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Bandini, Giulia. "Studies on fucosylation in Trypanosoma brucei." Thesis, University of Dundee, 2011. https://discovery.dundee.ac.uk/en/studentTheses/c74554c1-f4d3-4bb3-aa31-899fcf507e11.
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The biosynthesis of GDP-Fucose, the activated donor for fucose, has been recently shown to be essential in the parasite Trypanosoma brucei. Fucose is a common sugar modification on eukaryotic glycan structures, but it has not been well described in trypanosomatids. To elucidate the role of fucose in T. brucei we searched for putative fucosyltransferases in this parasite. A single putative T. brucei fucosyltransferase (TbFT) was identified and recombinantly expressed in Escherichia coli. The protein was active and structural characterization of its reaction product identified it as a GDP-Fuc: ß-D-galactose a-1,2-fucosyltransferase with preference for Galß1,3GlcNAc containing structures as glycan acceptors. A procyclic form conditional null mutant for TbFT was generated and this glycosyltransferase shown to be essential for parasite growth in vitro, with the mutant cells displaying a slightly abnormal morphology and an apparent reduction in the surface high molecular weight glycoconjugate complex. Here we also describe the various experimental approaches that were used to try to identify the fucosylated glycocojugates in T. brucei. Lastly, to better understand the biosynthesis of GDP-Mannose, the starting metabolite for the biosynthesis of GDP-Fuc, we biochemically characterized T. brucei phosphomannomutase (TbPMM). Here we show this enzyme could interconvert not only mannose-phosphates, but also glucose-phosphates.
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Keeley, Tyler S. "Investigating the Roles of Fucosylation and Calcium Signaling in Melanoma Invasion." Scholar Commons, 2018. https://scholarcommons.usf.edu/etd/7535.
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Melanoma is the deadliest form of skin cancer. Prognosis for early stage melanoma patients is excellent, and surgery is often curative for these patients. However, once patients have presented with invasive disease, the average 5-year survival rate drops significantly from over 90% to between 10 and 15%. Several therapies have been developed to target a commonly mutated oncogene BRAF, or its downstream effectors. Unfortunately, while these treatments show robust initial response, most patients relapse within a year. Moreover, therapy-resistant tumors are often more invasive and metastatic. Therefore, it is important to investigate the molecular mechanisms underlying melanoma invasion and metastasis, and to prevent melanoma cell dissemination and metastatic progression. Invadopodia are proteolytic membrane protrusions used by metastatic cancer cells to degrade the extracellular matrix and to facilitate cancer cell invasion and metastasis. In my thesis research I have focused on protein fucosylation and store-operated calcium entry, two separate mechanisms involved in invadopodial regulation.
Post translational modifications of proteins are essential for their structure and function. Many cell surface proteins require modifications such as glycosylation for protein-protein interactions, cell adhesion, and signal transduction. Fucosylation is a form of glycosylation that adds L-fucose on glycan structures of proteins. There is evidence indicating that fucosylation plays an important but cancer-type and branching dependent role in cancer progression. Emerging evidence indicates that the fucose salvage pathway and protein fucosylation are altered during melanoma progression and metastasis. Here, we report that the fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation by promoting α(1,2) fucosylation of cell surface proteins. The activation of the fucose salvage pathway decreases invadopodia numbers and inhibits the proteolytic activity of invadopodia in WM793 melanoma cells. Inhibiting fucokinase, one of the critical enzymes in the fucose salvage pathway, in melanoma cells abrogates L-fucose-mediated inhibition of invadopodia, suggesting dependence on the fucose salvage pathway. The inhibition of invadopodia formation by L-Fucose treatment or fucokinase overexpression could be rescued by treatment with α(1,2), but not α(1,3/4) fucosidase, implicating an α(1,2) fucose linkage-dependent inhibitory effect. The ectopic expression of FUT1, an α(1,2) fucosyltransferase, is sufficient to inhibit invadopodia formation and ECM degradation. Our findings indicate that the fucose salvage pathway can inhibit invadopodia formation, and consequently, invasiveness in melanoma via α(1,2) fucosylation. Re-activation of this pathway in melanoma could be useful for preventing melanoma invasion and metastasis.
Calcium is a critical second messenger involved in a multitude of biological processes from cell proliferation to muscle contraction. In melanoma, previous studies have found that activation of the store operated calcium entry (SOCE) channel promotes tumor invasion and metastasis, in vitro and in xenograft models. The expression levels of STIM1, an essential component of the store operated calcium channels, has been found to increase with later stages of melanoma. In melanoma cell lines, the over expression of STIM1 enhances invadopodia number whereas STIM1 knockdown inhibits invadopodia formation. Similarly, gelatin degradation activity is enhanced with STIM1 overexpression and abrogated with STIM1 knockdown, implicating STIM1 as an important factor in the regulation of invadopodia formation and melanoma invasion. Though the studies published have shown a significant role of STIM1 in tumor progression, a robust transgenic animal model has not yet been established. Here, we developed a novel transgenic mouse model which, upon 4-hydroxytamoxifen (4OHT) treatment, induces the BRAFV600E mutation and PTEN, STIM1, and STIM2 deletions in melanocytes via an inducible Cre-lox system. Our investigation found that the loss of STIM1 exacerbates tumor growth and results in tumor formation significantly more quickly than STIM1 wild type mice. Whereas PCR analysis of 4OHT-treated skin showed deletion of STIM1 and PTEN, immunohistochemical staining of these genes in tumors did not convincingly demonstrate complete deletion. Therefore, it remains to be determined whether the effects we observed are due to STIM1 and STIM2 loss. These findings need to be corroborated in the future.
Our studies focus on two important mechanisms required for melanoma progression and metastasis. We found that α(1,2) fucosylation is able to inhibit invadopodia formation, and melanoma cell invasion. The reestablishment of α(1,2) fucosylation in melanoma could potentially be exploited to inhibit melanoma metastasis. Additionally, early evidence points to STIM1 having a tumor suppressive role in melanoma oncogenesis and tumor growth based on the transgenic mouse model. Although the phenotype is unexpected, further investigation of this model will likely provide important insight for the complicate roles of SOCE in melanoma initiation and progression.
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Pennarubia, Florian. "Analyses biochimiques et fonctionnelles de protéines cibles de POFUT1." Thesis, Limoges, 2018. http://www.theses.fr/2018LIMO0075.
