Lisa K; Al-Taei, Saly; Clayton, Aled; Mason, Malcolm D; Staffurth, John; Tabi, Zsuzsanna

    2015-06-01

    Immune responses contribute to the success of radiotherapy of solid tumors; however, the mechanism of triggering CD8(+) T-cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8(+) T-cell stimulation. We established a cross-presentation model in which DCs present a naturally expressed oncofetal tumor antigen (5T4) from irradiated DU145 prostate cancer cells to 5T4-specific T cells. The aim was to establish which immunogenic signals are important in radiation-induced cross-presentation. Radiation (12 Gy) caused G2-M cell-cycle arrest and cell death, increased cellular 5T4 levels, high-mobility protein group-B1 (HMGB1) release, and surface calreticulin and heat-shock protein-70 (Hsp70) expression in DU145 cells. DCs phagocytosed irradiated tumor cells efficiently, followed by upregulation of CD86 on phagocytic DCs. CD8(+) 5T4-specific T cells, stimulated with these DCs, proliferated and produced IFNγ. Inhibition of HMGB1 or the TRIF/MyD88 pathway only had a partial effect on T-cell stimulation. Unlike previous investigators, we found no evidence that DCs carrying Asp299Gly Toll-like receptor-4 (TLR4) single-nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, pretreatment of tumor cells with Hsp70 inhibitors resulted in a highly statistically significant and robust prevention of antigen cross-presentation and CD86 upregulation on DCs cocultured with irradiated tumor cells. Blocking the Hsp70 receptor CD91 also abolished cross-presentation. Together, the results from our study demonstrate that irradiation induces immunologically relevant changes in tumor cells, which can trigger CD8(+) T-cell responses via a predominantly Hsp70-dependent antigen cross-presentation process. PMID:25678582

  3. Targeting immune effector molecules to human tumor cells through genetic delivery of 5T4-specific scFv fusion proteins.

    Myers, Kevin A; Ryan, Matthew G; Stern, Peter L; Shaw, David M; Embleton, M Jim; Kingsman, Susan M; Carroll, Miles W

    2002-11-01

    Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effector functions. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extracellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4. This TAA is expressed by a wide variety of carcinomas and is associated with metastasis and poorer clinical outcome. We have engineered retroviral constructs that produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effector moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFv-HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers. PMID:12386827

  4. Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

    Shaw, D M; Embleton, M J; Westwater, C; Ryan, M G; Myers, K A; Kingsman, S M; Carroll, M W; Stern, P L

    2000-12-15

    The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs. PMID:11113573

  5. Identification of pre- and post-treatment markers, clinical, and laboratory parameters associated with outcome in renal cancer patients treated with MVA-5T4

    RobertAmato

    2013-07-01

    Full Text Available The recent approvals of immunotherapeutic agents (Sipuleucel-T and Ipilimumab for the treatment of different solid tumors gave a boost to the growing cancer immunotherapy field, even though few immunotherapy studies have demonstrated convincingly that there is a direct link between the predicted mode of action of an immunological compound and therapeutic benefit. MVA-5T4 (Trovax® is a novel vaccine combining the tumor-associated antigen 5T4 to an engineered vector-modified vaccinia Ankara (MVA. MVA helps to express the oncofetal 5T4 antigen and subsequently trigger a tumor-directed immune reaction. The safety and clinical benefit reported in multiple phase I and II clinical trials using MVA-5T4 were encouraging; immune responses were induced in almost all treated patients, and associations between 5T4-specific cellular or humoral responses and clinical benefit were reported in most of the nine phase II trials. In particular, clinical studies conducted in renal cell carcinoma (RCC patients have demonstrated an association between 5T4-specific (but not MVA antibody responses and enhanced survival. This review describes the clinical studies using MVA-5T4 conducted in RCC that convincingly demonstrated that an antigen-specific immune response induced by vaccination is associated with enhanced patient survival and is not simply a function of the general “health” of patients. We will also provide our expert opinions on possible future better-designed clinical trials based on relevant biomarkers. In addition, various combinations of MVA-5T4 and different and newer immunomodulator agents with promising clinical benefit will be discussed.

  6. Oncofetal fibronectins in oral carcinomas

    Mandel, U; Gaggero, B; Reibel, J;

    1994-01-01

    -B-containing isoform and the oncofetal FN isoform derived by O-glycosylation, in oral squamous cell carcinomas, premalignant lesions, and normal oral mucosa. A selective expression of the ED-B-containing isoform was demonstrated in close relation to the invading carcinoma (38/38), whereas there was virtually no...... staining in submucosa underlying premalignant lesions (1/11) and normal epithelium (0/5). The ED-B-containing FN showed close co-distribution and staining pattern with the oncofetal isoform derived by O-glycosylation. These results demonstrate that accumulation of FN adjacent to oral carcinomas includes...... both the ED-B-containing isoform and the isoform derived by O-glycosylation. Although both the change in primary structure and glycosylation of FN create conformational and immunologically detectable changes, the functional consequences in association with invasive carcinoma are poorly understood at...

  7. UniProt search blastx result: AK288692 [KOME

    Full Text Available AK288692 J090060K04 Q13641|TPBG_HUMAN Trophoblast glycoprotein precursor (5T4 oncofetal... trophoblast glycoprotein) (5T4 oncotrophoblast glycoprotein) (5T4 oncofetal antigen) (M6P1) - Homo sapiens (Human) 0 ...

  8. Overexpression and potential targeting of the oncofoetal antigen 5T4 in malignant pleural mesothelioma.

    Al-Taei, Saly; Salimu, Josephine; Lester, Jason F; Linnane, Seamus; Goonewardena, Madusha; Harrop, Richard; Mason, Malcolm D; Tabi, Zsuzsanna

    2012-08-01

    Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treatments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this study was to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen in MPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and -histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtained from the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma of patients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing was determined using an HLA-A2(+), CD8(+) T-cell line, developed against the 5T4(17-25) peptide. We report here that cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothelin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF. Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but not on reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8(+) T-cells were able to kill four out of six HLA-A2(+) MPM cell lines but not an HLA-A2(-) cell line, demonstrating immune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 is a potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed on mesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated into a Phase II clinical trial applying 5T4-targeted therapies in MPM patients. PMID:22498111

  9. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  10. Measurement of pancreatic oncofetal antigen by immunoradiometric assay method

    Ohnami, Shumpei; Zeze, Fujio; Kuroda, Tamaki; Nakata, Hajime

    1988-01-01

    A commercially available immunoradiometric assay kit for measuring pancreatic oncofetal antigen (POA) in the serum was fundamentally and clinically evaluated. Fundamental results for reproducibility, recovery, and dilution of the kit were satisfactory enough to use it in the clinical setting. Serum levels of POA ranged between 3.4 U/ml and 23.2 U/ml in 137 normal subjects. Eighteen U/ml, observed in 95 % of the subjects, was defined as the cutoff level of POA. Positive rate of POA was 47 % (9/19) for esophageal cancer, 32 % (10/31) for lung cancer, 39 % (3/13) for breast cancer, 27 % (10/37) for gastric cancer, 64 % (35/55) for hepatocellular carcinoma, 45 % (9/20) for biliary cancer, 75 % (12/16) for pancreatic cancer, 41 % (12/29) for colorectal cancer, 14 % (4/28) for cervical cancer, and 35 % (6/17) for ovarian cancer. It was high as well for benign diseases, such as liver cirrhosis, duodenal ulcer, and chronic renal failure. The sole measurement of POA failed to differentiate benign from malignant diseases. In cases of pancreatic cancer, higher serum POA levels and increased positive rate of POA were associated with clinical progression. (Namekawa, K.).

  11. A novel unbalanced de novo translocation der(5t(4;5(q26;q21.1 in adult T-cell precursor lymphoblastic leukemia

    Kjeldsen Eigil

    2012-05-01

    Full Text Available Abstract We here describe a novel unbalanced de novo translocation der(5t(4;5(q26;q21.1 in a 39-year-old male diagnosed with acute T-cell lymphoblastic leukemia. Bone marrow (BM was massively infiltrated with 85 % highly proliferative polymorphic T-cell precursors. Immunologically, the malignant cells stained positive for CD7, CD34, intracytoplasmic CD3+, TdT + and negative for CD3 and CD5. G-banded chromosome analysis of BM cells showed the normal karyotype 46,XY[25] whereas BAC-based aCGH analysis revealed partial gain of 4q and partial loss of 5q. Multicolor karyotyping confirmed the presence of an unbalanced der(5t(4;5 as the sole structural abnormality. Subsequent high-resolution oligonucleotide-based aCGH analysis showed that the der(5t(4;5(q26;q21.1 resulted in partial trisomy of 4q26qter (117,719,015-190,613,014 and partial monosomy of 5q21.1qter (100,425,442-180,857,866 and that there was no indication of any gene disruptions resulting from the breakages. Interphase FISH analysis using BAC-based specific probes for 4q26 and 5q21.1 confirmed the breakpoints and revealed approximately 80 % abnormal cells accordingly. At 4q26 the MIR1973 gene is located centromeric to the breakpoint in the copy number neutral region and the TRAM1L1 gene is located within the gained region. At 5q21.1 the genes ST8SIA4 and MIR548p are located centromeric to the breakpoint and no known genes up to approximately 1 Mb telomeric to the breakpoint in the copy number loss region. Interestingly, only the gene ST8SIA4 at 5q21.1 have been implicated in T-cell regulation as it encodes one of the key enzymes for polysialysation of surface proteins on dendritic cells which are important regulators for T-cell proliferation. The der(5t(4;5 is thought to play a crucial role in the pathogenesis of acute T-ALL due to either gain of 4q, the loss of 5q, or deregulation of genes in proximity to the breakpoints.

  12. Active treatment of murine tumors with a highly attenuated vaccinia virus expressing the tumor associated antigen 5T4 (TroVax) is CD4+ T cell dependent and antibody mediated.

    Harrop, Richard; Ryan, Matthew G; Myers, Kevin A; Redchenko, Irina; Kingsman, Susan M; Carroll, Miles W

    2006-09-01

    5T4 is a tumor associated antigen that is expressed on the surface of a wide spectrum of human adenocarcinomas. The highly attenuated virus, modified vaccinia Ankara, has been engineered to express human 5T4 (h5T4). In a pre-clinical murine model, the recombinant virus (TroVax) induces protection against challenge with CT26-h5T4 (a syngeneic tumor line expressing h5T4). Anti-tumor activity is long lived, with protection still evident 6 months after the final vaccination. In a therapeutic setting, injection of mice with TroVax results in a reduction in tumor burden of >90%. Depletion of CD8+ T cells has no effect upon therapy in the active treatment model, whereas depletion of CD4+ T cells completely abrogates anti-tumor activity. In a prophylactic setting, depletion of CD4+ and CD8+ T cells after the induction of a h5T4 immune response has no deleterious effect on protection following challenge with CT26-h5T4. In light of these studies, the role of antibodies in protection against tumor challenge was investigated. 5T4 specific polyclonal serum decreased tumor burden by approximately 70%. Thus, we conclude that CD4+ T cells are essential for the induction of a protective immune response and that antibodies are the likely effector moiety in this xenogeneic murine tumor model. PMID:16311730

  13. Enzyme immunoassay of pancreatic oncofetal antigen (POA) as a marker of pancreatic cancer.

    Nishida, K; Sugiura, M; Yoshikawa, T; Kondo, M

    1985-01-01

    For the quantitative measurement of pancreatic oncofetal antigen (POA), an enzyme immunoassay for POA has been developed, and is based on the sandwich method using antibody-coupled glass beads and enzyme (peroxidase)-labelled antibody. Serum POA concentrations were increased significantly in patients with pancreatic cancer, but not in those with chronic pancreatitis or other miscellaneous diseases, or in normal subjects. It is concluded that the enzyme immunoassay could be used for the assay ...

  14. Sperm protein 17 is an oncofetal antigen: a lesson from a murine model.

    Arnaboldi, F; Menon, A; Menegola, E; Di Renzo, F; Mirandola, L; Grizzi, F; Figueroa, J A; Cobos, E; Jenkins, M; Barajon, I; Chiriva-Internati, Maurizio

    2014-10-01

    Sperm protein 17 (Sp17) was originally identified in the flagellum of spermatozoa and subsequently included in the subfamily of tumor-associated antigens known as cancer-testes antigens (CTA). Sp17 has been associated with the motility and migratory capacity in tumor cells, representing a link between gene expression patterns in germinal and tumor cells of different histological origins. Here we review the relevance of Sp17 expression in the mouse embryo and cancerous tissues, and present additional data demonstrating Sp17 complex expression pattern in this murine model. The expression of Sp17 in embryonic as well as adult neoplastic cells, but not normal tissues, suggests this protein should be considered an "oncofetal antigen." Further investigations are necessary to elucidate the mechanisms and functional significance of Sp17 aberrant expression in human adult cells and its implication in the pathobiology of cancer. PMID:24811209

  15. Lin28B is an oncofetal circulating cancer stem cell-like marker associated with recurrence of hepatocellular carcinoma.

    Shu-Wen Cheng

    Full Text Available By using an expressed sequence tag bioinformatic algorithm, we identified that Lin28 homolog B (Lin28B may have an oncofetal expression pattern which may facilitate detecting cancer cells in adults. It is also reported to be a potential marker for cancer stem cells. Therefore, we sought to verify oncofetal-stemness characters of Lin28B and test its potential as a circulating cancer stem cell-like marker in adult HCC patients. Lin28B mRNA was examined in a panel of fetal tissue, adult tissue and tumors. Lin28B was over-expressed or knocked down in HepG2 cells to evaluate its potential as a stem cell-like marker. RT-qPCR for Lin28B was performed in the peripheral blood mononuclear cells from patients with HCC receiving surgery (n=96 and non-HCC controls (n=60 and analyzed its clinical significance. Lin28B showed an oncofetal expression pattern. Its overexpression could upregulate stemness markers (OCT4, Nanog and SOX2 and enhance tumorsphere formation in vitro. Lin28B knockdown had opposite effects. Circulating Lin28B was detected in peripheral blood mononuclear cells in 3 cases (5% of non-HCC controls and 32 cases (33.3% of HCC patients. In HCC patients, circulating Lin28B was associated with high tumor grade (P=0.046, large size (P=0.005, high AJCC stage (P=0.044 and BCLC stage (P=0.017. Circulating Lin28B was significantly associated with decreased recurrence-free survival (P<0.001. Circulating Lin28B separated early stage HCC into 2 recurrence-free survival curves (P=0.003. In multivariate analysis, circulating Lin28B was an independent variable associated with early recurrence (P=0.045 and recurrence in early stage HCC (P=0.006. In conclusion, the oncofetal gene Lin28B is a potential oncofetal cancer-stem-cell-like circulating tumor cell marker that correlates with HCC recurrence after hepatectomy. Circulating Lin28B could refine early AJCC stages. Our finding supports the possible use of a TNMC (C for circulating tumor cells staging system

  16. Lin28B is an oncofetal circulating cancer stem cell-like marker associated with recurrence of hepatocellular carcinoma.

    Cheng, Shu-Wen; Tsai, Hung-Wen; Lin, Yih-Jyh; Cheng, Pin-Nan; Chang, Yu-Chung; Yen, Chia-Jui; Huang, Hsuan-Pang; Chuang, Yun-Pei; Chang, Ting-Tsung; Lee, Chung-Ta; Chao, Anning; Chou, Cheng-Yang; Chan, Shih-Huang; Chow, Nan-Haw; Ho, Chung-Liang

    2013-01-01

    By using an expressed sequence tag bioinformatic algorithm, we identified that Lin28 homolog B (Lin28B) may have an oncofetal expression pattern which may facilitate detecting cancer cells in adults. It is also reported to be a potential marker for cancer stem cells. Therefore, we sought to verify oncofetal-stemness characters of Lin28B and test its potential as a circulating cancer stem cell-like marker in adult HCC patients. Lin28B mRNA was examined in a panel of fetal tissue, adult tissue and tumors. Lin28B was over-expressed or knocked down in HepG2 cells to evaluate its potential as a stem cell-like marker. RT-qPCR for Lin28B was performed in the peripheral blood mononuclear cells from patients with HCC receiving surgery (n=96) and non-HCC controls (n=60) and analyzed its clinical significance. Lin28B showed an oncofetal expression pattern. Its overexpression could upregulate stemness markers (OCT4, Nanog and SOX2) and enhance tumorsphere formation in vitro. Lin28B knockdown had opposite effects. Circulating Lin28B was detected in peripheral blood mononuclear cells in 3 cases (5%) of non-HCC controls and 32 cases (33.3%) of HCC patients. In HCC patients, circulating Lin28B was associated with high tumor grade (P=0.046), large size (P=0.005), high AJCC stage (P=0.044) and BCLC stage (P=0.017). Circulating Lin28B was significantly associated with decreased recurrence-free survival (P<0.001). Circulating Lin28B separated early stage HCC into 2 recurrence-free survival curves (P=0.003). In multivariate analysis, circulating Lin28B was an independent variable associated with early recurrence (P=0.045) and recurrence in early stage HCC (P=0.006). In conclusion, the oncofetal gene Lin28B is a potential oncofetal cancer-stem-cell-like circulating tumor cell marker that correlates with HCC recurrence after hepatectomy. Circulating Lin28B could refine early AJCC stages. Our finding supports the possible use of a TNMC (C for circulating tumor cells) staging system in HCC

  17. Involvement of O-glycosylation defining oncofetal fibronectin in epithelial-mesenchymal transition process

    Freire-de-Lima, Leonardo; Gelfenbeyn, Kirill; Ding, Yao;

    2011-01-01

    The process termed "epithelial-mesenchymal transition" (EMT) was originally discovered in ontogenic development, and has been shown to be one of the key steps in tumor cell progression and metastasis. Recently, we showed that the expression of some glycosphingolipids (GSLs) is down-regulated during...... EMT in human and mouse cell lines. Here, we demonstrate the involvement of GalNAc-type (or mucin-type) O-glycosylation in EMT process, induced with transforming growth factor ß (TGF-ß) in human prostate epithelial cell lines. We found that: (i) TGF-ß treatment caused up-regulation of oncofetal...... fibronectin (onfFN), which is defined by mAb FDC6, and expressed in cancer or fetal cells/tissues, but not in normal adult cells/tissues. The reactivity of mAb FDC6 requires the addition of an O-glycan at a specific threonine, inside the type III homology connective segment (IIICS) domain of FN. (ii) This...

  18. Discovery and diagnostic value of a novel oncofetal protein: glypican 3.

    Wang, Sean K; Zynger, Debra L; Hes, Ondrej; Yang, Ximing J

    2014-11-01

    Glypican 3 is a membrane-bound heparan sulfate proteoglycan, which has recently been identified as a marker for liver cancer and germ cell malignancies. Individuals with loss-of-function mutations for the glypican 3 gene exhibit Simpson-Golabi-Behmel syndrome, a rare X-linked overgrowth disorder. Expression of glypican 3 mRNA and protein is normally silenced in most adult organs and may reappear during malignant transformation. In the past few years, immunohistochemical and molecular characteristics of glypican 3 in hepatocellular carcinoma have been elucidated. More recently, glypican 3 has been emerging as a new diagnostic marker for germ cell tumors and especially testicular and ovarian yolk sac tumors. However, in other tumors such as renal cell carcinomas, squamous cell carcinomas, and melanomas, studies disagree on the level of glypican 3 expression. Finally, there is the controversial notion of glypican 3 as a tumor suppressor gene. In this review article, we update current knowledge on glypican 3 expression in normal and neoplastic tissues, evaluate its utility as a tumor marker in clinical practice, and explore its role as a novel oncofetal protein with clinical implications. Our focus is on the diagnostic value of glypican 3 in germ cell tumors and other neoplasms in addition to hepatocellular carcinoma. In conclusion, glypican 3 has been proven to be a useful immunohistochemical marker in distinguishing yolk sac tumors, choriocarcinomas, and Wilms tumors from other malignancies histologically mimicking these primitive tumors. Clinically, we recommend that glypican 3 be used as part of a panel of markers in subtyping testicular germ cell tumors. PMID:25299314

  19. Glycosylation Engineering of Glycoproteins

    Sadamoto, Reiko; Nishimura, Shin-Ichiro

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

  20. Use of the uteroglobin platform for the expression of a bivalent antibody against oncofetal fibronectin in Escherichia coli.

    Elisa Ventura

    Full Text Available Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646-26654. Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19 toward the oncofetal fibronectin (B-FN, a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG, 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.

  1. The oncofetal protein IMP3: a novel grading tool and predictor of poor clinical outcome in human gliomas.

    Del Gobbo, Alessandro; Vaira, Valentina; Ferrari, Lucia; Patriarca, Carlo; Di Cristofori, Andrea; Ricca, Dario; Caroli, Manuela; Rampini, Paolo; Bosari, Silvano; Ferrero, Stefano

    2015-01-01

    Morphologic criteria illustrated in WHO guidelines are the most significant prognostic factor in human gliomas, but novel biomarkers are needed to identify patients with a poorer outcome. The present study examined the expression of the oncofetal protein IMP3 in a series of 135 patients affected by high-grade (grade III and IV) gliomas, correlating the results with proliferative activity, molecular parameters, and clinical and follow-up data. Overall, IMP3 expression was higher in glioblastomas (68%) than in grade III tumors (20%, P disease-free survival than negative ones (P = 0.0002 and P = 0.006, resp.). IMP3 expression was significantly associated with the absence of mutations of IDH1 gene (P = 0.0001) and with the unmethylated phenotype of MGMT in high-grade gliomas (P = 0.004). High Ki67 levels were correlated with better prognosis in glioblastomas but IMP3 expression was not correlated with the proliferation index. These findings confirm the role of IMP3 as a marker of poor outcome, also in consideration of its association with IDH1 wild-type phenotype and MGMT unmethylated status. The data suggest that IMP3 staining could identify a subgroup of patients with poor prognosis and at risk of recurrence in high-grade gliomas. PMID:25695077

  2. Monoclonal antibodies specific for oncofetal antigen – immature laminin receptor protein: Effects on tumor growth and spread in two murine models

    McClintock, Shannon D.; Warner, Roscoe L.; Ali, Saqib; Chekuri, Apurupa; Dame, Michael K.; Attili, Durga; Knibbs, Randall K; Aslam, Muhammad Nadeem; Sinkule, Joseph; Morgan, Alton Charles; Barsoum, Adel; Smith, Lauren B.; Beer, David G.; Johnson, Kent J.; Varani, James

    2015-01-01

    The oncofetal antigen – immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While...

  3. Oncofetal Epigenetic Bivalency in Breast Cancer Cells: H3K4 and H3K27 Tri-Methylation as a Biomarker for Phenotypic Plasticity.

    Messier, Terri L; Boyd, Joseph R; Gordon, Jonathan A R; Stein, Janet L; Lian, Jane B; Stein, Gary S

    2016-11-01

    Alterations in the epigenetic landscape are fundamental drivers of aberrant gene expression that contribute to cancer progression and pathology. Understanding specific modes of epigenetic regulation can be used to identify novel biomarkers or targets for therapeutic intervention to clinically treat solid tumors and leukemias. The bivalent marking of gene promoters by H3K4me3 and H3K27me3 is a primary mechanism to poise genes for expression in pluripotent embryonic stem cells (ESC). In this study we interrogated three well-established mammary cell lines to model epigenetic programming observed among breast cancer subtypes. Evidence is provided for a distinct bivalent signature, activating and repressive histone marks co-residing at the same gene promoter, in the MCF7 (ESR/PGR+) luminal breast cancer cell line. We identified a subset of genes, enriched for developmental pathways that regulate cellular phenotype and signaling, and partially recapitulate the bivalent character observed in ESC. We validated the biological relevance of this "oncofetal epigenetic" signature using data from ESR/PGR+ tumor samples from breast cancer patients. This signature of oncofetal epigenetic control is an informative biomarker and may provide novel therapeutic targets, selective for both recurring and treatment-resistant cancers. J. Cell. Physiol. 231: 2474-2481, 2016. © 2016 Wiley Periodicals, Inc. PMID:26916849

  4. Comparing Prothrombin induced by vitamin K absence-II (PIVKA-II) with the oncofetal proteins Glypican-3, Alpha feto protein and Carcinoembryonic antigen in diagnosing hepatocellular carcinoma among Egyptian patients

    Iman A. Abd El Gawad; Mossallam, Ghada I.; Noha H. Radwan; Elzawahry, Heba M; Niveen M. Elhifnawy

    2014-01-01

    Background: Hepatocellular carcinoma (HCC) is usually asymptomatic in the early stage and does not show elevated alpha-feto protein (AFP). AFP shows 60–80% sensitivity in diagnosing HCC. Glypican3 (GPC-3) is an oncofetal protein that is only detected in HCC cells but not in benign liver tissues, while Carcinoembryonic antigen (CEA) is expressed in various neoplasms including HCC. Although, it is not specific for HCC. Prothrombin induced by vitamin K absence-II (PIVKA-II) is an abnormal ...

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

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

    1991-12-01

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

  6. Lubrication by glycoprotein brushes.

    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.

  7. Monoclonal antibodies specific for oncofetal antigen – immature laminin receptor protein: Effects on tumor growth and spread in two murine models

    McClintock, Shannon D; Warner, Roscoe L; Ali, Saqib; Chekuri, Apurupa; Dame, Michael K; Attili, Durga; Knibbs, Randall K; Aslam, Muhammad Nadeem; Sinkule, Joseph; Morgan, Alton Charles; Barsoum, Adel; Smith, Lauren B; Beer, David G; Johnson, Kent J; Varani, James

    2015-01-01

    The oncofetal antigen – immature laminin receptor protein (OFA/iLRP) has been linked to metastatic tumor spread for several years. The present study, in which 2 highly-specific, high-affinity OFA/iLRP-reactive mouse monoclonal antibodies were examined for ability to suppress tumor cell growth and metastatic spread in the A20 B-cell leukemia model and the B16 melanoma model, provides the first direct evidence that targeting OFA/iLRP with exogenous antibodies can have therapeutic benefit. While the antibodies were modestly effective at preventing tumor growth at the primary injection site, both antibodies strongly suppressed end-organ tumor formation following intravenous tumor cell injection. Capacity of anti-OFA/iLRP antibodies to suppress tumor spread through the blood in the leukemia model suggests their use as a therapy for individuals with leukemic disease (either for patients in remission or even as part of an induction therapy). The results also suggest use against metastatic spread with solid tumors. PMID:25799942

  8. Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6

    Stadler, Christiane R.; Bähr-Mahmud, Hayat; Plum, Laura M.; Schmoldt, Kathrin; Kölsch, Anne C.; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    abstract The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need. We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties. Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicated by 6PHU3 treatment and survival of mice is significantly prolonged. Tumors of 6PHU3-treated mice are strongly infiltrated with activated CD4+, CD8+ T cells and TEM type cells but not Tregs and display a general activation of a mostly inflammatory phenotype. These effects are only observed upon bispecific but not monospecific engagement of 6PHU3. Together with the exceptionally cancer cell selective expression of the oncofetal tumor marker CLDN6, this provides a safeguard with regard to toxicity. In summary, our data shows that the concept of T-cell redirection combined with that of highly selective targeting of CLDN6-positive solid tumors is effective. Thus, exploring 6PHU3 for clinical therapy is warranted. PMID:27141353

  9. Phosphorylation of the multidrug resistance associated glycoprotein

    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 MgCl2 was enhanced a minimum of 2-fold by 10 μM 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 [γ-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance

  10. Phosphorylation of the multidrug resistance associated glycoprotein.

    Mellado, W; Horwitz, S B

    1987-11-01

    Drug-resistant cell lines derived from the mouse macrophage-like cell line J774.2 express the multidrug resistance phenotype which includes the overexpression of a membrane glycoprotein (130-140 kilodaltons). Phosphorylation of this resistant-specific glycoprotein (P-glycoprotein) in intact cells and in cell-free membrane fractions has been studied. The phosphorylated glycoprotein can be immunoprecipitated by a rabbit polyclonal antibody specific for the glycoprotein. Phosphorylation studies done with partially purified membrane fractions derived from colchicine-resistant cells indicated that (a) phosphorylation of the glycoprotein in 1 mM MgCl2 was enhanced a minimum of 2-fold by 10 microM cAMP and (b) the purified catalytic subunit of the cAMP-dependent protein kinase (protein kinase A) phosphorylated partially purified glycoprotein that was not phosphorylated by [gamma-32P]ATP alone, suggesting that autophosphorylation was not involved. These results indicate that the glycoprotein is a phosphoprotein and that at least one of the kinases responsible for its phosphorylation is a membrane-associated protein kinase A. The state of phosphorylation of the glycoprotein, which is a major component of the multidrug resistance phenotype, may be related to the role of the glycoprotein in maintaining drug resistance. PMID:3427052

  11. Isolation of glycoproteins from brown algae.

    Surendraraj, Alagarsamy; Farvin Koduvayur Habeebullah , Sabeena; Jacobsen, Charlotte

    2015-01-01

    The present invention relates to a novel process for the isolation of unique anti-oxidative glycoproteins from the pH precipitated fractions of enzymatic extracts of brown algae. Two brown seaweeds viz, Fucus serratus and Fucus vesiculosus were hydrolysed by using 3 enzymes viz, Alcalase, Viscozyme and Termamyl and the glycoproteins were isolated from these enzyme extracts.

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

    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, [5,6-3H]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

  13. Comparing Prothrombin induced by vitamin K absence-II (PIVKA-II) with the oncofetal proteins Glypican-3, Alpha feto protein and Carcinoembryonic antigen in diagnosing hepatocellular carcinoma among Egyptian patients

    Background: Hepatocellular carcinoma (HCC) is usually asymptomatic in the early stage and does not show elevated alpha-feto protein (AFP). AFP shows 60-80% sensitivity in diagnosing HCC. Glypican3 (GPC-3) is an oncofetal protein that is only detected in HCC cells but not in benign liver tissues, while Carcinoembryonic antigen (CEA) is expressed in various neoplasms including HCC. Although, it is not specific for HCC. Prothrombin induced by vitamin K absence-II (PIVKA-II) is an abnormal prothrombin protein that is increased in the serum of HCC patients. It has higher sensitivity and specificity compared to AFP. The aim of this study is to compare the clinical utility of PIVKA-II with GPC-3, AFP and CEA in diagnosing HCC. Prothrombin induced by vitamin K absence-II Patients and methods: This study included 40 patients with HCC, 10 patients with cirrhosis as a benign control group, and 10 apparently healthy volunteers as normal controls. Serum samples were subjected to routine laboratory investigations, measurement of CEA, AFP using MEIA technique (Axsym), glypican3, and PIVKA-II using ELISA technique in the sera of all patients and controls. Results: All markers showed the highest results in the HCC group. Higher concentrations of PIVKA- II were detected in patients with splenomegaly, and in tumors with size (>3 cm). Combination of Glypican-3 and PIVKA-II showed the highest sensitivity, while GPC-3 alone and combination of GPC-3 and AFP showed the highest specificity to differentiate HCC from liver cirrhosis and normal controls. GPC-3, PIVKAII, and combination of both showed the highest sensitivity, while GPC-3 alone showed the highest specificity to differentiate HCC from liver cirrhosis. Conclusion: Glypican-3 is the only oncofetal antigen that showed comparable high diagnostic accuracy as PIVKA-II in diagnosing HCC among Egyptian patients.

  14. Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

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

    2000-01-01

    A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as...

  15. Glycoprotein biosynthesis by human normal platelets

    Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation

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

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

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

    Mellado, W.

    1988-01-01

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

  18. Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for virion localization of glycoprotein H.

    Klupp, B G; Fuchs, W; Weiland, E; Mettenleiter, T.C.

    1997-01-01

    Herpesviruses contain a number of envelope glycoproteins which play important roles in the interaction between virions and target cells. Although several glycoproteins are not present in all herpesviruses, others, including glycoproteins H and L (gH and gL), are conserved throughout the Herpesviridae. To elucidate common properties and differences in herpesvirus glycoprotein function, corresponding virus mutants must be constructed and analyzed in different herpesvirus backgrounds. Analysis o...

  19. Isolation of glycoproteins from brown algae

    2015-01-01

    The present invention relates to a novel process for the isolation of unique anti-oxidative glycoproteins from the pH precipitated fractions of enzymatic extracts of brown algae. Two brown seaweeds viz, Fucus serratus and Fucus vesiculosus were hydrolysed by using 3 enzymes viz, Alcalase, Viscozyme...

  20. Salivary agglutinin/glycoprotein-340/DMBT1

    Ligtenberg, Antoon J M; Veerman, Enno C I; Nieuw Amerongen, Arie V;

    2007-01-01

    Salivary agglutinin (SAG), lung glycoprotein-340 (gp-340) and Deleted in Malignant Brain Tumours 1 (DMBT1) are three names for identical proteins encoded by the dmbt1 gene. DMBT1/SAG/gp-340 belongs to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins, a superfamily of secreted o...

  1. Intracellular trafficking of P-glycoprotein

    Fu, Dong; Arias, Irwin M.

    2011-01-01

    Overexpression of P-glycoprotein (P-gp) is a major cause of multidrug resistance in cancer. P-gp is mainly localized in the plasma membrane and can efflux structurally and chemically unrelated substrates, including anticancer drugs. P-gp is also localized in intracellular compartments, such as ER, Golgi, endosomes and lysosomes, and cycles between endosomal compartments and the plasma membrane in a microtubular-actin dependent manner. Intracellular trafficking pathways for P-gp and participat...

  2. Influenza Hemagglutinin and Neuraminidase Membrane Glycoproteins*

    Gamblin, Steven J.; Skehel, John J.

    2010-01-01

    Considerable progress has been made toward understanding the structural basis of the interaction of the two major surface glycoproteins of influenza A virus with their common ligand/substrate: carbohydrate chains terminating in sialic acid. The specificity of virus attachment to target cells is mediated by hemagglutinin, which acquires characteristic changes in its receptor-binding site to switch its host from avian species to humans. Anti-influenza drugs mimic the natural sialic acid substra...

  3. Solid phase group specific absorbants in assays for glycoproteins

    The focus of this paper is on several technical advances in the assays for glycoprotein hormones and enzymes that have been achieved by use of the solid phase carbohydrate specific adsorbant concanavalin-A. Puriffication of glycoprotein radioligand after labelling by the chloramine-T method is readily accomplished using a small column of agarose bound concanavalin-A which separates glycoprotein radioligand from radioiodide and radiolabelled unadsorbed contaminants. After concanavalin-A column chromatography, radiolabelled glycoprotein hormone preparations exhibited improved binding to antibodies and tissue receptors. To increase the effective sensitivity of radioimmunoassays for glycoproteins, agarose bound concanavalin-A is used to extract and concentrate the glycoproteins from various biologic samples. For example, the effective sensitivity for the detection of human thyrotropin in serum was improved approximately 5 fold by using concanavalin-A concentrates of 1.5 ml of serum. Partial purification of the glycoprotein dopamine-β-hydroxylase from serum using agarose bound concanavalin-A resulted in separation of the serum factors that interfere with the measurement of enzyme activity. We conclude that in assays for glycoproteins, concanavalin-A is useful for purification of radioligand, for preparation of concentrates of glycoproteins from biologic samples, and for separation of glycoproteins from various interfering factors contained in biologic samples prior to radioligand or radioenzyme assay. (orig.)

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

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

    2014-01-01

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

  5. Expression of Rh Glycoproteins in the Mammalian Kidney

    Han, Ki-Hwan; Kim, Hye-Young; Weiner, I. David

    2009-01-01

    Ammonia metabolism is a fundamental process in the maintenance of life in all living organisms. Recent studies have identified ammonia transporter family proteins in yeast (Mep), plants (Amt), and mammals (Rh glycoproteins). In mammalian kidneys, where ammonia metabolism and transport are critically important for the regulation of systemic acid-base homeostasis, basolateral Rh B glycoprotein and apical/basolateral Rh C glycoprotein are expressed along the distal nephron segments. Data from ex...

  6. Complex formation of platelet thrombospondin with histidine-rich glycoprotein.

    Leung, L L; Nachman, R L; Harpel, P C

    1984-01-01

    Thrombospondin and histidine-rich glycoprotein are two proteins with diverse biological activities which have been associated with human platelets and other cell systems. Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human platelet thrombospondin formed a complex with purified human plasma histidine-rich glycoprotein. The formation of the thrombospondin-histidine-rich glycoprotein complex was specific, concentration dependent, and saturable. Significant bindin...

  7. Analysis of the cleavage site of the human immunodeficiency virus type 1 glycoprotein: requirement of precursor cleavage for glycoprotein incorporation.

    Dubay, J W; Dubay, S R; Shin, H. J.; Hunter, E

    1995-01-01

    Endoproteolytic cleavage of the glycoprotein precursor to the mature SU and TM proteins is an essential step in the maturation of retroviral glycoproteins. Cleavage of the precursor polyprotein occurs at a conserved, basic tetrapeptide sequence and is carried out by a cellular protease. The glycoprotein of the human immunodeficiency virus type 1 contains two potential cleavage sequences immediately preceding the N terminus of the TM protein. To determine the functional significance of these t...

