Dissertations / Theses: 'Biochemistry of glycoproteins'

1

Jefferies, W. A. "Lymphocyte surface glycoproteins." Thesis, University of Oxford, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355757.

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2

Duffy, Iain. "Analysis of measles virus glycoproteins." Thesis, Queen's University Belfast, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324842.

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3

Wood, Sarah Louise. "Glycoproteins of the chromaffin granule membrane." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/19427.

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4

Howe, T. "Biosynthesis of serum glycoproteins by isolated rat hepatocytes." Thesis, Bucks New University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371224.

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5

Burdge, Graham Charles. "Biochemical analysis of proteolytic fragments from desmosomal glycoproteins." Thesis, University of Southampton, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.290426.

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6

Hemming, Richard John. "Radioautographical and biochemical studies on nucleoplasmic glycoproteins." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41298.

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EM radioautography was used to examine the tissue distribution of cells exhibiting nucleoplasmic labeling after being exposed to $ sp3$H-sugars or $ sp{35}$S-sulphate to indicate the general extent of the occurrence of nucleoplasmic glycoproteins within animal cells. The observation of some degree of such labeling in virtually all cells in tissues of three animal species suggests that nucleoplasmic glycoproteins are a common cellular feature. To better define the distribution and nature of the putative labeled nucleoplasmic glycoproteins, cultured cells were used as a model cell type for both quantitative EM radioautographic and biochemical studies. After exposure to $ sp3$H-sugars, all three lines of cultured cells examined exhibited significant nucleoplasmic reaction in which the euchromatin, heterochromatin and nucleoli were all labeled to some extent. Studies on isolated, envelope-depleted nuclei from myeloma cells confirmed that the molecules in the nucleoplasm itself were the source of the radioautographic reaction observed over nuclei. Biochemical analyses of fractions of isolated nuclei indicated that much of the label resided in nuclear matrix glycoproteins of different molecular weights. Lectin binding studies on nuclear matrix fractions revealed the presence of galactose, fucose, and/or sialic acid residues in proteins. Glycosidase experiments indicated that some but not all of these glycoproteins had N-linked sidechains.

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Smith, Kevin David. "The site-specific glycosylation patterns of serum and membrane glycoproteins." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315282.

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8

Brown, Vivienne Alison. "A study of cell surface glycoproteins in chronic lymphocytic leukaemia." Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/12092.

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9

Zamani, Mohammad Reza. "The role of glycoproteins in neural plasticity in domestic chick." Thesis, Open University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293893.

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10

Uysal, Hamdi. "Extracellular matrix glycoproteins of skin fibroblasts in tuberous sclerosis." Thesis, University of Nottingham, 1995. http://eprints.nottingham.ac.uk/13094/.

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The first main objective of this project was to isolate cellular fibronectin from cultures of skin fibroblasts derived from tuberous sclerosis (TS) patients and normals and to compare their structures, paying particular attention to the content and pattern of carbohydrate (glycosylation). The second main objective of this project was to establish the expression and localisation of glycoproteins fibronectin, laminin and tenascin of the extracellular matrix (ECM) in cultures of skin fibroblasts derived from patients with TS and normal individuals. In order to achieve these objectives, fibroblasts were established from primary cultures of skin explants of patients with TS. Control cells were cultured from skin explants donated by people not known to be suffering from any disorder. The purification of cellular fibronectin was achieved from conditioned medium of skin fibroblasts of TS patients and control fibroblasts using Prosep-gelatin affinity chromatography and gel filtration chromatography techniques. Analysis of purified cellular fibronectin by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) revealed that the carbohydrate portion of the fibronectin molecule was made up of galactose, mannose, glucosamine, galactosamine, sialic acid and fucose. An increased concentration of sialic acid, galactosamine, glucosamine, galactose and mannose was observed in purified fibronectin derived from neck and ungual fibromas of patients with TS. To provide a total increase of carbohydrates more than two fold in comparison to normal fibroblastsderived fibronectin. Purified cellular fibronectin from conditioned medium of fibroblasts grown from skin lesions of different TS patients and from normal skin fibroblasts did not express HNK-1 (anti-leu 7) carbohydrate epitope. Normal skin fibroblasts showed an altered morphology and less confluence when grown on cell culture plates coated with cellular fibronectin derived from TS fibroblasts compared with control fibronectin. This may be a consequence of an altered glycosylation of this protein. The amino acid composition of the purified fibronectin from TS fibroblasts was very similar to that purified fibronectins from normal fibroblasts and to standard commercial plasma and cellular fibronectins. Laminin and tenascin were partially purified from conditioned cell culture medium demonstrating their synthesis and secretion into the cell culture medium by dermal skin fibroblasts. Expression and distribution of fibronectin, tenascin and laminin by established TS and normal skin fibroblasts using immunofluorescence, ELISA, and flow cytometry techniques were analysed and presented qualitative and quantitatively in this thesis. Increased expression and altered distribution of fibronectin and tenascin were observed in the fibroblasts derived from ungual fibroma lesion of a TS patient, but not in fibroblasts of neck fibroma, forehead plaque lesion or unaffacted skin of TS patients in comparison to control fibroblasts. However, increased expression and altered distribution of laminin were observed in neck fibroma-derived fibroblasts in contrast to fibronectin and tenascin. Laminin expression was not changed in ungual fibroma and forehead plaque lesion-derived fibroblasts in comparison to control fibroblasts. Altered distribution of fibronectin was well observed by immunofluorescence particularly in large cells of ungual fibroma. Similar differences were observed with laminin of cells from neck fibroma of TS patients. These results suggest the abnormal assembly of ECM in different TS skin lesions. Abnormal migration of cells during early embryonic development and the hardening of tissues associated with TS may result from abnormal assembly of the ECM. Alterations in distribution and structure of these adhesive glycoproteins may cause functional disruption in their binding and interactions with cells and ECM macromolecules. Studies of these changes in the ECM components may contribute to the understanding of the mechanisms involved in the aetiology of hardened tissues of TS.

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Craig, Sarah Jane. "The analysis of complex glycoproteins by capillary electrophoresis and mass spectrometry." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283361.

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12

Rankine, Susan M. "Changes in glycosylation on antibodies between normal patients and those with rheumatoid arthritis." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.258151.

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13

Peiser, Leanne. "The role of the macrophage scavenger receptor in host defence." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393470.

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14

Youakim, Adel. "Tumour- and differentiation-associated changes in the carbohydrate structure of glycoproteins from human colonic cells." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75826.