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La O-fucosylation, catalysée par Pofut1, est une glycosylation rare qui consiste en l’ajout d’un fucose O-lié sur la sérine ou la thréonine d’une séquence consensus (C2X4(S/T)C3), portée par un domaine EGF-like (ELD) d’une glycoprotéine membranaire ou sécrétée. Notre analyse de la lignée murine Pofut1cax/cax, hypomorphe pour le gène Pofut1, a révélé une hypertrophie musculaire post-natale associée à une diminution du pool de cellules satellites. Ce phénotype est en partie associé à un défaut d’interaction entre les récepteurs NOTCH hypo-O-fucosylés des myoblastes dérivés de cellulessatellites (MDCS) et leurs ligands DSL, ce qui aboutit à une plus faible activation de la signalisation Notch. D’autres protéines potentiellement impliquées dans la myogenèse peuvent également être la cible de POFUT1. C’est notamment le cas de la protéine Wnt inhibitory factor 1 (WIF1), qui dispose de cinq ELDs, dont deux sont potentiellement aptes à recevoir un O-fucose (ELDs III et V). Par une approche phylogénétique, nous avons montré la conservation de ces deux sites de O-fucosylation et de deux sites de N-glycosylation chez la plupart des bilatériens. Nos expériences démontrent l’occupationde tous ces sites, excepté le site de O-fucosylation de l’ELD V, chez la protéine WIF1 murine. La capacité de l’ELD III, produit de manière isolée, à recevoir un fucose O-lié a été démontrée après O-fucosylation in vitro, par l’association de cycloaddition azide-alcyne assistée au cuivre (CuAAC) et de spectrométrie de masse en mode MRM. Cette nouvelle approche expérimentale a par la suite été standardisée et sa sensibilité évaluée en comparant deux autres ELDs (ELDs 12 et 26 de NOTCH1) connus pour être O-fucosylés mais présentant des affinités différentes pour POFUT1. De façonsurprenante, l’ELD V de WIF1 ne peut être O-fucosylé, probablement en raison d’un clash stérique entre cet ELD et POFUT1, prévenant ainsi leur interaction. L’analyse de la protéine WIF1 entière a confirmé les résultats obtenus sur les ELDs isolés et démontre l’occupation des deux sites de N-glycosylation. Enfin, nos résultats montrent également l’importance de ces deux N-glycanes, mais également celle du O-fucose de l’ELD III, pour une sécrétion optimale de la protéine WIF1 murineThe, Pofut1-catalyzed O-fucosylation, is a rare glycosylation which consists of the addition of an O-linked fucose to the serine or threonine of a consensus sequence (C2X4(S/T)C3), carried by an EGF-like domain (ELD) of a membrane or secreted glycoprotein. Our analysis of the murine line Pofut1cax/cax, hypomorphic for the Pofut1 gene, revealed post-natal muscle hypertrophy associated with a decrease in the satellite cell pool. This phenotype was partly associated with a lack of interaction between hypo-O-fucosylated NOTCH receptors of satellite cell-derived myoblasts (SCDM) and their DSL ligands, which resulted in a lower activation of Notch signaling. Other proteins potentially involved in myogenesis may also be the target of POFUT1. This is indeed the case for the protein Wnt inhibitory factor 1 (WIF1), which has five ELDs, whose only two are potentially able to receive an O-fucose (ELDs III and V). Using a phylogenetic approach, we showed in most bilaterians that these two O-fucosylation sites and two N-glycosylation sites were conserved. Our experiments showed theoccupation of all these sites, except for the O-fucosylation site of murine WIF1 protein ELD V. The ability of the ELD III, produced as an isolated protein, to receive O-linked fucose was demonstrated after an in vitro O-fucosylation by combination of copper-catalysed azide-alkyne cycloaddition (CuAAC) and MRM-mass spectrometry. This new experimental approach was then standardized and its sensitivity was evaluated by comparing two other ELDs (NOTCH1 ELDs 12 and 26) known to beO-fucosylated but with different affinities for POFUT1. Surprisingly, WIF1's ELD V could not be O-fucosylated, probably due to a steric clash between this ELD and POFUT1, thus preventing their interaction. The analysis of the full-length WIF1 protein confirmed our results obtained with isolated ELDs and demonstrated the occupation of the two N-glycosylation sites. Finally, our results also showed the importance of these two N-glycans, but also the importance of ELD III’s O-fucose, foroptimal secretion of the murine WIF1 protein
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Javaud, Christophe. "La Fucosylation distale et proximale chez Bos taurus : Structure et expression des gènes FUT4, FUT9 et FUT8." Limoges, 2002. http://www.theses.fr/2002LIMO0057.
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Chez les mammifères, le fucose est greffé à la périphérie des glycannes en position α1,2, α1,3 ou α1,4 lors des dernières étapes de la biosynthèse des oligosaccharides, ou plus préocement en α1,6 sur le premier GlcNAc du groupement chitobiose proximal des N-glycannes exclusivement. Au cours de ce travail, nous avons procédé à une étude approfondie de la fucosylation proximale en clonant le gène FUT8 bovin codant une α6-fucosyltransférase. Ce gène se démarque des autres gènes de fucosyltransférases par sa structure codante multiexonique. De plus, son découpage exon/intron s'avère très différent de celui de l'homme, de la drosophile et du nématode Caenorhabditis elegans. Il existe une excellente corrélation entre l'organisation des exons codant du gène FUT8b et les différents domaines de la protéines. Ces résultats corroborent donc la théorie de la formation ancestrale des gènes par brassage d'exons ("exon shuffling") développée par Gilbert en 1987. Chez Bos taurus, un seul gène codant une α3-fucosyltransférase a été isolé à ce jour. Il s'agit du gène futb (Oulmouden et al. , 1997), orthologue homologue du cluster de gènes humains FUT3-FUT5-FUT6. Alors que l'activité α4-fucosyltransférase responsable de la synthèse des antigènes Le(a) est contrôlée par les produits des gènes FUT3 et FUT5, il a été logiquement postulé que cette activité enzymatique était restreinte aux seules espèces possédant le cluster de gènes Lewis, les hominoi͏̈des (homme et chimpanzé). Après l'identification par Raychoudhury (1997) de l'activité α4-fucosyltransférase chez le rat, et suite aux travaux de Dupuy et al. (2002), montrant que l'α4-fucosylation était en réalité apparue beaucoup plus précocement chez les primates, nous avons entrepris avec succès la recherche d'antigène Le(a) chez le vertébré Bos taurus. Nous avons ensuite isolé deux gènes, bFUT4 et bFUT9 codant des enzymes possédant des aptitudes particulières, contrairement à leurs homologues humaines, pour le transfert du fucose en α1,4. L'α4-fucosylation pourrait donc être plus largement distribuée chez les vertébrés. De plus, par deux mécanismes distincts, chacun de ces gènes est en mesure de coder potentiellement une protéine soluble raccourcie de sa portion transmembranaire, inapte au transfert de fucose, mais dont la fonction de lectine reste à explorer
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Barber, Alistair John. "A behavioural investigation of the function of glycoprotein fucosylation in learning and memory in the day-old domestic chicken." Thesis, Open University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236514.
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Joly, Caroline. "Régulation de l'activité α(1,4)fucosyltransférase au cours du développement végétatif et floral du tabac (Nicotiana tabacum cv Xanthi)." Limoges, 2002. http://www.theses.fr/2002LIMO0056.
