Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-03-10T13:25:39Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese juliana vasconcelos.pdf 2.pdf: 2676689 bytes, checksum: 3be9c8d80ebc1f51f2479aaec4fe30ce (MD5) / Made available in DSpace on 2017-03-10T13:25:39Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese juliana vasconcelos.pdf 2.pdf: 2676689 bytes, checksum: 3be9c8d80ebc1f51f2479aaec4fe30ce (MD5) Previous issue date: 2016-02-26 / CNPQ / O câncer de mama é o tipo de tumor que mais acomete as mulheres no mundo. No Brasil estimam-se para 2016/2017, 57.960 novos casos de câncer de mama por ano. O desenvolvimento destes tumores envolve um mecanismo complexo e multifatorial, onde glicosiltransferases e microRNAs (miRNAs) desempenham papeis importantes. O objetivo deste estudo foi avaliar a galactosiltransferase 7 (GALNAC7) e o miRNA 30c no carcinoma ductal invasivo mamário (CDI). Nosso estudo demonstrou, uma baixa expressão do antígeno Tn nos tumores mamários, diante da baixa expressão da GalNAC7 e do seu carboidrato-substrato N-acetil-galactosamina (GalNAc). O estudo evidenciou, também, que o gene GALNT7 não apresenta influência significativa quanto a um prognostico reservado para CDI, diante de sua baixa expressão gênica e proteica, demostrando que o mecanismo de O-glicosilação na formação de mucinas pode ser estimulado, também, por outros genes da família GALNT, porém observamos que o gene GALNT7 pode ser modulado pelo o miRNA 30c frente ao aumento de sua expressão nos tumores estudados, levantando a hipótese de um novo alvo diagnóstico e terapêutico via modulação da expressão de genes da glicosilação. A análise dos dados clínicopatologicos, não demonstrou correlação significativa, com exceção da expressão de GalNAc e tamanho do tumor. Diante da análise do segmento clínico de cada paciente, na curva de sobrevida, não houve resultados significativos quando comparados com os subtipos moleculares, estadiamento clínico, idade e tamanho do tumor. Assim, nossos resultados sugerem que o miRNA 30c pode ser um potencial regulador da expressão do gene GALNT7 e que assim favorece a menor expressão da enzima GALNAC7 levando a uma menor inserção do carboidrato GalNAc nos glicoconjugados de superfície de células de carcinoma ductal invasivo. / Breast cancer is the most common cancer in women in world. In Brazil, it is estimated 57,960 new cases of mammary cancers per year in 2016/2017. The development of these tumors comprises a complex and multifactorial mechanism where glycosyltransferase and microRNAs (miRNAs) play important roles. This study aimed to evaluate the galactosyltransferase 7 (GALNAC7) and the miRNA-30c in mammary invasive ductal carcinoma (IDC). Our results showed a low expression of Tn antigen in mammary tumors resulting from the low expression of GALNAC7 which leads to a low insertion of its saccharidesubstrate N-acetyl-galactosamine (GalNAc) in tumor cell glycoconjugates. The study also evidenced that the gene GALNT7 did not influenced the poor prognostic of IDC, since a low gene expression and, consequently, a low protein expression was observed. Such fact indicates that other genes of the GALNT family may stimulate the O-glycosylation. However, we observed that miRNA-30c might modulate GALNT7 expression and can be a new potential target for diagnosis and therapeutic via modulation of the expression of glycosylation genes. Among the clinocopathologic data, only GalNAc expression and tumor size present a correlation. Patient’s survival curve did not present correlation with tumor molecular subtyping, clinic staging, age and tumor size. Our results suggest that miRNA-30c may be a potential regulator of the GALNT7 gene expression and so favoring a lower expression of the enzyme GALNAC7 leading to a lower insertion of the saccharide GalNAc in glycoconjugates of cell surface in invasive ductal carcinoma.