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The structure of the carbohydrate associated with glycoproteins was examined in various human colonic tumour cells in culture following incubation with labeled sugars. Glycopeptides obtained by pronase digestion of the cell surface glycoproteins of three human colon tumours (HCT-8R, CaCo-2, and HCT-15) were compared with those from cells (CCL 239) derived normal adult colon. The tumour cells contain large molecular weight fucose-labeled glycopeptides that are absent in the CCL 239 cells. The large molecular weight fucose- and glucosamine-labeled glycopeptides from HCT-8R cells contain mainly mild alkali labile O-linked oligosaccharides, whereas those isolated from CaCo-2 cells contain primarily N-linked polylactosaminoglycans which bind to Datura stramonium agglutinin-agarose and are sensitive to endo-$ beta$-galactosidase.
Differentiation of CaCo-2 cells to polarized cells containing brush border enzymes characteristic of enterocytes is accompanied by a decrease in the relative proportion of fucose- and glucosamine-labeled N-linked polylactosaminoglycans-containing glycopeptides. These polylactosaminoglycans are found on a restricted set of glycoproteins of M$ sb{ rm r}$ 100,000-130,000. In undifferentiated cells, these glycoproteins contain a greater proportion of polylactosaminoglycans than those from differentiated cells.

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Ferreira, Sandra S. "Improving the rational design of antifreeze glycoproteins through identification of the parameters that influence ice recrystallization inhibition." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28336.

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Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes isolated from Antarctic and Atlantic Teleost fish. These compounds have the ability to depress the freezing points of solutions as well inhibit ice crystal growth, thereby protecting fish from cryoinjury and death. Although native AFGPs have considerable promise as cryoprotectants, their limited bioavailability, cytotoxicity, biological instability and lengthy chemical syntheses have precluded their Widespread use. Consequently, Ben et al. have designed non-cytotoxic stable C-linked AFGPs with comparable ice recrystalization inhibition (RI) to that of the natural compounds. This work also revealed that amongst glycopeptides with the same amino acid backbone, the overall recrystalization inhibition activity correlates to the hydration of the carbohydrate moiety. This interesting relationship lead to question if whether the hydration values of carbohydrates could be used to predict the activity of synthetic antifreeze glycoproteins. If true, it would greatly improve the rational design of synthetic analogues. Therefore the relationship between hydration and ice recrystalization inhibition was studied using small molecule carbohydrates. These results were also compared to the current standard in cryoprotection of tissues and cells, dimethyl sulfoxide, and C-linked carbohydrate derivatives. The study elucidates that there is a more accurate parameter then hydration to predict the ice recrystalization inhibition of carbohydrates, and it was defined as the Hydration Index . Furthermore, the investigation of carbohydrates with respect to RI was extended to the disaccharide present in the native antifreeze glycoproteins, D-galactose-beta(1-3)-N-acetyl-galactosamine. Its vital importance in AFGPs is apparent as no synthetic AFGP (syAFGP) with a carbohydrate other then that found in the native disaccharide has been able to surpass the activity of the natural compounds. Yet the intrinsic properties of the disaccharide, separated from the peptide, have never been studied in terms of RI activity. Therefore, the synthesis of the native disaccharide and analysis of its RI activity would help to determine how much of a factor it is for the overall activity of the natural AFGP. This work also creates the opportunity to study how other aspects of D-galactose-beta(1-3)-N-acetyl-galactosamine effects ice recrystalization inhibition, such as the C2 N-acetyl moiety and the regiochemistry of the glycosidic linkage. Another key component in improving the rational design of syAFGPs as cryoprotectants is to distinguish between the structural motifs necessary for ice recrystalization inhibition and thermal hysteresis. In 2004, Nishimura et al. published such work on the essential components of AFGPs for thermal hysteresis. They reported that even small changes to the native AFGPs can cause a loss of thermal hysteresis, for example exchanging the naturally occurring threonine residues for serine. Unfortunately, they did not test any of their compounds for recrystallization inhibition. Opportunely, the synthesis towards an antifreeze glycoprotein which contains serine residues in place of threonine residues is presented, with the future goal of testing for its RI activity. The overall objective of this dissertation is to probe the characteristics that dictate antifreeze glycoprotein activity in terms of ice recrystalization inhibition, with the goal of providing useful information towards the rational design of potent non-toxic cryoprotectants.

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16

Chen, Yuning. "Biochemical Characterization of Hydroxyproline-rich Glycoproteins in the Arabidopsis Root Cell Wall." Ohio University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1352912989.

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17

Dong, Wen. "Extensin Peroxidase Identification and Characterization in Solanum lycopersicum." Ohio University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1425894387.

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18

Marshall, Jocelyn R. "A Study of the Impact of Membrane Organization of Glycosphingolipid E-selectin Ligands and Glycoproteins on Head and Neck Cancer Cell Adhesion to Vascular Endothelium." Ohio University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1313073422.

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19

Liang, Yan. "Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation." Ohio University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1343410954.

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20

Ward, J. "The biochemisry of the attatchment and release of the variant surface glycoprotein of Trypanosoma brucei brucei." Thesis, University of Cambridge, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.383373.

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21

Al-Kassab, A. S. K. "Glycoprotein production by human breast cancer tissue." Thesis, University of Nottingham, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.356554.

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22

Clark, Melanie Jane. "The structure of MRC OX-2 membrane glycoprotein." Thesis, University of Oxford, 1985. https://ora.ox.ac.uk/objects/uuid:4a98764e-7d08-4cda-a3aa-f50409c63383.

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The mouse monoclonal antibody MRC OX-2 recognises a rat cell surface glycoprotein of M_R about 45,000 which is expressed on brain and lymphoid tissues. The OX-2 antigen is of interest because its tissue distribution and biochemical properties are similar to Thy-1, a cell surface antigen which is known to have a structure resembling an immunoglobulin domain. In this thesis, the structure of OX-2 antigen is determined to evaluate whether it shares sequence homology with Thy-1 and immunoglobulins. OX-2 antigen was purified from rat brain by monoclonal antibody affinity chromatography, and several tryptic peptides were isolated and sequenced. An oligonucleotide probe was designed from the peptide sequence and used to isolate OX-2 cDNA clones from a thymocyte cDNA library. Several cDNA clones were analysed to derive the complete sequence of OX-2 antigen. The mature protein is 248 amino acids long, comprising of two extracellular domains that show homology with immunoglobulin, followed by a transmembrane segment and a short cytoplasmic region. The N-terminal domain fits best with V-regions while the second domain is like an Ig C-domain. Thus the structure is similar to an Ig light chain or one chain of the T cell receptor. Using Southern blot analysis, no rearrangement of the OX-2 gene could be detected in rat tissues which express the protein. Homologues of the OX-2 gene were indentified in Southern blots of mouse and human DNA, and the human OX-2 gene was isolated from a genomic library. Partial characterisation of the human gene showed that domain I is encoded by a single exon, the sequence of which is highly conserved between rat and human. Preliminary S1 nuclease mapping of rat OX-2 mRNA indicates that not all transcripts are capable of encoding the normal OX-2 protein. Abnormal transcripts which differ from the normal message at exon splice sites are abundant in RNA from lymphoid tissues, but absent from brain.

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23

Chai, Wengang. "New approaches to mass spectrometric characterisation of glycoprotein oligosaccharides." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315393.

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24

Cayrol, Romain. "Study of class I and class II P-glycoprotein chimeras." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19436.