  8. Glycoprotein component of plant cell walls

    The primary wall surrounding most dicotyledonous plant cells contains a hydroxyproline-rich glycoprotein (HRGP) component named extensin. A small group of glycopeptides solubilized from isolated cell walls by proteolysis contained a repeated pentapeptide glycosylated by tri- and tetraarabinosides linked to hydroxyproline and, by galactose, linked to serine. Recently, two complementary approaches to this problem have provided results which greatly increase the understanding of wall extensin. In this paper the authors describe what is known about the structure of soluble extensin secreted into the walls of the carrot root cells

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

    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)

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

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

    1980-01-01

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

  11. Glycoprotein Quality Control and Endoplasmic Reticulum Stress

    Qian Wang

    2015-07-01

    Full Text Available The endoplasmic reticulum (ER supports many cellular processes and performs diverse functions, including protein synthesis, translocation across the membrane, integration into the membrane, folding, and posttranslational modifications including N-linked glycosylation; and regulation of Ca2+ homeostasis. In mammalian systems, the majority of proteins synthesized by the rough ER have N-linked glycans critical for protein maturation. The N-linked glycan is used as a quality control signal in the secretory protein pathway. A series of chaperones, folding enzymes, glucosidases, and carbohydrate transferases support glycoprotein synthesis and processing. Perturbation of ER-associated functions such as disturbed ER glycoprotein quality control, protein glycosylation and protein folding results in activation of an ER stress coping response. Collectively this ER stress coping response is termed the unfolded protein response (UPR, and occurs through the activation of complex cytoplasmic and nuclear signaling pathways. Cellular and ER homeostasis depends on balanced activity of the ER protein folding, quality control, and degradation pathways; as well as management of the ER stress coping response.

  12. Role of envelope glycoproteins in intracellular virus maturation

    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 125I-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

  13. Solubilization of glycoproteins of envelope viruses by detergents

    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-β-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis. Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines

  14. Dominance of a Nonpathogenic Glycoprotein Gene over a Pathogenic Glycoprotein Gene in Rabies Virus▿

    Faber, Milosz; Faber, Marie-Luise; Li, Jianwei; Preuss, Mirjam A. R.; Schnell, Matthias J.; Dietzschold, Bernhard

    2007-01-01

    The nonpathogenic phenotype of the live rabies virus (RV) vaccine SPBNGAN is determined by an Arg→Glu exchange at position 333 in the glycoprotein, designated GAN. We recently showed that after several passages of SPBNGAN in mice, an Asn→Lys mutation arose at position 194 of GAN, resulting in GAK, which was associated with a reversion to the pathogenic phenotype. Because an RV vaccine candidate containing two GAN genes (SPBNGAN-GAN) exhibits increased immunogenicity in vivo compared to the si...

  15. Pumping of drugs by P-glycoprotein

    Litman, Thomas; Skovsgaard, Torben; Stein, Wilfred D

    2003-01-01

    The apparent inhibition constant, Kapp, for the blockade of P-glycoprotein (P-gp) by four drugs, verapamil, cyclosporin A, XR9576 (tariquidar), and vinblastine, was measured by studying their ability to inhibit daunorubicin and calcein-AM efflux from four strains of Ehrlich cells with different...... levels of drug resistance and P-gp content. For daunorubicin as a transport substrate, Kapp was independent of [P-gp] for verapamil but increased strictly linearly with [P-gp] for vinblastine, cyclosporin A, and XR9576. A theoretical analysis of the kinetics of drug pumping and its reversal shows that...... rather, in serial, i.e., a drug that is pumped from the cytoplasmic phase has to pass the preemptive route upon leaving the cell. Our results are consistent with the Sauna-Ambudkar two-step model for pumping by P-gp. We suggest that the vinblastine/cyclosporin A/XR9576-binding site accepts daunorubicin...

  16. Raman optical activity of proteins and glycoproteins

    Raman optical activity (ROA), measured in this project as a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarised incident laser light, offers the potential to provide more information about the structure of biological molecules in aqueous solution than conventional spectroscopic techniques. Chapter one contains a general discussion of the relative merits of different spectroscopic techniques for structure determination of biomolecules, as well as a brief introduction to ROA. In Chapter two a theoretical analysis of ROA is developed, which extends the discussion in chapter one. The spectrometer setup and sample preparation is then discussed in chapter three. Instrument and sample conditions are monitored to ensure that the best results are obtained. As with any experimental project problems occur, which may result in a degradation of the spectra obtained. The cause of these problems was explored and remedied whenever possible. Chapter four introduces a brief account of protein, glycoprotein and carbohydrate structure and function, with a particular emphasis on the structure of proteins. In the remaining chapters experimental ROA results on proteins and glycoproteins, with some carbohydrate samples, from a wide range of sources are examined. For example, in chapter five some β-sheet proteins are examined. Structural features in these proteins are examined in the extended amide III region of their ROA spectra, revealing that ROA is sensitive to the rigidity or flexibility inherent in proteins. Chapter six concentrates on a group of proteins (usually glycoproteins) known as the serine proteinase inhibitors (serpins). Medically, the serpins are one of the most important groups of proteins of current interest, with wide-ranging implications in conditions such as Down's syndrome, Alzheimer's disease, and emphysema with associated cirrhosis of the liver. With favourable samples and conditions ROA may offer the

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

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

    2016-05-01

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

  18. Determination of site-specific glycan heterogeneity on glycoproteins

    Kolarich, Daniel; Jensen, Pia Hønnerup; Altmann, Friedrich;

    2012-01-01

    site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco......The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their...

  19. Regulation of glycoprotein synthesis in yeast by mating pheromones

    In Saccharomyces cerevisiae, glycosylated proteins amount to less than 2% of the cell protein. Two intensively studied examples of yeast glycoproteins are the external cell wall - associated invertase and the vacuolar carboxypeptidase Y. Recently, it was shown that the mating pheromone, alpha factor, specifically and strongly inhibits the synthesis of N-glycosylated proteins in haploid a cells, whereas O-glycosylated proteins are not affected. In this paper, the pathways of glycoprotein biosynthesis are summarized briefly, and evidence is presented that mating pheomones have a regulatory function in glycoprotein synthesis

  20. Regenerated bacterial cellulose microfluidic column for glycoproteins separation.

    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. PMID:26686130

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

    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 [3H]glucosamine hydrochloride to label glycoproteins, then exposed to oxygen radicals generated by chelated ferric iron and ascorbic acid. Marked stimulation of glycoprotein release was observed after a 30-min exposure to the oxygen radical-generating system, and the effect was inhibited by mannitol. The stimulatory effect of hydroxyl radical was not accompanied by leakage of intracellular lactate dehydrogenase. Parallel experiments with human granulocytes activated with f-Met-Leu-Phe and coincubated with gallbladder explants revealed similar results. These results indicate that oxygen radicals, especially the hydroxyl radical (OH), are capable of stimulating rapid release of mucous-type glycoproteins from gallbladder epithelium

  2. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Daniel L Glauser

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

  3. KDN-containing glycoprotein from loach skin mucus.

    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

  4. P-Glycoprotein-ATPase Modulation: The Molecular Mechanisms

    Li-Blatter, Xiaochun; Beck, Andreas; Seelig, Anna

    2012-01-01

    P-glycoprotein-ATPase is an efflux transporter of broad specificity that counteracts passive allocrit influx. Understanding the rate of allocrit transport therefore matters. Generally, the rates of allocrit transport and ATP hydrolysis decrease exponentially with increasing allocrit affinity to the transporter. Here we report unexpectedly strong down-modulation of the P-glycoprotein-ATPase by certain detergents. To elucidate the underlying mechanism, we chose 34 electrically neutral and catio...

  5. Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36)

    Holmes, Roger S.

    2012-01-01

    Platelet glycoprotein 4 (CD36) (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3]) is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs) and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, mal...

  6. P-glycoprotein and its Role in Treatment Resistance

    Göğcegöz Gül, Işıl; Eryılmaz, Gül; Karamustafalıoğlu, K. Oğuz

    2016-01-01

    Polypharmacy which has often used to increase efficacy of treatment and to prevent resistance in psychiatry may lead to pharmacokinetic and pharmacodynamic drug interactions. One of the intensively studied topic in recent years to clarify the mechanism of drug interactions, in the pharmacokinetic area is p-glycoprotein related drug-drug and drug-food interactions. The interactions of some drugs with p-glycoprotein which is a carrier protein, can lead to a decrease in the bioavailability of th...

  7. P-GLYCOPROTEIN QUANTITATION IN ACUTE LEUKEMIA

    Mali in Nikougoftar

    2003-06-01

    Full Text Available Multi drug resistance(MDR is a major problem in the treatment of cancer and hemalological malignancies. This resistance is multi factorial and is the result of decreased intra cellular drug accumulation. This is partly due to the presence of a 170KD intra membranous protein termed P-glycoprotein(P-gp that is an energy-dependent efflux pump which has increased expression on drug-resistance cells. In this study we identified the presence of P-gp by staining with Fluorescent Iso Thio Cyanate (FITC conjugated anti P-gp in acute leukemia patients and flow cytometry in addition to performing immunophenotype analysis and French, American British (FAB classification. Results revealed that one fifth of leuke¬mic patients expressed P-gp and this phenotype was more prevalent in Acute Undifferentiated Leukemia(AUL and Acute Myelogenous Leukemia (AML than in Acute Lymphoblastic Leukemia(ALL. Other findings showed a logical rela¬tionship between this phenotype and age groups. There was not any association between P-gp+ phenotype and FAB and Immunophenotyping sub classification, but there was a linear relationship between CD34 and CD7 expression and P-gp+ phenotype. The accumulation of P-gp molecule that was stated as Mean Fluores¬cence Intensity (MFI on the blasts1 membrane of AUL and AML patients showed marked increase in comparison to ALL. Furthermore MFI in P-gp+ relapsed patients was much more than P-gp+ pretreatment patients.

  8. P-glycoprotein targeted nanoscale drug carriers

    Li, Wengang

    2013-02-01

    Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug carrying processes that shuttle the drugs out of tumor cells. Thus, P -gp inhibitors have attracted a lot of attention as they can stop cancer drugs from being pumped out of target cells with the consumption of ATP. Using quantitive structure activity relationship (QSAR), we have successfully synthesized a series of novel P -gp inhibitors. The obtained dihydropyrroloquinoxalines series were fully characterized and then tested against bacterial and tumor assays with over-expressed P -gps. All compounds were bioactive especially compound 1c that had enhanced antibacterial activity. Furthermore, these compounds were utilized as targeting vectors to direct drug delivery vehicles such as silica nanoparticles (SNPs) to cancerous Hela cells with over expressed P -gps. Cell uptake studies showed a successful accumulation of these decorated SNPs in tumor cells compared to undecorated SNPs. The results obtained show that dihydropyrroloquinoxalines constitute a promising drug candidate for targeting cancers with MDR. Copyright © 2013 American Scientific Publishers All rights reserved.

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

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

    2014-11-01

    Colorectal cancer is one of the leading causes of death due to cancer worldwide. Therefore, the identification of high-specificity and -sensitivity biomarkers for the early detection of colorectal cancer is urgently needed. Post-translational modifications, such as glycosylation, are known to play an important role in cancer progression. In the present work, we used a quantitative proteomic technique based on (18)O stable isotope labeling to identify differentially expressed N-linked glycoproteins in colorectal cancer tissue samples compared with healthy colorectal tissue from 19 patients undergoing colorectal cancer surgery. We identified 54 up-regulated glycoproteins in colorectal cancer samples, therefore potentially involved in the biological processes of tumorigenesis. In particular, nine of these (PLOD2, DPEP1, SE1L1, CD82, PAR1, PLOD3, S12A2, LAMP3, OLFM4) were found to be up-regulated in the great majority of the cohort, and, interestingly, the association with colorectal cancer of four (PLOD2, S12A2, PLOD3, CD82) has not been hitherto described. PMID:25247386

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

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

    1987-10-01

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

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

    Fun-In Wang

    2015-06-01

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

  12. Glycoprotein 2 antibodies in Crohn's disease.

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

    2013-01-01

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

  13. Intracellular localization of hydroxyproline-rich glycoprotein biosynthesis

    The structural proteins of plant cell walls are glycoproteins characterized by O-glucosidic linkages to hydroxyproline or serine. Proline, not hydroxyproline, is the translatable amino acid in hydroxyproline-rich glycoproteins (HRGP). Hydroxylation and arabinosylation of proline are sequential, post-translational events. Because of this, there is no a priori reason for expecting HRGP synthesis to follow the well-established route for secretory and plasma membrane (PM) glycoproteins, i.e., from endoplasmic reticulum (ER) via the Golgi apparatus (GA) to the PM. In this paper, two plausible alternatives for HRGO secretion are examined. Because a feature of the majority of dicotyledons is overlapping GA and PM regions in sucrose density gradients, the authors have used two monocotyledonous systems to determine the distribution of HRGP and enzyme activity

  14. Genetic Analysis of Glycoprotein Gene of Indonesian Rabies Virus

    Heru Susetya

    2015-10-01

    Full Text Available The amino acid sequences of the Glycoprotein gene (G gene of field rabies virus SN01-23 from Indonesiawas determined. This isolate showed homology of 93% in the ectodomain of the Glycoprotein gene to that of theRC-HL strain, which is used for production of animal vaccine in Japan. The high identity in the ectodomainbetween this field isolate and strain RC-HL suggest that the rabies animal vaccine used in Japan will be effectivefor rabies street viruses in Indonesia. Result of phylogenetic analysis using the nucleotide sequences of the Ggenes of rabies street viruses showed that SN01-23 from Indonesia is more closely related to a rabies virus fromChina than to viruses from Thailand and Malaysia. This genetic data and historical background suggest thatrabies viruses in China had been transferred to Indonesia through dogs brought by humans migrating from Chinato Indonesia.Keywords : Rabies virus, Glycoprotein gene, Ectodomain, Phylogenetic analysis

  15. P-glycoprotein and Its Role in Treatment Resistance

    Isil Gogcegoz Gul

    2016-03-01

    Full Text Available Polypharmacy which has often used to increase efficacy of treatment and to prevent resistance in psychiatry may lead to pharmacokinetic and pharmacodynamic drug interactions. One of the inten-sively studied topic in recent years to clarify the mechanism of drug interactions, in the pharmacoki-netic area is p-glycoprotein related drug-drug and drug-food interactions. The interactions of some drugs with p-glycoprotein which is a carrier protein, can lead to a decrease in the bioavailability of these drugs and reduction in passage through the blood-brain barrier. In this review, the role of p-glycoprotein on drug pharmacokinetics and bioavailability of psychiatric drugs are discussed. [Psikiyatride Guncel Yaklasimlar - Current Approaches in Psychiatry 2016; 8(1: 19-31

  16. Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

    Kovacs, J A; Powell, F; Edman, J C;

    1993-01-01

    hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development of...... antigen is a good candidate for development as a vaccine to prevent or control P. carinii infection. We have cloned and sequenced seven related but unique genes encoding the major surface glycoprotein of rat P. carinii. Partial amino acid sequencing confirmed the identity of these genes. Based on Southern......The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus this...

  17. Synthetic glycopeptides and glycoproteins with applications in biological research

    Ulrika Westerlind

    2012-05-01

    Full Text Available Over the past few years, synthetic methods for the preparation of complex glycopeptides have been drastically improved. The need for homogenous glycopeptides and glycoproteins with defined chemical structures to study diverse biological phenomena further enhances the development of methodologies. Selected recent advances in synthesis and applications, in which glycopeptides or glycoproteins serve as tools for biological studies, are reviewed. The importance of specific antibodies directed to the glycan part, as well as the peptide backbone has been realized during the development of synthetic glycopeptide-based anti-tumor vaccines. The fine-tuning of native chemical ligation (NCL, expressed protein ligation (EPL, and chemoenzymatic glycosylation techniques have all together enabled the synthesis of functional glycoproteins. The synthesis of structurally defined, complex glycopeptides or glyco-clusters presented on natural peptide backbones, or mimics thereof, offer further possibilities to study protein-binding events.

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

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

    2014-03-15

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

  19. Intestinal mucus and juice glycoproteins have a liquid crystalline structure

    X-ray diffraction patterns have been obtained from the following components of canine gastrointestinal tract: (1) native small intestine mucus layer; (2) the precipitate of the flocks formed in the duodenal juice with decreasing pH; (3) concentrated solutions of glycoproteins isolated from the duodenal juice. The X-ray patterns consist of a large number of sharp reflections of spacings between about 100 and 4 A. Some reflections are common for all components studied. All the patterns are interpreted as arising from the glycoprotein molecules ordered into a liquid crystalline structure. (author)

  20. Intestinal mucus and juice glycoproteins have a liquid crystalline structure

    Denisova, E.A.; Lazarev, P.I.; Vazina, A.A.; Zheleznaya, L.A.

    1985-11-05

    X-ray diffraction patterns have been obtained from the following components of canine gastrointestinal tract: (1) native small intestine mucus layer; (2) the precipitate of the flocks formed in the duodenal juice with decreasing pH; (3) concentrated solutions of glycoproteins isolated from the duodenal juice. The X-ray patterns consist of a large number of sharp reflections of spacings between about 100 and 4 A. Some reflections are common for all components studied. All the patterns are interpreted as arising from the glycoprotein molecules ordered into a liquid crystalline structure.

  1. Genetic Analysis of Glycoprotein Gene of Indonesian Rabies Virus

    Heru Susetya; Ito Naoto; Makoto Sugiyama; Nobuyuki Minamoto

    2015-01-01

    The amino acid sequences of the Glycoprotein gene (G gene) of field rabies virus SN01-23 from Indonesiawas determined. This isolate showed homology of 93% in the ectodomain of the Glycoprotein gene to that of theRC-HL strain, which is used for production of animal vaccine in Japan. The high identity in the ectodomainbetween this field isolate and strain RC-HL suggest that the rabies animal vaccine used in Japan will be effectivefor rabies street viruses in Indonesia. Result of phylogenetic an...

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

    Korecka, Lucie [Department of Analytical Chemistry, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic)]. E-mail: lucie.korecka@upce.cz; Jezova, Jana [Department of Analytical Chemistry, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Bilkova, Zuzana [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Benes, Milan [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Namesti 2, 162 06 Prague (Czech Republic); Horak, Daniel [Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho Namesti 2, 162 06 Prague (Czech Republic); Hradcova, Olga [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Slovakova, Marcela [Department of Biological and Biochemical Sciences, University of Pardubice, Namesti Cs. Legii 565, 532 10 Pardubice (Czech Republic); Laboratoire Physicochimie Curie, UMR 168 CNRS/Institute Curie, Paris Cedex 05 (France); Viovy, Jean-Louis [Laboratoire Physicochimie Curie, UMR 168 CNRS/Institute Curie, Paris Cedex 05 (France)

    2005-05-15

    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.

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

    David Clark

    2012-01-01

    Full Text Available 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.

  4. Human Milk Glycoproteins Protect Infants Against Human Pathogens

    Liu, Bo; Newburg, David S.

    2013-01-01

    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 ...

  5. Engineered CHO cells for production of diverse, homogeneous glycoproteins

    Yang, Zhang; Wang, Shengjun; Halim, Adnan; Schulz, Morten Alder; Frodin, Morten; Rahman, Shamim H.; Vester-Christensen, Malene Bech; Behrens, Carsten; Kristensen, Claus; Vakhrushev, Sergey Y.; Bennett, Eric Paul; Wandall, Hans H.; Clausen, Henrik

    2015-01-01

    genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of desired glycosylation, such as human-like alpha 2,6-linked sialic acid capping. This engineering approach will aid the production of glycoproteins with improved properties and therapeutic...

  6. Synthesis of cell envelope glycoproteins of Cryptococcus laurentii.

    Schutzbach, John; Ankel, Helmut; Brockhausen, Inka

    2007-05-21

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

  7. Glycoprotein secretion in a tracheal organ culture system

    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 14C-glucosamine and 3H-fucose-labelled glycoprotein. The response was dose-dependent. Similar results were obtained after a second exposure to pilocarpine. However, no dose response was observed. Morphological analyses of the tracheal epithelial secretory cells by Alcian Blue/Periodic Acid Schiff staining showed a significant decrease in the total number of Alcian Blue staining cells and an increase in the mixed cell population after a single exposure to pilocarpine. Second stimulation with the drug showed that the trachea was able to respond again, this time with a further decrease in the number of Alcian Blue staining cells and a decrease in the PAS staining cells as well. Carbohydrate analyses after the first simulation with pilocarpine showed increased levels of N-acetyl neuraminic acid and the neutral carbohydrates, fucose and galactose, in the precipitated glycoproteins

  8. Direct chemical modification and voltammetric detection of glycans in glycoproteins

    Trefulka, Mojmír; Paleček, Emil

    2014-01-01

    Roč. 48, NOV2014 (2014), s. 52-55. ISSN 1388-2481 R&D Projects: GA ČR(CZ) GAP301/11/2055 Institutional support: RVO:68081707 Keywords : Glycoproteins * Chemical modification * Os(VI)L complexes Subject RIV: BO - Biophysics Impact factor: 4.847, year: 2014

  9. Glycoprotein expression by adenomatous polyps of the colon

    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.

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

    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.

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

    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

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

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

    2016-08-01

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

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

    Katayama, Takane; Fujita, Kiyotaka; Yamamoto, Kenji

    2005-05-01

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

  14. Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells

    Westra, DF; Glazenburg, KL; Harmsen, MC; Tiran, A; Scheffer, AJ; Welling, GW; The, TH; WellingWester, S

    1997-01-01

    In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell sur

  15. BAT3 guides misfolded glycoproteins out of the endoplasmic reticulum.

    Jasper H L Claessen

    Full Text Available Secretory and membrane proteins that fail to acquire their native conformation within the lumen of the Endoplasmic Reticulum (ER are usually targeted for ubiquitin-dependent degradation by the proteasome. How partially folded polypeptides are kept from aggregation once ejected from the ER into the cytosol is not known. We show that BAT3, a cytosolic chaperone, is recruited to the site of dislocation through its interaction with Derlin2. Furthermore, we observe cytoplasmic BAT3 in a complex with a polypeptide that originates in the ER as a glycoprotein, an interaction that depends on the cytosolic disposition of both, visualized even in the absence of proteasomal inhibition. Cells depleted of BAT3 fail to degrade an established dislocation substrate. We thus implicate a cytosolic chaperone as an active participant in the dislocation of ER glycoproteins.

  16. TROPHOBLASTIC β1 – GLYCOPROTEIN SYNTHESIS IN SEROPOSITIVE PREGNANT WOMEN

    R. N. Bogdanovich

    2005-01-01

    Full Text Available Abstract. The level of trophoblastic β1 – glycoprotein (SP–1 was determined in the blood sera of 200 healthy pregnant women and 184 women with threatened abortions in term till 20 weeks of pregnancy. In group of women experiencing recurrent abortions in 38 % cases antibodies to chorionic gonadotropin, in 39,5 % cases antibodies to phospholipids, in 25,5 % – antibodies to tireoglobulin were revealed in significant amounts. In 20,65 % lupus anticoagulant was found. The majority of women in this group had changes in homeostasis. The presence of autoantibodies during pregnancy is the unfavourable factor in the development of placental insufficiency. This is proved by the decreased secretion of trophoblastic β1 – glycoprotein – a marker of the fetal part of placenta. (Med. Immunol., 2005, vol.7, № 1, pp. 85588

  17. Comparison of glycoprotein expression between ovarian and colon adenocarcinomas

    Multhaupt, H A; Arenas-Elliott, C P; Warhol, M J

    1999-01-01

    distinguishing between these 2 entities. CONCLUSION: A panel of monoclonal antibodies against cytokeratins 7 and 20 antigens, CA125, and carcinoembryonic antigen is useful in differentiating serous and endometrioid adenocarcinomas of the ovary from colonic adenocarcinomas. Mucinous ovarian adenocarcinomas cannot......, carcinoembryonic antigen, and cytokeratins 7 and 20 to detect tumor-associated glycoproteins and keratin proteins in ovarian and colonic carcinomas. RESULTS: CA125, carcinoembryonic antigen, and cytokeratins 7 and 20 can distinguish between colonic and serous or endometrioid adenocarcinomas of the ovary in both...... primary and metastatic lesions. Mucinous ovarian adenocarcinomas differed in that they express carcinoembryonic antigen and cytokeratins 7 and 20 and weakly express CA125. The other glycoprotein antigens were equally expressed by ovarian and colonic adenocarcinomas and therefore were of no use in...

  18. Pregnancy-specific glycoprotein function, conservation and receptor investigation

    O'Riordan, Ronan T

    2014-01-01

    Pregnancy-specific glycoproteins (PSGs) are highly glycosylated secreted proteins encoded by multi-gene families in some placental mammals. They are carcinoembryonic antigen (CEA) family and immunoglobulin (Ig) superfamily members. PSGs are immunomodulatory, and have been demonstrated to possess antiplatelet and pro-angiogenic properties. Low serum levels of these proteins have been correlated with adverse pregnancy outcomes. Objectives: Main research goals of this thesis were: 1). To attempt...

  19. Tumor specific glycoproteins and method for detecting tumorigenic cancers

    The detection of tumour specific glycoproteins (TSGP) in human sera often indicates the presence of a malignant tumour in a patient. The distinguishing characteristics of TSGP isolated from the blood sera of cancer patients are described in detail together with methods of TSGP isolation and purification. Details are also given of radioimmunoassay techniques capable of detecting very low levels of serum TSGP with high specificity. (U.K.)

  20. Emerging Technologies for Making Glycan-Defined Glycoproteins

    Wang, Lai-Xi; Lomino, Joseph V.

    2011-01-01

    Protein glycosylation is a common and complex posttranslational modification of proteins, which expands functional diversity while boosting structural heterogeneity. Glycoproteins, the end products of such a modification, are typically produced as mixtures of glycoforms possessing the same polypeptide backbone but differ in the site of glycosylation and/or in the structures of pendant glycans, from which single glycoforms are difficult to isolate. The urgent need for glycan-defined glycoprote...

  1. Specificity analysis of lectins and antibodies using remodeled glycoproteins

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

    2009-01-01

    Due to their ability to bind specifically to certain carbohydrate sequences, lectins are a frequently used tool in cytology, histology, and glycan analysis but also offer new options for drug targeting and drug delivery systems. For these and other potential applications, it is necessary to be certain as to the carbohydrate structures interacting with the lectin. Therefore, we used glycoproteins remodeled with glycosyltransferases and glycosidases for testing specificities of lectins from Ale...

  2. Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein

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

    2003-01-01

    Multiple sclerosis is a chronic disease of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal loss. The immunopathogenesis of demyelination in multiple sclerosis involves an autoantibody response to myelin oligodendrocyte glycoprotein (MOG), a type I transmembrane protein located at the surface of CNS myelin. Here we present the crystal structures of the extracellular domain of MOG (MOGIgd) at 1.45-Å resolution and the complex of ...

  3. Expression of Pneumocystis jirovecii Major Surface Glycoprotein in Saccharomyces cerevisiae

    Kutty, Geetha; England, Katherine J.; Kovacs, Joseph A.

    2013-01-01

    The major surface glycoprotein (Msg), which is the most abundant protein expressed on the cell surface of Pneumocystis organisms, plays an important role in the attachment of this organism to epithelial cells and macrophages. In the present study, we expressed Pneumocystis jirovecii Msg in Saccharomyces cerevisiae, a phylogenetically related organism. Full-length P. jirovecii Msg was expressed with a DNA construct that used codons optimized for expression in yeast. Unlike in Pneumocystis orga...

  4. Selective modulation of P-glycoprotein-mediated drug resistance

    Bebawy, M; Morris, M B; Roufogalis, B. D.

    2001-01-01

    Multidrug resistance associated with the overexpression of the multidrug transporter P-glycoprotein is a serious impediment to successful cancer treatment. We found that verapamil reversed resistance of CEM/VLB 100 cells to vinblastine and fluorescein-colchicine, but not to colchicine. Chlorpromazine reversed resistance to vinblastine but not to fluorescein-colchicine, and it increased resistance to colchicine. Initial influx rates of fluorescein-colchicine were similar in resistant and paren...

  5. Interaction of Common Azole Antifungals with P Glycoprotein

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

    2002-01-01

    Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and ex...

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

    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

  7. Radioactive fucose as a tool for studying glycoprotein secretion

    A. Haddad

    1998-02-01

    Full Text Available The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose, [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined. Vitreous samples were also processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE and histological sections of retina, ciliary body and lens (the eye components around the vitreous body were processed for radioautography. The specific activity (counts per minute per microgram of protein of the glycoproteins labeled with [3H]-fucose was always much higher than that of the proteins labeled with any of the other monosaccharides or any of the amino acids. There was a good correlation between the specific activity of the proteins labeled by any of the above precursors and the density of the vitreous protein bands detected by fluorography. This was also true for the silver grain density on the radioautographs of the histological sections of retina, ciliary body and lens. The contribution of radioautography (after [3H]-fucose administration to the elucidation of the biogenesis of lysosomal and membrane glycoproteins and to the determination of the intracellular process of protein secretion was reviewed. Radioactive fucose is the precursor of choice for studying glycoprotein secretion because it is specific, efficient and practical for this purpose

  8. Thermodynamics and kinetics of P-glycoprotein-substrate interactions

    Äänismaa, Päivi

    2007-01-01

    P-glycoprotein (Pgp, ABCB1) is a transmembrane protein, which extrudes a large number of structurally diverse compounds out of the cell membrane at the expense of ATP hydrolysis. The overexpression of Pgp strongly contributes to multidrug resistance, which hampers the chemotherapy of cancer and some other drug-treatable diseases. Therefore, the general aim of this thesis was to quantitatively characterize the thermodynamics and the kinetics of Pgp-substrate interactions. Specif...

  9. Solid-phase group-specific adsorbants in assays for glycoproteins

    The focus of the paper is on several technical advances in the assays for glycoprotein hormones and enzymes that have been achieved by the use of the solid-phase cabohydrate-specific adsorbant concanavalin-A. Purification of glycoprotein radioligand after labelling by the Chloramine-T method is readily accomplished using a small column of agarose-bound concanavalin-A which separates glycoprotein radioligand from radioiodide and radiolabelled unadsorbed contaminants. After concanavalin-A column chromatography, radiolabelled glycoprotein hormone preparations exhibited improved binding to antibodies and tissue receptors. To increase the effective sensitivity of radioimmunoassays for glycoproteins, agarose-bound concanavalin-A is used to extract and concentrate the glycoproteins from various biological samples. For example, the effective sensitivity for the detection of human thyrotropin in serum was improved approximately 5-fold by using concanavalin-A concentrates of 1.5ml of serum. Partial purification of the glycoprotein dopamine-β-hydroxylase from serum using agarose-bound concanavalin-A resulted in separation of the serum factors that interfere with the measurement of enzyme activity. We conclude that in assays for glycoproteins, concanavalin-A is useful for purification of radioligand, for preparation of concentrates of glycoproteins from biological samples and for separation of glycoproteins from various interfering factors contained in biological samples before radioligand or radioenzyme assay. (author)

  10. Glycoprotein fucosylation is increased in seminal plasma of subfertile men

    Beata Olejnik

    2015-04-01

    Full Text Available Fucose, the monosaccharide frequent in N- and O-glycans, is a part of Lewis-type antigens that are known to mediate direct sperm binding to the zona pellucida. Such interaction was found to be inhibited in vitroby fucose-containing oligo- and polysaccharides, as well as neoglycoproteins. The objective of this study was to screen seminal plasma proteins of infertile/subfertile men for the content and density of fucosylated glycoepitopes, and compare them to samples of fertile normozoospermic subjects. Seminal proteins were separated in polyacrylamide gel electrophoresis and blotted onto nitrocellulose membrane and probed with fucose-specific Aleuria aurantia lectin (AAL. Twelve electrophoretic bands were selected for quantitative densitometric analysis. It was found that the content, and especially the density of fucosylated glycans, were higher in glycoproteins present in seminal plasma of subfertile men. No profound differences in fucosylation density were found among the groups of normozoospermic, oligozoospermic, asthenozoospermic, and oligoasthenozoospermic subfertile men. According to the antibody probing, AAL-reactive bands can be attributed to male reproductive tract glycoproteins, including prostate-specific antigen, prostatic acid phosphatase, glycodelin and chorionic gonadotropin. Fibronectin, α1 -acid glycoprotein, α1 -antitrypsin, immunoglobulin G and antithrombin III may also contribute to this high fucosylation. It is suggested that the abundant fucosylated glycans in the sperm environment could interfere with the sperm surface and disturb the normal course of the fertilization cascade.

  11. Characterization of an estrogen-induced oviduct membrane glycoprotein

    During estrogen-induced chick oviduct differentiation a number of N-linked membrane glycoproteins are induced as judged by GDP-[14C]Man labeling of endogenous acceptors, 125I-con A labeling as well as coomassie blue and PAS staining of SDS polyacrylamide gels. The authors have begun to characterize one of these glycoproteins having an M/sub r/ of 91 KDa. The protein has been purified via preparative SDS-PAGE and electroelution. The purified protein migrates as a single band on analytical SDS-PAGE and comigrates with an endogenous membrane glycoprotein labeled with GDP-[14C]Man. Amino acid analysis indicates a high proportion of GLU and ASP residues (110 and 66 moles respectively). N-terminal sequence analysis by gas phase instrumentation yielded the following: X-X-VAL-ASP-VAL-ASP-ALA-THR-VAL-GLU-GLU-ASP-GLU. The protein contains about 2% neutral sugar including 6 mol Man, 2 mol Gal, 1 mol Fuc, 4 mol GlcNAc, 1 mol GalNAc and 1 mol sialic acid per mole of protein. The presence of the GalNAc residue suggests the protein contains an O-linked oligosaccharide moiety in addition to the N-linked chain(s). The detailed structure of the carbohydrate moieties is currently under investigation

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

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

    2016-01-01

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

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

    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

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

    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

  15. Nipah virus infection and glycoprotein targeting in endothelial cells

    Maisner Andrea

    2010-11-01

    Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

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

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

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

    Tollefsen, D M; Pestka, C A

    1985-01-01

    Heparin cofactor II is a plasma protein that inhibits thrombin rapidly in the presence of either heparin or dermatan sulfate. We have determined the effects of two glycosaminoglycan-binding proteins, i.e., histidine-rich glycoprotein and platelet factor 4, on these reactions. Inhibition of thrombin by heparin cofactor II and heparin was completely prevented by purified histidine-rich glycoprotein at the ratio of 13 micrograms histidine-rich glycoprotein/microgram heparin. In contrast, histidi...

  18. Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity

    Horvat, B; Multhaupt, H A; Damjanov, I

    1993-01-01

    in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate...... the complexity of glycoconjugate synthesis in mouse vagina and reveal the distinct cycle-specific patterns of individual glycoprotein expression. These cyclic glycoproteins could serve as vaginal biochemical markers for the specific phases of the estrous cycle....

  19. Intracellular localization of Crimean-Congo Hemorrhagic Fever (CCHF virus glycoproteins

    Fernando Lisa

    2005-04-01

    Full Text Available Abstract Background Crimean-Congo Hemorrhagic Fever virus (CCHFV, a member of the genus Nairovirus, family Bunyaviridae, is a tick-borne pathogen causing severe disease in humans. To better understand the CCHFV life cycle and explore potential intervention strategies, we studied the biosynthesis and intracellular targeting of the glycoproteins, which are encoded by the M genome segment. Results Following determination of the complete genome sequence of the CCHFV reference strain IbAr10200, we generated expression plasmids for the individual expression of the glycoproteins GN and GC, using CMV- and chicken β-actin-driven promoters. The cellular localization of recombinantly expressed CCHFV glycoproteins was compared to authentic glycoproteins expressed during virus infection using indirect immunofluorescence assays, subcellular fractionation/western blot assays and confocal microscopy. To further elucidate potential intracellular targeting/retention signals of the two glycoproteins, GFP-fusion proteins containing different parts of the CCHFV glycoprotein were analyzed for their intracellular targeting. The N-terminal glycoprotein GN localized to the Golgi complex, a process mediated by retention/targeting signal(s in the cytoplasmic domain and ectodomain of this protein. In contrast, the C-terminal glycoprotein GC remained in the endoplasmic reticulum but could be rescued into the Golgi complex by co-expression of GN. Conclusion The data are consistent with the intracellular targeting of most bunyavirus glycoproteins and support the general model for assembly and budding of bunyavirus particles in the Golgi compartment.

  20. Purification of a herpes simplex virus Type 1 specific glycoprotein

    The need for a sensitive and discriminating test to screen the sera of patients for previous infections of herpes simplex virus Type 1 (HSV-1), Type 2 (HSV-2) or both, has required the purification of type-specific antigens from both virus types. Work was conducted to purify such an antigen from HSV-1, for which glycoprotein C (gC-1) was selected as the most suitable antigen. Preparative polyacrylamide gel electrophoresis (Prep-PAGE) was used as an initial step in separating HSV-1 infected cell proteins, and two cycles of Prep-PAGE were sufficient to produce a solution of gC-1 free of other HSV-1 glycoproteins, but still containing a number of non-glycosylated proteins. Wheat germ lectin affinity chromatography was used to remove the non-glycosylated proteins from this solution of gC-1, but the gC-1 would not elute from the lectin under normal conditions. Difficulties encountered in eluting gC-1 from wheat germ lectin may have been caused by the use of sodium dodecyl sulphate (SDS) to solubilize the proteins prior to Prep-PAGE. For this reason, the wheat germ lectim affinity chromatography was repeated using HSV-1 membrane proteins solubilized in Triton X-100, which resulted in the purification of a mixture of HSV-1 glycoproteins from non-glycosylated proteins. Helix pomatia lectim affinity chromatography of HSV-1 membrane proteins solubilized in Triton X-100 did not selectively purify gC-1. During this experiments the HSV-1-infected cells were labelled with [3H]glucosamine and information as well as data is given on this labelling methods and auto radiographic analysis

  1. Mannostatin A, a new glycoprotein-processing inhibitor

    Mannostatin A is a metabolite produced by the microorganism Streptoverticillium verticillus and reported to be a potent competitive inhibitor of rat epididymal α-mannosidase. When tested against a number of other arylglycosidases, mannostatin A was inactive toward α- and β-glucosidase and galactosidase as well as β-mannosidase, but it was a potent inhibitor of jack bean, mung bean, and rat liver lysosomal α-mannosidases, with estimated IC50'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 (IC50 of about 10-15 nM with p-nitrophenyl α-D-mannopyranoside as substrate, and about 90 nM with [3H]mannose-labeled GlcNAc-Man5GlcNAc 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

  2. Interaction of tamoxifen with the multidrug resistance P-glycoprotein.

    Callaghan, R; Higgins, C F

    1995-01-01

    Tamoxifen is an anti-oestrogen which is currently being assessed as a prophylactic for women at high risk of breast cancer. Taxoxifen has also been shown to reverse multidrug resistance in P-glycoprotein (P-gp)-expressing cells, although the mechanism of action is unknown. In this study we demonstrate that tamoxifen interacts directly with P-gp. Plasma membranes from P-gp-expressing cells bound [3H]tamoxifen in a specific and saturable fashion. A 180 kDa membrane protein in these membranes, l...