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-10-22T16:02:34Z No. of bitstreams: 2 Dissertação - Maycon Carvalho Ribeiro - 2014.pdf: 1028131 bytes, checksum: 34ecaebcaac3768726bacc1c6c4f694b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-10-22T16:03:56Z (GMT) No. of bitstreams: 2 Dissertação - Maycon Carvalho Ribeiro - 2014.pdf: 1028131 bytes, checksum: 34ecaebcaac3768726bacc1c6c4f694b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-22T16:03:56Z (GMT). No. of bitstreams: 2 Dissertação - Maycon Carvalho Ribeiro - 2014.pdf: 1028131 bytes, checksum: 34ecaebcaac3768726bacc1c6c4f694b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-08-04 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is an important industrial enzyme for being the only one able to convert starch and related glucans in cyclic oligosaccharides called cyclodextrins (CDs). The arrangement of the glucose units in the formation of CD results in a molecule with the shape of a cone, with hydrophobic interior and hydrophilic surface. This arrangement of glucose molecules in CDs allows its use as a host molecule in the formation of inclusion complexes with organic and inorganic compounds. This mechanism is advantageous in protecting the guest molecule from light, heat and oxidizing conditions and also enable the "dissolution" of compounds of low solubility in aqueous media. Cyclodextrins are used from the food industry to the pharmaceutical, in controlled drug delivery systems and immobilization of toxic compounds for environmental protection. The CGTases are mainly produced by bacteria of the genus Bacillus, found degrading starch rich substrates. The aim of this study was to identify, isolate, select and characterize strains of CGTase-producing bacteria from soil samples from different regions of Brazil as well as calculate the enzymatic production of these bacteria on low-cost substrates. With this work, it was possible to identify 17 bacteria producing cyclodextrin glycosyltransferase enzyme, with nine of them had values above 1.5 for enzymatic production. Of these, all were characterized as gram positive Bacillus. Bioprospecting of bacteria in soils of different cultures led to the identification of bacteria that may be used in studies for the production of cyclodextrin glycosyltransferase and subsequent implementation by various industries. / Ciclodextrina glicosiltransferase (CGTase, EC 2.4.1.19) é uma enzima industrial importante, sendo a única capaz de converter o amido e glucanos afins em oligossacarídeos cíclicos chamados ciclodextrinas (CDs). O arranjo das unidades de glicose na formação da CD resulta em uma molécula com a forma de cone, com interior hidrofóbico e superfície hidrofílica. Este arranjo das moléculas de glicose permite seu uso como molécula hospedeira na formação de complexos de inclusão com compostos orgânicos e inorgânicos. Este mecanismo é vantajoso na proteção de moléculas contra luz, calor e condições oxidantes e também possibilita melhorar a solubilidade de compostos hidrofóbicos. Ciclodextrinas são utilizadas desde a indústria de alimentos até a farmacêutica, em sistemas de liberação controlada de drogasse e imobilização de compostos tóxicos para proteção ambiental. As CGTase são principalmente produzidas por bactérias do gênero Bacillus, encontradas degradando substratos ricos em amido. O objetivo deste estudo foi identificar, isolar, selecionar e caracterizar linhagens de bactérias produtoras de CGTase a partir de amostras de solo de diferentes regiões do Brasil bem como calcular o índice enzimático destas bactérias em substratos de baixo custo. Com a realização deste trabalho, foi possível identificar 17 bactérias produtoras da enzima ciclodextrina glicosiltransferase, sendo que nove delas apresentaram valores para índice enzimático acima de 1,5. Destas, todas foram caracterizadas como sendo Bacillus gram positivos. A bioprospecção de bactérias em solos de diferentes culturas possibilitou a identificação de bactérias que poderão ser usadas em estudos para a produção da ciclodextrina glicosiltransferase e posterior aplicação pelas mais diversas indústrias.

Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator.