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The Multidrug drug resistance (MDR) phenotype is a major limitation to chemotherapy. The MDR phenotype arises when class I P-glycoproteins (P-gps) are overexpressed in cultured cells in vitro and in tumor cells in vivo. P-gps are encoded in rodents by three genes: mdr1, mdr2 and mdr3. Class I P-gps (Mdrl, Mdr3) confer drug resistance upon transfection in non-resistant cells and act as ATP dependent drug efflux pumps, class II P-gps (Mdr2) do not convey MDR but act as lipid flippases in the canalicular membranes of hepatocytes. P-gps are composed of twelve transmembrane domains and two nucleotide binding sites. The membrane spanning domains (MSD) of Pgp show the lowest level of similarity amongst family members and are postulated to be responsible for the different substrate specificities of the two classes of P-gp. In this study, chimeric cDNAs were constructed between class I and class II P-gps. The cDNAs were optimized for expression and study in yeast (Saccharomyces cerevisiae and Pichia pastoris) expression systems. The ATPase activity of the expressed constructs was studied by vanadate induced trapping of nucleotides.

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25

Bond, Tamara Dawn. "Volume-regulated ion channels with their interaction with P-glycoprotein." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339361.

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26

Yuahasi, Katia Kioko. "Mecanismos de formação da LDL eletronegativa (LDL-): efeito da glicoxidação e da lipólise." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-09122016-143030/.

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A fração plasmática da lipoproteína de baixa densidade (LDL) é formada por partículas de diferentes tamanhos, carga e densidade. Baseada na diferença de carga das partículas, a LDL pode ser subfracionada em LDL nativa (nLDL) e LDL eletronegativa (LDL‾). A LDL‾ está presente no plasma e possui propriedades aterogênicas e pró-inflamatórias, assim como, possui menor concentração de antioxidantes lipossolúveis, maior concentração de dienos conjugados, alterações conformacionais da apoliproteína B-100 e menor afinidade para o receptor da LDL em comparação com a nLDL. Concentrações elevadas de LDL‾ têm sido observadas em pacientes com alto risco para o desenvolvimento de doenças cardiovasculares, incluindo hipercolesterolemia familiar, diabetes. Considerando-se que os mecanismos envolvidos na formação endógena da LDL‾ ainda não estão elucidados, neste estudo foi investigado o efeito da glicoxidação e da lipólise sobre a partícula LDL para avaliar a contribuição destes processos para a formação da LDL‾ in vitro e in vivo. As modificações químicas da LDL e da imunorreatividade com anticorpo monoclonal anti-LDL‾ foi analisada antes e depois da incubação do plasma com lipoproteína lipase (LPL) ou fosfolipase A2 (PLA2), assim como mimetizando-se a lipólise intravascular. Além disso, na lipólise in vivo foi monitorado no periodo pós-prandial em indivíduos normolipidêmicos para investigar a LDL‾ formada endogenamente. A contribuição da glicoxidação para a geração de LDL‾ foi avaliada in vitro pela incubação da LDL com glicose. O efeito da glicoxidação endógena foi monitorada pela medida, ex-vivo, dos os produtos de glicação avançada (AGEs) e LDL‾ no plasma de pacientes diabéticos tipo I (DM I), tipo II (DM II) e indivíduos intolerantes à glicose (IGT). O processo de glicação não enzimática, in vitro, resultou no aumento da concentração de LDL‾. Os indivíduos dos grupos DM I, DM II e IGT apresentaram concentrações plasmáticas elevadas de LDL‾ em relação aos seus respectivos controles, enquanto observou-se aumento de AGEs apenas nos grupos DM I e DM II. O processo de lipólise in vitro mediado pela LPL e PLA2, induziu aumento significante da concentração de LDL‾; entretanto, somente pela ação da LPL foi associada com modificações oxidativas. Em concordância, o processo de lipólise in vivo (pós-prandial) também promoveu aumento significativo da concentração de LDL‾ associado com modificações oxidativas. Conclusão, nossos dados mostram que, glicoxidação e de lipólise, poderiam contribuir na formação da LDL‾ in vivo.
The low density lipoprotein (LDL) fraction in blood plasma is formed by particles with different size, charge and density. Based on particle charge differences, LDL fraction may be separated into native (nLDL) and electronegative (LDL‾) subfractions. LDL‾ is present in blood plasma and has atherogenic and proinflammatory properties, as well as, lower concentrations of lipid soluble antioxidants, higher content of conjugated dienes, conformational alterations of apolipoprotein B-100 and lower affinity by LDL receptor in comparison to nLDL. Increased LDL‾ concentrations have been found in subjects with high risk for cardiovascular diseases, including those with familiar hypercholesterolemia, diabetes and hyperlipidemia. Considering that the mechanisms involved in the endogenous generation of LDL‾ are not yet well elucidated, in this study the effect of glucoxidation and lipolysis of LDL particles was investigated in order to evaluate their contribution to in vitro e in vivo LDL‾ formation. LDL chemical modifications and its reactivity towards a monoclonal anti-LDL‾ antibody were analyzed before and after incubation of either plasma or LDL with lipoprotein lipase (LPL) or phospholipase A2 (PLA2) as an in vitro lipolysis biomimetic system. Moreover, in vivo lipolysis was monitored at the post-prandial period in normolipidemic subjects to investigate LDL‾ endogenously formed. The contribution of glucoxidation to LDL‾ generation was evaluated in vitro by incubating LDL with glucose. The effect of endogenous glucoxidation was monitored by ex-vivo measurement of advanced glycation end products (AGES) and LDL‾ in blood plasma of type I (DM I) and II (DM II) diabetic patients, as well as, in subjects with glucose intolerance (IGT). The in vitro non-enzymatic glycation resulted in increased LDL‾ formation. The DM I, DM II and IGT groups showed higher LDL‾ concentrations than the respective control groups, while AGEs were increased only in DM I e DM II groups. The in vitro lipolysis mediated by LPL and PLA2 induced a significant increase of LDL‾; however, only LPL action was also associated to LDL oxidative modification. In accordance, in vivo lipolysis (post-prandial) also promoted a significant increase of LDL‾ levels associated to LDL oxidative modification. In conclusion, our data show that both, glycoxidation and lipolysis, could contribute to in vivo LDL‾generation.

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27

Allan, Linda M. "Studies on the surface epitopes of the Trypanosoma brucei variant surface glycoprotein." Thesis, University of Cambridge, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.335046.

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28

Bowers, Katherine Elisabeth. "Sorting of CD4 and the SIV envelope glycoprotein in the endocytic pathway." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286733.

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29

Spiciarich, David. "Chemical Glycoproteomics for Identification and Discovery of Glycoprotein Alterations in Human Cancer." Thesis, University of California, Berkeley, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10688096.

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Changes in glycosylation have long been appreciated to be part of the cancer phenotype; sialylated glycans are found at elevated levels on many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to cell surface sialylation are not well characterized, specifically in bona fide human cancer. Metabolic and bioorthogonal labeling methods have previously enabled enrichment and identification of sialoglycoproteins from cultured cells and model organisms. The goal of this work was to develop technologies that can be used for detecting changes in glycoproteins in clinical models of human cancer.