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Les motifs Lewis(a), générés par les α4-fucosyltransférases, interviennent principalement en tant que signaux de reconnaissance et de communication entre cellules. Néanmoins, les fonctions de l'α4-fucosylation des protéines dans les cellules végétales restent à définir. Nous nous sommes engagés dans l'étude de l'expression de l'activité α(1,4)fucosyltransférase et de la distribution des glycotopes Lewis(a) durant le développement du tabac. Au cours du développement végétatif, l'α4-fucosyltransférase est une enzyme non seulement exprimée de façon ubiquiste mais aussi d'un point de vue spatio-temporel. Nous montrons également que cette activité enzymatique est régulée au cours d'évènements particuliers comme l'élongation cellulaire et la lignification. L'importance de cette régulation est confirmée par l'étude de plants de tabac, exprimant l'α3/4-fucosyltransférase FUT3 humaine. Ces plants présentent un retard de croissance lié à un défaut d'élongation cellulaire. Un phénotype sauvage peut, néanmoins, être rétabli par un apport en gibbérellines. L'étude du développement floral a révélé un profil d'expression de l'activité α(1,4)fucosyltransférase spécifique du grain du pollen. L'α4-fucosyltransférase endogène est régulée comme une protéine "tardive" lors de la microgamétogénèse. De plus, les N-glycoprotéines α4-fucolisées semblent essentielles lors de la germination du grain de pollen et la croissance du tube pollinique. Une dé-régulation de l'activité α(1,4)fucosyltransférase, par surexpression de la FUT3 humaine, induit des altérations structurales et fonctionnelles des grains de pollen. En conclusion, nous montrons que les glycotopes Lewis(a) constituent des facteurs modulant la conformation et/ou de la fonction des glycoprotéines. Ces protéines joueraient, par ailleurs, des rôles clés dans les mécanismes aussi variés que fondamentaux que sont l'élongation cellulaire, la lignification et le développement du grain de pollen
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Audfray, Aymeric. "La protéine-O-fucosyltransférase 1 (Pofut1) : caractérisation fonctionnelle et régulation de la voie de signalisation de Notch au cours de la myogenèse." Limoges, 2008. https://aurore.unilim.fr/theses/nxfile/default/9265bb72-4572-4eac-8191-98d5650a5fea/blobholder:0/2008LIMO4051.pdf.
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La myogenèse du muscle squelettique est un processus complexe dont certaines étapes sont régulées par la voie de signalisation de Notch. Chez les Mammifères, la présence de O‑fucosylglycannes sur la partie extracellulaire des récepteurs Notch influence l’activation de la voie de signalisation. Leur synthèse débute par l’action de la O‑fucosyltransférase Pofut1. Au cours de ce travail de thèse, nous avons mis en évidence, par mutagenèse dirigée, l’implication du site conservé de N‑glycosylation N65 pour l’intégrité structurale de Pofut1. Ceci constitue un nouvel exemple de l’influence de la N‑glycosylation dans le repliement des glycoprotéines. Des analyses combinant l’utilisation de la lignée de cellules myoblastiques C2C12 et le suivi de l’expression des gènes de la myogenèse, constructeurs des O-fucosylglycannes et des acteurs de la voie de Notch, par PCR quantitative en temps réel et à haut débit, nous ont permis de proposer un modèle décrivant le fonctionnement de la voie de signalisation de Notch durant la différenciation myogénique. Ce modèle met en évidence le rôle majeur de plusieurs acteurs de cette voie de signalisation comme Dll1, Notch3, Lfng et Pofut1. Il constitue un point de départ nécessaire aux analyses de la sur- et sous-expression de Pofut1 lors de la différenciation myogénique. Dans cette perspective, nous avons construit une souche de cellules C2C12 surexprimant Pofut1. A terme, les études sur les modifications de l’expression des gènes impliqués dans la biosynthèse des O-fucosylglycannes nous permettront de connaître plus précisément, les mécanismes moléculaires qui orchestrent la voie de signalisation de Notch dans le processus de myogenèseSkeletal myogenesis is a complex process in which some steps are regulated by Notch signaling pathway. In mammals, the presence of O-fucosylglycans on the extracellular domain of Notch receptors influences the activation of signalling pathway. Pofut1 is an O-fucosyltransferase that initiates O‑fucosylglycans synthesis. During this thesis work, we have identified, by site-directed mutagenesis, the involvement of the conserved N-glycosylation site at position 65, N65, in Pofut1 structural integrity. This is an additional example of the influence of N-glycosylation in glycoprotein folding. Analysis using myogenic C2C12 cell line, and real-time quantitative PCR allowed us to study expression of genes involved in myogenesis, Notch signaling and O-fucosylglycans synthesis. We consequently suggest a model which defines the mechanism of Notch signaling during myogenic differentiation. This model highlights the essential role of several actors of this signalling pathway including Dll1, Notch3, Lfng and Pofut1. It constitutes a necessary starting point for further studies concerning Pofut1 over- and down-expression, during myogenic differentiation. Therefore, we created a C2C12 cell line over-expressing Pofut1. Ultimately, studies on expression modifications of genes implicated in O‑fucosylglycan biosynthesis will enable us to more precisely understand molecular mechanisms that orchestrate Notch signalling in the process of myogenesis
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Fitchette, Anne-Catherine. "Immunolocalisation de la xylosylation et le la fucosylation des glycannes complexes dans l'appareil de Golgi des cellules de sycomore (Acer pseudoplatanus L. )." Rouen, 1993. http://www.theses.fr/1993ROUES003.
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Les glycanes complexes portés par les glycoprotéines végétales diffèrent de ceux rencontrés chez les mammifères par l'absence d'acide sialique et par la présence d'un résidu fucose α1,3 lié au GlcNAc de la partie réductrice de l'unité chitobiose et d'un xylose lié en β1,2 au β-mannose. La séquence des évènements de maturation des glycanes n'est pas aussi bien connue dans la cellule végétale que pour la cellule animale. Dans cette étude, nous nous sommes principalement intéressés aux xylosyl- et fucosyl-transférases spécifiques aux plantes et intervenant dans la glycosylation tardive. Nous avons préparé des anticorps dirigés contre les glycanes végétaux contenant des résidus xylose ou fucose. Par immunodétection à l'aide de ces anticorps, nous avons visualisé la distribution subcellulaire des glycoprotéines portant ces glycanes complexes dans les cellules de sycomore. De plus, cette approche immunocytochimique a permis une localisation indirecte des xylosyl- et fucosyl-transférases en détectant les glycanes produits par ces enzymes dans les empilements golgiens des cellules de sycomore. Nos résultats indiquent que les glycanes complexes N-liés aux glycoprotéines vacuolaires ou pariétales sont xylosylés principalement dans le Golgi médian, alors que leur fucosylation est un évènement tardif intervenant dans le Golgi trans
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Loriol, Céline. "Les O-fucosyltransférases : caractérisation des enzymes bovines et étude préliminaire du rôle de Pofut1 murine dans la différenciation de la cellule musculaire." Limoges, 2006. http://aurore.unilim.fr/theses/nxfile/default/1c0efd0e-f3ae-4389-9e61-7d992890c88d/blobholder:0/2006LIMO0055.pdf.