Abstract Lysyl hydroxylase (EC 1.14.11.4, procollagen-lysine, 2-oxyglutarate, 5-dioxygenase, Plod) catalyzes the hydroxylation of certain lysine residues in collagens and in other proteins with collagenous domains. Three lysyl hydroxylase isoforms have been cloned from human and rat. The importance of lysyl hydroxylase 1 in collagen biosynthesis is demonstrated by the heritable disorder, Ehlers-Danlos syndrome type VI, which is characterized by joint laxity, progressive scoliosis, muscle hypotonia, scleral fragility and rupture of the ocular globe. An alternatively spliced form of lysyl hydroxylase 2 seems to function as a telopeptide lysyl hydroxylase. Lysyl hydroxylase 3 has three enzyme activities, lysyl hydroxylase, hydroxylysyl galactosyltransferase (EC 2.4.1.50), and galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66) activities that have been demonstrated earlier with in vitro experiments. In this thesis study, the cDNAs of mouse lysyl hydroxylase isoforms 1, 2 and 3 were cloned and characterized and the gene structures of lysyl hydroxylase 2, Plod2, and lysyl hydroxylase 3, Plod3, were determined. Mouse lysyl hydroxylase isoforms were found to be highly homologous to the corresponding human isoforms and they were approximately 60% identical with each other. The mouse Plod3 gene has 19 exons as do the human PLOD1 and PLOD3 genes, and mouse Plod2, like the human PLOD2, has 20 exons including one alternatively spliced extra exon. The mouse isoforms were also found to have distinct tissue distributions. Phylogenetic analysis revealed that the lysyl hydroxylase genes have evolved from an ancestral gene through two gene duplication events. Lysyl hydroxylase 3 was demonstrated to be the oldest isoform, which is further supported by the association of glycosyltransferase activities with lysyl hydroxylase 3 and with the only lysyl hydroxylase of Caenorhabditis elegans. The roles of the different enzyme activities of lysyl hydroxylase 3 were determined in vivo by generating three genetically modified lysyl hydroxylase 3 mouse lines. The analysis of these mouse lines demonstrated that lysyl hydroxylase 3 possesses at least lysyl hydroxylase and glucosyltransferase activities in vivo and it functions as the main, if not the only glucosyltransferase during embryogenesis. The absence of lysyl hydroxylase 3 and, especially, its glucosyltransferase activity results in the abnormal glycosylation of type IV collagen, and thus causes a severe basement membrane defect leading to death during early development. By contrast, lysyl hydroxylase activity had no effect on embryonic development, but caused changes in the structure of the epidermal basement membrane and changes in collagen fibril organization and probably in their interactions.

Candida albicans est une levure saprophyte présente dans la flore digestive humaine. Elle peut néanmoins devenir pathogène chez des individus immunodéficients et causer des infections sévères associées à de forts taux de mortalité. La paroi de C. albicans, en contact avec l’hôte, contient des β-1,2 oligomannosides (β-Man) liés à de multiples molécules pariétales telles que le phospholipomannane (PLM) ou le phosphopeptidomannane (PPM). Ces β-Man sont présents dans les espèces les plus pathogènes de Candida (principalement C. albicans, mais également C. glabrata et C. tropicalis) et sont considérés comme des facteurs de virulence. L’identification d’une famille de 9 gènes codant pour des β-mannosyltransférases (CaBmt) a permis une meilleure compréhension du rôle de 6 de ces enzymes. Des études de génétique inverse ont montré que la β-1,2-mannosylation du PPM était assurée par les enzymes CaBmt1 à 4, tandis que CaBmt5 et 6 étaient impliquées dans celle du PLM. Une première enzyme responsable de l’initiation de la β-mannosylation du PPM, CaBmt1, a donc été caractérisée dans l’équipe grâce à l’étude de l’activité d’une forme recombinante soluble.L’objectif de cette thèse est de caractériser l’activité et la structure de CaBmt3, l’enzyme qui initie la polymérisation des β-Man suite à l’action de CaBmt1, pour mieux comprendre le mécanisme catalytique des β-1,2-mannosyltransférases. Ainsi, nous avons précisément identifié le substrat accepteur de CaBmt3 et défini ses paramètres enzymatiques. Par une approche combinant la diffusion des rayons X aux petits angles (SAXS), la modélisation moléculaire in silico et la mutagenèse dirigée de protéines recombinantes, nous proposons un modèle structural et catalytique de CaBmt3 qui pourrait être étendu à l’ensemble de la famille. En parallèle, nous avons montré que des iminosucres mono- et multivalents étaient capables de moduler l’activité des β-mannosyltransférases. Enfin, nous avons amorcé le travail sur une dernière enzyme, CaBmt4, qui est susceptible de polymériser le β-Man initié par CaBmt1 et CaBmt3. Pour conclure, ces travaux offrent une meilleure compréhension de la β-mannosylation du PPM de C. albicans. Ces études fonctionnelles, couplées aux avancées structurales, pourraient conduire à l’élaboration d’inhibiteurs de CaBmt et développer ainsi de nouvelles approches thérapeutiques contre les candidoses invasives. / Candida albicans is a saprophytic yeast of human gastro-intestinal tract. It can however become pathogenic in immunocompromised individuals and cause severe infections associated with high mortality rates. The cell wall of