In Chapter 1 of this dissertation, I present an overview of the structures and functions of glycans and their relationship to cancer progression. I also discuss applications of in vivo bioorthogonal labeling in model organisms and how in humans, the significant regulatory and ethical barriers associated with introducing chemically altered sugars into people have hindered it. Finally, I review mass spectrometry-based proteomics and how it can be applied to clinical glycoproteomics.

In Chapter 2, I demonstrate the first application of this bioorthogonal labeling in a glycoproteomics platform applied to human tissues cultured ex vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of functionalized derivatives of N-acetyl mannosamine, the sialic acid biosynthetic precursor. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of human clinical relevance.

Secretome proteins play important roles in regulation of many physiological processes and show utility as potential biomarkers and for noninvasive diagnostics and treatment monitoring. In Chapter 3, I discuss a platform for identifying sialoglycoproteins that were secreted in the conditioned media from bioorthogonally labeled human prostate tissue slice cultures. This platform could be used to identify disease biomarkers in a faithful clinical model of human disease.

Mutations in granulocyte colony-stimulating factor 3 receptor (CSF3R), also known as G-CSFR, occur in the majority of patients with chronic neutrophilic leukemia (CNL) and are more rarely present in other kinds of leukemia. In Chapter 4, I discuss novel variants in CSF3R at asparagine residue N610, one of which was germline. Interestingly, these N610 substitutions are potently oncogenic and result in ligand-independent receptor activation. They confer activation of the JAK-STAT signaling pathway and concurrent sensitivity to JAK kinase inhibitors. The N610 residue is part of a consensus N-linked glycosylation motif in the receptor. Detailed mass spectrometry analysis demonstrates that this site is occupied by both complex and complex bisecting glycans. Further analysis demonstrates that N610 is the primary site of sialylation of the receptor. This study demonstrates that membrane-proximal N-linked glycosylation is critical for maintaining the ligand dependence of the receptor. Furthermore, it expands the repertoire of potently oncogenic mutations in CSF3R that are therapeutically targetable.

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30

Keyston, Rebecca. "Homotypic and heterotypic adhesion mediated by biliary glycoprotein, BGP." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=23900.

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Glycophosphatidyl-inositol (GPI)-linked carcinoembryonic antigen (CEA) family members, including CEA itself, are absent in rodents and seem to have evolved recently. We have shown previously that CEA-CEA binding required for intercellular adhesion involves a double reciprocal interaction between the N-terminal (N domain) and internal A3B3 domains of anti-parallel molecules on apposed cell surfaces. To answer the question of whether this binding mechanism also evolved with CEA, the adhesive binding mechanism for transmembrane biliary glycoprotein (BGP), presumably more primordial, was investigated. Since CEA and BGP can be expressed on the same cells, and can have opposite effects on the cell phenotype, heterotypic interactions between them were also of interest. A construct with a deletion (17 amino acids) in the N domain of BGP was created which is unable to mediate intercellular adhesion. This $ Delta$NBGP construct, along with BGP splice variant containing only the N domain and various chimeric CEA/NCAM (neural cell adhesion molecule) constructs, was used to establish the fact that both BGP-BGP and BGP-CEA binding are obligate N-N domain. Thus fundamentally different molecular adhesion mechanisms exist in the CEA family.

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Kwan, Tony 1972. "Structural and functional analysis of the mouse Mdr3 P-glycoprotein." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38069.

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Multidrug resistance in certain tumours in vivo and in cultured cells in vitro is caused by the overexpression of P-glycoprotein. Pgps are encoded by a small family of three genes in rodents (mdr1, mdr2, mdr3 ) and two in humans (MDR1, MDR2), and belong to the larger superfamily of ABC transporters, characterized by their ability to transport various substrates at the expense of ATP. Pgps are comprised of 12 transmembrane (TM) domains, some of which are involved in the binding of these drugs, and two intracellular nucleotide-binding (NB) domains that catalyze ATP hydrolysis. Communication occurs between these two regions as drug binding can significantly activate the ATPase activity, and the signalling is thought to occur through the highly conserved intracellular (IC) loops linking the individual TM domains. Using a random mutagenesis approach, mutations were identified in IC1 affecting both substrate specificity and drug-stimulated ATPase activity of Pgp. The finding that mutations in IC1 can modulate the activities of two separate and distinct regions supports the proposed role for the IC loops. Purification of Pgp mutants to a high degree allowed for the further biochemical characterization of selected mutants in IC1. The findings support a role for IC1 in signalling between the two regions: (1) from the TM domains to the NB sites to activate ATPase activity, and (2) in the opposite direction to effect drug release. The analysis of mutations in TM11 provides more support that this helix is involved in protein-substrate interactions and forms part of the drug-binding site. The formation and maintenance of an active drug-binding site through the proper organization and orientation of TM helices are critical to the function of Pgp. Despite the large body of functional data through the characterization of the many naturally occurring and genetically engineered mutants of Pgp, high-resolution structural information of the drug binding site and the overall structur

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32

Anderson, C. M. "Glycoprotein structure of components C2 and factor B of the human complement system." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375210.

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33

Rana, Tahir. "Synthesis of a C-linked antifreeze glycoprotein building block containing an internal olefin." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28186.

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Antifreeze glycoproteins (AFGPs) are polypeptide compounds found in the blood of Antarctic notothenioids and northern cod. These substances inhibit the growth and proliferation of ice in fish inhabiting sub-zero conditions. The structure of AFGPs, and the mechanism of ice growth inhibition at the molecular level have been a source of intense debate. Work in our laboratory involves the synthesis of AFGP analogues in an effort to discern the mechanism of ice growth inhibition and enhance antifreeze attributes for potential therapeutic applications. We have previously synthesized C-linked AFGP analogues possessing antifreeze properties (TH, RI and DIS). One such analogue, [OGG] 4[Gal], has exhibited recrystallization inhibition (RI) activity comparable to native AFGPs. An important structural feature of [OGG]4[Gal] is the presence of an amide bond. We report herein the synthesis of a C-linked AFGP building block with an internal olefin as an amide isostere, in an effort to elucidate the function of the amide bond in the recrystallization inhibition activity of [OGG]4[Gal].

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Holdstock, Jolyon Guy. "Intracellular mechanisms mediating transcriptional regulation and secretion of the pituitary glycoprotein hormone alpha-subunit." Thesis, Queen Mary, University of London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283413.

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35

Yule, Fiona Sara Bridget. "The correlation between the glycosylation of Alpha-1-acid glycoprotein and healing in wounds." Thesis, University of Strathclyde, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366836.

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36

Minuk, Jeffrey. "Studies on the intracellular sorting of the two myelin-associated glycoprotein isoforms." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=23414.