  3. Increased expression of mucinous glycoprotein KL-6 in human pterygium

    Kase, S; Kitaichi, N; Furudate, N.; Yoshida, K.

    2006-01-01

    Pterygia represent growth onto the cornea of fibrovascular tissue continuous with the conjunctiva.1 KL-6 (Krebs von den Lunge-6) is a high molecular weight mucinous glycoprotein, and the monoclonal antibody reacts with the sugar moiety of MUC-1.2,3 We have reported that measurement of serum KL-6 levels is useful for the diagnosis and management of uveitis patients with sarcoidosis.4,5 The aim of this study was to examine the expression of KL-6, and Ki-67, a proliferation marker, in normal hum...

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

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

    2015-01-01

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

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

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

    1991-01-01

    The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes. A portion of this gG gene has been expressed as a fusion protein in Escherichia coli. Expression was regulated by a lambda phage pL promoter. The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography. Amino acid sequence analysis confirmed the N terminus of the purified protein. Mice immunized with recombina...

  6. Effect of P-glycoprotein on flavopiridol sensitivity

    Boerner, S. A.; Tourne, M E; Kaufmann, S.H.; Bible, K C

    2001-01-01

    Flavopiridol is the first potent inhibitor of cyclin-dependent kinases (CDKs) to enter clinical trials. Little is known about mechanisms of resistance to this agent. In order to determine whether P-glycoprotein (Pgp) might play a role in flavopiridol resistance, we examined flavopiridol sensitivity in a pair of Chinese hamster ovary cell lines differing with respect to level of Pgp expression. The IC 50 s of flavopiridol in parental AuxB1 (lower Pgp) and colchicine-selected CHRC5 (higher Pgp)...

  7. Radioactive fucose as a tool for studying glycoprotein secretion

    Haddad, A

    1998-01-01

    The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose, [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined....

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

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

    1999-10-01

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

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

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

    2015-01-14

    Recent evidence suggests an important role for natural honey in modulating immune response. To identify active components responsible, this study investigated the immunomodulatory properties of glycoproteins and glycopeptides fractionated from Ziziphus honey. Honey proteins/peptides were fractionated by size exclusion chromatography into five peaks with molecular masses in the range of 2-450 kDa. The fractionated proteins exhibited potent, concentration-dependent inhibition of reactive oxygen species production in zymosan-activated human neutrophils (IC50 = 6-14 ng/mL) and murine macrophages (IC50 = 2-9 ng/mL). Honey proteins significantly suppressed the nitric oxide production by LPS-activated murine macrophages (IC50 = 96-450 ng/mL). Moreover, honey proteins inhibited the phagocytosis latex bead macrophages. The production of pro-inflammatory cytokines IL-1β and TNF-α by human monocytic cell line in the presence of honey proteins was analyzed. Honey proteins did not affect the production of IL-1β; however, TNF-α production was significantly suppressed. These findings indicated that honey glycoproteins and glycopeptides significantly interfere with molecules of the innate immune system. PMID:25496517

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

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

    2005-10-01

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

  11. Application of monolithic affinity HPLC column for rapid determination of malt glycoproteins

    Benkovská, D. (Dagmar); Flodrová, D. (Dana); Bobálová, J. (Janette)

    2013-01-01

    The aim of this study was to optimize separation and enrichment of barley malt glycoproteins on a monolithic ConA affinity HPLC column. ConA-bound proteins were separated on SDS-PAGE and identified using MALDI-TOF/TOF MS after chymotryptic digestion. Our proteomic analysis allowed successful determination of several putative malt glycoproteins.

  12. Tomato spotted wilt virus glycoproteins exhibit trafficking and localization signals that are functional in mammalian cells

    Kikkert, M.; Verschoor, A.; Kormelink, R.; Rottier, P.; Goldbach, R.

    2001-01-01

    The glycoprotein precursor (G1/G2) gene of tomato spotted wilt virus (TSWV) was expressed in BHK cells using the Semliki Forest virus expression system. The results reveal that in this cell system, the precursor is efficiently cleaved and the resulting G1 and G2 glycoproteins are transported from th

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

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

    2016-06-01

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

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

    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 [14C]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 [14C]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 x 104 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

  15. Protective effect of Cardiospermum halicacabum leaf extract on glycoprotein components on STZ-induced hyperglycemic rats

    Chinnadurai Veeramani; Khalid S Al-Numair; Mohammed A Alsaif; Govindasamy Chandramohan; Nouf S Al-Numair; Kodukkur Viswanathan Pugalendi

    2012-01-01

    Objective: To investigate the protective role of Cardiospermum halicacabum (C. halicacabum) leaf extract on glycoprotein metabolism in streptozotocin (STZ)-induced diabetic rats. Methods:Diabetes was induced in male albino Wistar rats by intraperitonial administration of STZ. TheC. halicacabum leaf extract (CHE) was administered orally to normal and STZ-diabetic rats for 45 days. The effects of C. halicacabum leaf extract (CHE) on plasma and tissue glycoproteins (hexose, hexosamine, fucose and sialic acid) were determined. Results: The levels of plasma and tissues glycoproteins containing hexose, hexosamine and fucose were significantly increased in STZ-induced diabetic rats. In addition, the level of sialic acid significantly increased in plasma and liver while decreased in kidney of STZ-induced diabetic rats. After administration of CHE to diabetic rats, the metabolic alteration of glycoprotein reverted towards normal levels.Conclusions:The present study indicates that the CHE possesses a protective effect on abnormal glycoprotein metabolism in addition to its antihyperglycemic activity.

  16. Determination of P-Glycoprotein Expression by Flow Cytometry in Hematological Malignancies

    Berkay Saraymen

    2016-03-01

    Full Text Available Objective: Determination the expression of P-glycoprotein is especially problematic for normal tissues because immuno­logical methods are limited in terms of sensitivity. We aimed to determine the expression of P-glycoprotein and CD34 by flow cytometry, and to evaluate the level of expression of P-glycoprotein and CD34 with unresponsive to treatment in pa­tients diagnosed with hematologic malignancy. Methods: Our study included fifty patients diagnosed with acute myeloblastic leukemia and acute lymphoblastic leuke­mia, and twenty healthy controls who were admitted to Erci­yes University Hematology-Oncology Hospital. The suspend­ed cells from bone marrow samples of patients and the pe­ripheral blood samples of healthy people were marked with P-glycoprotein phycoerythrin and CD34 FITC or PerCP Cy 5.5; and then surface expression was measured by means of flow cytometry. Results: In 6 of 30 acute myeloblastic leukemia patients P-glycoprotein and CD34 expression, in 6 of 20 acute lympho­blastic leukemia patients P-glycoprotein, in 5 of them CD34 expression were determined. A significant relation between P-glycoprotein and CD34 expressions in acute myeloblas­tic leukemia and acute lymphoblastic leukemia bone marrow samples was reported. Conclusion: Our data indicate that flow cytometry is more reliable, precise and faster than molecular methods for mea­suring P-glycoprotein expression and suggests the pos­sibility of a significant relationship between P-glycoprotein and CD34 expressions in acute myeloblastic leukemia and acute lymphoblastic leukemia bone marrow samples. The blast cells expressing CD34 on their surface along with P-glycoprotein simultaneously show that multi drug resistance 1 gene is mostly active in immature cells.

  17. Human platelet glycoprotein IX: An adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures

    The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (Mr, 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex. GPIb (Mr, 165,000). Using antibody screening, the authors cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage λgt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A)+ RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acids followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined

  18. Molecular characterization of glycoprotein antigens on surface of Treponema pallidum: comparison with nonpathogenic Treponema phagedenis biotype Reiter.

    Moskophidis, M; F. Müller

    1984-01-01

    Four glycoproteins of Treponema pallidum were identified by intrinsic [14C]glucosamine labeling. Only two glycoproteins were demonstrated in T. phagedenis biotype Reiter with the same technique. Glycoproteins of both treponemes were characterized as antigens and shown to be localized within the outer membranes of the microorganisms.

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

    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

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

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

    2016-01-01

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

  1. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    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.

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

    Schmidt, A. E., E-mail: schmidt@omrb.pnpi.spb.ru; Shvetsov, A. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation); Kuklin, A. I. [Joint Institute for Nuclear Research (Russian Federation); Lebedev, D. V.; Surzhik, M. A.; Sergeev, V. R.; Isaev-Ivanov, V. V. [National Research Center “Kurchatov Institute”, Konstantinov Petersburg Nuclear Physics Institute (Russian Federation)

    2016-01-15

    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.

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

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

    2010-03-01

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

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

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

    2009-03-15

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

  5. Crystal Structure of the Human Cytomegalovirus Glycoprotein B.

    Heidi G Burke

    2015-10-01

    Full Text Available 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.

  6. Protective Role of α2HS-Glycoprotein in HBV-Associated Liver Failure

    Xue-Gong Fan

    2011-06-01

    Full Text Available n this study, levels of plasma α2-Heremans-Schmid glycoprotein, serum tumor necrosis factor-α, serum liver function parameters and short-term mortality were measured in 100 hepatitis B patients. Release of interleukin-6 and tumor necrosis factor-α from the lipopolysaccharide-stimulated peripheral blood mononuclear cells in the presence/absence of spermine and α2-Heremans-Schmid glycoprotein were analyzed by enzyme-linked immunosorbent assay to determine the significance and potential mechanism of α2-Heremans-Schmid glycoprotein in hepatitis B virus-associated liver damage. Results showed that serum α2-Heremans-Schmid glycoprotein levels in acute-on-chronic liver failure patients were significantly lower than that in chronic hepatitis B patients or healthy controls (p < 0.05. A negative dependence between serum human α2-Heremans-Schmid glycoprotein and tumor necrosis factor-α levels was observed. Interleukin-6 and tumor necrosis factor-α levels in the lipopolysaccharide-induced peripheral blood mononuclear cell supernates were significantly reduced by spermine and/or α2-Heremans-Schmid glycoprotein. The latter two proteins jointly inhibited cytokine release. These observations suggest that plasma α2-Heremans-Schmid glycoprotein is an independent marker of liver damage and a prognostic indicator of hepatitis B virus chronicity. It may reduce liver inflammation by partially inhibiting release of inflammatory factors from activated peripheral blood mononuclear cells.

  7. THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

    A. V. Shulkin

    2015-09-01

    Full Text Available On the basis of the analysis of published data the role of P-glycoprotein, carrier protein, in rational pharmacotherapy in cardiology was shown on the example of its substrates – digoxin, antiplatelet agents and anticoagulants. Determination of C3435T polymorphism of multidrug resistance gene (MDR1, encoding P-glycoprotein, in pharmacotherapy with digoxin, antiplatelet drugs (clopidogrel tikagrelol, prasugrel and anticoagulants (dabigatran etexilate, rivaroxaban, edoxaban is not feasible in routine practice. Drug in- teractions have clinical implications for the efficacy and safety of pharmacotherapy in coadministration of these drugs with P-glycoprotein substrates, inducers and inhibitors.

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

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

    1988-02-01

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

  9. Purification of the envelope glycoproteins of western equine encephalitis virus by glass wool column chromatography.

    Yamamoto, K.; Simizu, B

    1980-01-01

    Glass wool column chromatography was used for separation of the two glycoproteins of western equine encephalitis virus. Cross-contamination of each protein separated was confirmed to be negligible by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  10. A 220-kilodalton glycoprotein in yeast extract inhibits Staphylococcus aureus adherence to human endothelial cells.

    Elliott, D.A.; Hatcher, V B; Lowy, F D

    1991-01-01

    A 220-kDa glycoprotein from yeast extract causes a twofold decrease in S. aureus adherence to human endothelial cells in vitro. Medium constituents can have a significant effect on bacterial adherence interactions.

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

    Holling, R A; Guskey, L E

    1984-07-01

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

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

    Shaw, P J; Hills, G J

    1984-06-01

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

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

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

    1996-01-01

    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 modi...

  14. Optimization of irinotecan chronotherapy with P-glycoprotein inhibition

    Filipski, Elisabeth; Berland, Elodie [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Ozturk, Narin [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Istanbul University Faculty of Pharmacy, Department of Pharmacology, Beyazit TR-34116, Istanbul (Turkey); Guettier, Catherine [Assistance Publique-Hôpitaux de Paris, Unité de Chronothérapie, Département de Cancérologie, Hôpital Paul Brousse, Villejuif F-94807 (France); Horst, Gijsbertus T.J. van der [Department of Genetics, Erasmus University Medical Center, 3000 CA Rotterdam (Netherlands); Lévi, Francis [INSERM, U776 “Rythmes biologiques et cancers”, CAMPUS CNRS, 7 rue Guy Môquet, F-94801 Villejuif (France); Univ Paris-Sud, UMR-S0776, Orsay F-91405 (France); Assistance Publique-Hôpitaux de Paris, Unité de Chronothérapie, Département de Cancérologie, Hôpital Paul Brousse, Villejuif F-94807 (France); 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.

  15. Comparative Studies of Vertebrate Platelet Glycoprotein 4 (CD36

    Roger S. Holmes

    2012-09-01

    Full Text Available Platelet glycoprotein 4 (CD36 (or fatty acyl translocase [FAT], or scavenger receptor class B, member 3 [SCARB3] is an essential cell surface and skeletal muscle outer mitochondrial membrane glycoprotein involved in multiple functions in the body. CD36 serves as a ligand receptor of thrombospondin, long chain fatty acids, oxidized low density lipoproteins (LDLs and malaria-infected erythrocytes. CD36 also influences various diseases, including angiogenesis, thrombosis, atherosclerosis, malaria, diabetes, steatosis, dementia and obesity. Genetic deficiency of this protein results in significant changes in fatty acid and oxidized lipid uptake. Comparative CD36 amino acid sequences and structures and CD36 gene locations were examined using data from several vertebrate genome projects. Vertebrate CD36 sequences shared 53–100% identity as compared with 29–32% sequence identities with other CD36-like superfamily members, SCARB1 and SCARB2. At least eight vertebrate CD36 N-glycosylation sites were conserved which are required for membrane integration. Sequence alignments, key amino acid residues and predicted secondary structures were also studied. Three CD36 domains were identified including cytoplasmic, transmembrane and exoplasmic sequences. Conserved sequences included N- and C-terminal transmembrane glycines; and exoplasmic cysteine disulphide residues; TSP-1 and PE binding sites, Thr92 and His242, respectively; 17 conserved proline and 14 glycine residues, which may participate in forming CD36 ‘short loops’; and basic amino acid residues, and may contribute to fatty acid and thrombospondin binding. Vertebrate CD36 genes usually contained 12 coding exons. The human CD36 gene contained transcription factor binding sites (including PPARG and PPARA contributing to a high gene expression level (6.6 times average. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate CD36 gene with vertebrate

  16. Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

    1983-01-01

    Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance ...

  17. Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

    Puddington, L; Woodgett, C; Rose, J. K.

    1987-01-01

    The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

  18. Specificity and affinity of binding of herpes simplex virus type 2 glycoprotein B to glycosaminoglycans.

    Williams, R K; Straus, S E

    1997-01-01

    Herpes simplex virus type 2 (HSV-2) interacts with cell surface glycosaminoglycans during virus attachment. Glycoprotein B of HSV-2 can potentially mediate the interaction between the virion and cell surface glycosaminoglycans. To determine the specificity, kinetics, and affinity of these interactions, we used plasmon resonance-based biosensor technology to measure HSV-2 glycoprotein binding to glycosaminoglycans in real time. The recombinant soluble ectodomain of HSV-2 gB (gB2) but not the s...

  19. Inhibitors of glycoprotein processing alter T-cell proliferative responses to antigen and to interleukin 2.

    Wall, K A; Pierce, J D; Elbein, A D

    1988-01-01

    Most of the cell-surface molecules involved in T-cell immune responses are N-linked glycoproteins. We have investigated the effects of inhibitors of glycoprotein processing on specific T-cell functions, with the dual aims of examining the functional role of carbohydrate and of testing the usefulness of such compounds as immunomodulators. Treatment of a cloned murine helper T-cell line with these inhibitors differentially affects the proliferative response of the cell, depending upon the natur...

  20. Aggregate structure of hydroxyproline-rich glycoprotein (HRGP) and HRGP assisted dispersion of carbon nanotubes

    Wegenhart Ben; Tan Li; Held Michael; Kieliszewski Marcia; Chen Liwei

    2006-01-01

    AbstractHydroxyproline-rich glycoproteins (HRGP) comprise a super-family of extracellular structural glycoproteins whose precise roles in plant cell wall assembly and functioning remain to be elucidated. However, their extended structure and repetitive block co-polymer character of HRGPs may mediate their self-assembly as wall scaffolds by like-with-like alignment of their hydrophobic peptide and hydrophilic glycopeptide modules. Intermolecular crosslinking further stabilizes the scaffold. Th...

  1. The quality control of glycoprotein folding in the endoplasmic reticulum, a trip from trypanosomes to mammals

    A.J. Parodi

    1998-05-01

    Full Text Available The present review deals with the stages of synthesis and processing of asparagine-linked oligosaccharides occurring in the lumen of the endoplasmic reticulum and their relationship to the acquisition by glycoproteins of their proper tertiary structures. Special emphasis is placed on reactions taking place in trypanosomatid protozoa since their study has allowed the detection of the transient glucosylation of glycoproteins catalyzed by UDP-Glc:glycoprotein glucosyltransferase and glucosidase II. The former enzyme has the unique property of covalently tagging improperly folded conformations by catalyzing the formation of protein-linked Glc1Man7GlcNAc2, Glc1Man8GlcNac2 and Glc1Man9GlcNAc2 from the unglucosylated proteins. Glucosyltransferase is a soluble protein of the endoplasmic reticulum that recognizes protein domains exposed in denatured but not in native conformations (probably hydrophobic amino acids and the innermost N-acetylglucosamine unit that is hidden from macromolecular probes in most native glycoproteins. In vivo, the glucose units are removed by glucosidase II. The influence of oligosaccharides in glycoprotein folding is reviewed as well as the participation of endoplasmic reticulum chaperones (calnexin and calreticulin that recognize monoglucosylated species in the same process. A model for the quality control of glycoprotein folding in the endoplasmic reticulum, i.e., the mechanism by which cells recognize the tertiary structure of glycoproteins and only allow transit to the Golgi apparatus of properly folded species, is discussed. The main elements of this control are calnexin and calreticulin as retaining components, the UDP-Glc:glycoprotein glucosyltransferase as a sensor of tertiary structures and glucosidase II as the releasing agent.

  2. Activation of the glycoprotein hormone alpha-subunit promoter by a LIM-homeodomain transcription factor.

    Roberson, M S; Schoderbek, W E; Tremml, G; Maurer, R A

    1994-01-01

    Recently, a pituitary-specific enhancer was identified within the 5' flanking region of the mouse glycoprotein hormone alpha-subunit gene. This enhancer is active in pituitary cells of the gonadotrope and thyrotrope lineages and has been designated the pituitary glycoprotein hormone basal element (PGBE). In the present studies, we sought to isolate and characterize proteins which interact with the PGBE. Mutagenesis experiments identified a 14-bp imperfect palindrome that is required for bindi...

  3. Inhibition of glycoprotein processing blocks assembly of spicules during development of the sea urchin embryo

    1990-01-01

    Previous studies have implicated an 130-kD glycoprotein containing complex, N-linked oligosaccharide chain(s) in the process of spicule formation in sea urchin embryos. To ascertain whether the processing of high mannose oligosaccharides to complex oligosaccharides is necessary for spiculogenesis, intact embryos and cultures of spicule-forming primary mesenchyme cells were treated with glycoprotein processing inhibitors. In both the embryonic and cell culture systems 1- deoxymannojirimycin (1...

  4. Identification of a Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Variant Resistant to Cold Inactivation▿ †

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

    2009-01-01

    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 sel...

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

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

    2015-01-01

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

  6. Protective Effect of Dodonaea viscosa (L) Against Lead Acetate Induced Altered Glycoprotein Profiles in Rats

    Sivanesan, D.; Selvi, A. V. Veera Thamarai; Bhakyaraj, R.; Arunachalam, T.

    2009-01-01

    The present study was undertaken to examine the inhibitory effect of crude leaves of Dodonaea viscosa (L) on lead acetate induced synthesis of glycoproteins and sialic acid in liver and plasma. Enhanced synthesis of glycoproteins (protein - bound hexose and protein - bound hexosamine) and sialic acid levels were found in liver and plasma of the lead acetate poisoned rats. Administration of crude leaves of D.viscosa (100 mg/100 g body weight P.O.) effectively suppressed the synthesis of glycop...

  7. Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

    Dalton, J.P.; Strand, M.; Mangold, B.L.; Dean, D.A.

    1986-01-01

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

  8. Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

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

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

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

    1988-06-01

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

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

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

    2016-03-01

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

  11. Molecular docking studies with rabies virus glycoprotein to design viral therapeutics

    Tomar N

    2010-01-01

    Full Text Available The genome of rabies virus encodes five proteins; the nucleoprotein, the phosphoprotein, the matrix protein, the glycoprotein, and the RNA-dependent RNA polymerase. Among these, the glycoprotein is the most important as it is the major contributor to pathogenicity and virus neutralizing antibody response. Keeping in mind that glycoprotein is the only protein exposed on the surface of virus and is thought to be responsible for the interaction with the cell membrane, it was attempted to target glycoprotein by a ligand polyethylene glycol 4000, which blocks its active site, as seen by molecular operating environment software, so that it may be possible to prevent the spread of virus into the host. The ligand polyethylene glycol 4000 was retrieved from Research Collaboratory for Structural Bioinformatics protein data bank by providing the glycoprotein sequence to the databank. In this study it was observed that the ligand was successfully docked on a major portion of antigenic site II of glycoprotein by mimicking the virus neutralizing antibodies. This knowledge may be important for the development of novel therapies for the treatment of rabies and other viral diseases in the future.

  12. Glycoproteins and Glycosylation Site Assignments in Cereal seed Proteomes

    Dedvisitsakul, Plaipol

    The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications. Glycosy......The study of plant proteomes is important to further the understanding of biological processes and enhance the agronomical and nutritional value of crops and food products. To gain deeper understanding on the proteome level, it is important to characterize posttranslational modifications...... supplementing cotton wool with ZIC-HILIC in a microcolumn (called ZIC-cotton). This approach reduced co-enrichment of non-glycosylated peptides and allowed glycoppeptide identification from large protein mixtures. It was applied for glycoprotein identification and glycosylation site assignment in wheat albumin...... and barley aleurone layer proteins. By sitespecific glycosylation labeling and LC-MS/MS analysis, 76 different glycosylation sites within 65 wheat albumin proteins were identified using a combination of ZIC-cotton and cotton wool. In addition, ZIC-cotton has been also applied to proteins produced from...

  13. Antibody Derived Peptides for Detection of Ebola Virus Glycoprotein.

    Luis Mario Rodríguez-Martínez

    Full Text Available 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.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.Antibody fragments might represent a useful, effective, and low cost alternative to full-length antibodies in Ebola related capture and diagnostics applications.

  14. Multidrug resistance factor - glycoprotein P in rheumatoid arthritis

    I P Kolosova

    2003-02-01

    Full Text Available Objective. To assess expression of P-glycoprotein (Pgp on peripheral blood (PB lymphocytes and its changes during therapy in pts with rheumatoid arthritis (RA. Methods. 51 RA pts and 11 healthy donors (control group were examined. 35 pts were followed up. 20 of them were treated with methotrexate (MT and 15 with glucocorticosteroid (GCS pulse therapy (PT. Pgp expression was examined with flow cytofluorometry with monoclonal antibodies (UIC2 PE, Immunotech. Results. Pgp expression on PB lymphocytes in RA was significantly more prominent (29,3 29,9 % than in control group (2,5 2,0 %, P<0,01. Pgp expression did not depend on pts age and sex, duration and stage of the disease, presence or absence history of disease modifying drugs therapy. PT with GCS but not MT significantly decreased Pgp expression (from 57,2±27,0 % to 28,8135,2 %, r<0,05. Conclusion. RA patients have increased Pgp expression, which is probably biologically sensible but clinically unfavourable response of immunocompetent cells to durable application of such foreign substances as medications. PT with GCS decrease Pgp expression on lymphocytes while treatment with MT does not change it.

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

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

    2016-01-15

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

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

    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.

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

    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 (125I) 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

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

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

    2016-05-01

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

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

    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

  20. Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine

    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.

  1. Effect of Urea on Activity and Conformation of a Glycoprotein

    WEI Xiang; WANG Xiaoyun; ZHOU Bo; ZHOU Haimeng

    2006-01-01

    The changes of the activity and conformation of Aspergillus niger phytase in urea were detected by farultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays. The results show that no enzyme activity can be detected after phytase is incubated for 10 h in 3.0 mol/L urea, even though at this urea concentration, less than 20% of the tertiary and secondary structures in the native enzyme changed. The inactivation reaction kinetics is found to be a monophasic first-order reaction, but the unfolding is a biphasic process consisting of two first-order reactions. The inactivation rates of the free enzyme and the substrate-enzyme complex are much faster than the conformational changes during urea denaturation. All of the results indicate that, as a glycoprotein, phytase's activity is strongly dependent on its conformational integrity. The phytase active sites seem to be located in a limited region in the molecule and display more conformational fragility and flexibility to denaturants than enzyme molecular structure as a whole.

  2. Myelin-associated Glycoprotein gene and brain morphometry in schizophrenia

    Daniel Felsky

    2012-05-01

    Full Text Available Myelin and oligodendrocyte disruption may be a core feature of schizophrenia pathophysiology. The purpose of the present study was to localize the effects of previously identified risk variants in the myelin associated glycoprotein gene on brain morphometry in schizophrenia patients and healthy controls. 45 schizophrenia patients and 47 matched healthy controls underwent clinical, structural magnetic resonance imaging, and genetics procedures. Gray and white matter cortical lobe volumes along with subcortical structure volumes were calculated from T1-weighted MRI scans. Each subject was also genotyped for the two disease-associated MAG single nucleotide polymorphisms (rs720308 and rs720309. Repeated measures general linear model analysis found significant region by genotype and region by diagnosis interactions for the effects of MAG risk variants on lobar gray matter volumes. No significant associations were found with lobar white matter volumes or subcortical structure volumes. Follow-up univariate general linear models found the AA genotype of rs720308 predisposed schizophrenia patients to left temporal and parietal gray matter volume deficits. These results suggest that the effects of the MAG gene on cortical gray matter volume in schizophrenia patients can be localized to temporal and parietal cortices. Our results support a role for MAG gene variation in brain morphometry in schizophrenia, align with other lines of evidence implicating MAG in schizophrenia, and provide genetically-based insight into the heterogeneity of brain imaging findings in this disorder.

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

    Andrea Picchianti-Diamanti

    2014-03-01

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

  4. Selenoprotein P. A selenium-rich extracellular glycoprotein.

    Burk, R F; Hill, K E

    1994-10-01

    Selenoprotein P is a glycoprotein that has been purified from rat and human plasma. In selenium-replete rats it contains 65% of the plasma selenium and its concentration is 25-30 mg protein/L. In selenium-deficient rats its concentration is life of 75Se in selenoprotein P is 3 to 4 h, indicating a rapid turnover. Purified rat selenoprotein P contains 7.5 +/- 1 selenium atoms per molecule as selenocysteine. The sequence of the cloned cDNA predicts 10 selenocysteine residues, which suggests that the protein in plasma is a modification of the predicted one. Deduced amino acid sequence identity between rats and humans is 72%. The 3' untranslated region of selenoprotein P cDNA contains two predicted stem loops of the type essential for selenocysteine incorporation. Northern analysis indicates that selenoprotein P is expressed by many tissues. Hepatic selenoprotein P mRNA level, but not its transcription, decreases during selenium deficiency. The decrease is less than the decrease of glutathione peroxidase mRNA, however. Selenoprotein P is postulated to serve as an extracellular oxidant defense because its presence correlates with selenium protection of selenium-deficient rats against diquat-induced lipid peroxidation and liver necrosis. More research will be required to test this hypothesis and to establish the biochemical function of selenoprotein P. PMID:7931697

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

    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

  6. Analysis of Determinants in Filovirus Glycoproteins Required for Tetherin Antagonism

    Kerstin Gnirß

    2014-04-01

    Full Text Available The host cell protein tetherin can restrict the release of enveloped viruses from infected cells. The HIV-1 protein Vpu counteracts tetherin by removing it from the site of viral budding, the plasma membrane, and this process depends on specific interactions between the transmembrane domains of Vpu and tetherin. In contrast, the glycoproteins (GPs of two filoviruses, Ebola and Marburg virus, antagonize tetherin without reducing surface expression, and the domains in GP required for tetherin counteraction are unknown. Here, we show that filovirus GPs depend on the presence of their authentic transmembrane domains for virus-cell fusion and tetherin antagonism. However, conserved residues within the transmembrane domain were dispensable for membrane fusion and tetherin counteraction. Moreover, the insertion of the transmembrane domain into a heterologous viral GP, Lassa virus GPC, was not sufficient to confer tetherin antagonism to the recipient. Finally, mutation of conserved residues within the fusion peptide of Ebola virus GP inhibited virus-cell fusion but did not ablate tetherin counteraction, indicating that the fusion peptide and the ability of GP to drive host cell entry are not required for tetherin counteraction. These results suggest that the transmembrane domains of filoviral GPs contribute to tetherin antagonism but are not the sole determinants.

  7. 3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

    Hiroshi Fujise

    2004-01-01

    Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

  8. Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics

    Yu-Hsuan Chiang; Yu-Jen Wu; Ya-Ting Lu; Kuan-Hung Chen; Tzu-Chun Lin; Chen, Yu-Kuang H.; Ding-Tzai Li; Fong-Ku Shi; Ching-Chuan Chen; Jue-Liang Hsu

    2011-01-01

    In this study, a commercially available fluorescent dye, Lissamine rhodamine B sulfonyl hydrazine (LRSH), was designed to specifically stain the glycoproteins in polyacrylamide gels. Through the periodate/Schiff base mechanism, the fluorescent dye readily attaches to glycoproteins and the fluorescence can be simultaneously observed under either 305 nm or 532 nm excitation therefore, the dye-stained glycoproteins can be detected under a regular UV transilluminator or a more elegant laser-based...

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

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

    1983-01-01

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

  10. The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells.

    Lincke, C R; Broeks, A; the, I; Plasterk, R H; Borst, P

    1993-01-01

    P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs. The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown. In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract. We have studied the tissue-specific expression of Caenorhabditis elegans P-glycoprotein genes pgp-1 and pgp-3 by transformation of nematodes with pgp-lacZ gene fusion constr...

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

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

    1988-05-03

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

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

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

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

    PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellium were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H] glucosamine- or [3H]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-β-galactosidase, 40-45% of the [3H]glucosamine of [3H]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 [3H]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 [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain

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

    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, gXt, synthesized in the presence of tromantadine still incorporate 3H-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 gXt, on SDS-PAGE. The gXt 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 gXt, 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, gCt, 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 gCt indicate that the O-linked disaccharide NAcGal-Galactose is present

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

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

    2013-01-01

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

  16. Chaperone requirements for biosynthesis of the trypanosome variant surface glycoprotein.

    Mark C Field

    Full Text Available BACKGROUND: Trypanosoma brucei does not respond transcriptionally to several endoplasmic reticulum (ER stress conditions, including tunicamycin or dithiothreitol, indicating the absence of a conventional unfolded protein response. This suggests divergent mechanisms for quality control (QC of ER protein folding and export may be present in trypanosomes. As the variant surface glycoprotein (VSG represents approximately 90% of trypanosome plasma membrane protein, it is possible that VSG has evolved to fold efficiently to minimize ER folding burden. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate the presence of a QC system by pharmacological inhibition of the trypanosome 26S proteasome. This indicates active proteasome-mediated VSG turnover as approximately 2.5 fold more VSG is recovered from cell lysates following MG132 inhibition. An in silico scan of the trypanosome genome identified 28 open reading frames likely to encode polypeptides participating in ER nascent chain maturation. By RNA interference we monitored the importance of these gene products to proliferation, VSG abundance and cell morphology. 68% of the cohort were required for normal proliferation, and depletion of most of these factors resulted in increased VSG abundance, suggesting involvement in ERQC and degradation. CONCLUSIONS/SIGNIFICANCE: The retention of genes for, and the involvement of many gene products in, VSG folding indicates a substantial complexity within the pathways required to perform this role. Counterintuitively, for a super-abundant antigen VSG is apparently made in excess. The biosynthetic excess VSG appears to be turned over efficiently by the proteasome, implying that considerable VSG is rejected by the trypanosome ERQC mechanism. Accordingly, the VSG polypeptide is not well optimized for folding, as only approximately 30% attains the native state. Finally as much of the core ERQC system is functionally conserved in trypanosomes, the pathway has an ancient

  17. Alternative promoter usage of the membrane glycoprotein CD36

    Whatling Carl

    2006-03-01

    Full Text Available Abstract Background CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation. Results We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters. Conclusion Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.

  18. Structural insights into the antigenicity of myelin oligodendrocyte glycoprotein

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

    2003-01-01

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

  19. Membrane topology analysis of HIV-1 envelope glycoprotein gp41

    Xiao Dan

    2010-11-01

    Full Text Available Abstract Background The gp41 subunit of the HIV-1 envelope glycoprotein (Env has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD. An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP that is only active in the cytoplasm. The tag protein (HaloTag and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. Results In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells, the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. Conclusions It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

  20. Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

    Oliveira M.C.

    1999-01-01

    Full Text Available Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6% presented hypogonadism (which was central in 45 cases and primary in 2, 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01 and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02. Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01 as well as free testosterone levels (r = -0.53, P<0.01. In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

  1. Interaction of Common Azole Antifungals with P Glycoprotein

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

    2002-01-01

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

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

    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%α-helix, 24% β-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% α-helix, 10% β-sheet, 81% disordered structures. These data were in good agreement with the 11% α-helix, 18% β-sheet, 61% β-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3

  3. P-glycoprotein acts as an immunomodulator during neuroinflammation.

    Gijs Kooij

    Full Text Available BACKGROUND: Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system in which autoreactive myelin-specific T cells cause extensive tissue damage, resulting in neurological deficits. In the disease process, T cells are primed in the periphery by antigen presenting dendritic cells (DCs. DCs are considered to be crucial regulators of specific immune responses and molecules or proteins that regulate DC function are therefore under extensive investigation. We here investigated the potential immunomodulatory capacity of the ATP binding cassette transporter P-glycoprotein (P-gp. P-gp generally drives cellular efflux of a variety of compounds and is thought to be involved in excretion of inflammatory agents from immune cells, like DCs. So far, the immunomodulatory role of these ABC transporters is unknown. METHODS AND FINDINGS: Here we demonstrate that P-gp acts as a key modulator of adaptive immunity during an in vivo model for neuroinflammation. The function of the DC is severely impaired in P-gp knockout mice (Mdr1a/1b-/-, since both DC maturation and T cell stimulatory capacity is significantly decreased. Consequently, Mdr1a/1b -/- mice develop decreased clinical signs of experimental autoimmune encephalomyelitis (EAE, an animal model for multiple sclerosis. Reduced clinical signs coincided with impaired T cell responses and T cell-specific brain inflammation. We here describe the underlying molecular mechanism and demonstrate that P-gp is crucial for the secretion of pro-inflammatory cytokines such as TNF-alpha and IFN-gamma. Importantly, the defect in DC function can be restored by exogenous addition of these cytokines. CONCLUSIONS: Our data demonstrate that P-gp downmodulates DC function through the regulation of pro-inflammatory cytokine secretion, resulting in an impaired immune response. Taken together, our work highlights a new physiological role for P-gp as an immunomodulatory molecule and reveals a possible

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

    Chang, Shan-Yan [Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Liu, Fu-Feng, E-mail: fufengliu@tju.edu.cn, E-mail: ysun@tju.edu.cn; Dong, Xiao-Yan; Sun, Yan, E-mail: fufengliu@tju.edu.cn, E-mail: ysun@tju.edu.cn [Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072 (China)

    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.

  5. Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum

    Bhattacharyya Suchita

    2011-01-01

    Full Text Available Abstract The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1. However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER, Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP, it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.

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

    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.