C. albicans, in contact with the host, contains β-1,2 oligomannosides (β-Man) linked to several parietal molecules such as phospholipomannan (PLM) and phosphopeptidomannan (PPM). These β-Man are found in the most pathogenic Candida species (primarily C. albicans, but also in non-albicans species such as C. glabrata and C. tropicalis) and are considered as virulence factors. The identification of a family of 9 genes coding for β-mannosyltransferases (CaBmt) led to a better understanding of the role of 6 of these enzymes. Reverse genetics studies showed that CaBmt1-4 were responsible for the β-1,2-mannosylation of PPM, whereas CaBmt5-6 were involved in the β-1,2-mannosylation of PLM. A first enzyme responsible for the initiation of the PPM’s β-mannosylation, CaBmt1, was characterized in the lab by studying the activity of its recombinant soluble form.The goal of this thesis is to characterize both activity and structure of CaBmt3, the enzyme initiating β-Man polymerization following CaBmt1’s activity, in order to further the understanding of β-1,2-mannosyltransferases catalytic mechanism. Thus, we precisely identified CaBmt3 acceptor substrate and characterized its enzymatic parameters. Combining small angle X-ray diffraction (SAXS), in silico molecular modelization and site-directed mutagenesis of recombinant proteins, we propose a structural and catalytic model of CaBmt3 which could be extended to the whole family. In parallel, we showed that mono- and multivalent iminosugars were able to modulate β-mannosyltransferases activity. Finally, we started to work on one last enzyme, CaBmt4, which could potentially polymerize the β-Man initiated by CaBmt1 and CaBmt3.In conclusion, the synthesis of these investigations offers a better understanding of the β-mannosylation processes occurring on the PPM of C. albicans. Functional studies, in conjunction with structural advances could ultimately lead to the development of CaBmt inhibitors in order to design new therapeutic approaches for the management of invasive candidiasis.

Les glycosyltransférases sont responsables de la biosynthèse d’oligosaccharides, de polysaccharides et de glycoconjugués. Etant donné le nombre croissant de processus biologiques reliés aux saccharides, il existe un grand intérêt pour la compréhension des rôles biologiques des ces saccharides et étudier leurs applications thérapeutiques potentielles. Ce projet concerne la conception, la synthèse, l’évaluationbiologique et l’analyse structurale de deux nouveaux types d’inhibiteurs de glycosyltransférases analogues du substrat donneur naturel. L’analogie repose sur l’incorporation d’une unité pyridine ou amino-acide neutre comme mime du motif pyrophosphate afin de fournir des substrats capables de pénétrer les cellules pour des applications potentielles in cellulo ou in vivo. Les synthèses d’inhibiteurs neutres de GTs ont été réalisées en utilisant une combinaison de réactions de conjugaison créant une liaison O-glycosidique, amide ou triazole. Au total, 26 inhibiteurs neutres de GTs ont ainsi été synthétisés. L’évaluation de l’inhibition pour cinq galactosyltransférases et une GlcNAc-transférase(OGT) a révélé des inhibitions modestes de l’ordre du micromolaire. La co-cristallisation des meilleurs inhibiteurs avec l’une de ces galactosyltransférases a démontré que le motif pyridine neutre chélate le cation manganèse impliqué dans le site catalytique de l’enzyme. Cependant, le motif galactose est orienté vers l’extérieur du site catalytique et loin de la position initiale du substart naturel (UDP-Gal) etindique donc un nouveau mode de liaison. Le concept d’inhibiteur neutre a aussi été examiné sur un système modèle de membrane pour leur perméation membranaire. / Glycosyltransferase is an important class of enzyme in living organisms responsible for the biosynthesis of oligosaccharides, polysaccharides and glycoconjugates. As more and more carbohydrate related biological processes are elucidated, there is great interest to define the biological roles of a given carbohydrate and examine its potential therapeutic applications. The present study reports the design, synthesis, biological evaluation and structural analysis of two novel types of glycosyltransferase donor substrate analogues. The design rationale is to make analogues of sugar nucleotide diphosphate substrates, but incorporating a ‘neutral’ pyridine or amino-acid moiety as thepyrophosphate surrogate in order to provide cell permeable substrates for potential in cellulo or in vivo applications. The syntheses of “neutral” GTs inhibitors were performed using a combination of conjugations through O-glycoside bond, amide bond or triazole functionalities. A total number of 26 “neutral” GT inhibitors were prepared. The evaluation of inhibition towards five galactosyltransferasesand one GlcNAc-transferase (OGT) revealed moderate inhibitions in the micromolar range. More interestingly, co-crystallyzation could be achieved for the most potent compounds in complex with a glycosyltransferase. The designed ‘neutral’ pyridine linker could chelate the manganese cation involved in the enzyme catalytic site. Whereas the sugar head-group was oriented away from the position found inthe related complex with natural substrate (UDP-Gal) indicating a new binding mode. The concept of ‘neutral’ inhibitor was examined by an artificial cell membrane penetration test.