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Myelin-associated glycoprotein (MAG) is amongst the earliest expressed myelin specific proteins. Its precise function remains unknown, though evidence supports a role for cell-cell adhesion. MAG may also act as a spacer molecule and could be involved in signal transduction. MAG is expressed as two isoforms, designated as L-MAG and S-MAG. Both share identical extracellular and transmembrane domains but differ in their cytoplasmic domains. L-MAG is expressed earlier during myelination than S-MAG. These features, as well as others, suggest that the isoforms have different functions. To confirm this hypothesis, both isoforms were expressed transiently and stably in Madin-Darby canine kidney (MDCK) epithelial cells and the localization of the isoforms was studied. In both transiently and stably transfected cells, L-MAG sorted primarily to the basolateral membrane. In single transfected cells, S-MAG sorted primarily to the apical membrane. When groups of adjacent cells became transiently transfected, S-MAG accumulated at areas of cell-cell contact within the basolateral membrane. In stably transfected cells S-MAG sorted to the basolateral membrane. The data suggest that L-MAG contains an invariable basolateral sorting signal, but that the sorting of S-MAG is dependent upon extrinsic factors, such as the co-expression by adjacent (contacting) cells. As MDCK cells sort the MAG isoforms differently, these data support the hypothesis that the MAG isoforms do perform different functions.

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37

Schiffner, Torben. "Refocusing antibody responses by chemical modification of vaccine antigens." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:2b2cac0f-6be3-4f91-96e5-9888e02780d4.

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The envelope glycoprotein (Env) of Human Immunodeficiency Virus 1 (HIV-1) has developed several immune-evasion mechanisms to avoid the induction of neutralising antibodies, including immunodominant non-neutralising epitopes, conformational flexibility of conserved epitopes, and spontaneous subunit dissociation, thus impeding vaccine development. Here, chemical modification of Env-based vaccine antigens is explored to overcome these obstacles. Firstly, covalent fixation of Env by chemical cross-linking was used to stabilise the conformationally flexible structure and prevent subunit dissociation. Cross-linked Env constructs showed reduced binding of many non-neutralising antibodies whilst largely maintaining antibody recognition by broadly neutralising antibodies. Compared to unmodified material, immunisation with some of these cross-linked proteins led to the induction of significantly increased antibody titres targeting the conserved CD4 binding site of Env despite similar overall antibody titres. These refocused antibody responses resulted in increased serum neutralising titres compared to animals receiving unmodified protein. Secondly, an epitope masking strategy was developed to reduce or eliminate the immunogenicity of neutralisation-irrelevant surfaces. This was achieved using site-selective addition of theoretically immunosilent glycoconjugates to lysine residues. Masking of model protein hen egg lysozyme (HEL) led to site-selective loss of antibody binding to the modification sites in vitro, which translated into refocusing of antibody responses from masked to unmasked epitopes in vivo. Mutant HIV-1 and influenza virus surface glycoproteins were designed that had lysine residues removed from close proximity to the respective broadly neutralising epitopes, but added throughout the remaining surface. Masking of these mutant proteins with second-generation glycoconjugates led to predictable perturbations of antibody binding in vitro. However, administration of these modified glycoproteins revealed unexpectedly that the masking glycans were highly immunogenic in vivo. Thus, this strategy may well prove useful if truly non-immunogenic glycoconjugates can be identified. Taken together, these chemical modifications of vaccine antigens may allow focused targeting of specific antigenic regions for increased B cell recognition, and may thus be a valuable tool for vaccine antigen design.

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Vilela, Marcelo Jose. "Monoclonal antibodies to desmosomal glycoprotein 1 : their contribution to cancer diagnosis and protein structure studies." Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.280459.

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39

Shell, Elizabeth. "Chemical Unfolding and Macromolecular Crowding of Alpha-1-Acid Glycoprotein." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1115047649.

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40

Hieber, A. David (Andrew David) 1966. "Characterisation of glycoprotein II from bovine adrenal medulla." Thesis, University of Auckland, 1993. http://hdl.handle.net/2292/1988.

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Glycoprotein II (GpII) is a glycoprotein isolated from the membranes of chromaffin granules in the adrenal medulla. The chromaffin granules of the adrenal medulla are responsible for the biosynthesis, storage and secretion of catecholamines, neuropeptides and various proteins. The abundance of chromaffin granules makes them an excellent model to further study the organelles specialized in the synthesis and secretion of hormones and neurotransmitters, from both endocrine and synaptic vesicles. When viewed by two-dimensional electrophoresis GpII is a heterogeneoug glycoprotein (80000-100000 daltons) consisting of two components, upper (GpIIa) and lower (GpIIb). Protein and immunological characterisation revealed that the two components of GpII are distinct from each other. Further molecular characterisation of GpIIa showed that it consisted of a 1224 base pair cDNA, encoding a 383 amino acid polypeptide, with a calculated molecular mass of 40500 daltons. This characterisation also revealed 19 potential glycosylation sites, with deglycosylation studies further demonstrating that an estimated 55% of the molecular mass of GpII is asparagine-linked carbohydrate. The presence of poly-N-acetyllactosamine carbohydrate groups on GpII was also demonstrated. The predicted amino acid sequence of GpIIa shares a 72% amino acid identity with the human lamp-l type protein, which belongs to a highly conserved group of lysosomal associated membrane glycoproteins (lamp proteins). As the C-terminal region of GpIIa was identical to the C-terminal region of lamp proteins, a synthetic peptide to the C-terminal region of GpII was used to raise antisera to this region. This antisera was then used to demonstrate that the C-terminal region of GpII wag present on chromaffin granules and facing the cytoplasm. This same C-terminal region on lamp proteins is known to be a cytoplasmic tail that is essential for the intracellular targeting of lamp proteins to the lysosome. A common feature to the pathways of both GpII and lamp proteins is their appearance on the cell surface followed by internalisation via endocytosis. As endocytosis of the chromaffin granule membrane is known to occur, the possibility that GpII was a marker for endocytosis was investigated. The presence of GpII within clathrin coated vesicles was shown, and evidence was presented to demonstrate that the C-terminal region of GpII interacts with adaptor proteins of clathrin coated vesicles. Adaptor proteins are known to mediate between the cytoplasmic domain of proteins internalised by endocytosis, and the outside coat of clathrin. The evidence presented would suggest that the cytoplasmic tail of GpII is a potential marker for endocytosis within the chromaffin granule membrane. The results presented in this study indicate that GpII is the secretory granule and species counterpart to the lamp proteins. This finding however raises some interesting questions regarding intracellular targeting between the lysosomes and secretory granules within the chromaffin cell.

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41

Dahabiyeh, Lina. "Targeted mass spectrometry for plasma glycoprotein profiling in pre-eclampsia." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/35445/.