  7. Glycoprotein IIIa Preparation for T-cell Stimulation

    GR Anani Sarab

    2005-10-01

    Full Text Available Chronic immune thrombocytopenic purpura (ITP is an autoimmune disease characterized by antiplatelet autoantibodis. The major target of the anti platelet antibodies is platelet membrane glycoprotein IIb/IIIa. In order to characterize the immunodominant epitopes in the structure of GPIIIa, the extracellular portions of GPIIIa will be expressed and purified. These antigens will be tested for antigenicity in further investigation. The first segment of GPIIIa which was considered for expression as a recombinant glutathion S-transferase (GST fusion proteins included IIIa22-262 which encompass amino acid residue 22-262 of the 762 amino acids of GPIIIa. A segment of GPIIIa complementary DNA (cDNA was subcloned into the 39 end of the Schistosoma japonicum GST gene in the bacterial expression plasmid vector, pGEX 6P-1 (Amersham Pharmacia Biotech. In summary, the expression plasmid vector, pGEX 6P-1 containing segment IIIa22-262 was introduced to E.coli. Saturated overnight culture was used and the bacterial cells were grown to log-phase. IPTG was added to the culture to induce overexpression of fusion protein and the cells were grown for an additional 1-3 hours. Bacterial lysate containing recombinant protein was prepared by sonication. The fusion protein was purified from total cell extract using glutathione-agarose beads. Specificity of the GST-fusion proteins was confirmed on Immunoblot probed with rabbit anti-GST polyclonal antibodies. PreScission Protease was used to remove the GST tag. Protein extract and purified products were analyzed by SDS gel electrophoresis. The recombinant GST-fusion protein IIIa22-262 was successfully expressed and purified in large quantities but the yield of the IIIa22-262 peptide after enzyme treatment was low. When a good yield is fully obtained, the purified protein segment will be used for T-cell stimulation in culture.

  8. Spatial Localization of the Ebola Virus Glycoprotein Mucin-Like Domain Determined by Cryo-Electron Tomography

    Tran, Erin E. H.; Simmons, James A.; Bartesaghi, Alberto; Shoemaker, Charles J.; Nelson, Elizabeth; Judith M White; Subramaniam, Sriram

    2014-01-01

    The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function.

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

    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. 3H-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

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

    Chen, Rui; Zou, Hanfa; Figeys, Daniel

    2016-06-01

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

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

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

    2016-01-01

    Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment. PMID:26459807

  12. Pharmacokinetic modeling of multidrug resistance P-glycoprotein transport of gamma-emitting substrates

    Bae, K. T.; Piwnica-Worms, D. [St. Louis, Washington Univ. (United States). Mallinckrodt Institute of Radiology. Lab. of Molecular Radiopharmacology]|[St. Louis, Washington Univ. (United States). Dept. of Molecular Biology and Pharmacology

    1997-06-01

    P-glycoprotein, the human multidrug resistance (MDR1) gene product, is an integral membrane protein expressed on the plasma membrane of MDR tumor cells and is the best characterized of a family of efflux transporters that confer chemotherapeutic resistance. The use of gamma-emitting {sup 99m}Tc-agents to image P-glycoprotein function in human tumors in vivo has been proposed. Net tumor cell content of {sup 99m}Tc-Sestamibi, {sup 99m}Tc-Tetrofosmin and several {sup 99m}Tc-Q-complexes ({sup 99m}Tc-Q58 and {sup 99m}Tc-Q63) are function of passive potential-dependent influx and MDR1 P-glycoprotein-mediated active extrusion. To better understand the overall fidelity of these P-glycoprotein substrates to report MDR activity in vivo in relation to tissue perfusion, a compartmental model of tracer pharmacokinetics was developed. Modeling indicates that tissue perfusion will impact pharmacokinetics in vivo in a manner that will tend to diminish P-glycoprotein-mediated phenotypic differences between tissues when they are perfusion-limited. However, dynamic imaging to extract efflux rate constants is independent of perfusion and may represent the highest quality methodology for collecting the desired information regarding activity of the efflux transporter. Much work remains to translate these concepts and biological targeting properties into clinical practice.

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

    Piwnica-Worms, D.; Vallabhaneni, V.R. [Washington Univ. Medical School, St. Louis, MO (United States); Kronauge, J.F. [Harvard Medical School, Boston, MA (United States)] [and others

    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.

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

    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

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

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

    2012-01-01

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

  16. Characterization of Intact Neo-Glycoproteins by Hydrophilic Interaction Liquid Chromatography

    Alice Pedrali

    2014-06-01

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

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

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

    2016-09-01

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

  18. Temporal pattern of incorporation of 3H precursors into pituitary glycoproteins and their subsequent release

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

  19. Prediction of P-glycoprotein expression and chemoresistant character of gliomas by SPECT

    Iuchi, Toshihiko; Togawa, Takashi; Oga, Masaru; Osato, Katsunobu [Chiba Cancer Center (Japan); Namba, Hiroki; Fujimoto, Shuichi

    2000-09-01

    In this prospective study of 25 malignant gliomas, we correlated the {sup 99m}Tc-MIBI uptake/{sup 201}Tl uptake ratio (MIBI/Tl) with the expression of P-glycoprotein in tumor tissue and the tumor's response to anticancer agents. All patients underwent {sup 99m}Tc-MIBI and {sup 201}Tl SPECT before surgery. Semiquantitative assessment of tracer uptake was performed using the ratio of radioactivity in the tumor relative to normal scalp. Immunohistochemical studies were performed on paraffin sections using an anti-P-glycoprotein monoclonal antibody, JSB-1. Chemosensitivity of the gliomas to following 12 anticancer agents: vincristine, vinblastine, vindesine, etoposide, irinotecan, daunomycin, adriamycin, aclarubicin, epirubicin, pirarubicin, actinomycin and mitoxantrone, was determined by an in vitro assay using surgical specimens, and chemosensitivity was expressed as the number of effective drugs. Gliomas expressing P-glycoprotein were significantly less chemosensitive than gliomas without the glycoprotein (p=0.010), and MIBI/Tl of gliomas expressing P-glycoprotein was significantly smaller than tumors without expression (p=0.008). From the prognostic point of view, gliomas showing MIBI/Tl of 0.6 or less had fewer effective drugs (p=0.008). However, MIBI/Tl was not effective at predicting overall survival in patients with malignant glioma. From these results, we concluded that efflux of {sup 99m}Tc-MIBI through P-glycoprotein could be evaluated by MIBI/Tl, and this index reflected well the chemoresistant character of malignant gliomas. (author)

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

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

    2011-01-01

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

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

    Chandipura virus glycoprotein ectodomain (Gth) was purified and crystallized at pH 7.5. X-ray diffraction data set was collected to a resolution of 3.1 Å. 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

  2. Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

    Tal-Singer, R; Peng, C.; Ponce de Leon, M; Abrams, W R; Banfield, B W; Tufaro, F; Cohen, G H; Eisenberg, R J

    1995-01-01

    The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the bacu...

  3. The Effects of Benzo(A) Pyrene Doxorubicin and Paclitaxel on P170 Glycoprotein

    COŞAN, Didem

    2001-01-01

    B(a)P is a mutagenic, carcinogenic and teratogenic substance. Paclitaxel and doxorubicin are antineoplastic drugs widely used in cancer treatment. The purpose of this study is to observe the effects of doxorubicin and paclitaxel on p170 glycoprotein in rat liver and kidney tissue after administration of B(a)P. As is well known, p170 glycoprotein is an indicator of drug resistance. We hypothesized that a combination of these antineoplastic drugs would cause lower p170 levels and thus would ha...

  4. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Beuy; Joob; Viroj; Wiwanitkit

    2014-01-01

    The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present.Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus.The in-depth analysis of the viral protein to find the binding site,active pocket is needed.Here,the authors analyzed the envelope glycoprotein GP2 from Ebola virus.Identification of active pocket and protein draggability within envelope glycoprotein GP2 from Ebola virus was done.According to this assessment,7 active pockets with varied draggability could be identified.

  5. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Beuy Joob; Viroj Wiwanitkit

    2014-01-01

    The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present. Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus. The in-depth analysis of the viral protein to find the binding site, active pocket is needed. Here, the authors analyzed the envelope glycoprotein GP2 from Ebola virus. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus was done. According to this assessment, 7 active pockets with varied druggability could be identified.

  6. Glycosylation Changes on Serum Glycoproteins in Ovarian Cancer May Contribute to Disease Pathogenesis

    Radka Saldova; Wormald, Mark R.; Dwek, Raymond A.; Rudd, Pauline M

    2008-01-01

    Ovarian cancer is the most lethal of all gynaecological cancers among women. Serum CA125 is the only biomarker that is used routinely and there is a need for further complementary biomarkers both in terms of sensitivity and specificity. N-glycosylation changes in ovarian cancer serum glycoproteins include a decrease in galactosylation of IgG and an increase in sialyl Lewis X (SLex) on haptoglobin β-chain, α1-acid glycoprotein and α1-antichymotrypsin. These changes are also present in chronic ...

  7. Structures of the Oligosaccharides of the Glycoprotein Coded by Early Region E3 of Adenovirus 2

    Kornfeld, Rosalind; Wold, William S. M.

    1981-01-01

    Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-3H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, f...

  8. Synthesis and localization of two sulphated glycoproteins associated with basement membranes and the extracellular matrix

    Hogan, B L; Taylor, A; Kurkinen, M; Couchman, J R

    1982-01-01

    Two sulphated glycoproteins (sgps) of apparent molecular weight (Mr) 180,000 and 150,000, are synthesized by murine PYS and PF HR9 parietal endoderm and Swiss 3T3 cells. The Mr 150,000 sgp has a similar chemical structure to the sulphated glycoprotein, C, synthesized and laid down in Reichert...... interactions and are not precursors or products of each other. They contain asparagine-linked oligosaccharides, but these are not the exclusive sites of sulphate labeling. Antiserum raised against the Mr 150,000 sgp C of Reichert's membranes has been used in an immunohistochemical analysis of rat skin. In...

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

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

    2015-05-01

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

  10. Identifying the viral genes encoding envelope glycoproteins for differentiation of Cyprinid herpesvirus 3 isolates

    Se Chang Park; Sang Phil Shin; Casiano Choresca Jr.; Ji Hyung Kim; Tristan Renault; Jee Eun Han; Jin Woo Jun

    2013-01-01

    Cyprinid herpes virus 3 (CyHV-3) diseases have been reported around the world and are associated with high mortalities of koi (Cyprinus carpio). Although little work has been conducted on the molecular analysis of this virus, glycoprotein genes identified in the present study seem to be valuable targets for genetic comparison of this virus. Three envelope glycoprotein genes (ORF25, 65 and 116) of the CyHV-3 isolates from the USA, Israel, Japan and Korea were compared, and interestingly, seque...

  11. Bovine herpesvirus glycoprotein D: a review of its structural characteristics and applications in vaccinology

    Alves Dummer, Luana; Pereira Leivas Leite, Fábio; van Drunen Littel-van den Hurk, Sylvia

    2014-01-01

    International audience The viral envelope glycoprotein D from bovine herpesviruses 1 and 5 (BoHV-1 and -5), two important pathogens of cattle, is a major component of the virion and plays a critical role in the pathogenesis of herpesviruses. Glycoprotein D is essential for virus penetration into permissive cells and thus is a major target for virus neutralizing antibodies during infection. In view of its role in the induction of protective immunity, gD has been tested in new vaccine develo...

  12. Electrophoretic demonstration of glycoproteins, lipoproteins, and phosphoproteins in human and bovine enamel

    Kirkeby, S; Moe, D; Bøg-Hansen, T C; Salling, E

    1990-01-01

    Enamel proteins from fully mineralized human molars and from bovine tooth germs were separated by electrophoresis. The gels were stained for detection of glycoproteins, lipoproteins, and phosphoproteins. Glycoproteins were shown by periodic acid-Schiff staining and lectin blotting. In mature human...... enamel a number of high molecular weight proteins could be demonstrated after ethylenediaminetetra-acetic acid demineralization and subsequent Triton X-100 extraction. These proteins are suggested to be lipoproteins. Phosphoproteins could only be visualized in enamel matrix from the tooth germs....

  13. A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection

    Foley, Heather D.; McGettigan, James P.; Siler, Catherine A.; Dietzschold, Bernhard; Schnell, Matthias J.

    2000-01-01

    To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were red...

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

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

    2016-03-01

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

  15. Characterisation of non-P-glycoprotein multidrug-resistant Ehrlich ascites tumour cells selected for resistance to mitoxantrone

    Nielsen, D; Eriksen, J; Maare, C;

    2000-01-01

    (i) value for P-glycoprotein-positive cells. However, whereas verapamil (50 microM) inhibited the ATPase activity of EHR2/MITOX microsomes, it stimulated the ATPase activity of microsomes derived from P-glycoprotein-positive cells. In conclusion, the resistance in EHR2/MITOX was multifactorial and appeared...

  16. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

    1996-01-01

    A histochemical avidin-biotin technique with three different alpha 1-acid glycoprotein glycoforms showed pronounced alterations in the cellular localization of two alpha 1-acid glycoprotein lectin-like receptors during cell differentiation in the developing rat testis. The binding of alpha 1-acid...

  17. Glycoproteins of coated pits, cell junctions, and the entire cell surface revealed by monoclonal antibodies and immunoelectron microscopy

    1983-01-01

    Topographical descriptions of three major plasma membrane glycoproteins of murine 3T3 cells were obtained by immunoelectron microscopy with monoclonal antibodies. A glycoprotein of Mr 80,000 was distributed throughout the total cell surface. A second of Mr 90,000 was concentrated in coated pits, and a third of Mr 100,000 was localized at cell junctions.

  18. P-glycoprotein activity in the blood-brain barrier is affected by virus-induced neuroinflammation and antipsychotic treatment

    Doorduin, Janine; de Vries, Erik F. J.; Dierckx, Rudi A.; Klein, Hans C.

    2014-01-01

    A large percentage of schizophrenic patients respond poorly to antipsychotic treatment. This could be explained by inefficient drug transport across the blood-brain barrier due to P-glycoprotein mediated efflux. P-glycoprotein activity and expression in the blood-brain barrier can be affected by inf

  19. Rhodocytin (aggretin) activates platelets lacking alpha(2)beta(1) integrin, glycoprotein VI, and the ligand-binding domain of glycoprotein Ibalpha

    Bergmeier, W; Bouvard, D; Eble, J A;

    2001-01-01

    collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha(2)beta(1) integrin. Here we show that the Cre/loxP-mediated loss of beta(1) integrin on mouse platelets has no effect on rhodocytin-induced platelet activation......Although alpha(2)beta(1) integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the......, excluding an essential role of alpha(2)beta(1) integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta(1)-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets...

  20. Use of λgt11 to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins

    A library of pseudorabies virus (PRV) DNA fragments was constructed in the expression cloning vector λgt11. 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 λgt11 vector, the cloned proteins were expressed in Escherichia coli as β-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 [14C]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

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

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

    2014-01-01

    Monoclonal antibodies (MAbs) against viral glycoproteins have important diagnostic and therapeutic applications. In most cases, the MAbs specific to viral glycoproteins are raised against intact virus particles. The biosynthesis of viral glycoproteins in heterologous expression systems such as bacteria, yeast, insect or mammalian cells is often problematic due to their low expression level, improper folding and limited stability. To generate MAbs against hantavirus glycoprotein Gc, we have us...

  2. Molecular characterization and baculovirus expression of the glycoprotein B of a seal herpesvirus (phocid herpesvirus-1).

    T.C. Harder (Timm); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractA glycoprotein B (gB) gene homologue was identified in a 5.4-kb BamHl genomic fragment of the phocid herpesvirus type-1 (PhHV-1) which represents a widespread and important pathogen of pinnipeds. Sequence analysis revealed a gB-specific open-reading frame comprising 881 amino acids. Phyl

  3. Human CRISP-3 binds serum alpha(1)B-glycoprotein across species

    Udby, Lene; Johnsen, Anders H; Borregaard, Niels

    2010-01-01

    CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such...

  4. Structural analysis of the carbohydrate chains of glycoproteins by 500-MHz 1H-NMR spectroscopy

    This thesis deals with the structural analysis by 500-MHz 1H-NMR spectroscopy of carbohydrate chains obtained from glycoproteins. In the chapters 1 to 6 the structural analysis of N-glycosidically linked carbohydrate chains is described. The chapters 7 to 10 describe the structural analysis of O-glycosidically linked carbohydrate chains. 381 refs.; 44 figs.; 24 tabs.; 7 schemes

  5. Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers

    Zhang, Xinzheng; Sheng, Ju; Austin, S. Kyle; Hoornweg, Tabitha E.; Smit, Jolanda M.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G.

    2015-01-01

    Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However,

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

    Yoshiki Yamaguchi

    2012-07-01

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

  7. Glycoprotein Biochemistry (Biosynthesis)--A Vehicle for Teaching Many Aspects of Biochemistry and Molecular Biology.

    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…

  8. Can celecoxib affect P-glycoprotein-mediated drug efflux? A microPET study

    De Vries, Erik F. J.; Doorduin, Janine; Vellinga, Namkje A. R.; Van Waarde, Aren; Dierckx, Rudi A.; Klein, Hans C.

    2008-01-01

    Introduction: P-glycoprotein (Pgp) is an efflux pump that protects vital organs like the brain from toxic substances, but which is also associated with therapy resistance. The anti-inflammatory drug celecoxib potentiates the efficacy of several cytostatic and neurotropic drugs that are known Pgp sub

  9. Carbon-11-labeled daunorubicin and verapamil for probing P-glycoprotein in tumors with PET

    Elsinga, PH; Franssen, EJF; Hendrikse, NH; Fluks, L; Weemaes, AMA; vanderGraaf, WTA; deVries, GE; Visser, GM; Vaalburg, W

    1996-01-01

    One of the mechanisms for multidrug resistance (MDR) of tumors is an overexpression of the P-glycoprotein (P-gp). The cytostatic agent daunorubicin and the modulator verapamil were labeled with C-11 to probe P-gp with PET. Methods: Carbon-11-daunorubicin was prepared from (CCH2N2)-C-11 with an aldeh

  10. Modification-specific proteomic analysis of glycoproteins in human body fluids by mass spectrometry

    Bunkenborg, Jakob; Hägglund, Per; Jensen, Ole Nørregaard

    2007-01-01

    -glycosylated proteins in body fluids and other complex samples. An approach for identification of N-glycosylated proteins and mapping of their glycosylation sites is described. In this approach, glycoproteins are initially selectively purified by lectin chromatography. Following tryptic digestion, glycopeptides are...

  11. Blood-brain barrier P-glycoprotein function is not impaired in early Parkinson's disease

    Bartels, A. L.; van Berckel, B. N. M.; Lubberink, M.; Luurtsema, G.; Lammertsma, A. A.; Leenders, K. L.

    2008-01-01

    The cause of Parkinson's disease (PD) is unknown. Genetic susceptibility and exposure to environmental toxins contribute to specific neuronal loss in PD. Decreased blood-brain barrier (BBB) P-glycoprotein (P-gp) efflux function has been proposed as a possible causative link between toxin exposure an

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

    Woo, Jung Hee; Neville, David M

    2003-08-01

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

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

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

    2009-01-01

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

  14. Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein

    Vliegenthart, J.F.G.; Rooijen, J.J.M. van; Kamerling, J.P.

    1998-01-01

    The primary structures of 32 sulfated di-, tri- and tetraantennary N-glycans of human Tamm-Horsfall glycoprotein (THP) have been determined. THP was isolated from the urine of one healthy male donor. The intact carbohydrate chains were released by PNGase-F and fractionated via FPLC on Resource Q, HP

  15. Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry

    Kamel El Omari

    2013-01-01

    Full Text Available Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1 at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

  16. Improved method for silver staining of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels

    Møller, H J; Poulsen, J H

    1995-01-01

    A method for detection of glycoproteins in thin sodium dodecyl sulfate polyacrylamide gels was developed by a combination of (i) initial periodic acid oxidation/Alcian blue staining and (ii) subsequent staining with silver nitrate. The procedure allowed detection of as little as 1.6 ng of alpha 1...

  17. Dynamics of multidrug resistance : P-glycoprotein analyses with positron emission tomography

    Hendrikse, NH; Vaalburg, W

    2002-01-01

    Multidrug resistance (MDR) is characterized by the occurrence of cross-resistance to a broad range of structurally and functionally unrelated drugs. Several mechanisms are involved in MDR. One of the most well-known mechanisms is the overexpression of P-glycoprotein (P-gp), encoded by the MDR1 gene

  18. A simple method to discriminate between beta(2)-glycoprotein I- and prothrombin-dependent lupus anticoagulants

    Simmelink, MJA; Derksen, RHWM; Arnout, J; De Groot, PG

    2003-01-01

    Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin. In the present study, we describe a method to

  19. Selective inhibition of herpes simplex virus glycoprotein synthesis by a benz-amidinohydrazone derivative

    1 H-benz[f]indene-1.3(2H)dione-bis-amidinohydrazone (benzhydrazone) inhibited incorporation of 14C-glucosamine, 14C-fucose and 14C-mannose into glycoproteins of HEp-2 cells infected with various strains of herpes simplex virus 1 (HSV-1) and impaired RNA and protein synthesis to a low extent. These biochemical effects are very similar to those induced by glycosylation inhibitors such as tunicamycin, D-glucosamine and 2-deoxy-D-glucose. In contrast to these inhibitors, benzhydrazone reduced HSV glycoprotein synthesis selectively since it did not significantly modify i) the saccharide uptake into glycoproteins of uninfected and of Sindbis virus-infected cells, ii) viral growth and cell fusion in paramyxovirus-infected cells, two activities which depend on viral glycoprotein synthesis. Benzhydrazone had only minor effects on the overall metabolism of uninfected cells, since it did not alter cell growth rate, and amino acid, uridine, and hexose incorporations were about 80 per cent those of untreated cells. (Author)

  20. Effect of luteolin on glycoproteins metabolism in 1, 2-dimethylhydrazine induced experimental colon carcinogenesis

    Manju Vaiyapuri

    2008-12-01

    carcinogenesis. Thus, the present study indicates that luteolin has protected the cell surface and maintained the structural integrity of the cell membranes during DMH induced colon carcinogenesis. Keywords: Colon cancer, 1, 2-dimethylhydrazine, luteolin, glycoproteins Received: 23 January 2009 / Received in revised form: 17 February 2009, Accepted: 28 February 2009, Published online: 3 March 2009

  1. Human intestinal P-glycoprotein activity estimated by the model substrate digoxin

    Larsen, U L; Hyldahl Olesen, L; Nyvold, Charlotte Guldborg;

    2007-01-01

    P-glycoprotein (Pgp) plays a part in the intestinal uptake of xenobiotics and has been associated with susceptibility to ulcerative colitis. The aim of this study was to examine Pgp activity in relation to age, gender, medical treatment (rifampicin or ketoconazole) and the multidrug resistance (M...

  2. Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

    Tomlinson, M. G.; Calaminus, S. D.; Berlanga, O.; Bori-Sanz, T.; Meyaard, L.; Watson, S. P.; Auger, J.M.

    2007-01-01

    Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR-gamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective: To determine why GPVI-FcR gamma signals poorly, or not at all, in response to

  3. St. John's Wort constituents modulate P-glycoprotein transport activity at the blood-brain barrier.

    Ott, M.; Huls, M.; Cornelius, M.G.; Fricker, G.

    2010-01-01

    PURPOSE: The purpose of this study was to investigate the short-term signaling effects of St. John's Wort (SJW) extract and selected SJW constituents on the blood-brain barrier transporter P-glycoprotein and to describe the role of PKC in the signaling. METHODS: Cultured porcine brain capillary endo

  4. Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

    Konings, WN; Poelarends, GJ

    2002-01-01

    Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural a

  5. Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta

    Ribeiro, D.M.O.G.; Borst, J.W.; Goldbach, R.W.; Kormelink, R.J.M.

    2009-01-01

    Recently, the Tomato Spotted Wilt Virus (TSWV) Gn and Gc glycoproteins were shown to induce the formation of (pseudo-) circular and pleomorphic membrane structures upon transient expression in plant cells. Furthermore, when singly expressed, Gc retains in the ER, while Gn is able to further migrate

  6. Low rate doses effects of gamma radiation on glycoproteins of transmembrane junctions in fibroblasts

    Glycoproteins of trans-membrane junctions are molecules that help to bind cells with the extracellular matrix. Integrins are the most important trans-membrane molecules among others. The damage of gamma radiation on those proteins could be an important early event that causes membrane abnormalities which may lead to cell malfunction and cancer induced by radiation due to cell dissociation. Randomized blocks with 3 repetitions of mouse embryo fibroblast cultures, were irradiated with Cobalt-60 gamma rays, during 20 days. Biological damage to glycoproteins and integrins was evaluated by cellular growth and fibroblast proliferative capacity. Integrins damage was studied by isolation by column immunoaffinity chromatography migrated on SDS-Page under reducing and non reducing conditions, and inhibition of integrins extracellular matrix adhesion by monoclonal antibodies effect. The dose/rate (0.05 Gy/day-0.2 Gy/day) of gamma given to cells did not show damage evidence on glycoproteins and integrins. If damage happened, it was repaired by cells very soon, was delayed by continuous cellular division or by glycoproteins characteristic of being multiple extracellular ligatures. Bio effects became more evident with an irradiation time greater than 20 days or a high dose/rate. (authors). 6 refs

  7. Development of oligoclonal nanobodies for targeting the tumor-associated glycoprotein 72 antigen

    Sharifzadeh, Zahra; Rahbarizadeh, Fatemeh; Shokrgozar, Mohammad Ali;

    2013-01-01

    The tumor-associated glycoprotein 72 (TAG-72) is a membrane mucin whose over-expression is correlated with advanced tumor stage and increased invasion and metastasis. In this study, we identified a panel of four nanobodies, single variable domains of dromedary heavy-chain antibodies that specific...

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

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

    2012-06-01

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

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

    Kopp, A; Mettenleiter, T C

    1992-05-01

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

  10. Feasibility and challenges in the development of immunocontraceptive vaccine based on zona pellucida glycoproteins.

    Choudhury, Sangeeta; Srivastava, Neelu; Narwal, P S; Rath, Archana; Jaiswal, Sonika; Gupta, Satish K

    2007-01-01

    The zona pellucida (ZP) glycoproteins play a crucial role during fertilization and thus are considered as important target antigens for the development of immunocontraceptive vaccines aiming to inhibit fertility at a pre-fertilization stage. In order to evaluate the immunocontraceptive potential of ZP glycoproteins, bonnet monkey (Macaca radiata) ZP2, ZP3 and ZP4 have been cloned and expressed using either E. coli or baculovirus expression systems. Active immunization studies with the recombinant ZP glycoproteins in female baboons (Papio anubis) and bonnet monkeys revealed curtailment of fertility. In order to minimize the ovarian pathology, synthetic peptides corresponding to B cell epitopes that are devoid of 'oophoritogenic' T cell epitopes were designed and their in vitro immunocontraceptive potential explored. There are several issues that need to be addressed before ZP glycoproteins based immunocontraceptive vaccines become feasible for use in humans. Nonetheless, the utility of such a vaccine is imminent for controlling wild life population. In this direction, active immunization of female non-descript dogs with recombinant canine ZP3 conjugated to diphtheria toxoid led to curtailment of fertility. Further, canine ZP3 has also been expressed in insect cells as a fusion protein with rabies virus glycoprotein G (RV-G), an antigen that is involved in providing protection against rabies. The immunogenicity of such a recombinant protein and its potential to curtail fertility was explored both in female mice and dogs. Simultaneously, DNA vaccine encoding canine ZP3 and RV-G have been made and evaluated for their immunogenicity. The results obtained so far, current shortcomings and the possible ways to circumvent these have been discussed in the present manuscript. PMID:17566293

  11. Baculovirus Coinfection Strategy for Improved Galactosylation of Recombinant Glycoprotein Produced by Insect Cell Culture

    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.

  12. Cloning and Sequence Analysis of Envelope Glycoprotein E1 Gene of Rubella Virus, JR23 Strain

    王志玉; 薛永磊; 王小凡; 宋艳艳; 温红玲

    2003-01-01

    To construct an expression vector containing the E1 glycoprotein gene of rubella virus for the study on the effectof mutation of the E1 gene glycoprotein and the analysis of phylogenetic differences of sequences, the gene encoding the E1envelope glycoprotein was amplified from rubella virus, Jinan strain JR23, by RT-PCR and ligated into PMD-18T vector.The clones that carried the E1 gene were identified after ampr selection and analysis of restriction enzyme digestion. After sequencing this gene was analyzed by Danstar and Winstar programs, and the map of phylogenetic tree was drawn. The clone of E1 glycoprotein was thus constructed. It was found that the sequence differences between JR23 strain and the TCRB strainfrom Japan and those between JR23 strain and Thomas strain of England were rather small with difference values of 0.9% and 1.2% respectively. Yet those between JR23 strain and BRD2 strain from Beijing and those between JR23 strain and XG379 strain from Hong Kong were comparatively larger with difference values of 7.6% and 7.3% respectively. The sequence of JR23 strain with other strains was less than 3% except the NC strain (3.7%). It concludes that the constructionof E1 glycoprotein gene offers an approach to study the relationship between structures and functions of E1 gene and its gene products. In the phylogenetic tree, it shows that there are significant differences in the sequences of rubella virus isolated in China, and this might be helpful to develop an effective subunit vaccine.

  13. Analysis of the apparent biphasic axonal transport kinetics of fucosylated glycoproteins

    Following intraocular injection of [3H]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 [35S]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 [3H]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

  14. Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein

    Lee, Jeffrey E.; Fusco, Marnie L.; Abelson, Dafna M.; Hessell, Ann J.; Burton, Dennis R. [Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Saphire, Erica Ollmann, E-mail: erica@scripps.edu [Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)

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

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

    2014-01-01

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

  16. Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

    McShane, Marisa P.; Longnecker, Richard

    2004-01-01

    Epstein–Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lin...

  17. Nucleotide sequence of a cDNA clone carrying the glycoprotein gene of infectious hematopoietic necrosis virus, a fish rhabdovirus.

    Koener, J F; Passavant, C W; Kurath, G; Leong, J

    1987-01-01

    The nucleotide sequence of the mRNA encoding the glycoprotein of infectious hematopoietic necrosis virus was determined from a cDNA clone containing the entire coding region. The G-protein cDNA is 1,609 nucleotides long (excluding the polyadenylic acid) and encodes a protein of 508 amino acids. The predicted amino acid sequence was compared with that of the glycoprotein of the Indiana and New Jersey serotypes of vesicular stomatitis virus and with the glycoprotein of rabies virus, using a com...

  18. A Novel Method for Detection of Glycoproteins on Sodium Dodecyl Sulphate Polyacrylamide Gel Using Radio-Iodinated Tyrosine

    Nalla, Amarnadh; Draz, Hossam M.; Dole, Anita;

    2009-01-01

    The aim of this study is to develop a novel method for detection of glycoproteins on polyacrylamide gel. In this method, radio-iodinated-tyrosine (125I-tyrosine) was conjugated to glycoprotein by schiff's base mechanism on the sodium dodecyl sulfate- polyacrylamide gel. Ovalbumin and Concanavalin A...... agent like Sodium Metabisulfite. The radio-iodinated glycoprotein on gel was scanned using a Multi-Photon Detection (MPD) scanner. The elechtrophoretic analysis of ovalbumin and Con A were performed and stained with Coomassie brilliant blue to identify total proteins, while MPD detection of...

  19. Role of p-glycoprotein expression in predicting response to neoadjuvant chemotherapy in breast cancer-a prospective clinical study

    Bhatia Ashima

    2005-09-01

    Full Text Available Abstract Background Neoadjuvant chemotherapy (NACT is an integral part of multi-modality approach in the management of locally advanced breast cancer. It is vital to predict response to chemotherapy in order to tailor the regime for a particular patient. The prediction would help in avoiding the toxicity induced by an ineffective chemotherapeutic regime in a non-responder and would also help in the planning of an alternate regime. Development of resistance to chemotherapeutic agents is a major problem and one of the mechanisms considered responsible is the expression of 170-k Da membrane glycoprotein (usually referred to as p-170 or p-glycoprotein, which is encoded by multidrug resistance (MDR1 gene. This glycoprotein acts as an energy dependent pump, which actively extrudes certain families of chemotherapeutic agents from the cells. The expression of p-glycoprotein at initial presentation has been found to be associated with refractoriness to chemotherapy and a poor outcome. Against this background a prospective study was conducted using C219 mouse monoclonal antibody specific for p-glycoprotein to ascertain whether pretreatment detection of p-glycoprotein expression could be utilized as a reliable predictor of response to neoadjuvant chemotherapy in patients with breast cancer. Patients and methods Fifty cases of locally advanced breast cancer were subjected to trucut® biopsy and the tissue samples were evaluated immunohistochemically for p-glycoprotein expression and ER, PR status. The response to neoadjuvant chemotherapy was assessed clinically and by using ultrasound after three cycles of FAC regime (cyclophosphamide 600 mg/m2, Adriamycin 50 mg/m2, 5-fluorourail 600 mg/m2 at an interval of three weeks. The clinical response was correlated with both the pre and post chemotherapy p-glycoprotein expression. Descriptive studies were performed with SPSS version 10. The significance of correlation between tumor response and p-glycoprotein

  20. Avian serum α1-glycoprotein, hemopexin, differing significantly in both amino acid and carbohydrate composition from mammalian (β-glycoprotein) counter parts

    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 α1-glycoprotein instead of a β1-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 125I concanavalin A and 125I wheat germ agglutinin binding suggest that chicken hemopexin has a mixture of three fucose-free N-linked bi- and triantennary oligosaccharides. In contrast, human hemopexin has give N-linked oligosaccharides and an additional O-linked glycan blocking the N-terminal threonine residue, while the rabbit form has four N-linked oligosaccharides. In keeping with the finding of a simpler carbohydrate structure, the avian hemopexin shows only a single band on polyacrylamide gel electrophoresis under both nondenaturing and denaturing conditions, whereas the hemopexins of the three mammalian species tested show several bands. In contrast, the isoelectric focusing pattern of chicken hemopexin is very complex, revealing at least nine bands between pH 4.0 and pH band 5.0, while the other hemopexins show a broad smear of multiple ill-defined bands in the same region.Results indicate the hemopexin of avians differs substantially from the hemopexins of mammals, which show a notable similarity with regard to carbohydrate structure and amino acid composition

  1. Avian serum. cap alpha. /sub 1/-glycoprotein, hemopexin, differing significantly in both amino acid and carbohydrate composition from mammalian (. beta. -glycoprotein) counter parts

    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.

  2. Predicting P-Glycoprotein-Mediated Drug Transport Based On Support Vector Machine and Three-Dimensional Crystal Structure of P-glycoprotein

    Bikadi, Zsolt; Hazai, Istvan; Malik, David; Jemnitz, Katalin; Veres, Zsuzsa; Hari, Peter; Ni, Zhanglin; Loo, Tip W.; Clarke, David M.; Hazai, Eszter; Mao, Qingcheng

    2011-01-01

    Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics,...

  3. Complementation of the Function of Glycoprotein H of Human Herpesvirus 6 Variant A by Glycoprotein H of Variant B in the Virus Life Cycle

    Oyaizu, Hiroko; Tang, Huamin; Ota, Megumi; Takenaka, Nobuyuki; Ozono, Keiichi; Yamanishi, Koichi; Mori, Yasuko

    2012-01-01

    Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6 variant A (HHV-6A) and HHV-6B, based on genetic, antigenic, and cell tropisms, although the homology of their entire genomic sequences is nearly 90%. The HHV-6A glycoprotein complex gH/gL/gQ1/gQ2 is a viral ligand that binds to the cellular receptor human CD46. Because gH has 94.3% amino acid identity between the variants, here we examined whether gH from one variant could compleme...

  4. Sestamibi is a substrate for MDR1 and MDR2 P-glycoprotein genes

    Joseph, Brigid; Malhi, Harmeet; Gupta, Sanjeev [Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann 625, 1300 Morris Park Avenue, NY 10461, Bronx (United States); Bhargava, Kuldeep K.; Palestro, Christopher J. [Division of Nuclear Medicine, Long Island Jewish Medical Center, New York (United States); Schilsky, Michael L. [Division of Liver Diseases, Mount Sinai School of Medicine, New York (United States); Jain, Diwakar [Division of Nuclear Cardiology, MCP-Hahnemann University School of Medicine, Philadephia (United States)

    2003-07-01

    Technetium-99m sestamibi has attracted interest for assessment of the function of P-glycoproteins, which are well expressed in the liver and have roles in biliary transport and the removal of chemotherapeutic drugs. To further examine the cross-reactivity of {sup 99m}Tc-sestamibi for P-glycoprotein family members, we conducted studies in animals. Hepatobiliary secretion of {sup 99m}Tc-sestamibi was determined in normal FVB/N mice, mutant mice with specific P-glycoprotein deficiencies in the FVB/N background, normal Long-Evans Agouti (LEA) rats, and Long-Evans Cinnamon (LEC) rats with abnormal copper transport and liver disease but intact P-glycoprotein expression. After intrasplenic injection, {sup 99m}Tc-sestamibi was rapidly incorporated in the mouse and rat liver, with maximal accumulation after 102{+-}31 and 109{+-}16 s, respectively (P=NS). In normal mice and rats, 55%{+-}11% and 55%{+-}6%, respectively, of the maximal sestamibi activity was retained in the liver after 1 h (P=NS). In double knockout mice lacking both mdr1a and mdr1b homologs of the human MDR1 (ABCB1) gene, 88%{+-}11% of maximal sestamibi activity was retained in the liver after 1 h (P<0.001). In knockout mice deficient in either mdr1a gene or mdr2 (ABCB4) gene, biliary sestamibi excretion was also impaired, although this impairment was relatively less pronounced in ABCB4-deficient mice than in double knockout mice lacking both ABCB1 gene homologs (P<0.03). Hepatobiliary sestamibi excretion in LEC rats was not different from that in control normal rats, despite the presence of significant liver disease in the former. Hepatobiliary sestamibi excretion requires P-glycoproteins and is unperturbed in chronic liver disease. Sestamibi appears to be a substrate for both ABCB1 and ABCB4 genes, although the former utilizes it far more efficiently. Assessment of P-glycoprotein activity with sestamibi should consider how regulation of ABCB1 and related family members might modulate sestamibi incorporation

  5. Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells

    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, 125I-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

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

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

    1983-01-01

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

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

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

    1983-03-01

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

  8. Use of P-glycoprotein gene probes to investigate anthelmintic resistance in Haemonchus contortus and comparison with Onchocerca volvulus

    Kwa, M.S.G.; Okoli, M.N.; Schulz-Key, H.; Okongkwo, P.O.; Roos, M.H.