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La O-fucosylation est l’ajout d’un fucose sur un résidu sérine ou thréonine compris dans deux types de domaines peptidiques, les EGF et les TSR. Cette modification post-traductionnelle dépend de deux enzymes, Pofut1 pour les EGF et Pofut2 pour les TSR. Nous avons étudié l’évolution des gènes Pofut1 et Pofut2, et avons démontré que ces gènes présents en un seul exemplaire, existaient déjà chez l'ancêtre des Bilatériens voire des Métazoaires, probablement sous forme morcelée. Une situation originale existe pour Pofut2, retrouvé chez un groupe de Protozoaires, les Apicomplexés. La structure des deux gènes chez le bovin et leur expression tissulaire ont été établies. Nous trouvons une situation inédite puisqu'il existe pour chacun d’entre eux, cinq variants transcriptionnels dont un seul code l'enzyme active, différemment exprimés selon les tissus bovins. Les enzymes Pofut1 et Pofut2 actives seraient présentes dans tous les tissus analysés, à l’exception notable des muscles squelettiques de l'adulte où des formes atypiques sont présentes. Les autres variants transcriptionnels, la plupart tronqués, auraient un rôle dans la régulation du taux d'expression des gènes. Pofut1 est la première fucosyltransférase à avoir été identifiée comme résidente du réticulum endoplasmique. D'un point de vue fonctionnel, nous avons démontré chez le bovin que cette glycosyltransférase, porteuse de deux N-glycanes vraisemblablement de type oligomannosidique, était correctement repliée et donc douée d'une activité enzymatique, à condition que son premier site de N-glycosylation soit occupé. L’activité des récepteurs Notch et de leurs ligands présents à la surface de nombreuses cellules est modulée par l’état de O-fucosylation de leurs domaines EGF. Etant donné l’implication de ces récepteurs dans la régulation de la myogenèse, la O-fucosylation doit y contribuer tout autant. Ainsi, nous avons débuté une étude des répercussions d’une modulation de l’expression de Pofut1 sur la différenciation de cellules primaires myoblastiques bovines et de cellules murines de la lignée C2C12. Nous montrons que la surexpression transitoire de l’enzyme Pofut1 murine retarde l’expression des facteurs de transcription myogéniques de la famille bHLH: Myf5, MyoD, Myogénine et MRF4. Ces résultats, encore préliminaires, ouvrent de nouvelles et passionnantes perspectives d’analyse de l’influence des O-fucosyltransférases au cours du processus de myogenèseO-fucosylation is the addition of a fucose on serine or threonine comprised in two types of peptidic domains, EGF and TSR. This post-translational modification depends on two enzymes. Pofut1 is responsible for O-fucosylation of EGF repeats, whereas Pofut2 adds fucose on TSR. We studied the evolution of Pofut1 and Pofut2 genes and demonstrated that these genes, present in a single copy, already existed in the ancestor of Bilaterians, or even Metazoans, probably in polyexonic form. An original situation exists for Pofut2, recovered in a group of Protozoans, the Apicomplexa. Structures of the two bovine genes and their tissular expression have been established. We find an original situation since it exists for each of them, five transcript variants of which only one encodes the active enzyme, differently expressed among bovine tissues. The Pofut1 and Pofut2 active enzymes would be present in all analyzed tissues, except for adult skeletal muscles where atypical forms are present. The other transcript variants, more or less truncated, probably play a role in regulating the expression level of these genes. Pofut1 is the first fucosyltransferase to have been identified as an endoplasmic reticulum resident. Functionally, we demonstrated for the bovine that this glycosyltransferase, bearing two N-glycans probably of oligomannosidic type, was correctly folded and therefore possessed an enzymatic activity, provided its first site of N-glycosylation is occupied. The activity of the Notch receptors and their ligands, present at the surface of numerous cells, is modulated by the state of O-fucosylation of their EGF domains. Considering the implication of these receptors in the regulation of myogenesis, Ofucosylation must contribute all as much there. Thus, we started a study of the repercussions of a modulation of Pofut1 expression on the differentiation of bovine primary muscular cells and murine cells of the C2C12 lineage. We show that the transient surexpression of the murine Pofut1 enzyme delays the expression of myogenic transcription factors belonging to bHLH family: Myf5, MyoD, Myogenine and MRF4. These preliminary results open new and exciting perspectives of analysing the influence of O-fucosyltransferases during the complex process of myogenesis
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Heu, Katy. "Caractérisation de la lignée murine hypomorphe Pofut1cax : un modèle d’étude in vivo du rôle de la O-fucosylation de Notch au cours de la myogenèse." Limoges, 2013. http://www.theses.fr/2013LIMO4033.
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La protéine O-fucosyltransférase 1 (POFUT1) greffe des résidus O-fucose sur des domaines EGF-like retrouvés sur diverses protéines dont les récepteurs Notch et leurs ligands. En plus de son rôle d’O-fucosyltransférase, POFUT1 possède aussi un rôle de chaperonne pour le récepteur Notch. La présence de résidus O-fucose ainsi que leur allongement par la -1,3-GlcNAc-transférase (lunatic fringe) influence la liaison de Notch avec ses ligands. La voie de signalisation Notch est connue pour son rôle dans divers processus biologiques. Dans le développement du muscle squelettique, la voie de signalisation Notch est impliquée dans le maintien et l’activation des cellules satellites et l’inhibition de la différenciation des myoblastes. L’inactivation de Pofut1 chez la souris est létale au stade embryonnaire à 9. 5jpc. Chez ces animaux, les anomalies constatées rappellent celles retrouvées chez des mutants d’acteurs de la voie de signalisation Notch (RBPJ et Présénilines). Récemment une mutation spontanée dans le gène murin Pofut1 qui se manifeste par l’insertion d’un élément IAP (intracisternal A particle) de la famille des rétrotransponsons dans l’intron 4 a été aractérisée comme un allèle hypomorphe Pofut1cax (Schuster-Gossler et al. 2009). Ce modèle constitue une nouvelle approche pour étudier le rôle de POFUT1 notamment au cours de la myogenèse. Dans cette perspective, à partir de ce modèle, une analyse morphométrique du muscle squelettique a été réalisée. Cette analyse a permis de mettre en évidence une hypertrophie musculaire à l’âge adulte associée à une diminution du nombre de cellules satellites. L’étude du programme myogénique de cultures primaires de cellules satellites issues de souris cax montre une différenciation précoce. Ces phénotypiques sont similaires à celles obtenues chez des mutants de la voie de signalisation Notch suggérant une régulation de la myogenèse par POFUT1 à travers la modulation de l’interaction du récepteur Notch avec ses ligandsThe Notch signaling pathway is an evolutionarily conserved pathway that is critical for tissue morphogenesis during development. Regulation of Notch signaling is involved in somitogenesis, muscle development, and the proliferation and cell fate determination of muscle stem cells. Notch receptors are modified by O-fucosylation of EGF-like repeats by protein O-fucosyltransferase 1 (POFUT1). Fringe enzymes add N-acetylglucosamine to Ofucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. Mice embryos lacking Pofut1 die with a phenotype indicative of global inactivation of Notch signaling. To address physiologic functions of O-fucose glycans in myogenesis, we examined mice with hypomorphic allele of Pofut1 (Pofut1cax), a spontaneous mutation in the Pofut1 gene caused by an insertion of an intracisternal A particle retrotransposon in the intron 4 (Schuster-Gossler et al. 2009). Phenotype characterization of this mouse line at the muscle level revealed muscle hypertrophy in adult and a depletion of satellite cells. Isolation and culture of these cells showed early differentiation. Taking together, these results suggest that cax mice phenocopy Notch signaling mutants. The combined data support a key role for Ofucose glycans in Notch regulation of myogenesis through modulation of Notch-ligand interactions
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Der, Vartanian Audrey. "Rôle(s) de la protéine O-fucosyltransférase 1 au cours de la différenciation myogénique." Thesis, Limoges, 2015. http://www.theses.fr/2015LIMO0048.