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Pre-eclampsia is a common hypertensive disorder of pregnancy that substantially affects maternal and neonatal morbidity and mortality worldwide. Despite decades of research, the aetiology of the disease remains poorly understood, and the clinical management of it is hampered by the lack of reliable diagnostic tests and effective therapy. Several screening tests have been suggested for the prediction of preeclampsia; however, none possess the sufficient specificity and sensitivity. Moreover, multiple pathways are known to be involved in the pathogenesis of pre-eclampsia, so it is very unlikely that a single or a small group of biomarkers will accurately predict the disease. In this thesis, two separate targeted LC-MS/MS methods were developed and validated to quantify plasma glycoproteins in pre- eclampsia to increase the pool of preeclampsia biomarker candidates and explore new pathways associated with the disease. The first method was hypothesis-driven and aimed to quantify the oxidation level of the plasma glycoprotein angiotensinogen (AGT), which has been proposed to be involved in the increased blood pressure characteristic of pre-eclampsia. The second method was hypothesis-generating and aimed to detect glycoprotein fold changes between different disease conditions using a simple and cost-effective conventional LC-MS/MS workflow. For both methods, a reproducible workflow for efficient glycoprotein/AGT extraction from human plasma was developed by coupling ConA lectin affinity chromatography with reversed-phase solid phase extraction fractionation (RP-SPE). Analysis of the enzymatically digested proteins was conducted using targeted LC-MS/MS working under the multiple reaction monitoring mode. For the quantification of the two distinct forms of AGT in the plasma (the sulphydryl-bridged oxidised form and the free thiol reduced form), a differential alkylation strategy was coupled with targeted LC-MS/MS to recognise and quantify the cysteine (Cys) peptides involved in the redox switch of AGT. The developed method enabled the reproducible detection of the two distinct forms of AGT in the plasma with CV% < 15%, and confirmation of the identity of the differentially alkylated Cys peptides was supported by LC-MS/MS. Analysis of clinical plasma samples using the developed method showed a significantly higher level of the oxidised AGT in pre-eclamptic women compared to gestational age-matched normotensive controls (P=0.008), whilst maintaining a similar total AGT level in the plasma. The research findings indicate that the elevated level of oxidised AGT rather than its total level might be a contributing factor to the hypertension characteristic of pre-eclampsia, and provide an extra line of evidence linking the oxidative state and the generation of reactive oxygen species with hypertension in pre-eclampsia. In the second part of the research, 54 clinically relevant glycoproteins were selected to be profiled by label-free targeted LC-MS/MS. Measurement of the analytical precision of the method revealed acceptable CV values for the majority of the assays (median CV 11.8%). Analysis of plasma samples collected from early- and late-onset preeclamptic women using the developed glycoprotein profiling methodology successfully identified significant changes in the level of several proteins in pre-eclampsia. Two of them, apolipoprotein D and kallikrein, are reported for the first time to be altered in the plasma of pre-eclamptic women suggesting that they could be further evaluated as novel biomarkers. Some pre-eclampsia-relevant pathways and biological processes, including iron transport and metabolism, coagulation, and lipid metabolism and oxidative stress were found to be altered in the disease. Moreover, different glycoproteins were changed in early-onset compared to late-onset pre-eclampsia which might reflect different pathophysiological mechanisms. Additionally, the method was applied to identify any altered glycoproteins in plasma samples from women with polycystic ovary syndrome (PCOS). These were subsequently compared with those found to be altered in pre-eclampsia, resulting in the proposal of possible underlying pathophysiological mechanisms that may explain the reported association between the two conditions, such as hypofibrinolysis and thrombophilia and iron overload. Moreover, the study detected, for the first time, significant changes in the plasma levels of vitronectin and insulin growth factor acid labile subunit, suggesting that these may also be further appraised as potential biomarkers for the diagnosis of PCOS. Taken together, the two targeted LC-MS/MS methods developed in this thesis provided relevant information regarding pre-eclampsia by identifying potential pre-eclampsia protein biomarkers. This sheds light on the different biochemical processes altered in the disease and points to possible pathophysiological mechanisms that might assist in explaining the link between PCOS and pre eclampsia, all of which should improve the understanding of the molecular mechanisms of the disease. The present work offers information that may play a key role in improving the health care of women with preeclampsia and serves as a foundational cornerstone for future work.

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Pereira, Elisângela Monteiro. "Modificação induzida por β2-glicoproteína I na resposta oxidativa de polimorfonucleares humanos durante a fagocitose." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-11012018-141639/.

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β2-glicoproteína I (β2GPI) é encontrada (200µg/mL) no plasma, 60% livre e 40% em lipoproteínas. Esta proteína de fase aguda, com afinidade por superfícies negativas pode ser clivada pela plasmina. Fragmentos são purificados como dímeros ou multímeros de β2GPI. Formas monomérica e dimérica de β2GPI foram purificadas de soro humano e identificadas por SDS-PAGE, imunoblot e ELISA Somente a forma monomérica foi detectada no teste ELISA Os efeitos de ambas as formas sobre o burst respiratório de polimorfonucleares humanos (PMN), estimulados in vitro com zymosan opsonizado, foram estudados por quimioluminescência amplificada por luminol ou lucigenina e citometria de fluxo, pela oxidação do OCFH. A forma monomérica inibiu a quimioluminescência amplificada por luminol (-43.6 x -7.33 AEU/s), mas não por lucigenina, e aumentou a oxidação de DCFH e a produção de Óxido Nítrico (•NO). É provável que o •No, via peroxinitrito, medeie os efeitos de β2GPI sobre o burst respiratório de PMN.
Circulating blood contains approximately 200µg/mL of β 2-glycoprotein I (β2GPI), either free (60%) or lipoprotein bound (40%). This acute phase protein, with affinity for negative surfaces, can be cleaved by plasmin. Fragments purify as dimeric or multimeric (β2GPI. Both (β2GPI forms were purified from human sera and identified by SDS-PAGE, immunoblotting and ELISA. ELISA reactivity was dependent on the monomeric status of (β2GPI. The effects of dimeric and monomeric (β2GPI upon respiratory burst of human polimorphonuclear neutrophils (PMN) stimulated in vitro with opsonized zymosan were studied. Respiratory burst was evaluated by luminol- or lucigenin-amplified chemiluminescence, or by DCFH oxidation (flow-cytometry assay). The monomeric, but not the dimeric form, inhibited the luminol chemiluminescence of zymosan-stimulated PMNs (-43.6 x -7.33 AEU/s). Lucigenin chemiluminescence was insensitive to (β2-GPI. Monomeric (β2GPI increases both DCFH oxidation and nitric oxide production. Nitric oxide, probably through peroxynitrite reactions, mediates (β2GPI effects upon PMNs respiratory burst.

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43

McNally, Tracy Jane. "An investigation of the role of antiphospholipid antibodies and #beta#2 glycoprotein-I in the modulation of haemostasis." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.339151.

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44

Wand, Nadina Ivanova. "Variant surface glycoprotein synthesis and cell cycle progression in Trypanosoma brucei." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:01bbdf34-8cb3-4942-a14d-d6ba3a3e669d.