    1998-01-01

    A P-glycoprotein gene probe from the sheep parasitic nematode Haemonchus contortus was developed and used to analyse restriction fragment length polymorphisms between susceptible isolates and isolates resistant to either benzimidazole; levamisole and benzimidazole; or benzimidazole, ivermectin and c

  9. Brain Barriers and a Subpopulation of Astroglial Progenitors of Developing Human Forebrain Are Immunostained for the Glycoprotein YKL-40

    Bjørnbak, Camilla; Brøchner, Christian B; Larsen, Lars A;

    2014-01-01

    YKL-40, a glycoprotein involved in cell differentiation, has been associated with neurodevelopmental disorders, angiogenesis, neuroinflammation and glioblastomas. We evaluated YKL-40 protein distribution in the early human forebrain using double-labeling immunofluorescence and immunohistochemistr...

  10. Autoradiographic detection of RNA and glycoprotein synthesis in epithelial cells of the oviduct in ovulated heifers

    Heifers (n=9) of the Black-Pied Holstein-Friesian breed were slaughtered on the 3rd, 6th and 9th day of the synchronized cycle, respectively (heat=day 0). Tissue samples from the ampullar part of the oviduct were collected immediately after slaughter and treated for histoautoradiographic (ARG) analyses. Investigated autoradiograms revealed an intensive synthesis of RNA in both cilliated as well as in secretory cells of the oviduct epithelium. The intensity of RNA synthesis remains comparable during the early luteal phase from the 3rd to the 9th day of the cycle. The glycoproteins, synthesized first in the Golgi apparatus in the supranuclear region, were seen to move to the apical region of the epithelial cells later on. The most intensive autoradiographic reaction of newly synthesized glycoproteins was detected on the 3rd day of the cycle without an evident change on the 6th day but decreasing on the 9th day

  11. Structural analysis of N- and O-glycans released from glycoproteins

    Jensen, Pia Hønnerup; Karlsson, Niclas G; Kolarich, Daniel; Packer, Nicolle H

    2012-01-01

    4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.......This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically...... released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography...

  12. Biochemical and immunologic heterogeneity of Ia glycoproteins isolated from a chronic lymphocytic leukemia

    Ia glycoproteins have been isolated from human chronic lymphocytic leukemic cells (CLL) by Lens culinaris chromatography and by filtration on ACA-34 Ultrogel. Ia antigenic activity, measured by inhibition of the cellular radioimmunoassay, was separated by gel filtration into 2 fractions, peak I and II. Monoclonal antibodies, produced against peak II glycoproteins, appear to recognize different antigenic determinants of Ia molecules. Monoclonal antibody 18a4 reacted with Ia molecules of peaks I and II, whereas monoclonal antibodies 18c2 and 18d5 reacted almost exclusively with peak II molecules both in the cellular radioimmunoassay and by immunoprecipitation. In addition to antigenic differences, minor variations in the apparent m.w. of the Ia polypeptide chains were observed between peaks I and II. These results indicate the existence of antigenically distinct subsets of Ia molecules that are separated by gel filtration

  13. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology.

    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

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

    Kellich, G; Ziegler, D

    1975-04-01

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

  15. Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Critical for Ebola Virus Replication in Nonhuman Primates▿

    Neumann, Gabriele; Geisbert, Thomas W.; Ebihara, Hideki; Geisbert, Joan B.; Daddario-DiCaprio, Kathleen M.; Feldmann, Heinz; Kawaoka, Yoshihiro

    2007-01-01

    Enveloped viruses often require cleavage of a surface glycoprotein by a cellular endoprotease such as furin for infectivity and virulence. Previously, we showed that Ebola virus glycoprotein does not require the furin cleavage motif for virus replication in cell culture. Here, we show that there are no appreciable differences in disease progression, hematology, serum biochemistry, virus titers, or lethality in nonhuman primates infected with an Ebola virus lacking the furin recognition sequen...

  16. Persistence of HIV-1 structural proteins and glycoproteins in lymph nodes of patients under highly active antiretroviral therapy

    Popovic, Mikulas; Tenner-Racz, Klara; Pelser, Colleen; Stellbrink, Hans-Jurgen; van Lunzen, Jan; Lewis, George; Kalyanaraman, Vaniambadi S.; Gallo, Robert C.; Racz, Paul

    2005-01-01

    Here we report a long-term persistence of HIV-1 structural proteins and glycoproteins in germinal centers (GCs) of lymph nodes (LNs) in the absence of detectable virus replication in patients under highly active antiretroviral therapy (HAART). The persistence of viral structural proteins and glycoproteins in GCs was accompanied by specific antibody responses to HIV-1. Seven patients during the chronic phase of HIV-1 infection were analyzed for the presence of the capsid protein (HIV-1p24), ma...

  17. Identification and Confirmation of biomarkers using an integrated platform for quantitative analysis of glycoproteins and their glycosylations

    Liu, Yashu; He, Jintang; Li, Chen; Benitez, Ricardo; Fu, Sherry; Marrero, Jorge; Lubman, David M

    2010-01-01

    Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. However, accurate diagnosis can be difficult as most of the patients who develop this tumor have symptoms similar to those caused by longstanding liver disease. Herein we developed an integrated platform to discover the glycoprotein biomarkers in early HCC. At first, lectin arrays were applied to investigate the differences in glycan structures on serum glycoproteins from HCC and cirrhosis patients. The in...

  18. Comparative studies on the analysis of glycoproteins and lipopolysaccharides by the gel-based microchip and SDS-PAGE

    Hsieh, Jung-Feng; Chen, Shui-Tein

    2006-01-01

    In order to determine time efficiency between the gel-based microchip (LabChip) and traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), glycoproteins and lipopolysaccharides were analyzed in this study. After 90 min of gel electrophoresis, glycoproteins (bovine serum albumin, lysozyme, ovalbumin, and apo-transferrin) and fluorescent lipopolysaccharides (LPS-O and LPS-S) under reducing conditions could be analyzed by SDS-PAGE, and it would take (including imaging ...

  19. Kinetic Validation of the Models for P-Glycoprotein ATP Hydrolysis and Vanadate-Induced Trapping. Proposal for Additional Steps

    Lugo, Miguel Ramón; Sharom, Frances Jane

    2014-01-01

    P-Glycoprotein, a member of the ATP-binding cassette (ABC) superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich d...

  20. Fucose and Sialic Acid Expressions in Human Seminal Fibronectin and α1-Acid Glycoprotein Associated with Leukocytospermia of Infertile Men

    Kratz, Ewa M.; Ricardo Faundez; Iwona Kątnik-Prastowska

    2011-01-01

    Introduction: The aim of this study was to compare fucose and sialic acid residue expression on fibronectin and α 1-acid glycoprotein in the seminal plasma of men suspected of infertility and suffering from leukocytospermia. Subjects and methods: Seminal ejaculates were collected from 27 leukocytospermic and 18 healthy, normozoospermic men. The relative degree of fucosylation and sialylation of fibronectin and α 1-acid glycoprotein was estimated by ELISA using fucose and sialic acid specific ...

  1. Significance of Serum L-fucose Glycoprotein as Cancer Biomarker in Head and Neck Malignancies without Distant Metastasis

    Shetty, Rathan K.S.; Bhandary, Satheesh Kumar; Kali, Arunava

    2013-01-01

    Background: Head and neck neoplasia is a major form of cancer in India, accounting for 30% of all cancers which occur in males and 11% of cancers which occur in females. Elevated serum L-fucose glycoprotein levels have been reported to be associated with neoplastic conditions involving various sites. Therefore, monitoring serum/tissue L-fucose glycoprotein levels could be a promising approach for the early diagnosis and prognosis of head neck cancers.

  2. Immunization with Cytomegalovirus Envelope Glycoprotein M and Glycoprotein N DNA Vaccines can Provide Mice with Complete Protection against a Lethal Murine Cytomegalovirus Challenge

    Huadong Wang; Yanfeng Yao; Chaoyang Huang; Quanjiao Chen; Jianjun Chen; Ze Chen

    2013-01-01

    Human cytomegalovirus virions contain three major glycoprotein complexes (gC Ⅰ,Ⅱ,Ⅲ),all of which are required for CMV infectivity.These complexes also represent major antigenic targets for anti-viral immune responses.The gC Ⅱ complex consists of two glycoproteins,gM and gN.In the current study,DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model.Humoral and cellular immune responses,spleen viral titers,and mice survival and body-weight changes were examined.The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection,whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response,and provided mice with complete protection against a lethal MCMV challenge.This study provides the first in vivo evidence that the gC Ⅱ (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.

  3. Targeting HIV-1 Envelope Glycoprotein Trimers to B Cells by Using APRIL Improves Antibody Responses

    Melchers M; Bontjer I; Tong T; Chung NP; Klasse PJ; Eggink D; Montefiori DC; Gentile M; Cerutti A; Olson WC; Berkhout B; Binley JM; Moore JP; Sanders RW

    2012-01-01

    An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The...

  4. Mucus glycoprotein biosynthesis in the human gall bladder: inhibition by aspirin.

    Rhodes, M; A. Allen; Lennard, T W

    1992-01-01

    Aspirin, which inhibits mucin secretion in the gastrointestinal tract prevents gall stone formation in animals and may reduce gall stone recurrence in man. This study examines the effect of aspirin on mucin synthesis in human gall bladder explants. Two hundred explants were cultured with 3H-glucosamine (74 kBq/ml) for 24 hours at 37 degrees C. Mucin and other glycoproteins were isolated by papain digestion (72 hours) and exhaustive dialysis (144 hours) to remove non-incorporated radioactivity...

  5. Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells

    Braakman, L.J.; Tatu, U.; Helenius, A

    1993-01-01

    Using influenza hemagglutinin (HA0) and vesicular stomatitis virus G protein as model proteins, we have analyzed the effects of dithiothreitol (DTT) on conformational maturation and transport of glycoproteins in the secretory pathway of living cells. While DTT caused reduction of folding intermediates and misfolded proteins in the endoplasmic reticulum (ER), it did not affect molecules that had already acquired a mature trimeric conformation, whether present in the ER or elsewhere. The conver...

  6. In vivo P-glycoprotein function before and after epilepsy surgery

    Bauer, Martin; Karch, Rudolf; Zeitlinger, Markus; Liu, Joan; Koepp, Matthias J.; Asselin, Marie-Claude; Sisodiya, Sanjay M; Hainfellner, Johannes A; Wadsak, Wolfgang; Mitterhauser, Markus; Müller, Markus; Pataraia, Ekaterina; Langer, Oliver

    2014-01-01

    Objectives: To study the functional activity of the multidrug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier of patients with temporal lobe epilepsy using (R)-[11C]verapamil (VPM)-PET before and after temporal lobe surgery to assess whether postoperative changes in seizure frequency and antiepileptic drug load are associated with changes in Pgp function. Methods: Seven patients with drug-resistant temporal lobe epilepsy underwent VPM-PET scans pre- and postsurgery. Patient...

  7. Structure and evolution of the mouse pregnancy-specific glycoprotein (Psg) gene locus

    Okumura Katsuzumi; Ball Melanie; Wynne Freda; Hori Tomomi; Dveksler Gabriela; Fischer Beate; McLellan Andrew S; Moore Tom; Zimmermann Wolfgang

    2005-01-01

    Abstract Background The pregnancy-specific glycoprotein (Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls (even-toed ungulates / hoofed mammals), there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to d...

  8. Pregnancy-specific Glycoprotein 1 Induces Endothelial Tubulogenesis through Interaction with Cell Surface Proteoglycans*

    Lisboa, Felipe A.; Warren, James; Sulkowski, Gisela; Aparicio, Marta; David, Guido; Zudaire, Enrique; Dveksler, Gabriela S.

    2010-01-01

    Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their...

  9. Anti-β2 Glycoprotein-I Antibody in Acute Myocardial Infarction

    Mohammad Shojaei; Abdolreza S. Jahromi; Hamideh Ebadat; Seyed H. Moosavy; Bita Seddigh; Abdolhossien Madani

    2011-01-01

    Problem statement: Ischemic cardiac manifestations have been reported in a various percentage of patients with anti-phospholipid antibodies. Data concerning the relation between anti- Phospholipid (aPL) antibodies and myocardial infarction in subjects without evidence of overt autoimmune disease are conflicting. Anti-beta2 glycoprotein-I (anti-beta2-GPI) antibody is detected in various diseases like rheumatoid arthritis, systemic lupus erythematosus and anti-phospholipid antibody syndrome. Th...

  10. Proteolysis of the Ebola Virus Glycoproteins Enhances Virus Binding and Infectivity▿

    Kaletsky, Rachel L.; Simmons, Graham; Bates, Paul

    2007-01-01

    Cellular cathepsins are required for Ebola virus infection and are believed to proteolytically process the Ebola virus glycoprotein (GP) during entry. However, the significance of cathepsin cleavage during infection remains unclear. Here we demonstrate a role for cathepsin L (CatL) cleavage of Ebola virus GP in the generation of a stable 18-kDa GP1 viral intermediate that exhibits increased binding to and infectivity for susceptible cell targets. Cell binding to a lymphocyte line was increase...

  11. Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria.

    Karav, S; Le Parc, A; Leite Nobrega de Moura Bell, JM; Frese, SA; Kirmiz, N; Block, DE; Barile, D.; Mills, DA

    2016-01-01

    Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobact...

  12. Mutational changes in the vesicular stomatitis virus glycoprotein affect the requirement of carbohydrate in morphogenesis.

    Chatis, P A; Morrison, T G

    1981-01-01

    The role of carbohydrate in the morphogenesis of vesicular stomatitis virus was studied, using the antibiotic tunicamycin to inhibit glycosylation. It has been reported previously (Gibson et al., J. Biol. Chem. 254:3600-3607, 1979) that the San Juan strain of vesicular stomatitis virus requires carbohydrate for efficient migration of the glycoprotein (G) to the cell surface and for virion formation, whereas the prototype or Orsay strain of vesicular stomatitis virus is less stringent in its c...

  13. Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta

    Bériot, Mathilde; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia; Beckers, Jean-François; Melo de Sousa, Noelita

    2014-01-01

    Background: This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDSPAGE and N-terminal microsequencing. Result...

  14. Effect of carbenoxolone on the synthesis of glycoproteins and DNA in rat gastric epithelial cells.

    van Huis, G A; Kramer, M.F.

    1981-01-01

    The influence of carbenoxolone on the synthesis of glycoproteins in the surface mucous cells and the production of new cells in the rat gastric mucosa was studied by means of a vascular perfusion system. The rate of incorporation of tritiated galactose, glucosamine, serine, and sulphate in surface mucous cells, studied by autoradiography, was not affected by the addition of carbenoxolone to the drinking water. The sugar composition (determined by gas-liquid chromatography) of the gastric glyc...

  15. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei.

    Zomerdijk, J C; Ouellette, M; ten Asbroek, A L; Kieft, R.; Bommer, A M; Clayton, C E; Borst, P

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site specific transcripts. The start of transcription was determined by hybridization and RNase protection analysis of nascent RNA. The 5' ends of the major transcripts coming from the initiation region m...

  16. The N-glycan Glycoprotein Deglycosylation Complex (Gpd) from Capnocytophaga canimorsus Deglycosylates Human IgG

    Renzi, Francesco; Manfredi, Pablo; Mally, Manuela; Moes, Suzanne; Jenö, Paul; Cornelis, Guy R

    2011-01-01

    Author Summary Capnocytophaga canimorsus are Gram-negative bacteria from the normal oral flora of dogs and cats. They cause rare but severe infections in humans that have been bitten or simply licked by a dog or cat. Fulminant septicemia and peripheral gangrene with a high mortality are the most common symptoms. A surprising feature of these bacteria is their capacity to feed by foraging the glycan moieties of glycoproteins from animal cells, including phagocytes. Here we show that C. canimor...

  17. Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF.

    Guillet Catherine; d'Hondt Véronique; Feyens Anne-Marie; Fages Christiane; Monville Christelle; Vernallis Ann; Gascan Hugues; Peschanski Marc

    2002-01-01

    Abstract Background The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures. Results Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were I...

  18. P-Glycoprotein Induction Ameliorates Colistin Induced Nephrotoxicity in Cultured Human Proximal Tubular Cells

    Lee, Sun-hyo; Kim, Jin-Sun; Ravichandran, Kameswaran; Gil, Hyo-Wook; SONG, HO-YEON; Hong, Sae-Yong

    2015-01-01

    The pathogenesis of colistin induced nephrotoxicity is poorly understood. Currently there are no effective therapeutic or prophylactic agents available. This study was aimed to determine the mechanism of colistin induced nephrotoxicity and to determine whether P-glycoprotein (P-gp) induction could prevent colistin induced nephrotoxicity. Colistin induced cell toxicity in cultured human proximal tubular cells in both dose and time dependent manner. Colistin provoked ROS in a dose dependent man...

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

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

    2016-04-01

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

  20. A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo

    Massberg, Steffen; Gawaz, Meinrad; Grüner, Sabine; Schulte, Valerie; Konrad, Ildiko; Zohlnhöfer, Dietlind; Heinzmann, Ulrich; Nieswandt, Bernhard

    2003-01-01

    Platelet adhesion and aggregation at sites of vascular injury is crucial for hemostasis but may lead to arterial occlusion in the setting of atherosclerosis and precipitate diseases such as myocardial infarction. A current hypothesis suggests that platelet glycoprotein (GP) Ib interaction with von Willebrand factor recruits flowing platelets to the injured vessel wall, where subendothelial fibrillar collagens support their firm adhesion and activation. However, so far this hypothesis has not ...

  1. Structural Modeling and Analysis of Pregnancy-Associated Glycoprotein-1 of Buffalo (Bubalus bubalis)

    Jerome Andonissamy; Singh, S. K.; Agarwal, S. K.

    2012-01-01

    The present study was conducted to design and analyze the structural model of buffalo pregnancy-associated glycoprotein-1 (PAG-1) using bioinformatics. Structural modeling of the deduced buffalo PAG-1 protein was done using PHYRE, CONSURF servers and its structure was subsequently constructed using MODELLER 9.9 and PyMOL softwares Buffalo PAG-1 structural conformity was analyzed using PROSA, WHATIF, and 3D-PSSM servers. Designed buffalo PAG-1 protein structure on BLAST analysis retrieved prot...

  2. Characterization and In Silico Analysis of Pregnancy-Associated Glycoprotein-1 Gene of Buffalo (Bubalus bubalis)

    Jerome A.; Singh, S. K.; Agarwal, S. K.; Mohini Saini; Ashwin Raut

    2011-01-01

    Pregnancy-Associated Glycoproteins (PAGs) are trophoblastic proteins belonging to the Aspartic proteinase family secreted by different placental cells of many mammalian species. They play a pivotal role in placentogenesis, foetomaternal unit remodeling, and implantation. The identification of the genes encoding those proteins will be helpful to unravel the intricate embryogenomic functions during pregnancy establishment. Considering importance of these proteins, the present study was undertak...

  3. Capsosiphon fulvescens glycoprotein inhibits AGS gastric cancer cell proliferation by downregulating Wnt-1 signaling

    Kim, Young-Min; KIM, IN-HYE; NAM, TAEK-JEONG

    2013-01-01

    Previously, we examined various apoptosis pathways in the AGS gastric cancer cell line using Capsosiphon fulvescens glycoprotein (Cf-GP). In this study, we focused on the downregulation of the Wnt-1 signaling pathway and cell cycle arrest. Upregulation of the Wnt signaling pathway has been observed in various cancer cells. The Wnt signal ligand acts in both canonical and non-canonical pathways. Among them, Wnt-1 was dependent on the canonical pathway. Here, we show inhibition of Wnt-1 signali...

  4. Targeted Reduction in Expression of Trypanosoma cruzi Surface Glycoprotein gp90 Increases Parasite Infectivity

    Málaga, Sergio; Yoshida, Nobuko

    2001-01-01

    A previous study had shown that the expression of gp90, a stage-specific surface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes, is inversely correlated with the parasite's ability to invade mammalian cells. By using antisense oligonucleotides complementary to a region of the gp90 gene implicated in host cell adhesion, we investigated whether the selective inhibition of gp90 synthesis affected the capacity of metacyclic forms to enter target cells. Parasites were incubated for 2...

  5. Characterization of Pneumocystis Major Surface Glycoprotein Gene (msg) Promoter Activity in Saccharomyces cerevisiae

    Kutty, Geetha; Shroff, Robert; Kovacs, Joseph A.

    2013-01-01

    Major surface glycoprotein (Msg), the most abundant cell surface protein of Pneumocystis, plays an important role in the interaction of this opportunistic pathogen with host cells, and its potential for antigenic variation may facilitate evasion of host immune responses. In the present study, we have identified and characterized the promoter region of msg in 3 species of Pneumocystis: P. carinii, P. jirovecii, and P. murina. Because Pneumocystis cannot be cultured, promoter activity was measu...

  6. Glycoproteins in probiotic bacteria - Exploring the glycosylation potential of Lactobacillus rhamnosus GG by genomics and glycoproteomics

    Tytgat, Hanne

    2015-01-01

    A wide variety of glycoconjugates covers the cell wall of prokaryotes, forming a unique species-specific barcode. These glycoconjugates are important to establish specific interactions with the environment. Most studies of bacterial glycobiology focus on pathogens. Here, the importance of glycosylation in the microbiota isolate and model probiotic strain Lactobacillus rhamnosus GG is investigated, with a focus on glycoproteins. To shed light on how and why L. rhamnosus GG glycosylates part of...

  7. Synthetic glycoprotein D-related peptides protect mice against herpes simplex virus challenge.

    Eisenberg, R J; Cerini, C P; Heilman, C J; Joseph, A. D.; Dietzschold, B; Golub, E; Long, D; Ponce de Leon, M; Cohen, G H

    1985-01-01

    Glycoprotein D (gD) of herpes simplex virus (HSV) protects mice from a lethal challenge by either HSV type 1 (HSV-1; oral) or HSV-2 (genital). We evaluated whether synthetic peptides representing residues 1 through 23 of gD (mature protein) can be used as a potential synthetic herpesvirus vaccine. The immunogenicity of the peptides was demonstrated by the biological reactivity of antipeptide sera in immunoprecipitation and neutralization assays. All sera which immunoprecipitated gD had neutra...

  8. Pharmacological properties of radiotracers that measure p-glycoprotein function and density

    Kannan, Pavitra

    2012-01-01

    Energy-dependent transporters of the ATP-binding cassette (ABC) family regulate the movement of molecules across cellular membranes. Several of these transporters are expressed in the endothelial cells of brain microvessels (blood-brain barrier) to protect brain tissue from exposure to toxins in the blood. Three of the most common ABC transporters at the blood-brain barrier are P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance protein 1 (M...

  9. Imaging the Function of P-Glycoprotein With Radiotracers: Pharmacokinetics and In Vivo Applications

    Kannan, P.; John, C; Zoghbi, SS; Halldin, C.; Gottesman, MM; Innis, RB; Hall, MD

    2009-01-01

    P-glycoprotein (P-gp), an efflux transporter, controls the pharmacokinetics of various compounds under physiological conditions. P-gp-mediated drug efflux has been suggested as playing a role in various disorders, including multidrug-resistant cancer and medication-refractory epilepsy. However, P-gp inhibition has had, to date, little or no clinically significant effect in multidrug-resistant cancer. To enhance our understanding of its in vivo function under pathophysiological conditions, sub...

  10. Doxycycline Induces Expression of P Glycoprotein in MCF-7 Breast Carcinoma Cells

    Mealey, Katrina L; Barhoumi, Rola; Burghardt, Robert C.; Safe, Stephen; Kochevar, Deborah T.

    2002-01-01

    P-glycoprotein (P-gp) overexpression by tumor cells imparts resistance to multiple antineoplastic chemotherapeutic agents (multiple drug resistance). Treatment of tumor cells with chemotherapeutic agents such as anthracyclines, epipodophyllotoxins, and Vinca alkaloids results in induction of P-gp expression. This study was performed to determine if clinically relevant antimicrobial drugs (i.e., drugs that are used to treat bacterial infections in cancer patients) other than antineoplastic age...

  11. In Situ Biochemical Demonstration That P-Glycoprotein Is a Drug Efflux Pump with Broad Specificity

    Chen, Yu; Simon, Sanford M.

    2000-01-01

    While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed popul...

  12. Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells

    Saksena, Seema; Priyamvada, Shubha; Kumar, Anoop; Akhtar, Maria; Soni, Vikas; Anbazhagan, Arivarasu Natarajan; Alakkam, Anas; Alrefai, Waddah A.; Dudeja, Pradeep K.; Gill, Ravinder K.

    2013-01-01

    Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether K...

  13. Human myeloid plasma membrane glycoprotein CD13 (gp150) is identical to aminopeptidase N.

    Look, A T; Ashmun, R A; Shapiro, L H; Peiper, S C

    1989-01-01

    To determine the primary structure of CD13, a 150-kD cell surface glycoprotein originally identified on subsets of normal and malignant human myeloid cells, we isolated the complete sequences encoding the polypeptide in overlapping complementary DNA (cDNA) clones. The authenticity of our cDNA clones was demonstrated by the ability of the coding sequences, subcloned in a retroviral expression vector, to mediate expression of bona fide CD13 molecules at the surface of transfected mouse fibrobla...

  14. An electrochemiluminescence assay for analysis of rabies virus glycoprotein content in rabies vaccines

    Smith, Todd G.; Ellison, James A.; Ma, Xiaoyue; Kuzmina, Natalia; William C. Carson; Rupprecht, Charles E.

    2013-01-01

    Vaccine potency testing is necessary to evaluate the immunogenicity of inactivated rabies virus (RABV) vaccine preparations before human or veterinary application. Currently, the NIH test is recommended by the WHO expert committee to evaluate RABV vaccine potency. However, numerous disadvantages are inherent concerning cost, number of animals and biosafety requirements. As such, several in vitro methods have been proposed for the evaluation of vaccines based on RABV glycoprotein (G) quality a...

  15. HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein

    Cara Andrea; Andreotti Mauro; Galluzzo Clementina; Verdoliva Antonio; Costi Roberta; Molinari Agnese; Dupuis Maria; Cianfriglia Maurizio; Di Santo Roberto; Palmisano Lucia

    2007-01-01

    Abstract Background The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To ad...

  16. The Pregnancy-Associated Glycoproteins: Biochemical Aspects and Clinical Application for Pregnancy Follow-up

    Melo De Sousa, Noelita; O. Barbato; Bella, Amina; Alvarez Oxiley, Andrea Vivian; Sulon, J.; Debenedetti, A.; Beckers, Jean-François

    2007-01-01

    Pregnancy diagnosis is an important part in reproduction management of ruminants. In the last years, a large polymorphic family of placenta-expressed proteins has been discovered in ruminant species and used for pregnancy diagnosis. Members of this family are named pregnancy-associated glycoproteins (PAG), being synthesized in the mono- and binucleate cells of the ruminant’s trophectoderm. Part of them are released in the maternal blood circulation where they can be assayed by different labor...

  17. Conformational epitopes of myelin oligodendrocyte glycoprotein are targets of potentially pathogenic antibody responses in multiple sclerosis

    Menge Til; Lalive Patrice H; von Büdingen H -Christian; Genain Claude P

    2011-01-01

    Abstract Background Myelin/oligodendrocyte glycoprotein (MOG) is a putative autoantigen in multiple sclerosis (MS). Establishing the pathological relevance and validity of anti-MOG antibodies as biomarkers has yielded conflicting reports mainly due to different MOG isoforms used in different studies. Because epitope specificity may be a key factor determining anti-MOG reactivity we aimed at identifying a priori immunodominant MOG epitopes by monoclonal antibodies (mAbs) and at assessing clini...

  18. Features of glycoproteins distribution in the pancreatic structures of newborn rats after prenatal antigenic influence

    Grinivetska N.V.

    2014-03-01

    Full Text Available Background. Abnormalities of digestion and absorption are the most common syndromes associated with the digestive system diseases in children. Pancreatic enzyme failure leads to violation of different metabolic processes, especially in neonates. Exocrine pancreas is sensitive to a variety of factors, including virus. It emphasizes the importance of investigation of pancreatic secretory activity in children who was born from mothers exposed to viral infection during pregnancy. Objective. The purpose is to determine the features of glycoproteins distribution in the pancreatic structures of newborn rats after prenatal antigenic influence. Methods. Animals were divided into four groups: the 1st – intact, the 2nd – intrafetal injection of the antigen, the 3rd – animals administered with antigen into the amniotic fluid, and the 4th group – control (intrafetal injection of a normal salt solution. As antigen we used Vaxigrip vaccine. Results. It was revealed that on the14th day after birth antigen-exposed animals were characterized by the increase of glycoproteins (++ in a connective tissue capsule of pancreas and decreased content of glycoproteins in the cytoplasm of acinar cells and ducts comparing with the intact group (++/+. Taken together this data evidence the reduction of the synthetic activity of acinar cells after intrafetal administration of the antigen. This may cause a predisposition for the development of dyspepsia and food allergy. Further work is planned to investigate the dynamics of glycosaminoglycans redistribution in different parts of pancreas after prenatal antigenic administrations. Citation: Grinivetska NV. [Features of glycoproteins distribution in the pancreatic structures of newborn rats after prenatal antigenic influence]. Morphologia. 2014;8(1:30-4. Ukrainian.

  19. Macroporous cryogels with immobilized enzymes and affinity ligands for glycoprotein analysis

    Křenková, Jana; Foret, František

    Universidad de La Laguna, 2013. s. 232. [International Symposium on Electro- and Liquid Phase-separation Techniques /20./. 06.10.2013-09.10.2013, Puerto de la Cruz, Tenerife] R&D Projects: GA ČR(CZ) GBP206/12/G014; GA ČR(CZ) GAP301/11/2055 Grant ostatní: GA AV ČR(CZ) M200311201 Institutional support: RVO:68081715 Keywords : glycoprotein * cryogel * enzymes * mass spectrometry Subject RIV: CB - Analytical Chemistry, Separation

  20. A Gene Optimization Strategy that Enhances Production of Fully Functional P-Glycoprotein in Pichia pastoris

    Bai, Jiangping; Swartz, Douglas J.; Protasevich, Irina I.; Brouillette, Christie G; Harrell, Patina M.; Hildebrandt, Ellen; Gasser, Brigitte; Mattanovich, Diethard; Ward, Andrew; Chang, Geoffrey; Urbatsch, Ina L.

    2011-01-01

    Background Structural and biochemical studies of mammalian membrane proteins remain hampered by inefficient production of pure protein. We explored codon optimization based on highly expressed Pichia pastoris genes to enhance co-translational folding and production of P-glycoprotein (Pgp), an ATP-dependent drug efflux pump involved in multidrug resistance of cancers. Methodology/Principal Findings Codon-optimized “Opti-Pgp” and wild-type Pgp, identical in primary protein sequence, were rigoro...

  1. Impact of small molecules immunosuppressants on P-glycoprotein activity and T-cell function

    Llaudó Vallmajor, Inés; Cassis, L.; Torras Ambròs, Joan; Bestard Matamoros, Oriol; Franquesa, M.; Cruzado, Josep Ma.; Cerezo, G.; Castaño Boldú, Esther; Pétriz, J; Herrero Fresneda, Immaculada; Grinyo Boira, Josep M.; Lloberas Blanch, Núria

    2012-01-01

    Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several dr ugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different T- cell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrol...

  2. Homology between the glycoproteins of vesicular stomatitis virus and rabies virus.

    Rose, J. K.; Doolittle, R. F.; Anilionis, A; Curtis, P J; Wunner, W H

    1982-01-01

    We compared the predicted amino acid sequences of the vesicular stomatitis virus and rabies virus glycoproteins by using a computer program which provides an optimal alignment and a statistical significance for the match. Highly significant homology between these two proteins was detected, including identical positioning of one glycosylation site. A significant homology between the predicted amino acid sequences of vesicular stomatitis virus and influenza virus matrix proteins was also found.

  3. Protein and Site Specificity of Fucosylation in Liver-Secreted Glycoproteins

    Pompach, Petr; Ashline, David J.; Brnáková, Z.; Benicky, J.; Sanda, M.; Goldman, R.

    2014-01-01

    Roč. 13, č. 12 (2014), s. 5561-5569. ISSN 1535-3893 R&D Projects: GA MŠk LH13051; GA ČR GAP206/12/0503 Grant ostatní: Charles Univ.(CZ) UNCE_204025/2012 Institutional support: RVO:61388971 Keywords : fucose * glycoproteins * liver * site specificity Subject RIV: CE - Biochemistry Impact factor: 4.245, year: 2014

  4. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen;

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k...

  5. Effect of P-glycoprotein modulators on the pharmacokinetics of camptothecin using microdialysis

    Tsai, T H; Lee, C. H.; Yeh, P H

    2001-01-01

    By performing microdialysis, this study investigated the pharmacokinetics of unbound camptothecin in rat blood, brain and bile in the presence of P-glycoprotein mediated transport modulators (cyclosporin A, berberine, quercetin, naringin and naringenin). Pharmacokinetic parameters of camptothecin were assessed using a non-compartmental model.Camptothecin rapidly crosses the blood-brain barrier (BBB) within 20 min after camptothecin administration. The disposition of camptothecin in rat bile a...

  6. Recombinant Glycoprotein Vaccines for Human Immunodeficiency Virus-Infected Children and Their Effects on Viral Quasispecies

    Essajee, Shaffiq M.; Yogev, Ram; Pollack, Henry; Greenhouse, Bryan; Krasinski, Keith; Borkowsky, William

    2002-01-01

    In individuals infected with human immunodeficiency virus type 1 (HIV-1), specific immunity is associated with a more diverse viral repertoire and slower disease progression. Attempts to enhance antiviral immunity with therapeutic vaccination have shown that recombinant glycoprotein (RGP) vaccines are safe, well tolerated, and immunogenic, but the effect of RGP vaccines on the viral repertoire is unknown. We evaluated diversification of the viral envelope in 12 HIV-infected children who recei...

  7. Bovine herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg

    Franceschi Valentina; Capocefalo Antonio; Cavirani Sandro; Donofrio Gaetano

    2013-01-01

    Abstract Background Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus, belonging to Rhadinovirus genus, with no clear association with disease. However, there is increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle. BoHV-4 Open Reading Frame 8 (ORF8) codifies for glycoprotein B (gB) that shows a heterodimeric structure, composed of two subunits and covalently linked by disulfide bonds and responsible for host cell adhesion through binding to hepara...

  8. Glycoprotein synthesis in the Golgi apparatus of spermatids during spermiogenesis of the rat

    During steps 1-7 of spermiogenesis the Golgi apparatus contributes to the formation of the acrosomic system which develops at the surface of the nucleus. Later, in step 8, the Golgi apparatus detaches from the acrosome and remains suspended in the elongated cytoplasm until it degenerates during step 16. Using 3H-fucose as a tracer and the radioautographic technique, we observed that the Golgi apparatus incorporates the tracer and delivers the labeled glycoproteins to the developing acrosomic system during steps 1-7 of spermiogenesis, to multivesicular bodies during steps 1-9, and to the remaining cytoplasm and plasma membrane during steps 1-15. Throughout these steps of spermiogenesis the Golgi apparatus does not show major changes in structure; it is composed of a cortex made up of connected stacks of saccules and a medulla showing a loose aggregate of vesicular profiles. Glycoprotein synthesis in this Golgi apparatus, before and after it contributes lysosomal glycoproteins to the growing acrosomic system, was quantitatively assessed in electron microscope EM radioautographs of tissue sections from animals sacrificed at 1, 4, 8, and 24 h of 3H-fucose injection. The incorporation of the labeled sugar was found to remain quantitatively similar during steps 1-15 of spermiogenesis, and therefore, no shift in glycoprotein synthesis took place following separation of the Golgi apparatus from the acrosomic system. Throughout these steps, fucose molecules are first incorporated in the cortex of the organelle and subsequently transported to the medulla, where they temporarily accumulate before being delivered, depending on the step of spermiogenesis, to the acrosomic system, to the multivesicular bodies, and also, presumably, to the plasma membrane

  9. Bunyaviruses with segmented glycoprotein genes and methods for generating these viruses

    Kortekaas, J.A.; Wichgers Schreur, P.J.; Oreshkova, N.D.; Moormann, R. J. M.