Full textAbstract:
Au cours de la myogenèse post-natale, la voie de signalisation de Notch participe au développement et à la régénération du muscle squelettique chez les mammifères. Elle permet le maintien de l'état prolifératif des myoblastes, contrôle la quiescence des cellules satellites in vivo et préserve une sous-population de cellules de réserve indifférenciées in vitro. L' activation de la voie et l'interaction du récepteur Notch avec ses ligands est dépendante de leur entité glucidique, notamment de leurs O-fucosylglycannes. La synthèse de ces derniers est initiée par la protéine O-fucosyltransférase 1 (Pofut1) qui greffe un O-fucose sur des domaines peptidiques particuliers appelés EGF-like. Bien que les acteurs moléculaires de la différenciation myogénique aient été largement étudiés par la communauté scientifique, la contribution de la glycosylation des protéines dans ce processus reste peu documentée. Une approche expérimentale in vitro basée sur l'utilisation de la lignée myoblastique murine C2C12 nous a permis d'identifier une expression importante de Pofut1 dans les cellules de réserve tandis qu' elle est restreinte dans les myotubes durant la différenciation myogénique. Plusieurs lignées de cellules C2C12 ont été générées pour qu' elles expriment de manière stable et différentielle Pofut1. Elles permettent ainsi d' évaluer l' importance du niveau d' expression de Pofut1 sur la différenciation myogénique.La sous-expression de Pofut1 réduit l' activation de la voie de signalisation de Notch conduisant à une entrée précoce des myoblastes dans le programme myogénique. Ceci a pour conséquence la dépletion des cellules de réserve Pax7+/MyoD- au profit d' une augmentation du nombre de myotubes. Des études morphométriques ont révélé un défaut d' accrétion nucléaire dans les myotubes sous-exprimant Pofut1, caractéristique d' une altération de la fusion secondaire. Ces observations sont accompagnées d' une diminution significative de l' expression du récepteur à l' interleukine 4 dans les cellules de reserve sous-exprimant Pofut1. Les lignées cellulaires ré-exprimant Pofut1 présentent une activation de la voie de signalisation de Notch et un processus de fusion myoblastique correctement restaurés.Ces travaux de thèse ont mis en exergue pour la première fois le rôle essentiel de Pofut1 dans le devenir cellulaire et la fusion des myoblastes au cours de la différenciation myogéniqueDuring post-natal myogenesis, Notch signaling pathway is involved in the development and regeneration of skeletal muscle in mammals. It maintains progenitor cell properties during the development of the myogenic lineage and controls the transition of satellite cells from a quiescent to an active state and preserves a subpopulation of reserve cells, in cell culture, in an undifferentiated state. The interaction between Notch and its ligands and the activation of this signaling is mainly controlled by the activity of protein O-fucosyltranferase 1 (Pofut1) and thus by the O-fucosylation state of the EGF-like repeats.Although the molecular players in myogenic differentiation have been extensively studied by the scientific community, the contribution of glycosylated proteins in this process remains poorly documented. An experimental in vitro study based on the C2C12 mouse myoblast cell line allowed us to identify a high expression of Pofut1 in reserve cells while a low expression was found in myotubes during myogenic differentiation. Several C2C12 cell lines were generated to express Pofut1 at different levels. They were used to evaluate the contribution of Pofut1 expression to the myogenic differentiation.The knockdown of Pofut1 repressed Notch signaling pathway activation leading to an earlier entrance of myoblasts in myogenic program. This resulted in the depletion of reserve cells Pax7+/MyoD- and an increase in the number of myotubes. Morphometric analysis revealed a nuclear accretion defect in Pofut1 knockdown myotubes. A significant decrease in the expression of the interleukin-4 receptor in Pofut1 knockdown reserve cells was also observed. Cell lines re-expressing correctly Pofut1 restored Notch signaling pathway and subsequently myoblast fusion process.This thesis work highlights, for the first time, the crucial role of Pofut1 in the cell fate decision and the fusion of myoblasts during myogenic differentiation
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Léonard, Renaud. "Contribution à l'étude de l'α(1,4)-fucosylation chez les végétaux : Purification partielle de l'α(1,4)-fucosyltransferase de S. alba et clonage du gène correspondant chez A. thaliana." Limoges, 2001. http://www.theses.fr/2001LIMO0051.