La paroi végétale assure des fonctions biologiques majeures définissant la singularité des plantes ; elle est également à l'origine de multiples applications en tant que ressource agro-alimentaire, source de biomatériaux ou encore pour la production de biocarburants. Malgré cette importance fondamentale et pratique de la paroi végétale, la connaissance de sa biosynthèse apparaît à ce jour toujours très limitée. En effet, la faible abondance des glycosyltransférases (GTs) responsables de sa biosynthèse, l'absence de substrat spécifique et les difficultés à obtenir certains nucléotides-sucres nécessaires aux tests enzymatiques, a souvent rendu difficile les approches de biochimie classiques. Cependant, le séquençage de génomes (Arabidopsis thaliana, Oryza sativa, Poplar populus), la création de banques de mutants d'insertion et la classification des activités glycosyltransférases dans la base de données CAZy (www.cazy.org) sont autant d'outils récents ayant permis des avancées significatives vers la compréhension de la biosynthèse de la paroi des végétaux. Le CERMAV a participé à ce type d'avancée en 2009, en publiant une liste de 24 gènes candidats, nommés « NGT » pour « Nouvelles GlycosylTransférases », présentant des signatures caractéristiques des glycosyltransférases. Afin de démontrer l'implication des gènes NGT dans les processus d'édification de la paroi végétale, nous avons développé une approche de génomique fonctionnelle, analysant en parallèle des lignées mutantes d'Arabidopsis altérées pour les gènes NGT et testant l'activité GT de ces protéines exprimées en systèmes hétérologues. Durant mes travaux de thèse j'ai pu caractériser 15 lignées mutantes à l'état homozygote pour 7 des 24 gènes NGT. Ces lignées homozygotes ont été criblées afin de rechercher un phénotype d'altération du développement ou de la composition en sucres de leur paroi qui soit corrélé à l'altération des gènes NGT. Ce travail de criblage a conduit à s'intéresser plus particulièrement aux mutants ngt1-1 et ngt1-2 altérés pour le gène NGT1 (At5g28910). La caractérisation des lignées mutantes ngt1-1 et ngt1-2 a permis de quantifier un phénotype de croissance foliaire réduit de 38%, par comparaison au développement des feuilles de la plante sauvage. Par ailleurs, la caractérisation biochimique de la paroi des mutants a révélé des réductions significatives et quantitatives de l'arabinose, du galactose et du rhamnose dans la paroi des mutants, ainsi que des modifications qualitatives marquées principalement des arabinanes. L'altération des arabinanes a d'ailleurs pu être confirmée par microscopie après immuno-marquage de sections d'hypocotyle de mutants à l'aide des anticorps monoclonaux LM6 et LM13 dirigés contre des épitopes α-1,5-arabinanes. Il a pu être montré également que la complémentation des mutants par une construction 35S::NGT1 permet de restaurer un phénotype sauvage à ces mutants. Par ailleurs, de façon à tester l'activité glycosyltransférase de la protéine NGT1, nous avons réalisé son expression en système hétérologue. A ce jour, malgré des résultats préliminaires encourageants, il n'a pas été possible de déterminer des conditions de tests permettant d'observer une activité glycosyltransférase suffisante et reproductible pour la protéine NGT1, que ce soit une activité fucosyltransférase (correspondant à la signature de la séquence du gène) ou bien une activité arabinosyltransférase (correspondant au phénotype biochimique des mutants ngt1). / The plant cell wall not only defines the unique biology of the plants but also have practical applications as feedstock for biomaterials and for the production of biofuels. Plant primary cell wall is mainly composed of cellulose, hemicelluloses and pectins. Significant progress has been made recently in identifying the enzymes involved in plant cell wall biosynthesis, but only a handful of those have been involved in pectin biosynthesis. With the aim of identifying new putative glycosyltransferases (GTs), in lab Hansen et al 2009 designed a bioinformatic strategy and identified a new group of 24 genes called “NGT” for (Novel Glycosyltransferase) which were considered “strong” candidates for putative glycosyltransferase activities. In order to determine the putative role