    2014-01-01

    Bunyaviruses with segmented glycoprotein precursor genes and methods for generating these viruses Abstract: The invention relates to a bunyavirus, in which separated (NSm)Gn and Gc coding regions are functionally present on two different genome segments, preferably a bunyavirus that comprises a total of at least 4 genome segments. The invention further relates to methods for producing said bunyavirus, and to a composition comprising said bunyavirus and a suitable excipient.

  10. Molecular models of human P-glycoprotein in two different catalytic states

    Tulkens Paul M

    2009-01-01

    Full Text Available Abstract Background P-glycoprotein belongs to the family of ATP-binding cassette proteins which hydrolyze ATP to catalyse the translocation of their substrates through membranes. This protein extrudes a large range of components out of cells, especially therapeutic agents causing a phenomenon known as multidrug resistance. Because of its clinical interest, its activity and transport function have been largely characterized by various biochemical studies. In the absence of a high-resolution structure of P-glycoprotein, homology modeling is a useful tool to help interpretation of experimental data and potentially guide experimental studies. Results We present here three-dimensional models of two different catalytic states of P-glycoprotein that were developed based on the crystal structures of two bacterial multidrug transporters. Our models are supported by a large body of biochemical data. Measured inter-residue distances correlate well with distances derived from cross-linking data. The nucleotide-free model features a large cavity detected in the protein core into which ligands of different size were successfully docked. The locations of docked ligands compare favorably with those suggested by drug binding site mutants. Conclusion Our models can interpret the effects of several mutants in the nucleotide-binding domains (NBDs, within the transmembrane domains (TMDs or at the NBD:TMD interface. The docking results suggest that the protein has multiple binding sites in agreement with experimental evidence. The nucleotide-bound models are exploited to propose different pathways of signal transmission upon ATP binding/hydrolysis which could lead to the elaboration of conformational changes needed for substrate translocation. We identified a cluster of aromatic residues located at the interface between the NBD and the TMD in opposite halves of the molecule which may contribute to this signal transmission. Our models may characterize different steps

  11. Development and Evidence for Efficacy of CMV Glycoprotein B Vaccine with MF59 Adjuvant

    Pass, Robert F.

    2009-01-01

    A vaccine comprised of recombinant cytomegalovirus (CMV) envelope glycoprotein B (gB) with MF59 adjuvant developed in the 1990s recently was recently found to have efficacy for prevention of CMV infection in a phase 2 clinical trial in young mothers. This review briefly considers the rationale for gB as a vaccine antigen, the history of this CMV gB vaccine and the data supporting vaccine efficacy.

  12. Synthesis of fucosyl-containing glycoproteins of the vitelline coat in oocytes of Ciona intestinalis (Ascidia).

    F.Rosati; Cotelli, F.; De Santis, R.; A. Monroy; Pinto, M. R.

    1982-01-01

    The sperm receptors of the ascidian oocyte are located at the outer surface of the vitelline coat (formerly called the chorion). The fucose residues are the receptor's most important components for sperm recognition and binding. We asked whether the fucosyl-containing glycoproteins of the vitelline coat are a product of the oocyte, the follicle cells, or the test cells. Ovaries of Ciona intestinalis were injected with L-[3H]fucose and the progress of its incorporation was followed by using au...

  13. Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

    Hirschberg, C B; Baker, R.M.; Perez, M.; Spencer, L A; Watson, D

    1981-01-01

    Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells ...

  14. The expression and regulation of pregnancy-specific glycoproteins in the mouse

    Williams, John Michael

    2013-01-01

    Pregnancy-Specific Glycoproteins (PSG) are the most abundant fetally expressed proteins in the maternal bloodstream at term. This multigene family are immunoglobulin superfamily members and are predominantly expressed in the syncytiotrophoblast of human placenta and in giant cells and spongiotrophoblast of rodent placenta. PSGs are encoded by seventeen genes in the mouse and ten genes in the human. Little is known about the function of this gene family, although they have been implicated in i...

  15. A New Rabies Vaccine Based on a Recombinant Orf Virus (Parapoxvirus) Expressing the Rabies Virus Glycoprotein

    Amann, Ralf; Rohde, Jörg; Wulle, Ulrich; Conlee, Douglas; Raue, Rudiger; Martinon, Olivier; Rziha, Hanns-Joachim

    2013-01-01

    The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protec...

  16. Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

    Artois, M.; Charlton, K M; Tolson, N D; Casey, G A; Knowles, M. K.; Campbell, J. B.

    1990-01-01

    Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and...

  17. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence

    Papaneri, Amy B.; Wirblich, Christoph; Cann, Jennifer A.; Cooper, Kurt; Peter B. Jahrling; Schnell, Matthias J.; Blaney, Joseph E.

    2012-01-01

    We are developing inactivated and live-attenuated rabies virus (RABV) vaccines expressing Ebola virus (EBOV) glycoprotein for use in humans and endangered wildlife, respectively. Here, we further characterize the pathogenesis of the live-attenuated RABV/EBOV vaccine candidates in mice in an effort to define their growth properties and potential for safety. RABV vaccines expressing GP (RV-GP) or a replication-deficient derivative with a deletion of the RABV G gene (RVΔG-GP) are both avirulent ...

  18. eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells.

    Xing Hua Tang

    Full Text Available We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs.Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.

  19. siRNA-based targeting of antiapoptotic genes can reverse chemoresistance in P-glycoprotein expressing chondrosarcoma cells

    Yang Jay

    2009-05-01

    Full Text Available Abstract Background High expression of P-glycoprotein is one of the well-known mechanisms of chemoresistance in chondrosarcomas. However, the role of antiapoptotic proteins, a common mechanism responsible for chemoresistance in other tumors, has not been well studied in chondrosarcomas. We examined the importance of P-glycoprotein and antiapoptotic proteins in the chemoresistance to doxorubicin of two Grade II chondrosarcoma cell lines, JJ012 and SW1353. Results We confirmed that both chondrosarcoma cell types expressed P-glycoprotein and antiapoptotic proteins (Bcl-2, Bcl-xL and XIAP. siRNA knockdown as well as pharmacologic inhibitors of cell survival proteins (Bcl-2, Bcl-xL and XIAP enhanced apoptosis of chemoresistant chondrosarcoma cells by up to 5.5 fold at 0.1 μmol and 5.5 fold at 1 μmol doxorubicin. These chemosensitizing effects were comparable to those of P-glycoprotein inhibition by siRNA or pharmacologic inhibitor. Conclusion These findings suggest that antiapoptotic proteins play a significant role in the chemoresistance of chondrosarcoma cells independent of P-glycoprotein. Based on the results, a new siRNA-based therapeutic strategy targeting antiapoptotic genes can be designed to overcome the chemoresistance of chondrosarcomas which is often conferred by P-glycoprotein.

  20. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D. (NIH)

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  1. Site-directed mutagenesis of boar proacrosin reveals residues involved in binding of zona pellucida glycoproteins.

    Jansen, S; Jones, R; Jenneckens, I; Marschall, B; Kriegesmann, B; Coadwell, J; Brenig, B

    1998-10-01

    Proacrosin, the zymogen form of the serine protease beta-acrosin, is thought to function as a secondary binding molecule between mammalian gametes during fertilization (Jansen et al., 1995: Int J Dev Biol 39, 501-510). The interaction involves strong ionic bonds between positively charged amino acids on proacrosin and negatively charged polysulphate groups on zona pellucida glycoproteins. In this investigation, we identified the basic residues on proacrosin that are important for this binding. Site-directed mutagenesis shows that two groups of amino acids comprising His47, Arg50, and Arg51 together with Arg250, Lys252, and Arg253 are crucial because their deletion or replacement severely reduces affinity for zona glycoproteins. Molecular models of proacrosin reveal that these residues are located along one face of the protein on two exposed surface loops that project over and around the catalytic site. These findings support the hypothesis that polysulphate binding sites on proacrosin are formed by a restricted number of basic amino acids on the surface of the protein, presenting a specific orientation that is complementary to negatively charged sulphate groups on zona glycoproteins. Identification and elucidation of the stereochemistry of these charged moieties will aid design of new kinds of nonsteroidal antifertility agents. PMID:9740326

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

    Goso, Yukinobu

    2016-03-01

    Hydrazine treatment is frequently used for releasing mucin-type O-glycans (O-glycans) from glycoproteins because the method provides O-glycans that retain a reducible GalNAc at their reducing end, which is available for fluorescent labeling. However, many O-glycans are degraded by "peeling" during this treatment. In the current study, it was found that malonic acid suppressed O-glycan degradation during hydrazine treatment of bovine fetuin or porcine gastric mucin in both the gas and liquid phases. This is paradoxical because the release of O-glycans from glycoproteins occurs under alkaline conditions. However, malonic acid seems to prevent the degradation through its acidic property given that other weak acids also prevented the degradation. Accordingly, disodium malonate did not suppress O-glycan degradation. Application of this method to rat gastric mucin demonstrated that the majority of the major O-glycans obtained in the presence of malonic acid were intact, whereas those obtained in the absence of malonic acid were degraded. These results suggest that hydrazine treatment in the presence of malonic acid would allow glycomic analysis of native mucin glycoproteins. PMID:26723492

  3. Glycosylation of dengue virus glycoproteins and their interactions with carbohydrate receptors: possible targets for antiviral therapy.

    Idris, Fakhriedzwan; Muharram, Siti Hanna; Diah, Suwarni

    2016-07-01

    Dengue virus, an RNA virus belonging to the genus Flavivirus, affects 50 million individuals annually, and approximately 500,000-1,000,000 of these infections lead to dengue hemorrhagic fever or dengue shock syndrome. With no licensed vaccine or specific antiviral treatments available to prevent dengue infection, dengue is considered a major public health problem in subtropical and tropical regions. The virus, like other enveloped viruses, uses the host's cellular enzymes to synthesize its structural (C, E, and prM/M) and nonstructural proteins (NS1-5) and, subsequently, to glycosylate these proteins to produce complete and functional glycoproteins. The structural glycoproteins, specifically the E protein, are known to interact with the host's carbohydrate receptors through the viral proteins' N-glycosylation sites and thus mediate the viral invasion of cells. This review focuses on the involvement of dengue glycoproteins in the course of infection and the virus' exploitation of the host's glycans, especially the interactions between host receptors and carbohydrate moieties. We also discuss the recent developments in antiviral therapies that target these processes and interactions, focusing specifically on the use of carbohydrate-binding agents derived from plants, commonly known as lectins, to inhibit the progression of infection. PMID:27068162

  4. The N-glycan glycoprotein deglycosylation complex (Gpd from Capnocytophaga canimorsus deglycosylates human IgG.

    Francesco Renzi

    2011-06-01

    Full Text Available C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases.

  5. Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae

    Dalton, J.P.; Strand, M.; Mangold, B.L.; Dean, D.A.

    1986-06-15

    The humoral immune response of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one-and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with sulfur-35 methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by the sera of infected mice. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.

  6. A UDP-glucose:glycoprotein glucose-1-phosphotransferase in embryonic chicken neural retina

    A subclass of cell-surface glycoproteins from embryonic chicken neural retina contains a high mannose-type oligosaccharide that terminates with glucose linked via a phosphodiester bond to penultimate mannose. This unusual oligosaccharide seems responsible for the glycoprotein attachments to the cell-surface baseplate ligatin. Using beta-32P-UDP-3H-glucose, we demonstrate in retinal homogenates the existence of a UDP-glucose:glycoprotein glucose-1-phosphotransferase (GlcPTase) that catalyzes the synthesis of such a linkage. Characterization of the doubly labeled product resulting from activity of the transferase reveals a family of endoglycosidase H-sensitive oligosaccharides displaying a cation-exchange profile similar to that of oligosaccharides derived from ligatin-associated proteins synthesized in vivo. Further analysis confirms that the incorporation of label is due to a terminal 3H-glucose joined via a 32P-phosphodiester linkage to carbon 6 of a penultimate mannose. We propose that GlcPTase may be a controlling enzyme for the targeting of certain newly synthesized proteins to the cell surface

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

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

    2009-06-01

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

  8. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

    Yagi, Hirokazu [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Nakamura, Masatoshi [National Institute of Agrobiological Sciences, Genetic Resources Conservation Research Unit, Genetic Resources Center (Japan); Yokoyama, Jun [Taiyo Nippon Sanso Corporation, Tsukuba Laboratories (Japan); Zhang, Ying; Yamaguchi, Takumi [National Institutes of Natural Sciences, Institute for Molecular Science and Okazaki Institute for Integrative Bioscience (Japan); Kondo, Sachiko [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan); Kobayashi, Jun [Yamaguchi University, Department of Biological and Environmental Sciences, Faculty of Agriculture (Japan); Kato, Tatsuya; Park, Enoch Y. [Shizuoka University, Laboratory of Biotechnology, Research Institute of Green Science and Technology (Japan); Nakazawa, Shiori [Nagoya University, Sugashima Marine Biological Laboratory, Graduate School of Science (Japan); Hashii, Noritaka; Kawasaki, Nana [National Institute of Health Sciences, Division of Biological Chemistry and Biologicals (Japan); Kato, Koichi, E-mail: kkato@phar.nagoya-cu.ac.jp [Nagoya City University, Faculty and Graduate School of Pharmaceutical Sciences (Japan)

    2015-06-15

    Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing {sup 15}N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a {sup 15}N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies.

  9. Terminal Mannose Residues in Seminal Plasma Glycoproteins of Infertile Men Compared to Fertile Donors

    Beata Olejnik

    2015-07-01

    Full Text Available The impact of seminal plasma components on the fertilization outcomes in humans is still under question. The increasing number of couples facing problems with conception raises the need for predictive biomarkers. Detailed understanding of the molecular mechanisms accompanying fertilization remains another challenge. Carbohydrate–protein recognition may be of key importance in this complex field. In this study, we analyzed the unique glycosylation pattern of seminal plasma proteins, the display of high-mannose and hybrid-type oligosaccharides, by means of their reactivity with mannose-specific Galanthus nivalis lectin. Normozoospermic infertile subjects presented decreased amounts of lectin-reactive glycoepitopes compared to fertile donors and infertile patients with abnormal semen parameters. Glycoproteins containing unveiled mannose were isolated in affinity chromatography, and 17 glycoproteins were identified in liquid chromatography-tandem mass spectrometry with electrospray ionization. The N-glycome of the isolated glycoproteins was examined in matrix-assisted laser desorption ionization mass spectrometry. Eleven out of 27 identified oligosaccharides expressed terminal mannose residues, responsible for lectin binding. We suggest that lowered content of high-mannose and hybrid type glycans in normozoospermic infertile patients may be associated with impaired sperm protection from preterm capacitation and should be considered in the search for new infertility markers.

  10. Types of oligosaccharide sulphation, depending on mucus glycoprotein source, corpus or antral, in rat stomach.

    Goso, Y; Hotta, K

    1989-01-01

    Radiolabelled mucus glycoprotein was obtained from tissue and a culture medium each of the corpus and antrum of rat stomach incubated with [35S]sulphate in vitro. Gel-filtration analysis of oligosaccharides liberated by alkaline-borohydride treatment from glycoproteins indicated that 35S-labelled oligosaccharides from the corpus vary considerably with respect to chain length whereas those from antral mucus glycoprotein are composed of small oligosaccharides. Examination of the reduced radiolabelled products obtained by HNO2 cleavage of the hydrazine-treated oligosaccharides indicated sulphate esters of N-acetylglucosamine to be present at three locations on a carbohydrate unit: [35S]sulphated monosaccharide (2,5-anhydromannitol 6-sulphate), [35S]sulphated disaccharide [galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate] and [35S]sulphated trisaccharide [fucosyl(alpha 1-2)-galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate]. Sulphated disaccharide and trisaccharide, possibly originating from the N-acetyl-lactosamine and fucosyl-N-acetyl-lactosamine sequences respectively, were detected in the corpus, especially as large oligosaccharides, but were present in the antrum in only very small amounts. The sulphated monosaccharide, however, most probably originating from 6-sulphated N-acetylglucosamine residues at non-reducing termini, was present in all oligosaccharide fractions in both the corpus and antrum. Images Fig. 4. Fig. 7. Fig. 8. PMID:2695066

  11. Glycosylation and sulphation of colonic mucus glycoproteins in patients with ulcerative colitis and in healthy subjects.

    Morita, H; Kettlewell, M G; Jewell, D P; Kent, P W

    1993-07-01

    Studies have been made of mucus glycoprotein biosynthesis in different regions of the lower gastrointestinal tract in normal patients and those with ulcerative colitis (UC), active or inactive, by means of 3H-glucosamine (3H-GlcNH2)--35S-sulphate double labelling of epithelial biopsy specimens under culture conditions. The time based rate of 3H-GlcNH2 labelling of mucus in rectal tissue was similar to that in active or inactive UC whereas the rate of 35SO4(2) labelling was significantly increased in active disease. The 3H specific activities measuring the amount of isotopic incorporation into surface and tissue mucus glycoproteins were increased in patients with active UC compared with normal or inactive subjects. The 35S specific activities did not differ significantly between patients with active UC and those in remission. In the rectum, glycosylation of mucus glycoproteins decreases with the increasing age of the patient. Regional differences in 3H-labelling of mucus components are reported for ascending colon, transverse colon, sigmoid colon, and rectum. Sulphation (35S-labelling) was higher in all parts of the colon in left sided UC. Results point to accelerated glycosylation of core proteins in the active phase of UC. PMID:8344580

  12. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule

    Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing 15N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a 15N-enrichment ratio of approximately 80 %. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies

  13. Separation and sugar component analysis of the oligosaccharides in the surface glycoproteins of newcastle disease virus

    The precursor glycoproteins HN0 and F0 in the surface spikes of Newcastle Disease Virus strain Ulster as produced by MDBK cells, were found to contain 10.4 and 11.9 weight per cent, respectively, of the sugars typical for N-glycosidically linked glycoprotein glycans. A molar ratio of D-mannose:D-galactose:L-fucose:N-acetyl-D-glucosamine approaching 1.0:1.1:0.5:1.0 was found for HN0, and of 1.0:0.7:0.3:0.6 for F0. By a sequence of degradation (with pronase, with endo-β-N-acetylglucosaminidase H [endo H], and by hydrazinolysis) and separation procedures (Concanavalin A-affinity and Biogel P-4 chromatography), the radiolabelled carbohydrate moieties of NDV HN0 and F0 (as oligosaccharitols) were separated into (at least) ten and eight fractions, respectively. Separate in vivo labelling with tritiated derivatives of the four sugars showed that both glycoproteins contain oligosaccharides of the oligomannosidic ('high mannose'), of the N-acetyllactosaminic ('complex'), as well as of the 'mixed type). The majority of the oligosaccharides in F0, but not of those in HNsub0, was found to be endo H-sensitive. (Author)

  14. Carbohydrates of influenza virus. I. Glycopeptides derived from viral glycoproteins after labeling with radioactive sugars

    The carbohydrate moiety of the influenza glycoproteins NA, HA1, and HA2 were analyzed by labeling with radioactive sugars. Analysis of glycopeptides obtained after digestion with Pronase indicated that there are at least two different types of carbohydrate side chains. The side chain of type I is composed of glucosamine, mannose, galactose, and fucose. It is found on NA, HA1, and HA2. The side chain of type II contains a high amount of mannose and is found only on NA and HA2. The molecular weights of the corresponding glycopeptides obtained from virus grown in chicken ambryo cells are 2,600 for type I and 2,000 for type II. The glycoproteins of virus grown in MDBK cells have a higher molecular weight than those of virus grown in chicken embryo cells, and there is a corresponding difference in the molecular weights of the glycopeptides. Under conditions of partial inhibition of glycosylation, virus particles were isolated that contained hemagglutinin with reduced carbohydrate content. Glycopeptide analysis indicated that this reduction is due to the lack of whole carbohydrate side chains and not to the incorporation of incomplete ones. This observation suggests that glycosylation of the viral glycoproteins involves en bloc transfer of the core sugars to the polypeptide chains

  15. Clearance and binding of radiolabeled glycoproteins by cells of the murine mononuclear phagocyte system

    The clearance and binding of radiolabeled lactoferrin and fast α2-macroglobulin were studied. Both glycoproteins cleared rapidly following intravenous injection in mice, and both bound specifically to discrete receptors on murine peritoneal macrophages. The simultaneous presence of excess, unlabeled ligands specific for receptors recognizing terminal fucose, mannose, N-acetylglucosamine or galactose residues did not inhibit the clearance or binding of either lactoferrin or fast-α2M. The clearance and binding of enzymatically defucosylated lactoferrin was indistinguishable from native lactoferrin, indicating that terminal α(1-3)-linked fucose on lactoferrin is not necessary for receptor recognition. The clearance and binding of two fast -α2M forms, α2M-trypsin and α2M-MeNH2 cross compete with each other. Saturation binding studies indicated that the total binding of mannosyl -BSA, fusocyl-BSA, and N-acetylglucosaminyl-BSA to macrophages activated by BCG was approximately 15% of the levels observed with inflammatory macrophages elicited by thioglycollate broth. Cross-competition binding studies demonstrated a common surface receptor mediated binding of all three neoglycoprotein ligands and was identical to the receptor on mononuclear phagocytes that binds mannosyl- and N-acetylglucosaminyl-terminated glycoproteins. These results suggest that difference between discrete states of macrophage function may be correlated with selective changes in levels of the surface receptor for mannose-containing glycoproteins

  16. Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

    Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC

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

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

    1986-01-01

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

  18. Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane.

    Bergmann, J E; Tokuyasu, K. T.; Singer, S. J.

    1981-01-01

    The intracellular pathway of biogenesis of the vesicular stomatitis virus transmembrane glycoprotein was investigated in situ by using indirect immunofluorescence of whole infected Chinese hamster ovary cells and immunoelectron microscopy of ultrathin frozen sections of infected cells. Transport of the glycoprotein was synchronized by using the temperature-sensitive virus mutant Orsay-45 and a temperature shift-down protocol. Sequential appearance of the glycoprotein in the rough endoplasmic ...

  19. A review on the relation between the brain-serum concentration ratio of drugs and the influence of P-glycoprotein

    Ejsing, Thomas Broeng; Morling, Niels; Linnet, Kristian

    2007-01-01

    This overview on the brain-serum relationship for drugs illustrates the importance of the drug transporter P-glycoprotein at the blood-brain barrier. Generally, an inverse relationship exists between the magnitude of the brain-serum ratio and the influence of P-glycoprotein. Concerning the pharma...... pharmacogenomics of P-glycoprotein, no clear effect of single nucleotide polymorphisms (SNPs) has been demonstrated in humans....

  20. Kinetic validation of the models for P-glycoprotein ATP hydrolysis and vanadate-induced trapping. Proposal for additional steps.

    Miguel Ramón Lugo

    Full Text Available P-Glycoprotein, a member of the ATP-binding cassette (ABC superfamily, is a multidrug transporter responsible for cellular efflux of hundreds of structurally unrelated compounds, including natural products, many clinically used drugs and anti-cancer agents. Expression of P-glycoprotein has been linked to multidrug resistance in human cancers. ABC transporters are driven by ATP hydrolysis at their two cytoplasmic nucleotide-binding domains, which interact to form a closed ATP-bound sandwich dimer. Intimate knowledge of the catalytic cycle of these proteins is clearly essential for understanding their mechanism of action. P-Glycoprotein has been proposed to hydrolyse ATP by an alternating mechanism, for which there is substantial experimental evidence, including inhibition of catalytic activity by trapping of ortho-vanadate at one nucleotide-binding domain, and the observation of an asymmetric occluded state. Despite many studies of P-glycoprotein ATPase activity over the past 20 years, no comprehensive kinetic analysis has yet been carried out, and some puzzling features of its behaviour remain unexplained. In this work, we have built several progressively more complex kinetic models, and then carried out simulations and detailed analysis, to test the validity of the proposed reaction pathway employed by P-glycoprotein for ATP hydrolysis. To establish kinetic parameters for the catalytic cycle, we made use of the large amount of published data on ATP hydrolysis by hamster P-glycoprotein, both purified and in membrane vesicles. The proposed kinetic scheme(s include a high affinity priming reaction for binding of the first ATP molecule, and an independent pathway for ADP binding outside the main catalytic cycle. They can reproduce to varying degrees the observed behavior of the protein's ATPase activity and its inhibition by ortho-vanadate. The results provide new insights into the mode of action of P-glycoprotein, and some hypotheses about the

  1. MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drags as judged by interference with nucleotide trapping

    Smith, A.J.; van Helvoort, A.; van Meer, G; Szabó, K.; Welker, E; Szakács, G; Váradi, A; Sarkadi, B.; Borst, P

    2000-01-01

    The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, p...

  2. Shedding of soluble glycoprotein 1 detected during acute Lassa virus infection in human subjects

    Momoh Mambu

    2010-11-01

    Full Text Available Abstract Background Lassa hemorrhagic fever (LHF is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. With a high rate of infection that may lead to morbidity and mortality, understanding how the virus interacts with the host's immune system is of great importance for generating vaccines and therapeutics. Previous work by our group identified a soluble isoform of the Lassa virus (LASV GP1 (sGP1 in vitro resulting from the expression of the glycoprotein complex (GPC gene 12. Though no work has directly been done to demonstrate the function of this soluble isoform in arenaviral infections, evidence points to immunomodulatory effects against the host's immune system mediated by a secreted glycoprotein component in filoviruses, another class of hemorrhagic fever-causing viruses. A significant fraction of shed glycoprotein isoforms during viral infection and biogenesis may attenuate the host's inflammatory response, thereby enhancing viral replication and tissue damage. Such shed glycoprotein mediated effects were previously reported for Ebola virus (EBOV, a filovirus that also causes hemorrhagic fever with nearly 90% fatality rates 345. The identification of an analogous phenomenon in vivo could establish a new correlate of LHF infection leading to the development of sensitive diagnostics targeting the earliest molecular events of the disease. Additionally, the reversal of potentially untoward immunomodulatory functions mediated by sGP1 could potentiate the development of novel therapeutic intervention. To this end, we investigated the presence of sGP1 in the serum of suspected LASV patients admitted to the Kenema Government Hospital (KGH Lassa Fever Ward (LFW, in Kenema, Sierra Leone that tested positive for viral antigen or displayed classical signs of Lassa fever. Results It is reasonable to expect that a narrow window exists for

  3. Profiling of Concanavalin A-Binding Glycoproteins in Human Hepatic Stellate Cells Activated with Transforming Growth Factor-β1

    Yannan Qin

    2014-11-01

    Full Text Available Glycoproteins play important roles in maintaining normal cell functions depending on their glycosylations. Our previous study indicated that the abundance of glycoproteins recognized by concanavalin A (ConA was increased in human hepatic stellate cells (HSCs following activation by transforming growth factor-β1 (TGF-β1; however, little is known about the ConA-binding glycoproteins (CBGs of HSCs. In this study, we employed a targeted glycoproteomics approach using lectin-magnetic particle conjugate-based liquid chromatography-tandem mass spectrometry to compare CBG profiles between LX-2 HSCs with and without activation by TGF-β1, with the aim of discovering novel CBGs and determining their possible roles in activated HSCs. A total of 54 and 77 proteins were identified in the quiescent and activated LX-2 cells, respectively. Of the proteins identified, 14.3% were glycoproteins and 73.3% were novel potential glycoproteins. Molecules involved in protein processing in the endoplasmic reticulum (e.g., calreticulin and calcium signaling (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase β-2 [PLCB2] were specifically identified in activated LX-2 cells. Additionally, PLCB2 expression was upregulated in the cytoplasm of the activated LX-2 cells, as well as in the hepatocytes and sinusoidal cells of liver cirrhosis tissues. In conclusion, the results of this study may aid future investigations to find new molecular mechanisms involved in HSC activation and antifibrotic therapeutic targets.

  4. Emerging Structural Insights into Glycoprotein Quality Control Coupled with N-Glycan Processing in the Endoplasmic Reticulum

    Tadashi Satoh

    2015-01-01

    Full Text Available In the endoplasmic reticulum (ER, the sugar chain is initially introduced onto newly synthesized proteins as a triantennary tetradecasaccharide (Glc3Man9GlcNAc2. The attached oligosaccharide chain is subjected to stepwise trimming by the actions of specific glucosidases and mannosidases. In these processes, the transiently expressed N-glycans, as processing intermediates, function as signals for the determination of glycoprotein fates, i.e., folding, transport, or degradation through interactions of a series of intracellular lectins. The monoglucosylated glycoforms are hallmarks of incompletely folded states of glycoproteins in this system, whereas the outer mannose trimming leads to ER-associated glycoprotein degradation. This review outlines the recently emerging evidence regarding the molecular and structural basis of this glycoprotein quality control system, which is regulated through dynamic interplay among intracellular lectins, glycosidases, and glycosyltransferase. Structural snapshots of carbohydrate-lectin interactions have been provided at the atomic level using X-ray crystallographic analyses. Conformational ensembles of uncomplexed triantennary high-mannose-type oligosaccharides have been characterized in a quantitative manner using molecular dynamics simulation in conjunction with nuclear magnetic resonance spectroscopy. These complementary views provide new insights into glycoprotein recognition in quality control coupled with N-glycan processing.

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

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

    2016-06-01

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

  6. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

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

    Sophia Böcker

    2015-07-01

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

  8. Genetic characterization of complete open reading frame of glycoprotein C gene of bovine herpesvirus 1

    Saurabh Majumder

    2013-10-01

    Full Text Available Aim: To characterize one of the major glycoprotein genes viz., glycoprotein C (gC; UL44, unique long region 44 of bovineherpesvirus 1(BoHV1 of Indian origin at genetic and phylogenetic level.Materials and Methods: A bovine herpesvirus 1 isolate viz., (BoHV1/IBR 216 II/ 1976/ India maintained at Division ofVirology, IVRI, Mukteswar was used for the current study. The DNA was extracted using commercial kit and the completeORF of gC gene was amplified, cloned, and sequenced by conventional Sanger sequencing method. The sequence wasgenetically and phylogenetically analysed using various bioinformatic tools. The sequence was submitted in the Genbankwith accession number Kc756965.Results: The complete ORF of gC gene was amplified and sequenced. It showed 100% sequence homology with referencecooper strain of BoHV1 and divergence varied from 0% to 2.7% with other isolates of BoHV1. The isolate under study haddivergence of 9.2%, 13%, 26.6%, and 9.2% with BoHV5 (Bovine herpesvirus 5, CvHV1 (Cervid herpesvirus 1, CpHV1(Caprine herpesvirus 1, and BuHV1 (Bubaline herpesvirus 1, respectively.Conclusion: This is the first genetic characterization of complete open reading frame (ORF of glycoprotein C gene (UL44 ofIndian isolate of BoHV1. The gC gene of BoHV1 is highly conserved among all BoHV1 isolates and it can be used as a targetfor designing diagnostic primers for the specific detection of BoHV1.

  9. Interactions between zona pellucida glycoproteins and sperm proacrosin/acrosin during fertilization.

    Howes, Liz; Jones, Roy

    2002-01-01

    Fertilization is one of the most specific and carefully regulated cell-cell interactions in the animal body and is determined to a large extent by compatibility between ligand and receptor molecules on the surface of each gamete. On the zona pellucida (ZP), sperm receptor activity is associated with glycoproteins ZP3 (primary receptor for acrosome-intact sperm) and ZP2 (secondary receptor for acrosome-reacted sperm) but their complementary binding proteins on sperm are less well defined. In this communication we review the evidence for proacrosin as a secondary ZP binding protein. Proacrosin/acrosin binds non-enzymically to ZP glycoproteins. Binding is a strong ionic interaction between polysulphate groups on ZP glycoproteins (probably on their carbohydrate moieties) and basic residues on the surface of proacrosin. The stereochemistry of the reactants is crucial and determines to a large extent the affinity of binding. Site-directed mutagenesis and a 3D-structural analysis of boar and ram acrosin have identified 2 clusters of basic residues potentially involved in binding. A polysulphonated anticancer drug, suramin, has been shown to bind strongly to proacrosin/acrosin and to inhibit sperm-egg binding in vitro. In the mouse model, 125I-ZP2 and 3H-suramin bind approximately 65% less effectively to acrosin 'null' sperm than to wild-type sperm. Neither ZP2 nor suramin bind to acrosome intact sperm and can, therefore, only exert their effects after exposure of the acrosomal contents. Overall, this combination of biochemical, genetic and functional data supports the hypothesis that proacrosin is a multifunctional protein with a significant role in retaining acrosome-reacted sperm on the ZP surface long enough to enable ZP penetration to begin. PMID:11730915

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

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

    2016-01-01

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

  11. Beta2-glycoprotein I dependent anticardiolipin antibodies and lupus anticoagulant in patients with recurrent pregnancy loss.

    Kumar K

    2002-01-01

    Full Text Available AIM: The present study was aimed to define the incidence of antiphospholipid antibodies of different types lupus anticoagulant (LAC, venereal disease research laboratory test (VDRL and Beta2-glycoprotein I dependent anticardiolipin antibodies Beta2 I aCL in our cohort of population experiencing recurrent pregnancy loss (RPL from Andhra Pradesh, South India. SETTING AND DESIGN: A referral case-control study at a tertiary centre over a period of 5 years. PARTICIPANTS: 150 couples experiencing 3 or more recurrent pregnancy losses with similar number of matched controls. MATERIAL AND METHODS: LAC activity was measured by the activated partial thromboplastin time (aPTT according to the method of Proctor and Rapaport with relevant modifications. VDRL analysis was performed by the kit method supplied by Ranbaxy Diagnostics Limited and Beta2 Glycoprotein I dependent anticardiolipin antibodies were estimated by ELISA kit (ORGen Tech, GmbH, Germany with human Beta2 Glycoprotein I as co-factor. STATISTICAL ANALYSIS: Statistical analysis was performed using Student′s t test. RESULTS: LAC activity was found positive in 11 women (10.28%. The mean +/- SE Beta2 I aCL concentration in the study group was 14.53 (micro/ml +/- 1.79 (range 0 to 90.4 micro/ml which was higher than the control group with a mean +/- SE of 7.26 (micro/ml +/- 0.40 (range 0 to 18 u/ml. The binding of the antibodies to the antigen was observed in 40.24% (n=33 of the cases compared to 6.09% (n=5 in controls. VDRL test was positive in 7(2.34% individuals (3 couples and 1 male partner and none among controls. CONCLUSIONS: The present study indicates the importance of antiphospholipid antibodies in women experiencing RPL and suggests the usefulness of screening for these antibodies as a mandatory routine for instituting efficient therapeutic regimens for a successful outcome of pregnancy.

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

    Badowska-Kozakiewicz, A M; Malicka, E

    2010-01-01

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

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

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

    2016-07-01

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

  14. Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.

    Shridhar Bale

    2011-11-01

    Full Text Available BACKGROUND: Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2 on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2 is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP(1,2 trimeric, viral surface spike. After entry into host endosomes, GP(1,2 is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown. PRINCIPAL FINDINGS: We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS of glycoproteins from two different ebolaviruses that although enzymatic priming of GP(1,2 is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP(1,2. Further, ELISA binding data of primed GP(1,2 to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion. SIGNIFICANCE: The results reveal that the ebolavirus GP(1,2 ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP(1,2 and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.

  15. Immunogenicity of an aphthovirus chimera of the glycoprotein of vesicular stomatitis virus.

    Grigera, P R; Garcia-Briones, M; Periolo, O; La Torre, J L; Wagner, R R

    1996-01-01

    An oligodeoxynucleotide coding for amino acids 139 through 149 of antigenic site A (ASA) of the VP1 capsid protein of the foot-and-mouth disease virus C3 serotype (FMDV C3) was inserted into three different in-frame sites of the vesicular stomatitis virus New Jersey serotype (VSV-NJ) glycoprotein (G) gene cDNA present in plasmid pKG97 under control of the bacteriophage T7 polymerase promoter. Transfection of these plasmids into CV1 cells coinfected with the T7 polymerase-expressing vaccinia v...

  16. Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model

    Andersson, O.; Badisco, L.; Hansen, A. H.;

    2014-01-01

    In earlier studies insects were proposed as suitable models for vertebrate blood–brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain...... vertebrates, the locust brain–barrier function is morphologically confined to one specific cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model...

  17. Anticardiolipin and anti-beta2-glycoprotein I antibodies in Behcet's disease.

    Kang, H. J.; Lee, Y. W.; Han, S H; Cho, H C; K.M. Lee

    1998-01-01

    To investigate prevalence of anticardiolipin antibodies (aCL) in patients with Behcet's disease (BD) and to determine whether they are related to anti-beta2-glycoprotein I antibodies (aGPI), we measured aCL and aGPI in 47 patients of BD and 14 patients of systemic lupus erythematosus (SLE). The levels of aCL and aGPI were determined by conventional enzyme immunoassay for both IgG and IgM classes. Twelve (25.5%) patients with BD were positive for IgG or IgM aCL and no patient was positive for ...

  18. Replication and virulence of pseudorabies virus mutants lacking glycoprotein gX.

    Thomsen, D R; Marchioli, C C; Yancey, R J; Post, L E

    1987-01-01

    Pseudorabies virus (PRV) glycoprotein gX accumulates in the medium of infected cells. In an attempt to study the function of gX, two viruses were constructed that lacked a functional gX gene. One virus, PRV delta GX1, was derived by insertion of the herpes simplex virus thymidine kinase gene into the gX-coding region. The other virus, PRV delta GXTK-, was derived by subsequent deletion of the inserted herpes simplex virus thymidine kinase gene. Both viruses replicated in cell cultures but pro...