Full textAbstract:
Parmi les oses qui entrent dans la composition des N-glycannes végétaux, le fucose peut être transféré respectivement selon une liaison α(1,3)- sur le chitobiose et/ou en α(1,4) sur un groupement de lacto-N-biose par des α(1,3) ou des α(1,4)-fucosyltransférases. Cependant, aucun gène d'α(1,4)-fucosyltransférase n'a été cloné à ce jour. Dans le but d'obtenir des données moléculaires sur les α(1,4)-fucosyltransférases végétales, une démarche de purification protéique et une approche de clonage de gènes ont été entreprises. La purification protéique a été réalisée sur des cellules en culture de Silene alba. Bien que partielle, elle a permis de caractériser l'enzyme végétale en termes de spécificités de substrats accepteurs et de mécanisme catalytique. Elle a également permis de proposer l'hypothèse de l'existence d'une forme soluble d'α(1,4)-fucosyltransférase chez cette espèce en plus d'une forme membranaire. Avec le modèle Arabidopsis thaliana, nous avons pu identifiér le premier gène végétal codant une α(1,4)-fucosyltransférase. Cette enzyme présente quelques similitudes avec les autres α(1,3/4)-fucosyltransférases connues et s'exprime dans les différents organes étudiés de la plante (feuilles, racines et fleurs). L'étude de la répartition tissulaire et cellulaire du produit de l'α(1,4)-fucosyltransférase, le motif Lewis a, confirme les résultats obtenus et montre en plus que les protéines α(1,4)-fucosylées sont essentiellement présentes à la surface de la cellule végétale et dans les différents organes étudiés,végétatifs comme reproducteursFucose of plant N-glycans is tranferred in α(1,3) linkage on chitobiose and/or in α(1,4) linkage on lacto-N-biose by α(1,3)- or α(1,4)-fucosyltransferases, respectively. Nevertheless, no plant α(1,4)-fucosyltransferase gene has been cloned yet. To get sequence information on plant α(1,4)-fucosyltransferases, we have undertaken both a protein purification procedure and a molecular biology approach. [. . . ]
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D, Nolfi. "Studio morfologico e funzionale dell’apparato di Golgi in relazione ad una struttura LTL-positiva nelle cellule di carcinoma prostatico DU145." Doctoral thesis, Università di Siena, 2020. http://hdl.handle.net/11365/1095701.
Full textAbstract:
The Golgi apparatus is a complex structure present in all eukaryotic cells. This organelle, which was first observed in 1989, still represents a fascinating enigma for much of its structural and functional peculiarity. Generally, the Golgi apparatus is known as the heart of the secretory pathway and glycosylation, which is one of the major post-traductional modifications.
Most of the reactions of the glycosylation pathway occur in the Golgi apparatus, where glycosyltransferases and glycosidases modify glucidic chains by adding or removing monosaccharides. All the steps follow a precise sequential order from the cis-Golgi to the trans-Golgi network, depending on the exclusive presence of peculiar enzymes in each Golgi compartment.
The sequential order of all these reactions is guaranteed by the morphological stability of the Golgi apparatus, which appears as many staked cisternae fused together forming a unique ribbon structure in vertebrates’ cells. The integrity of the Golgi ribbon is the result of a perfect harmonization between Golgi matrix proteins and the cytoskeleton.
Defect in glycosylation has been observed in many pathologies, such as in neurodegenerative disease and malignant transformation of cancer cells. Particularly, the Golgi apparatus of cancer cells loses its typical ribbon shape and splits in smaller vesicles scattered in the cytosol.
This thesis focuses on the defects of glycosylation in cancer cells, with a particular regard to fucosylation, since we observed a novel Golgi-derived α 1,2 fucosylated tubular structure exclusive of high proliferative cells. This structure, which was highlighted thanks to the α 1,2 fucose binding Lotus tetragonolobus lectin (LTL), seems to be responsible for a peculiar uptake system that lets many molecules, including LTL itself, enter the cell.
To better understand 1) the relation between the morphological scattering of the cisternae and the functionality of the Golgi apparatus and 2) the link between the Golgi disarrangement and the origin of the LTL-positive tubular system, we analysed cells from the human prostatic tumor cell line DU145 by confocal microscopy using the lectins LTL and AAA (Aleuria aurantia Agglutinin), both specific for fucose, and different antibodies able to mark the organelle.
Microscopic observations were parallelly performed with SDS-PAGE on DU145 extracts to analyze the localization of the Golgi proteins and glycans in the nuclear, cytoplasmic, membrane, and cytoskeleton fractions obtained using the Qproteome Cell Compartment Kit.
Furthermore, in order to evaluate the relationship between the Golgi apparatus/tubular system and the actin cytoskeleton, DU145 were left to grow in presence of Cytochalasin D, a fungal toxin capable to depolymerize the microfilaments.
Finally, we performed an uptake experiment to test the functionality of the tubular system, both in the presence and absence of cytochalasin.
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Lang, Robert [Verfasser], and Michael [Akademischer Betreuer] Vogeser. "An LC-MS/MS-based approach for analysis of site-specific core-fucosylation of low-concentrated glycoproteins in human serum using the biomarker prostate-specific antigen (PSA) as example / Robert Lang ; Betreuer: Michael Vogeser." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1176409727/34.
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Weng, Yi-Cheng, and 翁義程. "Calreticulin regulates FUT1 expression and beta1 integrin fucosylation in PC3 prostate cancer cells." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/65136801959781499618.
Full textAbstract:
碩士國立臺灣大學
生命科學系
104
Calreticulin (CRT) is a multifunctional ER chaperon protein and it had been revealed that knockdown of CRT may suppress cell proliferation and migration in J82 bladder cancer cell. We had further demonstrated that CRT enhanced the expression of fucosyltransferase1 (FUT1), which leads to an enhancement of fucosylation of beta1 integrin, and promote cell adhesion in J82 cells. Previous studies had shown that knockdown of CRT suppress cell proliferation and migration in PC3 prostate cancer cell as well. However, the effects by CRT on integrin fucosylation have not been revealed in prostate cancer so far. In this study, therefore, we attempted to investigate whether CRT affect FUT1 expression and integrin fucosylation in PC3 prostate cancer cells as well. Our results showed that FUT1 expression and beta1 integrin fucosylation are suppressed after knockdown of CRT. Futhermore, cell adhesion and integrin activation are down regulated as well. These observations imply that CRT might regulate prostate cancer metastasis through FUT1 and therefore could be a potential target for development of cancer treatments.
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Chun-I, Chen. "Glycomics Mapping & Sequencing of Colon Carcinomas by MS: Fucosylation on Extended Type 1 Chain." 2004. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-0507200412595800.
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Chen, Chun-I., and 陳駿逸. "Glycomics Mapping & Sequencing of Colon Carcinomas by MS: Fucosylation on Extended Type One Chain." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/63396719829498645607.