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The unicellular eukaryote Trypanosoma brucei causes African Sleeping sickness and multiplies extracellularly in the bloodstream of the infected host. The parasite evades antibody-mediated lysis by switching its Variant Surface Glycoprotein (VSG) coat. Blocking VSG synthesis results in an abrupt growth inhibition and a precise pre-cytokinesis cell cycle arrest, with an accumulation of cells with two nuclei and two kinetoplasts. Additionally, induction of VSG RNAi triggers a global block in translation, which is not due to a general decrease in transcript levels. The mechanism behind this translation arrest was investigated. It was observed that it correlated with a decrease in polysomes, indicating that translation was blocked at the level of initiation. It was also shown that the VSG RNAi-triggered growth inhibition was reversible, which suggests that this is not a lethal phenotype. The VSG221 RNAi-induced growth arrest could be alleviated if a second different VSG (VSG117), which was not recognised by the VSG221 RNAi, was expressed immediately downstream of the promoter of the active VSG221 Expression site. Further, it was possible to delete the telomeric VSG221 in these VSG double-expressors, leaving the cells completely reliant on the second complementing VSG117 gene. VSG117 expressed from a promoter-adjacent position in the active Expression site was shown to form a functional surface coat that protected the parasites from complement-mediated lysis in vitro. Transiently transfecting cells with anti-VSG221 morpholino oligonucleotides allowed us to specifically block translation of VSG221 mRNA without degrading it. This resulted in a pre-cytokinesis cell cycle arrest similar to that induced by VSG221 RNAi. This indicates that the VSG RNAi-triggered growth inhibition was due to a lack of VSG protein or its synthesis rather than the ablation of the abundant VSG mRNA. In addition, it was shown that blocking VSG synthesis reduced the rate of surface VSG internalisation in cells that were stalled precytokinesis, but had no effect on other endocytic markers. These experiments give us further insight into the importance of the protective VSG coat for pathogenicity in T. brucei.

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Tomotani, Ester Junko. "Imobilização da invertase em resina de troca iônica (tipo Dowex®): seu uso na modificação da sacarose." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-16082017-104536/.

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A invertase comercial (Bioinvert®) foi imobilizada por adsorção em resinas aniônicas do tipo Dowex® [1x8:50-400, 1x4:50-400 e 1x2:100-400, todos copolímeros estireno-divinilbenzênicos, porém de granulometria (50-400 mesh) e quantidades de ligações cruzadas diferentes (2-8%)] em meio aquoso. A melhor percentagem de adsorção da invertase nas resinas foi observada em pH 5,5 a 32°C, tendo o complexo Dowex®1x4-200/lnvertase apresentado índice de adsorção e coeficiente de imobilização iguais a 100%. Os parâmetros cinéticos e termodinâmicos foram determinados para a invertase solúvel e insolúvel de Bioinvert® e também para a invertase purificada (Fluka®). O complexo Dowex®1 x4-200/Bioinvert® apresentou-se estável durante as reações sem desprendimento da enzima do suporte. Os parâmetros termodinâmicos da forma solúvel e insolúvel da Fluka® foram idênticos aos do Bioinvert®, no entanto, após a imobilização apresentou uma redução de 28% na sua atividade. O estudo da atividade transferásica de ambas as formas de Bioinvert® em diferentes concentrações de sacarose foram analisadas através da cromatografia de camada delgada. A estabilidade operacional e de estocagem foi também determinada para o complexo Dowex®1x4-200/Bioinvert®.
The invertase (trademarked as Bioinvert®) solubilized in deionized water was immobilized by adsorption on anion exchange resins, collectively named Dowex®, [1x8:50-400, 1x4:50-400 and 1x2:100-400, styrene-divinylbenzene copolymers, with different granulometry (50-400mesh) and different degrees of cross-linking (2-8%)]. The best percentage of adsorption of invertase on resins was observed in pH 5.5 at 32°C and the complex Dowex®1x4-200/invertase has shown a coupling yield and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrosehydrolysis for both soluble and insoluble enzyme were evaluated to Bioinvert® and purified invertase purchased from Fluka®. The complex Dowex®/Bioinvert® was stable without any desorption of enzyme from the support during the reaction and having the thermodynamic parameters equal to the soluble formo However, the loss of activity for immobilized Fluka® was found to be 28% when compared to the soluble one. The transfructosylating activity of Bioinvert® in both forms in different concentrations of sucrose was investigated through TLC. In regard to insoluble Bioinvert® its storage and operational stability were also determined.

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46

Rodríguez, Torrecillas Ivan 1979. "Molecular basis to human P-glycoprotein reversion." Doctoral thesis, Universitat Pompeu Fabra, 2015. http://hdl.handle.net/10803/586099.

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ABC (ATP-binding cassette) transporters are involved in translocate a wide spectrum of molecules across the lipid bilayer and some of them are associated with various diseases. They also have an important role in multidrug resistance (MDR) in cancer, which has been a major obstacle in cancer chemotherapy and in the treatment of leishmaniasis. While diverse transporters may confer MDR phenotype in bacteria, in human it is mainly achieved by five ABC proteins, among them P-glycoprotein/(ABCB1). To understand the structural basis of P-gp inhibitory activity, to determine the ligand binding sites within P-gp and to guide the design of more potent P-gp inhibitors, we performed i) a 3D-QSAR model using CoMSIA on a set of sesquiterpenes, ii) molecular docking simulations of various compounds in a homology model of LMDR1, a P-gp-like transporter belonging to the Leishmania ABC family and iii) and a complete study of sesquiterpenes interaction with human P-gp.
Los transportadores ABC (ATP binding cassette), encargados de transportar un amplio espectro de moléculas a través de la bicapa lipídica, pueden estar asociados con diversas enfermedades. Juegan un papel importante en la multirresistencia a fármacos (MDR), obstáculo importante en la quimioterapia del cáncer y en el tratamiento de leishmaniasis. En bacterias varios transportadores pueden conferir el fenotipo MDR, en humanos son principalmente cinco, entre ellos la glicoproteína P/(ABCB1). Para comprender la base estructural de la actividad inhibidora de P-gp, determinar los sitios de unión de los ligandos a P-gp y diseñar inhibidores de P-gp más potentes, se realizó i) un modelo 3D-QSAR usando CoMSIA en un conjunto de sesquiterpenos, ii) simulaciones de acoplamiento molecular de varios compuestos en un modelo de homología de LMDR1, transportador de la P-gp perteneciente a la familia ABC de Leishmania y iii) un estudio completo de interacción entre sesquiterpenos y la P-gp humana.

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47

Fallahi, Firouzeh. "Characterization of epitopes on the rabies virus glycoprotein by selection and analysis of escape mutants." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/26898.

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Rabies virus, a negative sense single-stranded RNA virus, is the type member of the genus Lyssavirus of the Rhabdoviridae . The glycoprotein (G), which projects from the surface of the lyssavirus particle, is the only protein known to be capable of eliciting the production of neutralizing antibodies and knowledge of the antigenic nature of this protein is therefore important. Five different antigenic sites have been mapped on the G protein. In this study, the isolation of mutants resisting antibody neutralization (escape mutants) was attempted by a selection strategy employing three distinct strains of rabies: Evelyn Rokitnicki Abelseth (ERA), Big Brown Bat (BBB), and Silver Haired Bat (SHB). No escape mutants were generated from BBB and SHB but a total of seven independent ERA mutants were recovered using monoclonal antibodies (Mabs) directed against antigenic sites I and IIIa of the glycoprotein. (Abstract shortened by UMI.)

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48

Powell, Kevin F. H. (Kevin Frederick Herbert). "Gene sequencing and in vitro synthesis of the rotavirus non-structural glycoprotein." Thesis, University of Auckland, 1986. http://hdl.handle.net/2292/2385.