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

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

    2011-06-01

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

  20. Murine CD9 Is the Receptor for Pregnancy-specific Glycoprotein 17

    Waterhouse, Roseann; Ha, Cam; Dveksler, Gabriela S.

    2002-01-01

    Pregnancy-specific glycoproteins (PSGs) are a family of highly similar secreted proteins produced by the placenta. PSG homologs have been identified in primates and rodents. Members of the human and murine PSG family induce secretion of antiinflammatory cytokines in mononuclear phagocytes. For the purpose of cloning the receptor, we screened a RAW 264.7 cell cDNA expression library. The PSG17 receptor was identified as the tetraspanin, CD9. We confirmed binding of PSG17 to CD9 by ELISA, flow ...

  1. Characterization of receptors for murine pregnancy specific glycoproteins 17 and 23

    Sulkowski, G. N.; Warren, J; Ha, C. T.; Dveksler, G S

    2011-01-01

    In primates and rodents, trophoblast cells synthesize and secrete into the maternal circulation a family of proteins known as pregnancy specific glycoproteins (PSG). The current study was undertaken to characterize the receptor for two members of the murine PSG family, PSG17 and PSG23. Binding of recombinant PSG17 and PSG23 to CHO-K1 and L929 cells and their derived mutants was performed to determine whether these proteins bound to cell surface proteoglycans. We also examined binding of these...

  2. Anti-beta2 glycoprotein 1 and the anti-phospholipid syndrome.

    Keane, Pearse A

    2012-02-03

    PURPOSE: To describe a patient who presented with bilateral retinal vascular occlusion and the use of anti-beta2 glycoprotein 1 (GPI) antibody testing in the diagnosis of antiphospholipid syndrome. DESIGN: Observational case report. METHODS: Hematological investigations were performed on a 49-year-old man who presented with rapid onset of bilateral severe central retinal vein occlusion. RESULTS: Lupus anticoagulant and anticardiolipin antibody testing was negative. Markedly raised titers of anti-beta2 GPI antibodies were detected on two separate occasions. CONCLUSIONS: The raised titers of anti-beta2 GPI antibodies were considered to strongly suggest an underlying diagnosis of the antiphospholipid syndrome.

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

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

  4. Overexpression of the Rabies Virus Glycoprotein Results in Enhancement of Apoptosis and Antiviral Immune Response

    Faber, Milosz; Pulmanausahakul, Rojjanaporn; Hodawadekar, Suchita S.; Spitsin, Sergei; McGettigan, James P.; Schnell, Matthias J.; Dietzschold, Bernhard

    2002-01-01

    A recombinant rabies virus (RV) carrying two identical glycoprotein (G) genes (SPBNGA-GA) was constructed and used to determine the effect of RV G overexpression on cell viability and immunity. Immunoprecipitation analysis and flow cytometry showed that tissue culture cells infected with SPBNGA-GA produced, on average, twice as much RV G as cells infected with RV carrying only a single RV G gene (SPBNGA). The overexpression of RV G in SPBNGA-GA-infected NA cells was paralleled by a significan...

  5. Expression of the Thy-1 glycoprotein gene by DNA-mediated gene transfer.

    Evans, G A; Ingraham, H A; Lewis, K; Cunningham, K; Seki, T.; Moriuchi, T; Chang, H. C.; Silver, J; Hyman, R

    1984-01-01

    We isolated a gene encoding the Thy-1.2 glycoprotein from a recombinant library constructed from BALB/c mouse DNA. To evaluate the expression of this cloned gene in different genomic environments, we introduced it into cell lines derived from fibroblast, lymphoid, and neuronal tissues by DNA-mediated gene transfer. When integrated into the genome of mouse L cells, cell-surface Thy-1 can be detected with anti-Thy-1 monoclonal antibodies. These L-cell lines contain between two and four copies o...

  6. Molecular cloning and chemical synthesis of a region of platelet glycoprotein IIb involved in adhesive function.

    Loftus, J C; Plow, E F; Frelinger, A.L.; D'Souza, S E; Dixon, D; Lacy, J.; Sorge, J; Ginsberg, M H

    1987-01-01

    Membrane glycoprotein (GP) IIb-IIIa is a component of a platelet adhesive protein receptor. A region of the heavy chain of GPIIb, defined by the monoclonal antibody PMI-1, is involved in adhesion receptor function. We have localized and chemically synthesized this region of GPIIb. A cDNA clone that directs the synthesis of a fusion protein reactive with the PMI-1 antibody was isolated from a phage lambda gt11 expression library constructed with mRNA from an erythroleukemia (HEL) cell line. Th...

  7. Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry

    Calvano, Cosima D; Zambonin, Carlo G; Jensen, Ole Nørregaard

    2008-01-01

    Protein glycosylation is a common post-translational modification that is involved in many biological processes, including cell adhesion, protein-protein and receptor-ligand interactions. The glycoproteome constitutes a source for identification of disease biomarkers since altered protein...... glycosylation profiles are associated with certain human ailments. Glycoprotein analysis by mass spectrometry of biological samples, such as blood serum, is hampered by sample complexity and the low concentration of the potentially informative glycopeptides and -proteins. We assessed the utility of lectin...... cancer patients and healthy individuals to assess glycosylation site frequencies....

  8. Effects of Kaempferia parviflora extracts and their flavone constituents on P-glycoprotein function

    Patanasethanont, Denpong; Nagai, Junya; Yumoto, Ryoko; Murakami, Teruo; Sutthanut, Khaetthareeya; Sripanidkulchai, Bung-Orn; Yenjai, Chavi; Takano, Mikihisa

    2006-01-01

    The purpose of this study was to examine the effects of extracts and flavone derivatives from the rhizome of Kaempferia parviflora on P-glycoprotein (P-gp)-mediated transport in LLC-GA5-COL150, a transfectant cell line of a porcine kidney epithelial cell line LLC-PK1 with human MDR1 cDNA. Ethanol extract obtained from Kaempferia parviflora rhizome significantly increased the accumulation of rhodamine 123 and daunorubicin, P-gp substrates, in LLC-GA5-COL150 cells, but not in LLC-PK1 cells. The...

  9. Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D.

    Nicola, A V; Willis, S. H.; Naidoo, N N; Eisenberg, R J; Cohen, G H

    1996-01-01

    Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry. Truncated forms of gD lacking the transmembrane and cytoplasmic tail regions have been shown to bind to cells and block plaque formation. Using complementation analysis and a panel of gD mutants, we previously identified four regions of gD (regions I to IV) which are important for virus entry. Here, we used baculovirus vectors to overexpress truncated forms of wild-type gD from HSV type 1 (HSV-1) [gD-1(306t)] and ...

  10. Identification and Characterization of Glycoproteins on the Spore Surface of Clostridium difficile

    Strong, Philippa C. R.; Fulton, Kelly M.; Aubry, Annie; Foote, Simon; Twine, Susan M; Logan, Susan M.

    2014-01-01

    In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a num...

  11. HZ08 Reverse P-Glycoprotein Mediated Multidrug Resistance In Vitro and In Vivo

    Hu, Zheyi; Zhou, Zaigang; Hu, Yahui; Wu, Jinhui; Li, Yunman; Huang, Wenlong

    2015-01-01

    Background Multidrug efflux transporter P-glycoprotein (P-gp) is highly expressed on membrane of tumor cells and is implicated in resistance to tumor chemotherapy. HZ08 is synthesized and studied in order to find a novel P-gp inhibitor. Methods MDCK-MDR1 monolayer transport, calcein-AM P-gp inhibition and P-gp ATPase assays were used to confirm the P-gp inhibition capability of HZ08. Furthermore, KB-WT and KB-VCR cells were used to evaluate the P-gp inhibitory activity of HZ08 both in vitro a...

  12. P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity

    Mechetner, Eugene B.; Schott, Brigitte; Morse, Brian S.; Stein, Wilfred D.; Druley, Todd; Davis, Kenneth A.; Tsuruo, Takashi; Roninson, Igor B

    1997-01-01

    The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding t...

  13. Evaluation of P-Glycoprotein Inhibitory Potential Using a Rhodamine 123 Accumulation Assay

    Elodie Jouan; Marc Le Vée; Abdullah Mayati; Claire Denizot; Yannick Parmentier; Olivier Fardel

    2016-01-01

    In vitro evaluation of P-glycoprotein (P-gp) inhibitory potential is now a regulatory issue during drug development, in order to predict clinical inhibition of P-gp and subsequent drug–drug interactions. Assays for this purpose, commonly based on P-gp-expressing cell lines and digoxin as a reference P-gp substrate probe, unfortunately exhibit high variability, raising thus the question of developing alternative or complementary tests for measuring inhibition of P-gp activity. In this context,...

  14. Blockade of P-Glycoprotein Decreased the Disposition of Phenformin and Increased Plasma Lactate Level

    Choi, Min-Koo; Song, Im-Sook

    2016-01-01

    This study aimed to investigate the in vivo relevance of P-glycoprotein (P-gp) in the pharmacokinetics and adverse effect of phenformin. To investigate the involvement of P-gp in the transport of phenformin, a bi-directional transport of phenformin was carried out in LLC-PK1 cells overexpressing P-gp, LLC-PK1-Pgp. Basal to apical transport of phenformin was 3.9-fold greater than apical to basal transport and became saturated with increasing phenformin concentration (2–75 μM) in LLC-PK1-Pgp, s...

  15. Functional Impact of ABCB1 Variants on Interactions between P-Glycoprotein and Methadone

    Hung, Chin-Chuan; Chiou, Mu-Han; Teng, Yu-Ning; Hsieh, Yow-Wen; Huang, Chieh-Liang; Lane, Hsien-Yuan

    2013-01-01

    Methadone is a widely used substitution therapy for opioid addiction. Large inter-individual variability has been observed in methadone maintenance dosages and P-glycoprotein (P-gp) was considered to be one of the major contributors. To investigate the mechanism of P-gp’s interaction with methadone, as well as the effect of genetic variants on the interaction, Flp-In™-293 cells stably transfected with various genotypes of human P-gp were established in the present study. The RNA and protein e...

  16. Irradiation of rat brain reduces P-glycoprotein expression and function

    Bart, J; Nagengast, W B; Coppes, R P; Wegman, T D; Graaf, W.T.A. van der; Groen, H J M; Vaalburg, W; de Vries, E G E; Hendrikse, N H

    2007-01-01

    The blood–brain barrier (BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P-glycoprotein (P-gp), expressed on brain capillary endothelial cells, is part of the BBB. P-gp expression on capillary endothelium decreases 5 days after brain irradiation, which may reduce P-gp function and increase brain levels of P-gp substrates. To elucidate whether radiation therapy reduces P-gp expression and function in the brain, right hemispheres of rats wer...

  17. P-glycoprotein Dysfunction Contributes to Hepatic Steatosis and Obesity in Mice

    Vignault-Foucaud, Magali; soayfane, zeina; Menez-Berlioz, Cécile; Bertrand-Michel, Justine; Guy, Pascal; Martin, Pierre; Guillou, Hervé; Collet, Xavier; Lespine, Anne

    2011-01-01

    Although the main role of P-glycoprotein (Pgp) is to extrude a broad range of xenochemicals and to protect the organism against xenotoxicity, it also transports a large range of endogenous lipids. Using mice lacking Pgp, we have investigated the possible involvement of Pgp in lipid homeostasis in vivo. In a long term study, we have followed the food intake, body status and lipid markers in plasma and liver of wild-type and mdr1ab(-/-) mice over 35 weeks. Pgp-deficient mice showed excess weigh...

  18. Immunophilin-like TWISTED DWARF1 modulates auxin efflux activities of Arabidopsis p-glycoproteins

    BOUCHARD R; Bailly, A; Blakeslee, J.J.; VINCENZETTI V; Paponov, I.; Palme, K; S. Mancuso; Murphy, A.S.; Schulz, B.; Geisler, M.

    2006-01-01

    The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin trans...

  19. The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane

    Garrigues, Alexia; Escargueil, Alexandre E.; Orlowski, Stéphane

    2002-01-01

    P-glycoprotein (P-gp) is a plasma membrane ATP-binding cassette transporter, responsible for multidrug resistance in tumor cells. P-gp catalyzes the ATP hydrolysis-dependent efflux of numerous amphiphilic compounds of unrelated chemical structures. In the absence of any identified substrate, P-gp exhibits an apparently futile, basal ATPase activity. By using native membrane vesicles containing high amounts of P-gp, we show here that (i) this basal ATPase activity is tightly dependent on the p...

  20. P-glycoprotein efflux pump plays an important role in Trypanosoma cruzi drug resistance

    Campos, Mônica Caroline Oliveira; Castro-Pinto, Denise Barçante; Ribeiro, Grazielle Alves; Berredo-Pinho, Márcia Moreira; Gomes, Leonardo Henrique Ferreira; da Silva Bellieny, Myrtes Santos; Goulart, Carla Marins; Echevarria, Áurea; Leon, Leonor Laura

    2013-01-01

    Drug resistance in protozoan parasites has been associated with the P-glycoprotein (Pgp), an energy-dependent efflux pump that transports substances across the membrane. Interestingly, the genes TcPGP1 and TcPGP2 have been described in Trypanosoma cruzi, although the function of these genes has not been fully elucidated. The main goal of this work was to investigate Pgp efflux pump activity and expression in T. cruzi lines submitted to in vitro induced resistance to the compounds 4-N-(2-metho...

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

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

    2015-10-13

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

  2. A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

    Peter J Bredenbeek; Molenkamp, Richard; Spaan, Willy J. M.; Deubel, Vincent; Marianneau, Phillippe; Salvato, Maria S.; Moshkoff, Dmitry; Zapata, Juan; Tikhonov, Ilia; Patterson, Jean; Carrion, Ricardo; Ticer, Anysha; Brasky, Kathleen; Lukashevich, Igor S.

    2006-01-01

    The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa F...

  3. P-Glycoprotein (Abcb1) is involved in absorptive drug transport in skin

    Ito, Katsuaki; Nguyen, Hai Thien; Kato, Yukio; Wakayama, Tomohiko; Kubo, Yoshiyuki; Iseki, Shoichi; Tsuji, Akira

    2008-01-01

    The purpose of the present study was to investigate the role of P-glycoprotein (P-gp) in drug disposition in skin. The distribution of P-gp substrates (rhodamine 123 and itraconazole) to the skin after administration from the epidermal side was lower in P-gp gene knockout (mdr1a/1b-/-) mice than that in wild-type mice. Coadministration of propranolol, a P-gp inhibitor, decreased the distribution of itraconazole to the skin in wild-type mice, but not in mdr1a/1b-/- mice. These results suggest ...

  4. P-Glycoprotein (Abcb1) is involved in absorptive drug transport in skin

    Ito, Katsuaki; Nguyen, Hai Thien; Kato, Yukio; Wakayama, Tomohiko; Kubo, Yoshiyuki; Iseki, Shoichi; Tsuji, Akira

    2008-01-01

    The purpose of the present study was to investigate the role of P-glycoprotein (P-gp) in drug disposition in skin. The distribution of P-gp substrates (rhodamine 123 and itraconazole) to the skin after administration from the epidermal side was lower in P-gp gene knockout (mdr1a/1b-/- ) mice than that in wild-type mice. Coadministration of propranolol, a P-gp inhibitor, decreased the distribution of itraconazole to the skin in wild-type mice, but not in mdr1a/1b-/- mice. These results suggest...

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

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

    2007-01-01

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

  6. Distinct Glycoprotein O Complexes Arise in a Post-Golgi Compartment of Cytomegalovirus-Infected Cells

    Theiler, Regan N.; Compton, Teresa

    2002-01-01

    Human cytomegalovirus (CMV) glycoproteins H, L, and O (gH, gL, and gO, respectively) form a heterotrimeric disulfide-bonded complex that participates in the fusion of the viral envelope with the host cell membrane. During virus maturation, this complex undergoes a series of intracellular assembly and processing events which are not entirely defined (M. T. Huber and T. Compton, J. Virol. 73:3886-3892, 1999). Here, we demonstrate that gO does not undergo the same posttranslational processing in...

  7. Structure and expression of the human MDR (P-glycoprotein) gene family.

    Chin, J E; Soffir, R; Noonan, K E; Choi, K.; Roninson, I B

    1989-01-01

    The human MDR (P-glycoprotein) gene family is known to include two members, MDR1 and MDR2. The product of the MDR1 gene, which is responsible for resistance to different cytotoxic drugs (multidrug resistance), appears to serve as an energy-dependent efflux pump for various lipophilic compounds. The function of the MDR2 gene remains unknown. We have examined the structure of the human MDR gene family by Southern hybridization of DNA from different multidrug-resistant cell lines with subfragmen...

  8. Induction of liver alpha-1 acid glycoprotein gene expression involves both positive and negative transcription factors.

    Y. M. Lee; Tsai, W H; Lai, M Y; Chen, D S; Lee, S. C.

    1993-01-01

    Expression of the alpha-1 acid glycoprotein (AGP) gene is liver specific and acute phase responsive. Within the 180-bp region of the AGP promoter, at least five cis elements have been found to interact with trans-acting factors. Four of these elements (A, C, D, and E) interacted with AGP/EBP, a liver-enriched transcription factor, as shown by footprinting analysis and by an anti-AGP/EBP antibody-induced supershift in a gel retardation assay. Modification of these sites by site-directed mutage...

  9. Characterization of duck enteritis virus UL53 gene and glycoprotein K

    Yang Xiaoyuan; Wu Ying; Wang Mingshu; Cheng Anchun; Xiang Jun; Zhang Shunchuan; Zhu Dekang; Jia Renyong; Luo Qihui; Chen Zhengli; Chen Xiaoyue

    2011-01-01

    Abstract Background Most of the previous research work had focused on the epidemiology and prevention of duck enteritis virus (DEV). Whilst with the development of protocols in molecular biology, nowadays more and more information about the genes of DEV was reported. But little information about DEV UL53 gene and glycoprotein K(gK) was known except our reported data. Results In our paper, the fluorescent quantitative real-time PCR(FQ-RT-PCR) assay and nucleic acid inhibition test were used to...

  10. The Rabies Virus Glycoprotein Receptor p75NTR Is Not Essential for Rabies Virus Infection▿

    Tuffereau, Christine; Schmidt, Klaus; Langevin, Christelle; Lafay, Florence; Dechant, Georg; Koltzenburg, Martin

    2007-01-01

    Rabies virus glycoprotein (RVG) is known to be the only factor that mediates rabies infection. The neurotrophin receptor (p75NTR), through its cysteine-rich domain 1, is a specific receptor for RVG and neutralizes virus infectivity, but its role in virus infection has remained obscure. We used adult mouse dorsal root ganglion (DRG) neurons as a model to study the role of p75NTR in RV infection of primary neurons. We show that RV infects around 20% of DRG neurons, of which more than 80% are p7...

  11. Ligand and Structure-Based Classification Models for Prediction of P-Glycoprotein Inhibitors

    Klepsch, Freya; Vasanthanathan, Poongavanam; Ecker, Gerhard F

    2014-01-01

    The ABC transporter P-glycoprotein (P-gp) actively transports a wide range of drugs and toxins out of cells, and is therefore related to multidrug resistance and the ADME profile of therapeutics. Thus, development of predictive in silico models for the identification of P-gp inhibitors is of great interest in the field of drug discovery and development. So far in silico P-gp inhibitor prediction was dominated by ligand-based approaches because of the lack of high-quality structural informatio...

  12. P-glycoprotein expression as a predictor of response to neoadjuvant chemotherapy in breast cancer

    S Vishnukumar; Umamaheswaran, G.; D Anichavezhi; Indumathy, S.; C Adithan; Srinivasan, K.; D Kadambari

    2013-01-01

    Background: Chemoresistance is an important factor determining the response of tumor to neoadjuvant chemotherapy (NACT). P-glycoprotein (P-gp) expression-mediated drug efflux is one of the mechanisms responsible for multi-drug resistance. Our study was aimed to determine the role of P-gp expression as a predictor of response to NACT in locally advanced breast cancer (LABC) patients. Materials and Methods: P-gp expression was performed by real-time quantitative polymerase chain reaction [qRT-P...

  13. Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.

    Ryan Williams

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

  14. Efficient transduction of neurons using Ross River glycoprotein-pseudotyped lentiviral vectors

    Jakobsson, J; Nielsen, T Tolstrup; Staflin, K;

    2006-01-01

    , including the possibility to establish stable producer cell lines. After injection of RRV-LV expressing green fluorescent protein into different structures in the rat brain we found efficient transduction of both neurons and glial cells. By using two cell-type-specific promoters, neuron-specific enolase and...... human glial fibrillary acidic protein, we demonstrated cell-specific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells....

  15. Bovine Herpesvirus Type 4 Glycoprotein L Is Nonessential for Infectivity but Triggers Virion Endocytosis during Entry

    Lété, Céline; Machiels, Bénédicte; Stevenson, Philip G.; Vanderplasschen, Alain; Gillet, Laurent

    2012-01-01

    The core entry machinery of mammalian herpesviruses comprises glycoprotein B (gB), gH, and gL. gH and gL form a heterodimer with a central role in viral membrane fusion. When archetypal alpha- or betaherpesviruses lack gL, gH misfolds and progeny virions are noninfectious. However, the gL of the rhadinovirus murid herpesvirus 4 (MuHV-4) is nonessential for infection. In order to define more generally what role gL plays in rhadinovirus infections, we disrupted its coding sequence in bovine her...

  16. On the association of glycoprotein Ib and actin-binding protein in human platelets

    1985-01-01

    Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this prot...

  17. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites

    Cordon-Cardo, C.; O' Brien, J.P.; Casals, D.; Biedler, J.L.; Melamed, M.R.; Bertino, J.R. (Memorial Sloan-Kettering Cancer Center, New York, NY (USA)); Rittman-Grauer, L. (Hybritech, Inc., San Diego, CA (USA))

    1989-01-01

    Endothelial cells of human capillary blood vessels at the blood-brain and other blood-tissue barrier sites express P-glycoprotein as detected by mouse monoclonal antibodies against the human multidrug-resistance gene product. This pattern of endothelial cell expression may indicate a physiological role for P-glycoprotein in regulating the entry of certain molecules into the central nervous system and other anatomic compartments, such as the testes. These tissues, which limit the access of systemic drugs, are known pharmacologic sanctuaries for metastatic cancer. P-glycoprotein expression in capillary endothelium of brain and testes and not other tissues (i.e., kidney and placenta) may in part explain this phenomenon and could have important implications in cancer chemotherapy.

  18. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    Tiwari, Vaibhav [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Darmani, Nissar A.; Thrush, Gerald R. [Department of Basic Medical Sciences, College of Osteopathic Medicine of the Pacific and College of Optometry, Western University of Health Sciences, Pomona, CA 91766 (United States); Shukla, Deepak, E-mail: dshukla@uic.edu [Department of Ophthalmology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2009-12-18

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  19. An unusual dependence of human herpesvirus-8 glycoproteins-induced cell-to-cell fusion on heparan sulfate

    Human herpesvirus-8 (HHV-8) is known to interact with cell surface heparan sulfate (HS) for entry into a target cell. Here we investigated the role of HS during HHV-8 glycoproteins-induced cell fusion. Interestingly, the observed fusion demonstrated an unusual dependence on HS as evident from following lines of evidence: (1) a significant reduction in cell-to-cell fusion occurred when target cells were treated with heparinase; (2) in a competition assay, when the effector cells expressing HHV-8 glycoproteins were challenged with soluble HS, cell-to-cell fusion was reduced; and, (3) co-expression of HHV-8 glycoproteins gH-gL on target cells resulted in inhibition of cell surface HS expression. Taken together, our results indicate that cell surface HS can play an additional role during HHV-8 pathogenesis.

  20. Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

    Liu; Xueguang; Zheng; Huaidong; Guo; Xinshuo; Luo; Jin; Lin; Cuicui; Wang; Qiuyu

    2014-01-01

    [Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

  1. Structural and Antigenic Definition of Hepatitis C Virus E2 Glycoprotein Epitopes Targeted by Monoclonal Antibodies

    Giuseppe Sautto

    2013-01-01

    Full Text Available Hepatitis C virus (HCV is the major cause of chronic liver disease as well as the major indication for liver transplantation worldwide. Current standard of care is not completely effective, not administrable in grafted patients, and burdened by several side effects. This incomplete effectiveness is mainly due to the high propensity of the virus to continually mutate under the selective pressure exerted by the host immune response as well as currently administered antiviral drugs. The E2 envelope surface glycoprotein of HCV (HCV/E2 is the main target of the host humoral immune response and for this reason one of the major variable viral proteins. However, broadly cross-neutralizing monoclonal antibodies (mAbs directed against HCV/E2 represent a promising tool for the study of virus-host interplay as well as for the development of effective prophylactic and therapeutic approaches. In the last few years many anti-HCV/E2 mAbs have been evaluated in preclinical and clinical trials as possible candidate antivirals, particularly for administration in pre- and post-transplant settings. In this review we summarize the antigenic and structural characteristics of HCV/E2 determined through the use of anti-HCV/E2 mAbs, which, given the absence of a crystal structure of this glycoprotein, represent currently the best tool available.

  2. A recombinant canine distemper virus expressing a modified rabies virus glycoprotein induces immune responses in mice.

    Li, Zhili; Wang, Jigui; Yuan, Daoli; Wang, Shuang; Sun, Jiazeng; Yi, Bao; Hou, Qiang; Mao, Yaping; Liu, Weiquan

    2015-06-01

    Canine distemper virus (CDV) and rabies virus (RV) are two important pathogens of the dog. CDV, a member of the morbillivirus genus, has shown promise as an expression vector. The glycoprotein from RV is a main contributor to protective immunity and capable of eliciting the production of virus-neutralizing antibodies. In this study, we recovered an attenuated strain of canine distemper virus and constructed a recombinant virus, rCDV-RV-G, expressing a modified (R333Q) rabies virus glycoprotein (RV-G) of RV Flury strain LEP. RV-G expression by the recombinant viruses was confirmed. Furthermore, G was proved to be incorporated into the surface of CDV particles. While replication of the recombinant virus was slightly reduced compared with the parental CDV, it stably expressed the RV-G over ten serial passages. Inoculation of mice induced specific neutralizing antibodies against both RV-G and CDV. Therefore, the rCDV-RV-G has the potential as a vaccine that may be used to control rabies virus infection in dogs and other animals. PMID:25764477

  3. Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates.

    Chad E Mire

    Full Text Available The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV that expresses an individual filovirus glycoprotein (GP in place of the VSV glycoprotein (G. The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV GP; three animals received rVSV-wild type (wt vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.

  4. Prediction and identification of mouse cytotoxic T lymphocyte epitopes in Ebola virus glycoproteins

    Wu Shipo

    2012-06-01

    Full Text Available Abstract Background Ebola viruses (EBOVs cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8+ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2d-specific T cell epitopes in EBOV glycoproteins (GPs. Results Computer-assisted algorithms were used to predict H-2d-specific T cell epitopes in two species of EBOV (Sudan and Zaire GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV, GPCAGDFAF and LYDRLASTV (Zaire EBOV could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma. Conclusion Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.

  5. The Role of P-Glycoprotein in Transport of Danshensu across the Blood-Brain Barrier

    Peng-Fei Yu

    2011-01-01

    Full Text Available Danshensu (3-(3, 4-dihydroxyphenyl lactic acid, a water-soluble active component isolated from the root of Salvia miltiorrhiza Bunge, is widely used for the treatment of cerebrovascular diseases. The present study aims to investigate the role of P-glycoprotein in transport of Danshensu across the blood-brain barrier. Sprague-Dawley rats were pretreated with verapamil at a dose of 20 mg kg−1 (verapamil group or the same volume of normal saline (control group. Ninety minutes later, the animals were administrated with Danshensu (15 mg kg−1 by intravenous injection. At 15 min, 30 min, and 60 min after Danshensu administration, the levels of Danshensu in the blood and brain were detected by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS. The results showed that Danshensu concentrations in the brain of the rats pretreated with verapamil were significantly increased. In addition, the brain-plasma ratios of the group pretreated with verapamil were much higher than that of the control group. There was no difference in Danshensu level in plasma between the verapamil group and control group. The findings indicated that Danshensu can pass the blood-brain barrier, and P-glycoprotein plays an important role in Danshensu transportation in brain.

  6. Vaccinia Virus Recombinant Expressing Herpes Simplex Virus Type 1 Glycoprotein D Prevents Latent Herpes in Mice

    Cremer, Kenneth J.; Mackett, Michael; Wohlenberg, Charles; Notkins, Abner Louis; Moss, Bernard

    1985-05-01

    In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

  7. A monoclonal antibody to the platelet membrane IIb-IIIa glycoprotein complex

    A hybridoma that secretes monoclonal mouse anti-platelet antibody has been produced by fusing spleen cells from immunized Balb/c mice with NS-1 myeloma cells using polyethylene glycol. Bulk production of the antibody was accomplished in vivo by injecting the cloned hybridoma cells intraperitoneally into pristane-primed mice. The antibody was purified from the ascitic fluid and characterised as belonging to the IgG1 sub-class by straphylococcal protein A-sepharose affinity chromatography. Purity was ascertained by SDS PAGE and isoelectric focusing. The antibody inhibited clot retraction of normal human blood and platelet aggregation induced by ADP, thrombin or epinephrine. Equilibrium binding studies revealed that the number of antibody molecules bound per platelet varied between 21 000 and 67 000 with an average of 42 000. Immunoprecipitation experiments with radioactively labelled platelets showed that the antibody bound the IIb-IIa glycoprotein complex. Platelets of patients with Glanzmann's thrombasthenia (a hereditary defect in which these glycoproteins are deficient) failed to bind the antibody. Immunofluorescent techniques showed that the antigen was present on megakaryocytes and platelets but not on the other cells from defibrinated peripheral blood. The antibody was also able to inhibit specifically the binding of radiolabelled human fibrinogen to ADP-stimulated platelets

  8. Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

    The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4+ and T8+ cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4+ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo

  9. Analysis of ER-associated glycoprotein degradation using synthetic glycopeptide probes

    Quality control of proteins is an essential process for maintaining normal cell activity. It ensures that only correctly folded proteins are produced and terminally misfolded proteins are eliminated by degradation. ER-associated degradation (ERAD) of misfolded proteins is an important aspect of protein quality control system. Recent studies have revealed that glycoprotein glycans play significant roles in this process. It includes polyubiquitination, deglycosylation, and proteasomal degradation. In the present study, a systematic analysis of these steps was carried out using chemically synthesized glycopeptides. We revealed that N-linked glycopeptides are degraded by 20S proteasome, but with drastically reduced rate compared to non-glycosylated peptide. This result strongly suggests that deglycosylating activity of peptide:N-glycanase (PNGase) is important for the facile degradation of glycoproteins. Our study showed, for the first time, that PNGase cleaves truncated glycans as short as chitobiose from peptide. However, this cleavage required the presence of hydrophobic region nearby N-glycosylation site. Furthermore, analysis of interactions with F-box protein Fbs1 was conducted with fluorescent correlation spectroscopy (FCS). It was shown that the presence of Fbs1 perturb the activity of PNGase toward high-mannose-type glycopeptides

  10. Silencing of P-glycoprotein increases mortality in temephos-treated Aedes aegypti larvae.

    Figueira-Mansur, J; Ferreira-Pereira, A; Mansur, J F; Franco, T A; Alvarenga, E S L; Sorgine, M H F; Neves, B C; Melo, A C A; Leal, W S; Masuda, H; Moreira, M F

    2013-12-01

    Re-emergence of vector-borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P-glycoproteins (P-gps) are ATP-dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P-glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P-gps. The basal ATPase activity of ATP-binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P-gp (AaegP-gp) gene expression was detected in temephos-treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP-gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)-mediated silencing of the AaegP-gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide-induced P-gp expression can be involved in the modulation of insecticide efflux. PMID:23980723

  11. Transient expression of Fc-fused human glycoprotein 130 in Expi293F suspension cells.

    Zhao, Xiaozhi; Chen, Wei; Ge, Liyuan; Jiang, Wei; Tang, Bo; Zhang, Qing; Xu, Xiaoyu; Wang, Chong; Cao, Lin; Guo, Hongqian

    2016-08-01

    Human glycoprotein 130 (gp130) is a signal-transducing receptor for interleukin 6 (IL-6), whose signaling plays a critical role in chronic inflammation and cancer. The soluble form of gp130 specifically inhibits IL-6 trans-signaling. However, achieving high-level expression of a large glycoprotein such as gp130 is difficult. Here, we designed and constructed one Fc-gp130-pcDNA mammalian expression vector, with the mouse IgG2a Fc fragment added to the N-terminus of human gp130, which greatly increased the secretion of recombinant gp130 protein from Expi293F suspension cells. Recombinant fusion Fc-gp130 was easily and efficiently purified from the supernatant of transfected cells by one-step affinity chromatography. Moreover, Fc-gp130 could automatically form dimers by the disulfide bond. Fc-gp130 was confirmed as a more efficient IL-6 trans-signaling blocker by its higher biological activity against signal transducer and activator of transcription 3 (STAT3). This purified active Fc-gp130 could be used to develop valuable therapeutic agents against inflammatory diseases and cancers. PMID:27113713

  12. INFLUENCE OF ALPHA-1-ACID GLYCOPROTEIN UPON PRODUCTION OF CYTOKINES BY PERIPHERAL BLOOD MONONUCLEARS

    М. V. Osikov

    2014-07-01

    Full Text Available Abstract. Alpha-1-acid glycoprotein (orosomucoid is a multifunctional acute phase reactant belonging to the family of lipocalines from plasma alpha-2 globulin fraction. In present study, we investigated dosedependent effects of orosomucoid upon secretion of IL-1â, IL-2, IL-3, IL-4 by mononuclear cells from venous blood of healthy volunteers. Mononuclear cells were separated by means of gradient centrifugation, followed by incubation for 24 hours with 250, 500, or 1000 mcg of orosomucoid per ml RPMI-1640 medium (resp., low, medium and high dose. The levels of cytokine production were assayed by ELISA technique. Orosomucoid-induced secretion of IL-1â and IL-4 was increased, whereas IL-3 secretion was inhibited. IL-2 production was suppressed at low doses of orosomucoid, and stimulated at medium and high doses. The effect of alpha-1-acid glycoprotein upon production of IL-2, IL-3 and IL-4 was dose-dependent. Hence, these data indicate that orosomucoid is capable of modifying IL-1â, IL-2, IL-3, and IL-4 secretion by blood mononuclear cells.

  13. β2-Glycoprotein I Is a Cofactor for t-PA–Mediated Plasminogen Activation

    Bu, Chunya; Gao, Lei; Xie, Weidong; Zhang, Jainwei; He, Yuhong; Cai, Guoping; McCrae, Keith R

    2010-01-01

    Regulation of the conversion of plasminogen to plasmin by tissue-type plasminogen activator (t-PA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. Here, we report that the abundant plasma glycoprotein, β2-glycoprotein I (β2GPI), stimulates t-PA–dependent plasminogen activation in the fluid phase and within a fibrin gel. The region within β2GPI responsible for stimulating t-PA activity is at least partially contained within β2GPI domain V. β2GPI bound t-PA with high affinity (Kd ~ 20 nM), stimulated t-PA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (kcat/Km) of t-PA–mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of β2GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following β2GPI repletion. Finally, stimulation of t-PA–mediated plasminogen activity by β2GPI was inhibited by monoclonal anti-β2GPI antibodies, as well as by anti-β2GPI antibodies from patients with antiphospholipid syndrome (APS). These findings suggest that β2GPI may be an endogenous regulator of fibrinolysis. Impairment of β2GPI-stimulated fibrinolysis by anti-β2GPI antibodies may contribute to the development of thrombosis in patients with APS. PMID:19180513

  14. A Potato cDNA Encoding a Homologue of Mammalian Multidrug Resistant P-Glycoprotein

    Wang, W.; Takezawa, D.; Poovaiah, B. W.

    1996-01-01

    A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with S-15-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170-180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.

  15. Initial characterization of CST1, a Toxoplasma gondii cyst wall glycoprotein.

    Zhang, Y W; Halonen, S K; Ma, Y F; Wittner, M; Weiss, L M

    2001-01-01

    Toxoplasma gondii is an important protozoan pathogen of humans that can cause encephalitis in immunocompromised individuals such as those with AIDS. This encephalitis is due to reactivation of latent infection in T. gondii-seropositive patients. Latent organisms survive within tissue cysts, which are specialized parasitophorous vacuoles containing bradyzoites. The cyst wall of this structure is produced by modification of the parasitophorous vacuole by the parasite and is important in cyst survival. The components of the cyst wall have been poorly characterized. By using immunofluorescence and immunoelectron microscopy, we have identified a monoclonal antibody (MAb 93.18) that reacts with the cyst wall. This antibody recognizes a 116-kDa glycoprotein, which we have termed CST1, containing sugar residues that bind Dolichos biflorans lectin (DBA). CST1 is distinct from T. gondii antigen labeled with succinyl Triticum vulgare lectin (S-WGA) and represents the major DBA-binding component in T. gondii. The carbohydrate components of the tissue cyst, such as CST1, are probably important in both providing stability and facilitating persistence in its host. As is seen in the carbohydrate capsules of fungi, glycoproteins in the T. gondii cyst wall may protect cysts from the immune response of the host. Further characterization of the formation of the cyst wall and its components should lead to insights into the mechanism of tissue cyst persistence and may suggest novel therapeutic approaches to eliminate tissue cysts of this organism. PMID:11119543

  16. Development of a radioimmunoassay for human milk-fat-globule membrane glycoprotein

    A radioimmunoassay (RIA) was developed for the quantitative measurement of a glycoprotein which was purified from human milk-fat-globule membrane (MFGM) and termed MFGM-pg 70. The assay with a sensitivity of detecting 30 ng of MFGM-pg 70/ml was employed to quantitate the levels of MFGM-gp 70 shed in supernatants from primary cultures of normal and malignant human breast cells and from various established cell lines of human mammary including myoepithelial and fibroblast and non-mammary malignant epithelial cells. The RIA for MFGM-gp 70 showed that the amount of antigen shed was much higher in supernatants from normal mammary epithelial cells compared with their malignant counterparts grown in primary culture and with those from established cell lines of malignant mammary epithelial cells. The levels of MFGM-pg 70 in spent media were unaffected by the lactogenic hormones such as prolactin or insulin. The RIA for MFGM-gp 70 provides a sensitive and quantitative means to in vitro study the synthesis of a membrane glycoprotein from human mammary epithelium. (Auth.)