Full textAbstract:
碩士國立臺灣大學
生化科學研究所
92
Aberrant glycosylation is recognized as a common phenotype for tumor, which usually involves increased or decreased expression of lacto-serious type 1 -(3Gal1-3GlcNAc1-)n and/or type 2 -(3Gal1-4GlcNAc1-)n chain, concomitant with altered degree of sialylation and fucosylation. While the type 2 poly-N-aectyllactosamine (polyLacNAc) chain has been found in branched and/or extended form in both normal and cancer cells, the type 1 N-aectyllactosamine (LacNAc) chain is generally considered not to occur in an extended form. In the 90s, two papers have documented and characterized the presence of extended type 1 chains as Lea-Lea and Leb-Lea on lactosylceramides of human colonic adenocarcinoma cell line Colo205. The possible presence of similar epitope on extended type 1 chain of glycoproteins have been implicated by monoclonal antibody detection but no N- or O-glycans bearing this epitope from Colo205 or any other sources has ever been identified due to analytical difficulties. Our interest in Colo205 aims to develop tandem mass spectrometry (MS)-based sequencing strategy for facile discrimination of type 1 and 2 chain and, with it, to differentiate between Lex/y from Lea/b in glycomic applications. The dimeric Leb/Lea bearing glycosphingolipids (GSLs) from Colo205 were first used as standards to optimize and validate the MS/MS sequencing methodologies based on concerted use of ESI- and MALDI-MS, in conjunction with chemical and enzymatic derivatization. Next, glycomic maps of the neutral, mono-, and disialylated multifucosylated GSLs were derived from DEAE anion chromatography, for neutral fraction, larger GSLs carrying additional Le units as both linear and branched polyLacNAc chain and for sialylated GSLs, glycans with dimeric Lea were identified. Similar MS-based glycomic mapping on the released O-glycans coupled with further chromatographic fractionation led to identification of dimeric Lea and Lea-Lex epitopes preferentially extending from the 3- and 6-arms of core 1 structures, respectively. A different strategy was adopted for the even more complicated N-glycans in which endo--galactosidase was used to release non-reducing terminal glycan chains with internal non-fucosylated type 2 LacNAc unit that is susceptible to enzymatic cleavage. Extended type 1 chains were thus identified by MS/MS sequencing to be present among the N-glycans, conjugated to internal type 2 unit. In a slight variation, mannose binding lectin (MBL) which shows growth inhibitory activity toward a different adenocarcinoma cell line, SW1116, was used to pull out binding ligands from the N-glycan pools released from initially MBL-enriched SW1116 glycoproteins. Using the same MS methodologies developed, the MBL ligands were identified as Leb/a terminated polyLacNAc chains carried on the multiantennary complex type N-glycans.
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Mickael, Guillemineau. "Total Synthesis of the Tumor-Associated Carbohydrate Antigen Lewis A Lewis X Hexasaccharide and Selected Fragments." Thesis, 2012. http://hdl.handle.net/10214/3847.
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Carbohydrates constitute the most abundant class of natural products in the living world and they play various roles. They are notably involved in cell-cell interactions and immune reactions. It has been observed that tumor cells express, on their surface, unusual oligosaccharides named Tumor-Associated Carbohydrate Antigen (TACA). One TACA of interest to our research group is the Lewis A Lewis X hexasaccharide that is displayed on the surface of squamous lung carcinoma cells. Since carbohydrates are involved in immune reactions and can be recognized by antibodies, it becomes possible to design a carbohydrate-based vaccine against these tumor cells. This thesis describes the total synthesis of the TACA Lewis A Lewis X hexasaccharide and the preparation of two fragments: one tetra- and one pentasaccharide. These molecules were prepared as hexyl and aminohexylglycosides. In addition, the hexasaccharide was synthesized as a disulfide. This diversity of these synthons will allow conjugation to a protein, analysis by nuclear magnetic resonance techniques, and immobilization on gold of the antigen. Without doubt, this work is a significant contribution to the development of an anti cancer vaccine as it constitutes the first stage of the process.
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Liu, Ying-Chih, and 劉英志. "Sialylation and Fucosylation of Epidermal Growth Factor Receptor Suppress its Dimerization and Activation in Lung Cancer Cells." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/55633949371805189193.
Full textAbstract:
博士國立臺灣大學
生化科學研究所
99
Glycosylation is an important post-translational modification, which regulates proteins folding and functional expression. Previous studies have shown that, abnormal glycosylation in tumor cells can participate in cancer progression and malignancy. In the current study, we compared the sialylated and fucosylated proteins with alkynyl sugars in different lung cancer cell lines, CL1-0 and CL1-5, two subpopulations derived from the same parental cell line but with distinct invasiveness. Our initial experiments demonstrated that CL1-5, the highly aggressive cells, expressed more sialylated and fucosylated proteins than CL1-0. At next step, we identified the unique sialylated proteins in CL1-5. Among them, we chose epidermal growth factor receptor (EGFR) to further study the role of sialylation on its function. Based on glycan analysis, we validated the distinct sialylation level of EGFR in both cells. Interestingly, we also observed higher fucosylation of EGFR in CL1-5 than in CL1-0, corresponding to the comparison of whole proteins glycosylation in both cells using alkynyl fucose. Further study suggested that overexpression of sialyltranssferases in CL1-5 and
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Sui, Ning. "DELLA is O-Fucosylated by SPINDLY." Diss., 2016. http://hdl.handle.net/10161/13413.
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Plant growth and development are strictly regulated by internal hormonal signaling networks, which integrate and coordinate to promote plants’ adaptation and survival in the changing environment. Among the diverse hormones, gibberellins (GAs) are the phytohormones that regulate various processes, from seed germination to fruit development. The conserved plant-specific GRAS family protein DELLAs, key repressors in the GA signaling pathway, serve as the central coordinator of multiple signaling networks through physical interactions with many key transcription factors/regulators in other pathways.
Diverse DELLA-interacting proteins (DIPs) from different signaling pathways and various protein families have been identified in recent years. All the DIPs interact with the C-terminal GRAS domain of DELLA, however, the mechanism of how the GRAS domain interacts with diverse proteins remains a mystery. To solve this problem, I expressed a number of DELLA proteins in E.coli and obtained high-purity protein for biochemical and structural analysis.
As the central coordinator of plant growth and development, DELLA’s activity and stability are regulated by post-translational modifications. Our lab recently showed that SECRET AGENT (SEC) modulates the activity of DELLA through O-linked N-acetylglucosamine (O-GlcNAc) modification in Arabidopsis. Nevertheless, SEC’s paralog SPINDLY (SPY), a putative O-GlcNAc transferase (OGT) identified 20 years ago, does not have OGT activity, and serves as opposite role to SEC in GA signaling with an unknown mechanism.
Our lab made the breakthrough in uncovering the SPY function, and showed it promotes the O-fucosylation of DELLA in planta. I further proved that SPY is a novel protein O-fucosyltransferase through biochemical analysis. SPY specifically transfers O-fucose from GDP-fucose to its substrate peptide, and SPY mutant proteins showed reduced or abolished transferase activity. This is the first work to identify O-fucosylation of nuclear proteins in any organism. O-fucosylation of DELLA activates DELLA by promoting its interaction with DIPs, opposite to repression of interaction with O-GlcNAcylation. Previous studies showed that SPY is involved in multiple cellular pathways such as GA signaling, cytokinin signaling and the circadian clock. Thus, SPY plays an important role in regulating plant growth and development through O-fucosylation of key components in diverse intracellular pathways.
SPY orthologs are conserved in bacteria, protists, algae and plants, while SEC orthologs are also present in fungi and animals. SPY-like and SEC-like proteins share high sequence similarity, except that two key residues important for the OGT activity of SEC is missing in SPY. Structure analysis of SPY (or its orthologs) would greatly facilitate our understanding of its unique substrate specificity. Toward this goal, I expressed Arabidopsis SPY proteins (as well as bacterial SPY orthologs) in E.coli and obtained high-purity protein for structural analysis. I further identified lead conditions that produce needle-cluster crystals. While optimization would be required, these studies will ultimately reveal the structure of SPY and the architecture of the active site, to show how SPY interacts with GDP-fucose for the transferase activity.