of these NGT genes in plant cell wall biosynthesis, we designed a functional genomics strategy, analysing in parallel Arabidopsis T-DNA mutant lines and performing heterologous expression of candidate genes. I have characterized 15 homozygous mutant lines among the group of 24 putative NGT genes through PCR. We analysed the homozygous mutants for phenotypic alteration such as dwarfing or organ malformation and found that some of mutant lines have narrow leaves as compared to Wild type plants. In parallel I have carried out the cell wall chemical analysis of 12 homozygous mutant lines and did not get any strong difference in neutral monosaccharide composition. The detailed and complete analysis (chemical, expression and microscopic analysis) of all the above mentioned genes could have been time consuming and an overwhelming work, so I focused on At5g28910 (named NGT1) which harbours a fucosyltransferase peptide signature and on At5g14550 (named P), a gene belonging to the DUF266 gene family. Homozygous T-DNA mutant lines ngt1-1 and ngt1-2 lines were analyzed and showed a reduced growth phenotype (leaf area). Leaf area was quantified at various development stages using ImageJ, and showed a 38% reduction in mutants. Additionally, biochemical characterization of the cell wall was performed showing a reduction in neutral monosaccharide contents, like arabinose, rhamnose and galactose in mutant cell wall. Furthermore glycosyl linkage analysis of mutant lines ngt1-1 and ngt1-2 has shown that 5-Arabinofuranose (5-Araf) and 3,5-Arabinofuranose (3,5-Araf ) contents were decreased as compared to Wild type Col0 cell wall. These results were also confirmed by immunolabeling of stem cross section of mutant and wild type plants. The complementation of the mutant plants through Agrobacterium transformation resulted in the complete restoration of plant phenotype. Taken together, these data suggest that NGT1 could be an arabinosyltransferase. In order to characterize its biochemical activity, the NGT1 protein was heterologously expressed in Pichia pastoris. The recombinant protein was used to perform in vitro activity tests, but we were unable to demonstrate any neither fucosyltransferase (on the basis of peptide signature) nor arabinosyltransferase activity. In parallel to this study, I contributed to the heterologous expression and characterization of two biochemically characterized Arabidopsis GTs involved in xyloglucan synthesis: the fucosyltransferase (AtFUT1) and xylosyltransferase (AtXT1). I have successfully expressed a truncated and active form of AtFUT1, which represents an essential step for further structural studies that will be undertaken in the lab.

Uridine diphosphate glycosyltransferase (UGT) is a multigene family of enzymes responsible for catalyzing glycosylation of small hydrophobic molecules. Recently, a genomic analysis of the corn earworm (Helicoverpa zea) identified 45 different UGT genes. We discovered a UGT gene (UGT34) showing high levels of expression exclusively in the silk gland tissue. The expression levels of UGT34 were analyzed in different developmental stages and silk gland sub-segments, revealing that UGT34 is generally expressed at all larval instar stages and largely expressed in the middle and posterior subsegments of the silk glands. The soybean looper (Chrysodeixis includens), another noctuid moth species, was analyzed and found to have similar gene expression patterns. To determine UGT34 function RNA interference (RNAi) was used, but it revealed to be unsuccessful. Taken together, the present study implies that UGT34 plays an important role in silk glands, yet its molecular and physiological function needs to be determined by further study.

Yes / The polysialyltransferases are biologically important glycosyltransferase enzymes responsible for the biosynthesis of polysialic acid, a carbohydrate polymer that plays a critical role in the progression of several diseases, notably cancer. Having improved the chemical synthesis and purification of the fluorescently-labelled DMB-DP3 acceptor, we report optimisation and validation of a highly sensitive cell-free high-throughput HPLC-based assay for assessment of human polysialyltransferase activity.

No description available.