  17. Partial Characterization of a Vicilin-Like Glycoprotein from Seeds of Flowering Tobacco (Nicotiana sylvestris

    Jared Q. Gerlach

    2009-01-01

    Full Text Available A vicilin-like glycoprotein from the seeds of Nicotiana sylvestris, flowering tobacco, has been identified using nanoLC/ESI-MS/MS. Sequences from a fragment of protein demonstrated homology with vicilins from other members of the Solanaceae family, notably potato (Solanum demissum. Reducing and nonreducing SDS-PAGE analyses of the identified protein indicated that fragments resulting from in situ proteolytic processing are joined by intrachain disulphide bonds. Staining with Con A lectin was specifically inhibited by mannose suggested the presence of N-linked glycosylation which was confirmed by carbohydrate compositional analysis of PVDF-bound protein subunits. HPAEC-PAD analysis of the monosaccharides released from the glycoprotein by acid hydrolysis revealed glucosamine and mannose. N-acetylglucosamine termination of attached oligosaccharides was further verified by inhibitable WGA lectin staining. Immunostaining of PVDF-bound N. sylvestris proteins with antibodies against G. max total protein demonstrated cross-staining at masses corresponding to fragments from the proteolytically processed protein subunits.

  18. The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins

    Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with 125I-lectins, and by the metabolic incorporation of L-(3H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with 125I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells

  19. A reassessment of the evolutionary timescale of bat rabies viruses based upon glycoprotein gene sequences.

    Kuzmina, Natalia A; Kuzmin, Ivan V; Ellison, James A; Taylor, Steven T; Bergman, David L; Dew, Beverly; Rupprecht, Charles E

    2013-10-01

    Rabies, an acute progressive encephalomyelitis caused by viruses in the genus Lyssavirus, is one of the oldest known infectious diseases. Although dogs and other carnivores represent the greatest threat to public health as rabies reservoirs, it is commonly accepted that bats are the primary evolutionary hosts of lyssaviruses. Despite early historical documentation of rabies, molecular clock analyses indicate a quite young age of lyssaviruses, which is confusing. For example, the results obtained for partial and complete nucleoprotein gene sequences of rabies viruses (RABV), or for a limited number of glycoprotein gene sequences, indicated that the time of the most recent common ancestor (TMRCA) for current bat RABV diversity in the Americas lies in the seventeenth to eighteenth centuries and might be directly or indirectly associated with the European colonization. Conversely, several other reports demonstrated high genetic similarity between lyssavirus isolates, including RABV, obtained within a time interval of 25-50 years. In the present study, we attempted to re-estimate the age of several North American bat RABV lineages based on the largest set of complete and partial glycoprotein gene sequences compiled to date (n = 201) employing a codon substitution model. Although our results overlap with previous estimates in marginal areas of the 95 % high probability density (HPD), they suggest a longer evolutionary history of American bat RABV lineages (TMRCA at least 732 years, with a 95 % HPD 436-1107 years). PMID:23839669

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

    Sai Li

    2016-02-01

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

  1. The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release.

    Spiegel, Martin; Plegge, Teresa; Pöhlmann, Stefan

    2016-01-01

    Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle. PMID:27455305

  2. The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release

    Martin Spiegel

    2016-07-01

    Full Text Available Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV and severe fever with thrombocytopenia syndrome virus (SFTSV as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV. Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle.

  3. Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

    Arthur, L.O.; Pyle, S.W.; Nara, P.L.; Bess, J.W. Jr.; Gonda, M.A.; Kelliher, J.C.; Gilden, R.V.; Robey, W.G.; Bolognesi, D.P.; Gallo, R.C.

    1987-12-01

    The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4/sup +/ and T8/sup +/ cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4/sup +/ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.

  4. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry

    Jennifer Pasquier

    2015-06-01

    Full Text Available The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp. The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading, we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

  5. Controlled viral glycoprotein expression as a safety feature in a bivalent rabies-ebola vaccine.

    Papaneri, Amy B; Bernbaum, John G; Blaney, Joseph E; Jahrling, Peter B; Schnell, Matthias J; Johnson, Reed F

    2015-02-01

    Using a recombinant rabies (RABV) vaccine platform, we have developed several safe and effective vaccines. Most recently, we have developed a RABV-based ebolavirus (EBOV) vaccine that is efficacious in nonhuman primates. One safety feature of this vaccine is the utilization of a live but replication-deficient RABV construct. In this construct, the RABV glycoprotein (G) has been deleted from the genome, requiring G trans complementation in order for new infectious viruses to be released from the initial infected cell. Here we analyze this safety feature of the bivalent RABV-based EBOV vaccine comprised of the G-deleted RABV backbone expressing EBOV glycoprotein (GP). We found that, while the level of RABV genome in infected cells is equivalent regardless of G supplementation, the production of infectious virus is indeed restricted by the lack of G, and most importantly, that the presence of EBOV GP does not substitute for G. These findings further support the safety profile of this replication-deficient RABV-EBOV bivalent vaccine. PMID:25481284

  6. Immunogenicity and functional characterization of Leishmania-derived hepatitis C virus envelope glycoprotein complex

    Grzyb, Katarzyna; Czarnota, Anna; Brzozowska, Agnieszka; Cieślik, Anna; Rąbalski, Łukasz; Tyborowska, Jolanta; Bieńkowska-Szewczyk, Krystyna

    2016-01-01

    Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are the main inducers of a cross-neutralizing antibody response which plays an important role in the early phase of viral infection. Correctly folded and immunologically active E1E2 complex can be expressed in mammalian cells, though the production process might still prove restrictive, even if the immunological response of a vaccine candidate is positive. Here, we report a characterization and immunogenicity study of a full-length (fE1E2) and soluble version of the E1E2 complex (tE1E2) from genotype 1a, successfully expressed in the cells of Leishmania tarentolae. In a functional study, we confirmed the binding of both Leishmania-derived E1E2 complexes to the CD-81 receptor and the presence of the major epitopes participating in a neutralizing antibody response. Both complexes were proved to be highly immunogenic in mice and elicited neutralizing antibody response. Moreover, cross-reactivity of the mouse sera was detected for all tested HCV genotypes with the highest signal intensity observed for genotypes 1a, 1b, 5 and 6. Since the development of a prophylactic vaccine against HCV is still needed to control the global infection, our Leishmania-derived E1E2 glycoproteins could be considered a potential cost-effective vaccine candidate. PMID:27481352

  7. Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity

    Halaban, R.; Moellmann, G. (Yale Univ. School of Medicine, New Haven, CT (USA))

    1990-06-01

    Melanogenesis is regulated in large part by tyrosinase, and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmentated (B{sup lt}/B{sup lt}) coat versus the wild-type black (B/B). The authors show that the b locus codes for a glycoprotein with the activity of a catalase (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, they conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. The studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the B{sup lt} mutation renders the protein susceptible to rapid proteolytic degradation.

  8. Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity

    Melanogenesis is regulated in large part by tyrosinase, and defective tyrosinase leads to albinism. The mechanisms for other pigmentation determinants (e.g., those operative in tyrosinase-positive albinism and in murine coat-color mutants) are not yet known. One murine pigmentation gene, the brown (b) locus, when mutated leads to a brown (b/b) or hypopigmentated (Blt/Blt) coat versus the wild-type black (B/B). The authors show that the b locus codes for a glycoprotein with the activity of a catalase (catalase B). Only the c locus protein is a tyrosinase. Because peroxides may be by-products of melanogenic activity and hydrogen peroxide in particular is known to destroy melanin precursors and melanin, they conclude that pigmentation is controlled not only by tyrosinase but also by a hydroperoxidase. The studies indicate that catalase B is identical with gp75, a known human melanosomal glycoprotein; that the b mutation is in a heme-associated domain; and that the Blt mutation renders the protein susceptible to rapid proteolytic degradation

  9. Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus

    Ghiasi, Seyed Mojtaba; Salmanian, A H; Chinikar, S;

    2011-01-01

    glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed......A antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way...

  10. An Essential Role for the Proximal but Not the Distal Cytoplasmic Tail of Glycoprotein M in Murid Herpesvirus 4 Infection

    May, Janet S.; Smith, Christopher M.; Michael B Gill; Stevenson, Philip G.

    2008-01-01

    Murid herpesvirus-4 (MuHV-4) provides a tractable model with which to define common, conserved features of gamma-herpesvirus biology. The multi-membrane spanning glycoprotein M (gM) is one of only 4 glycoproteins that are essential for MuHV-4 lytic replication. gM binds to gN and is thought to function mainly secondary envelopment and virion egress, for which several predicted trafficking motifs in its C-terminal cytoplasmic tail could be important. We tested the contribution of the gM cytopl...

  11. Duck Enteritis Virus Glycoprotein D and B DNA Vaccines Induce Immune Responses and Immunoprotection in Pekin Ducks

    Zhao, Yan; Cao, Yongsheng; Cui, Lihong; Ma, Bo; Mu, Xiaoyu; Li, Yanwei; Zhang, Zhihui; Li, Dan; Wei, Wei; Gao, Mingchun; Wang, Junwei

    2014-01-01

    DNA vaccine is a promising strategy for protection against virus infection. However, little is known on the efficacy of vaccination with two plasmids for expressing the glycoprotein D (gD) and glycoprotein B (gB) of duck enteritis virus (DEV) in inducing immune response and immunoprotection against virulent virus infection in Pekin ducks. In this study, two eukaryotic expressing plasmids of pcDNA3.1-gB and pcDNA3.1-gD were constructed. Following transfection, the gB and gD expressions in DF1 ...

  12. Re-evaluation of the role of P-glycoprotein in in vitro drug permeability studies with the bovine brain microvessel endothelial cells.

    Hakkarainen, Jenni J; Rilla, Kirsi; Suhonen, Marjukka; Ruponen, Marika; Forsberg, Markus M

    2014-03-01

    1.  Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2.  BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3.  P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4.  In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates. PMID:23924297

  13. Identification of the Receptor-Binding Domain of the Spike Glycoprotein of Human Betacoronavirus HKU1

    Ou, Xiuyuan; Góes, Luiz Gustavo Bentim; Osborne, Christina; Castano, Anna; Holmes, Kathryn V.

    2015-01-01

    ABSTRACT Coronavirus spike (S) glycoproteins mediate receptor binding, membrane fusion, and virus entry and determine host range. Murine betacoronavirus (β-CoV) in group A uses the N-terminal domain (NTD) of S protein to bind to its receptor, whereas the β-CoVs severe acute respiratory syndrome CoV in group B and Middle East respiratory syndrome CoV in group C and several α-CoVs use the downstream C domain in their S proteins to recognize their receptor proteins. To identify the receptor-binding domain in the spike of human β-CoV HKU1 in group A, we generated and mapped a panel of monoclonal antibodies (MAbs) to the ectodomain of HKU1 spike protein. They did not cross-react with S proteins of any other CoV tested. Most of the HKU1 spike MAbs recognized epitopes in the C domain between amino acids 535 and 673, indicating that this region is immunodominant. Two of the MAbs blocked HKU1 virus infection of primary human tracheal-bronchial epithelial (HTBE) cells. Preincubation of HTBE cells with a truncated HKU1 S protein that includes the C domain blocked infection with HKU1 virus, but preincubation of cells with truncated S protein containing only the NTD did not block infection. These data suggest that the receptor-binding domain (RBD) of HKU1 spike protein is located in the C domain, where the spike proteins of α-CoVs and β-CoVs in groups B and C bind to their specific receptor proteins. Thus, two β-CoVs in group A, HKU1 and murine CoV, have evolved to use different regions of their spike glycoproteins to recognize their respective receptor proteins. IMPORTANCE Mouse hepatitis virus, a β-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another β-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory β-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the

  14. The inhibitory and combinative mechanism of HZ08 with P-glycoprotein expressed on the membrane of Caco-2 cell line

    Zhang, Yanyan; Hu, Yahui; Feng, Yidong; Kodithuwakku, Nandani Darshika; Fang, Weirong [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Li, Yunman, E-mail: yunmanlicpu@hotmail.com [State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing 210009 (China); Huang, Wenlong [Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009 (China)

    2014-01-15

    Recently, the research and development of agents to reverse the phenomenon of multidrug resistance has been an attractive goal as well as a key approach to elevating the clinical survival of cancer patients. Although three generations of P-glycoprotein modulators have been identified, poor clearance and metabolism render these agents too toxic to be used in clinical application. HZ08, which has been under investigation for several years, shows a dramatic reversal effect with low cytotoxicity. For the first time, we aimed to describe the interaction between HZ08 and P-glycoprotein in Caco-2 cell line in which P-glycoprotein is overexpressed naturally. Cytotoxicity and multidrug resistance reversal assays, together with flow cytometry, fluorescence microscopy and siRNA interference as well as Caco-2 monolayer transport model were employed in this study to evaluate the interaction between HZ08 and P-glycoprotein. This study revealed that HZ08 was capable of reversing adriamycin resistance mediated by P-glycoprotein as a result of intracellular enhancement of adriamycin accumulation, which was found to be superior to verapamil. In addition, we confirmed that HZ08 suppressed the transport of Rhodamine123 in the Caco-2 monolayer model but had little effect on P-glycoprotein expression. The transport of HZ08 was diminished by P-glycoprotein inhibitors (verapamil and LY335979) and its accumulation was increased via siRNA targeting MDR1 in Caco-2 cells. Furthermore, considering the binding site of P-glycoprotein, verapamil performed as a competitive inhibitor with HZ08. In conclusion, as a P-glycoprotein substrate, HZ08 inhibited P-glycoprotein activity and may share the same binding site of verapamil to P-glycoprotein. - Highlights: • The cytotoxicity and reversing effect of HZ08 was measured in Caco-2 cell line. • HZ08 inhibited the transport of Rhodamine123 across Caco-2 cell monolayer. • The efflux ratio of HZ08 was dropped when combined with P-glycoprotein

  15. Complementation of the Function of Glycoprotein H of Human Herpesvirus 6 Variant A by Glycoprotein H of Variant B in the Virus Life Cycle

    Oyaizu, Hiroko; Tang, Huamin; Ota, Megumi; Takenaka, Nobuyuki; Ozono, Keiichi; Yamanishi, Koichi

    2012-01-01

    Human herpesvirus 6 (HHV-6) is a T-cell-tropic betaherpesvirus. HHV-6 can be classified into two variants, HHV-6 variant A (HHV-6A) and HHV-6B, based on genetic, antigenic, and cell tropisms, although the homology of their entire genomic sequences is nearly 90%. The HHV-6A glycoprotein complex gH/gL/gQ1/gQ2 is a viral ligand that binds to the cellular receptor human CD46. Because gH has 94.3% amino acid identity between the variants, here we examined whether gH from one variant could complement its loss in the other. Recently, we successfully reconstituted HHV-6A from its cloned genome in a bacterial artificial chromosome (BAC) (rHHV-6ABAC). Using this system, we constructed HHV-6ABAC DNA containing the HHV-6B gH (BgH) gene instead of the HHV-6A gH (AgH) gene in Escherichia coli. Recombinant HHV-6ABAC expressing BgH (rHHV-6ABAC-BgH) was successfully reconstituted. In addition, a monoclonal antibody that blocks HHV-6B but not HHV-6A infection neutralized rHHV-6ABAC-BgH but not rHHV-6ABAC. These results indicate that HHV-6B gH can complement the function of HHV-6A gH in the viral infectious cycle. PMID:22647694

  16. Interaction of asialo von Willebrand factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation

    Von Willebrand factor (vWF) is necessary for the initial attachment of platelets to exposed subendothelium, particularly under flow conditions like those prevailing in the microcirculation. Little is known about its possible participation in subsequent events leading to formation of platelet thrombi at sites of vascular injury. The authors addressed this question by studying the mechanisms by which desialylated vWF induces platelet aggregation in the absence of any other stimulus. Asialo vWF, unlike the native molecule, does not require ristocetin to interact with platelets. They have shown here that binding of asialo vWF to platelets was accompanied by release of dense granule content and subsequent ADP-dependent fibrinogen binding to receptors on the glycoprotein (GP) IIb/IIIa complex. The initial interaction of asialo vWF with platelets was mediated by GPIb, as shown by blocking obtained with monoclonal antibody. Inhibition of this initial interaction completely abolished platelet aggregation induced by asialo vWF. This, however, did not block asialo vWF binding to platelets, but rather inhibited subsequent fibrinogen binding induced by asialo vWF. Therefore, the latter process was also essential for platelet aggregation under the conditions described. This study shows that asialo, and possibly native, vWF acts as a platelet agonist after its binding to GPIb and induces aggregation through a pathway dependent on GPIIb/IIIa-related receptors

  17. Enzymatic sulfation of gastric mucous glycoprotein in rat--changes in glycoprotein sulfotransferase activity with stress and anti-ulcer agent, sofalcone

    Enzymatic sulfation of mucous glycoprotein (GP) was studied in gastric mucosa of rat. After rat stomach was incubated with [35S]-sulfate, incorporation of radioactivity into gastric mucosal APS (adenosine 5'-phosphosulfate), PAPS (3'-phosphoadenosine 5'-phosphosulfate) and endogenous GPs could be detected. The degree of sulfation of endogenous GPs was highest in the macromolecular GP (peak I) and lowest in the low molecular GP (peak III). By using a crude preparation of GP sulfotransferase from rat gastric mucosa, the transfer of [35S]-sulfate from [35S]-PAPS into macromolecular mucous GP was determined as being the activity of sulfotransferase. The activity of GP sulfotransferase was mainly distributed in the microsomal fraction, and was proportional to the incubation time, substrate (mucous GP) concentration and [35S]-PAPS concentration. The enzyme activity was significantly higher in the corpus than that in the antral mucosa. The activity of GP sulfotransferase was significantly decreased at 6 h and was significantly increased at 12 h after the stress load, compared with that of the non-stressed condition. Anti-ulcer agent, sofalcone, increased the GP sulfotransferase activity under the stressed condition. On the other hand, cimetidine showed a significant inhibitory effect under the same condition. Changes in the GP sulfotransferase activity with stress and anti-ulcer agents were consistent with those in the incorporation of [35S]-sulfate into macromolecular mucous GP. These results suggest the importance of GP sulfotransferase as a key enzyme regulating the sulfation of mucous GP

  18. Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.

    Huemer, H P; Wang, Y; Garred, P; Koistinen, V; Oppermann, S

    1993-08-01

    Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR

  19. Bovine herpesvirus 4 glycoprotein B is indispensable for lytic replication and irreplaceable by VSVg

    Franceschi Valentina

    2013-01-01

    Full Text Available Abstract Background Bovine herpesvirus 4 (BoHV-4 is a gammaherpesvirus, belonging to Rhadinovirus genus, with no clear association with disease. However, there is increasing evidence of its secondary pathogenic role in cases of post-partum metritis in cattle. BoHV-4 Open Reading Frame 8 (ORF8 codifies for glycoprotein B (gB that shows a heterodimeric structure, composed of two subunits and covalently linked by disulfide bonds and responsible for host cell adhesion through binding to heparan sulfates associated with cellular proteoglycans. Here we describe the generation of several tagged soluble forms of gB ectodomain, in order to test their ability to neutralize BoHV-4 infection. Results The results show, however, that none of these soluble forms are able to block viral infectivity. To better understand the role of gB during BoHV-4 lytic replication, a recombinant BoHV-4 was generated by homologous recombination from a BoHV-4 cloned as a Bacterial artificial chromosome (BAC (pBAC-BoHV-4-A, in which most of the BoHV-4 gB ORF was substituted by the insertion of a DNA stuffer selectable cassette. The resulting recombinant BoHV-4 genome (pBAC-BoHV-4-AΔgB-KanaGalK was completely unable to reconstitute infectious replicating viral particles (Infectious Replicating Viral Particles, IRVPs and to replicate when transfected in permissive cell lines in comparison to its revertant clone (pBAC-BoHV-4-ΔgB-Rev or pBAC-BoHV-4-A parental clone. Conclusion This demonstrates that the BoHV-4 replicating cycle is dependent on gB. Moreover, when gB was deleted from a recombinant BoHV-4 genome delivering an heterologous glycoprotein, Vesicular Stomatitis Virus Glycoprotein (VSVg, VSVg was unable to complement gB. This study provides direct evidence that gB is necessary for BoHV-4 lytic replication.

  20. Characterization of intramolecular disulfide bonds and secondary modifications of the glycoprotein from viral hemorrhagic septicemia virus, a fish rhabdovirus

    Einer-Jensen, Katja; Nielsen, Thomas Krogh; Roepstorff, Peter; Lorenzen, Niels

    1998-01-01

    Viral hemorrhagic septicemia virus (VHSV) infections cause high losses in cultured rainbow trout in Europe. Attempts to produce a recombinant vaccine based on the transmembrane glycoprotein (G protein) have indicated that proper folding is important for the antigenicity and immunogenicity of the...

  1. Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation

    Sharma, Pawan; Tran, Thai; Stelmack, Gerald L; McNeill, Karol; Gosens, Reinoud; Mutawe, Mark M; Unruh, Helmut; Gerthoffer, William T; Halayko, Andrew J

    2008-01-01

    Airway smooth muscle (ASM) cells may contribute to asthma pathogenesis through their capacity to switch between a synthetic/proliferative and a contractile phenotype. The multimeric dystrophin-glycoprotein complex (DGC) spans the sarcolemma, linking the actin cytoskeleton and extracellular matrix. T

  2. Hydroxyproline-rich glycoproteins accumulate in pearl millet after seed treatment with elicitors of defense responses against Sclerospora graminicola

    Sujeeth, Neerakkal; Deepak, Shantharaj; Shailasree, Sekhar; Kini, Ramachandra K.; Shetty, Shekar H.; Hille, Jacques

    2010-01-01

    The accumulation of hydroxyproline-rich glycoproteins (HRGPs) was investigated after induction of resistance in pearl millet against downy mildew caused by Sclerospora graminicola. Treatment of susceptible pearl millet seeds with various biotic and abiotic elicitors resulted in increased HRGP conten

  3. Differential profiling studies of N-linked glycoproteins in glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor.

    Dai, Lan; Liu, Yashu; He, Jintang; Flack, Callie G; Talsma, Caroline E; Crowley, Jessica G; Muraszko, Karin M; Fan, Xing; Lubman, David M

    2011-10-01

    We have recently demonstrated that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes cancer stem cells (CSCs) in Glioblastoma Multiforme (GBM) through reduced proliferation and induced apoptosis. However, the detailed mechanism by which the manipulation of Notch signal induces alterations on post-translational modifications such as glycosylation has not been investigated. Herein, we present a differential profiling work to detect the change of glycosylation pattern upon drug treatment in GBM CSCs. Rapid screening of differential cell surface glycan structures has been performed by lectin microarray on live cells followed by the detection of N-linked glycoproteins from cell lysates using multi-lectin chromatography and label-free quantitative mass spectrometry analysis. A total of 51 and 52 glycoproteins were identified in the CSC- and GSI-treated groups, respectively, filtered by a combination of decoy database searching and Trans-Proteomic Pipeline (TPP) processing. Although no significant changes were detected from the lectin microarray experiment, 7 differentially expressed glycoproteins with high confidence were captured after the multi-lectin column including key enzymes involved in glycan processing. Functional annotations of the altered glycoproteins suggest a phenotype transformation of CSCs toward a less tumorigenic form upon GSI treatment. PMID:21898824

  4. Effect of dietary fat on hepatic liver X receptor expression in P-glycoprotein deficient mice: implications for cholesterol metabolism

    Lee Stephen D

    2008-06-01

    Full Text Available Abstract Pgp (P-glycoprotein, MDR1, ABCB1 is an energy-dependent drug efflux pump that is a member of the ATP-binding cassette (ABC family of proteins. Preliminary studies have reported that nonspecific inhibitors of Pgp affect synthesis and esterification of cholesterol, putatively by blocking trafficking of cholesterol from the plasma membrane to the endoplasmic reticulum, and that relative increases in Pgp within a given cell type are associated with increased accumulation of cholesterol. Several key efflux proteins involved in the cholesterol metabolic pathway are transcriptionally regulated by the nuclear hormone liver X receptor (LXR. Therefore, to examine the interplay between P-glycoprotein and the cholesterol metabolic pathway, we utilized a high fat, normal cholesterol diet to upregulate LXRα without affecting dietary cholesterol. Our research has demonstrated that mice lacking in P-glycoprotein do not exhibit alterations in hepatic total cholesterol storage, circulating plasma total cholesterol levels, or total cholesterol concentration in the bile when compared to control animals on either a normal (25% calories from dietary fat or high fat (45% calories from dietary fat diet. However, p-glycoprotein deficient mice (Mdr1a-/-/1b-/- exhibit increased hepatic LXRα protein expression and an elevation in fecal cholesterol concentration when compared to controls.

  5. In-vitro evaluation of the P-glycoprotein interactions of a series of potentially CNS-active Amaryllidaceae alkaloids

    Eriksson, André Huss; Rønsted, Nina; Jäger, Anna Katharina; Sendra, Júlia Rodríguez; Brodin, Birger

    2012-01-01

    Drug compounds interacting with the blood-brain barrier efflux transporter P-glycoprotein (P-gp) might have limited access to brain tissue. The aim of the present study was to evaluate whether nine potentially CNS-active Amaryllidaceae alkaloids of the crinine, lycorine and galanthamine types...... interact with P-gp....

  6. Structural and functional studies on a unique linear neutralizing antigenic site (G5) of the rabies virus glycoprotein.

    R.W.J. van der Heijden (Roger); J.P.M. Langedijk; J. Groen (Jan); F.G.C.M. Uytdehaag (Fons); R.H. Meloen; A.D.M.E. Osterhaus (Albert)

    1993-01-01

    textabstractThe core of a unique linear neutralization epitope (G5) on the glycoprotein of rabies virus, recognized by a virus-neutralizing mouse monoclonal antibody (MAb 6-15C4), was determined by Pepscan analysis. The G5 epitope was defined as an octapeptide (LHDFRSDE). The contribution of the ind

  7. Characterization of a new virus-neutralizing epitope that denotes a sequential determinant on the rabies virus glycoprotein.

    H. Bunschoten; M. Gore (Milind); I.J.Th.M. Claassen (Ivo); F.G.C.M. Uytdehaag (Fons); B. Dietzschold; W.H. Wunner; A.D.M.E. Osterhaus (Ab)

    1989-01-01

    textabstractTwo new monoclonal antibodies (MAbs) derived from mice immunized with the Pitman-Moore (PM) strain of rabies virus were used to identify and characterize two unique antigenic determinants on the rabies virus glycoprotein. One of the determinants, which defined an additional antigenic sit

  8. A new in vivo method to study P-glycoprotein transport in tumors and the blood-brain barrier

    Hendrikse, NH; de Vries, EGE; Eriks-Fluks, L; van der Graaf, WTA; Hospers, GAP; Willemsen, ATM; Vaalburg, W; Franssen, EJF

    1999-01-01

    Drug resistance is a major cause of chemotherapy failure in cancer treatment, One reason is the overexpression of the drug efflux pump P-glycoprotein (P-gp), involved in multidrug resistance (MDR), In vivo pharmacokinetic analysis of P-gp transport might identify the capacity of modulation by P-gp s

  9. Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk

    Wellenberg, G.J.; Verstraten, E.; Belak, S.; Verschuren, S.B.E.; Rijsewijk, F.A.M.; Peshev, R.; Oirschot, van J.T.

    2001-01-01

    A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were

  10. Purification and Partial Characterization of a Novel Anti-tumor Glycoprotein from Cultured Mycelia of Grifola frondosa.

    Cui, Fengjie; Zan, Xinyi; Li, Yunhong; Yang, Yan; Sun, Wenjing; Zhou, Qiang; Yu, Silian; Dong, Ying

    2013-10-25

    A novel glycoprotein GFG-3a with the molecular weight of 88.01 kDa and potent anti-tumor activity was isolated from the cultured mycelia of Grifola frondosa GF9801. GFG-3a was heat-sensitive with the decreasing anti-proliferative activity after treated from 56°C to 100°C for 10-120min. GFG-3a was a glycoprotein with O-glycosylation and contained 6.20% carbohydrate composed of D-arabinose, D-fructose, D-mannose, and D-glucose with a molar ratio of 1.33:4.51:2.46:1.00. FT-IR and NMR spectra proved that GFG-3a contained protein and carbohydrate portions with 3-O-methyl-galactose residues, (1→4)-linked β- galactose residues, and β-linked glucose residues. Circular dichroism (CD) revealed that GFG-3a was a predominantly β-sheet glycoprotein with a relatively small α-helical content. Protein sequencing and 3D model of GFG-3a were finally obtained by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODLE Workspace service. Our findings will be a reference for the further structure-activity relationship analysis of the mushroom glycoproteins. PMID:24512992

  11. Studies on glycoproteins produced by wild type and wheat germ agglutinin-resistant B16 mouse melanoma cells

    Two variants of B16 mouse melanoma cells have been selected in serum-free medium for their resistance to toxic levels of wheat germ agglutinin isolation 1 (WGA). Chromosome analysis and characteristic melanin production showed that the variants are derived from the parent mouse melanoma cell lines. However, the two variants were less tumorigenic in mice compared to the parent B16 mouse melanoma cells. The variants showed a marked decrease in cell agglutination with WGA. Cell agglutination with recin and peanut lectin was not different between the three cell lines, but the two variants showed a slight increase in agglutination with concanavalin A. The binding of 125I-labeled wheat germ agglutinin to the two variant cells was reduced compared to that of the parent cell. Glycoproteins secreted or shed by the three lines were isolated after growth in serum-free medium in the presence of [3He]glucosamine and bovine serum albumin (1%). These metabolically labeled products were fractionated on the basis of their interaction with WGA-Sepharose (2 mg/ml). The WGA-Sepharose affinity chromatographic data suggested a decrease in WGA-binding glycoprotein(s) secreted to the medium by the two variants. The WGA-bound glycoproteins from the two variants upon SDS-PAGE revealed three bands of approximate molecular weights, 92,000, 56,000, and 42,000, none of which were present in the parent cell line (50,000 molecular weight)

  12. CLASSICAL AND NOVEL FORMS OF MULTIDRUG-RESISTANCE AND THE PHYSIOLOGICAL FUNCTIONS OF P-GLYCOPROTEINS IN MAMMALS

    BORST, P; SCHINKEL, AH; SMIT, JJM; WAGENAAR, E; VANDEEMTER, L; SMITH, AJ; EIJDEMS, EWHM; BAAS, F; ZAMAN, GJR

    1993-01-01

    In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam. We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes. The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early deve

  13. Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics

    Yu-Hsuan Chiang

    2011-01-01

    Full Text Available In this study, a commercially available fluorescent dye, Lissamine rhodamine B sulfonyl hydrazine (LRSH, was designed to specifically stain the glycoproteins in polyacrylamide gels. Through the periodate/Schiff base mechanism, the fluorescent dye readily attaches to glycoproteins and the fluorescence can be simultaneously observed under either 305 nm or 532 nm excitation therefore, the dye-stained glycoproteins can be detected under a regular UV transilluminator or a more elegant laser-based gel scanner. The specificity and detection limit were examined using a standard protein mixture in polyacrylamide gels in this study. The application of this glycoprotein stain dye was further demonstrated using pregnancy urine samples. The fluorescent spots were further digested in gel and their identities confirmed through LC-MS/MS analysis and database searching. In addition, the N-glycosylation sites of LRSH-labeled uromodulin were readily mapped via in-gel PNGaseF deglycosylation and LC-MS/MS analysis, which indicated that this fluorescent dye labeling does not interfere with enzymatic deglycosylation. Hence, the application of this simple and specific dual-wavelength excitable dye staining in current glycoproteome research is promising.

  14. Identification of a Novel Virulence Determinant Within the E2 Structural Glycoprotein of Classical Swine Fever Virus

    Classical Swine Fever Virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829-837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant Pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus ...

  15. Intraventricularly applied D-galactosamine inhibits the incorporation of [3H]-fucose into rat brain glycoproteins

    In the rat, intraventricularly applied D-galactosamine (GalN) (10 or 20 μmoles) inhibited [3H]-fucose incorporation into hippocampal and subcortical glycoproteins by appr. 50% of the controls. The inhibitory effect occurred within 40 min after GalN administration and did not reach normal values even 16 h upon GalN injection. During first hours upon GalN administration the inhibition of fucose incorporation involved predominantly water-soluble and Triton-soluble glycoproteins, whereas the Triton-resistant fraction showed a delayed suppression. The results suggest that GalN suppresses the formation of membrane glycoproteins (because only 15% of fucose radioactivity remained in water-soluble fraction) which are believed to play an essential role in formation of long-term memory. Under these aspects, the amnesic effect of GalN on the retention of a brightness discrimination and the abolishment by uridine treatment, as observed in our laboratory, may be interpreted as a further support to the particular role of glycoproteins in the consolidation of a memory trace. (author)

  16. Comparison of three tissue fixatives on the immunoreactivity of mammalian P-glycoprotein antibodies to teleost tissues

    Hemmer, M.J. [Environmental Protection Agency, Gulf Breeze, FL (United States)]|[Univ. of Mississippi, University, MS (United States). Dept. of Pharmacology; Courtney, L.A. [Environmental Protection Agency, Gulf Breeze, FL (United States); Benson, W.H. [Univ. of Mississippi, University, MS (United States)

    1994-12-31

    Mammalian P-glycoprotein is a highly conserved integral membrane protein functioning as an energy dependent plasma membrane efflux pump which decreases the concentration of certain lipophilic aromatic compounds entering the cell by diffusion. Studies indicate that P-glycoprotein is capable of increased expression in response to certain chemical stressors and has demonstrated the ability to transport xenobiotic contaminants. Expression of a xenobiotic transporter in teleost species could play a significant role in conferring resistance to fish populations exposed to xenobiotic stressors and may serve as a potential indicator of species at risk to environmental contaminants. Past studies demonstrated a strong correlation between corresponding mammalian and teleost tissues showing immunoreactivity to specific mammalian P-glycoprotein antibodies. In this study, comparisons of staining pattern, intensity, and tissue specificity between Lillie`s, Bouin`s and Dietrich`s fixed tissues was determined in the sheepshead minnow, Cyprinodon variegatus, using monoclonal antibodies (mAbs) C219, C494 and JSB-1. Immunoreactivity of the mAbs was found to be fixative-dependent and results are presented illustrating the differential staining patterns and tissue specificity observed for each tissue, fixative, and antibody combination. These data indicate tissue fixation has a significant impact on P-glycoprotein immunoreactivity in teleost tissues and must be considered in the comparison and interpretation of results.

  17. Effect of the Contents and Form of Rabies Glycoprotein on the Potency of Rabies Vaccination in Cattle

    Piza AT

    2002-01-01

    Full Text Available One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached of rabies glycoprotein (G in the vaccine batches, and the virus-neutralizing antibodies (VNA titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001 between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.

  18. Induction of anti-beta(2)-glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes

    Van Os, G. M. A.; Meijers, J. C. M.; Agar, C.; Seron, M. V.; Marquart, J. A.; Akesson, P.; Urbanus, R. T.; Derksen, R. H. W. M.; Herwald, H.; Morgelin, M.; De Groot, P. G.

    2011-01-01

    Background: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-beta 2-glycoprotein I (beta 2-GPI) autoantibodies. beta 2-GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholip

  19. SAR Studies on Tetrahydroisoquinoline Derivatives : The Role of Flexibility and Bioisosterism To Raise Potency and Selectivity toward P-glycoprotein

    Capparelli, Elena; Zinzi, Laura; Cantore, Mariangela; Contino, Marialessandra; Perrone, Maria Grazia; Luurtsema, Gert; Berardi, Francesco; Perrone, Roberto; Colabufo, Nicola A.

    2014-01-01

    The development of P-glycoprotein (P-gp) ligands remains of considerable interest, mostly for investigating the protein's structure and transport mechanism. In recent years, many different generations of ligands have been tested for their ability to modulate P-gp activity. The aim of the present wor

  20. (11) C- and (18) F-Labeled Radioligands for P-Glycoprotein Imaging by Positron Emission Tomography.

    Cantore, Mariangela; Benadiba, Marcel; Elsinga, Philippus; Kwizera, Chantal; Dierckx, Rudi; Colabufo, Nicola A.; Luurtsema, Geert

    2016-01-01

    Abstract P-Glycoprotein (P-gp) is an efflux transporter widely expressed at the human blood-brain barrier. It is involved in xenobiotics efflux and in onset and progression of neurodegenerative disorders. For these reasons, there is great interest in the assessment of P-gp expression and function by