Dissertation
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"Diagnostic and Prognostic Capacity of Serum Glycan Nodes in Different Types of Cancer." Doctoral diss., 2018. http://hdl.handle.net/2286/R.I.49169.
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abstract: Glycans are monosaccharide-based heteropolymers that are found covalently attached to many different proteins and lipids and are ubiquitously displayed on the exterior surfaces of cells. Serum glycan composition and structure are well known to be altered in many different types of cancer. In fact, glycans represent a promising but only marginally accessed source of cancer markers. The approach used in this dissertation, which is referred to as “glycan node analysis”, is a molecularly bottom-up approach to plasma/serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, β1-6 branching, and core fucosylation as single analytical signals.
The diagnostic utility of this approach as applied to lung cancer patients across all stages as well as prostate, serous ovarian, and pancreatic cancer patients compared to certifiably healthy individuals, nominally healthy individuals and/or risk-matched controls is reported. Markers for terminal fucosylation, α2-6 sialylation, β1-4 branching, β1-6 branching and outer-arm fucosylation were most able to differentiate cases from controls. These markers behaved in a stage-dependent manner in lung cancer as well as other types of cancer. Using a Cox proportional hazards regression model, the ability of these markers to predict progression and survival in lung cancer patients was assessed. In addition, the potential mechanistic role of aberrant P/S glycans in cancer progression is discussed.
Plasma samples from former bladder cancer patients with currently no evidence of disease (NED), non-muscle invasive bladder cancer (NMIBC), and muscle invasive bladder cancer (MIBC) along with certifiably healthy controls were analyzed. Markers for α2-6 sialylation, β1-4 branching, β1-6 branching, and outer-arm fucosylation were able to separate current and former (NED) cases from controls; but NED, NMIBC, and MIBC were not distinguished from one another. Markers for α2-6 sialylation and β1-6 branching were able to predict recurrence from the NED state using a Cox proportional hazards regression model adjusted for age, gender, and time from cancer. These two glycan features were found to be correlated to the concentration of C-reactive protein, a known prognostic marker for bladder cancer, further strengthening the link between inflammation and abnormal plasma protein glycosylation.Dissertation/Thesis
Doctoral Dissertation Chemistry 2018
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Tan, Keng-Poo, and 陳瓊寶. "Fucosylation of LAMP-1 and LAMP-2 by FUT1 Correlates with the Lysosomal Positioning and Autophagic Flux of Breast Cancer Cells." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/49978591496419101600.
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博士國立陽明大學
微生物及免疫學研究所
104
Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. LAMP-1, lysosome-associated membrane protein-1, has been reported to carry alpha1,2-fucosylated LeY antigens in breast cancer cells; however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member LAMP-2 displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and LAMP-2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS3 and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER- breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mTORC1 and enhanced autophagosome-lysosome fusion. This might be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased rate of autophagic flux by decreasing mTOR signaling and increasing autolysosome formation.
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Calderon, Molina Angie Dayan. "Application of Human Glycosyltransferases in N-glycan Synthesis and Their Substrate Specificity Studies." 2016. http://scholarworks.gsu.edu/chemistry_diss/128.
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Glycoscience is important in many areas such as human health, energy and material science. Glycans have been shown to be involved in the pathophysiology of almost every major disease. Additional glycan structure knowledge is required to help advance personal medicine, and pharmaceutical developments, among others. For glycoscience to advance there is a need for large quantities of well-defined glycans and have quick access to glycosyltransferases for manipulating glycan synthesis.
Herein, we will cover our efforts on studying the substrate specificities of human glycosyltransferases such as FUT8 and Gn-T V, and their application on N-glycan synthesis. Complex asymmetric N-glycan isomer structures have been related to many diseases such as breast cancer, among others. Synthesis of complex asymmetric N-glycan isomer structures including: alpha-1,6 core-fucosylated, and tri-antennary structures can be achieved by taking advantage of the high specificity of glycosyltransferases that can work as unique catalyst to generate well-defined glycan structures.
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Luo, Wen-I. "The Role of Fucose in Learning and Memory: The Identification of a Fucosylprotein, Synapsin I, in the Rat Brain and the Characterization of its Fucosylation." Thesis, 2004. https://thesis.library.caltech.edu/7639/1/Luo-Wen-i-2004.pdf.
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Previous studies have shown that the glycoproteins containing the fucose moiety are involved in neuronal communication phenomena such as long-term potentiation and memory formation. These results imply that fucose containing glycoproteins might play an important role in learning and memory. To understand the role of fucose in neuronal communication, and the mechanisms by which fucose may be involved in information storage, the identification of fucosylproteins is essential. This report describes the identification and characterization of fucosylproteins in the brain, which will provide new insights into the role of the fucose involved molecular interactions.
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Freitas, Érica Nair André de. "Cloning of FUT8 gene and characterization of its expression in human cell lines." Master's thesis, 2018. http://hdl.handle.net/10362/59488.
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Altered glycosylation is a universal feature of cancer cells, and certain glycans are well-known markers of tumor progression. Glycans are involved in fundamental molecular and cell biology processes occurring in cancer, such as tumor cell dissociation and invasion, cell–matrix interactions, immune modulation, tumor angiogenesis, and metastasis formation.
Fucosylation, which comprises the transfer of a fucose residue to oligosaccharides and proteins, is regulated by many types of molecules, including fucosyltransferases. Fucosylation levels in normal colon is relatively low but increases during carcinogenesis.
In the present work, we developed a molecular tool, through an adenoviral expression system to express the fucosyltransferase FUT8. FUT8 enzyme is responsible for core alpha-(1,6)-fucosylation, which is reported to be increased in cancer, but its role in colorectal cancer (CRC) is still unknown. The FUT8-adenoviral system reported here was used to transduce CRC cell lines, and the resultant transduced cell lines were evaluated for the FUT8 expression, for alpha-(1,6)-fucosylation and tested for implications on the hallmarks of cancer: proliferation, invasion and metastasis.
Our data show that the overexpression of FUT8 increases the proliferation rate and increases the migration capacity of CRC cells. We also show that increased expression of FUT8 leads to changes of the expression of growth factors like TGF-β.
Our results suggest that increased FUT8 expression in CRC is associated with increased malignancy and further studies should be envisaged to understand the underlying mechanisms.
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Hellbusch, Christina. "Ein Knockout-Mausmodell für Congenital Disorder of Glycosylation-IIc: Defizienz des Golgi-GDP-Fucose-Transporters." Doctoral thesis, 2006. http://hdl.handle.net/11858/00-1735-0000-0006-AC49-1.
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