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1. Recombinant DNA techniques have been applied to the dsRNA genome of the bovine rotavirus Nebraska Calf Scours Diarrhoea virus (NCDV). The sequence of a full - length cloned copy of genomic segment 10 of NCDV has been determined using the Sanger dideoxynucleoside sequencing technique by subcloning cDNA into M13 vectors. 2. Genomic segment 10 codes for the non-structural protein NCVP5, a protein which appears to be involved in virus maturation (Estes et al., 1983). Determination of the nucleic acid sequence of the gene has enabled the amino acid sequence o f the bovine NCVPS protein to be inferred. Comparison of the inferred amino acid sequence with homologous sequences derived from other virus strains (Both et al., 1983c; Baybutt and McCrae, 1984; Okada et al., 1984; Ward et al., 1985) has enabled conserved regions of the molecule to be identified. A small region of the NCVPS protein has been identified (residues 131-161) which exhibits considerable variability between rotavirus strains. 3. A computer-based algorithm has been utilised to predict the folding pattern of NCDV gene 10 mRNA. This reveals a 'panhandle ' structure which differs from that proposed for the related gene of strain Wa rotavirus (Okada et al., 1984) but both molecules possess a common feature in that the initiation codon falls within a potentially-stable duplex formed with a portion of the 3 untranslated region. 4. A series of four site-directed deletion mutants of the cloned gene were constructed in order to investigate the functional significance of the three N-terminal hydrophobic regions of the NCVP5 protein. Two mutants were constructed using conveniently-located HindIII and BamHI restriction enzyme sites. The other two mutants were generated using M13 vectors and synthetic oligonucleotides. These modifications yielded genes coding for proteins in which portions of the first and second hydrophobic regions had been deleted. 5. DNA corresponding to the 'wild-type' coding region was inserted into an SP6 transcription vector to enable mRNA to be produced in vitro. This mRNA, when incubated in a reticulocyte lysate, directed the synthesis of a protein of the correct size (20 K). The addition of dog pancreatic microsomes to the reaction yielded a protein product (29 K) of a size consistent with the glycosylated ('wild-type') form of the NCVP5 protein. 6. The four variant forms of the NCVP5 gene were also inserted into SP6 transcription vectors and the protein products synthesised by the resulting mRNAs studied. All four mRNAs directed synthesis of variant protein products of the anticipated size. 7. The ability of the four variant proteins to become glycosylated and to associate with membranes was investigated. The topology of the proteins in the membrane was examined by digestion with proteolytic enzymes. Variant proteins altered in the first or second hydrophobic regions retained their ability to associate with membranes, suggesting that the third hydrophobic region, which was not altered, might play a role in membrane association. 8. A model for the disposition of NCVPS in the endoplasmic reticulum is proposed in which the first hydrophobic region is located within the lumen of the endoplasmic reticulum, the second hydrophobic region spans the membrane and the third hydrophobic sequence associates independently with the membrane from the cytoplasmic side leaving the C-terminus of the molecule exposed to the cytoplasm. The model proposed accounts for the experimental observations but is in conflict with current mechanisms proposed for the insertion of proteins into membranes. (Wickner and Lodish, 1985).
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49

De, Champlain Annick. "Study of the key residues in close proximity to the ATP molecule in the nucleotide binding domains of P-glycoprotein by cysteine scanning mutagenesis." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80249.

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The three dimensional structure of P-gp nucleotide binding sites is unknown but a high resolution crystal structure was solved for the homodimer of HiSP, the ATP binding subunit of the bacterial ABC transporter histidine permease. The main goal was to test the hypothesis that the nucleotide binding domains (NBD) of P-gp are structurally and functionally conserved with HisP with respect to key residues in intimate contact with the ATP molecule and possibly involved in catalysis. The strategy adopted involves the insertion of cysteine residues at the desired position in a cysteine-less protein backbone. A set of 14 mutants was generated this way for each NBD. The effects of the mutations on drug transport are tested with a cytotoxicity assay using the anti-fungal drug FK506. A431C/A1074C and T435C/T1078C showed the same level of activity as the wild-type while the other residues were completely or partially defective. (Abstract shortened by UMI.)

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50

Ma, Jerome H. Y. "Atomistic studies of the dynamics of P-glycoprotein and its ligands." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:e2e2bbe0-d4ae-4351-b339-c8e02ef3d3d9.

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Abstract:

A signifficant obstacle facing the healthcare industry is the phenomenon of multidrug resistance (MDR) in which a cell acquires simultaneous resistance to many unrelated drugs that it has never been exposed to. At the molecular level, MDR can be characterised by a reduction of intracellular drug levels due to their active efflux by multidrug transporters such as P-glycoprotein (Pgp). Pgp is able to efflux a phenomenally wide variety of chemically unrelated drugs and causal relationships have been established between its expression and the acquisition of MDR to numerous anticancer and central nervous system (CNS) drugs. There has thus been much effort to understand the molecular biology of Pgp and how it functions. However, many aspects of its functioning remain unclear. From a drug discovery viewpoint, we have yet to fully understand what features make some drugs susceptible to Pgp-mediated efflux (substrates) and what makes others able to inhibit Pgp function (inhibitors). From a mechanistic viewpoint, it is still uncertain what the exact nature of Pgp's binding site is, the role of ATP binding and hydrolysis in transport and how both of these interplay with ligand binding. The work presented in this thesis attempts to answer these questions from two perspectives. Firstly the mouse Pgp crystal structure [PDB 3G60] was used as a unique starting point for molecular dynamics (MD) simulations to characterise the dynamics and conformational exibility of Pgp, properties believed to be integral to its function. The simulations revealed Pgp to be a highly dynamic molecule at both its transmembrane (TM) and nucleotide binding domains (NBDs). The latter exhibited a conformational asymmetry that supports the Constant Contact model of ATPase activity. In the presence of the Pgp substrate, daunorubicin, the NBDs exhibited tighter asymmetric dimerisation leading to increased affinity for ATP. In contrast, the presence of the Pgp inhibitor, QZ59-RRR led to NBD conformational changes that reduced their affinity for ATP. Thus providing an appealing mechanism for how QZ59-RRR inhibits Pgp ATPase activity. MD simulation was also used to provide atomic-detail interpretations of multiple binding stoichiometries of drug and lipid molecules observed by collaborator-led mass spectrometry experiments. This also provided opportunity to validate the Pgp simulations against novel experimental data. The second strand of the thesis explored the membrane permeation dynamics of CNS therapeutics in order to identify differences in protonation states, conformations, orientations and membrane localisation that might distinguish those that are Pgp substrates and from those that are not. These properties were studied using complementary MD simulation and nuclear magnetic resonance (NMR) techniques. The simulations revealed a novel set of criteria that in uence the likelihoodof a drug to 'flip-flop' across a membrane, a behaviour that may make drugs more susceptible to Pgp efflux. These observations were broadly consistent with the NMR experiments. However, the NMR data also highlighted limitations in the simulation approaches used in this thesis and emphasised the need to also consider the kinetics of permeation in addition to its thermodynamics.

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Dissertations / Theses on the topic 'Biochemistry of glycoproteins'

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