Abstract Collagens and collagenous proteins undergo several post-translational modifications that are important for their structure and functions. Lysine hydroxylation produces hydroxylysines, which are important for collagen cross-link formation and provide attachment sites for galactose and glucosylgalactose. Glycosylated hydroxylysines are crucial for embryonic development and the assembly of certain collagen types. They may also facilitate interactions between collagen and adjacent molecules as well as control the diameter of collagen fibrils. Lysine hydroxylation is catalyzed by three lysyl hydroxylases (LH1, LH2 and LH3). In addition to lysyl hydroxylase activity, LH3 possesses collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. In this study, polyclonal antibodies against the lysyl hydroxylase isoforms were produced for protein level studies to localize the expression and understand the functions of the different isoenzymes. The results indicated ubiquitous expression during embryonic development compared to the more restricted, cell and tissue specific expression patterns observed in adult mouse tissues. Differences were seen also in the alternative splicing of LH2 during embryogenesis and between tissue types. Analyses of the subcellular localization revealed that LH3 is also present in extracellular space. Tissue and cell specific differences were noted in the distribution of LH3 between cellular compartments. Substrate analysis suggested an additional and novel role for LH3 as an enzyme catalyzing lysine modifications of collagenous proteins in the extracellular space. The importance of LH1 and LH2 has been highlighted in Ehlers-Danlos type VI and Bruck syndromes, respectively. In this study, the lysyl hydroxylase 3 gene was linked to a heritable disorder for the first time. Urinary screening revealed a patient that lacked a glucosylgalactosyl derivative of a pyridinium cross-link. The GGT activity levels measured from the patient’s serum and lymphoblastoid cells were also reduced, which suggested a defect in the lysyl hydroxylase 3 gene. Genetic analyses revealed two mutations, one in each allele of LH3 in this compound heterozygous patient. Recombinant mutant proteins showed defects in lysyl hydroxylase and collagen glycosyltransferase activities, respectively. In conclusion, it was shown that a defect in LH3 catalyzed modifications leads to a novel disorder, which shares features with many other connective tissue disorders.

Glucosyltransferases, or GTs, are enzymes which perform glucosylation reactions. These glucosylation reactions involve attaching a UDP-activated glucose molecule to acceptor molecules specific to the enzyme. The products of these reactions are observed to have a myriad of effects on metabolic processes, including stabilization of structures, solubility modification, and regulation of compound bioavailability. The enzyme which our lab focuses its research on is a flavonol-specific 3-O-GT found in Citrus paradisi, or grapefruit. This enzyme is part of the class of enzymes known as flavonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Citrus paradisi 3-O-GT in an attempt to modify its substrate specificity and regiospecificity. In this poster, we report our findings thus far concerning the addition of specific residues to the 3-O-GT's amino acid sequence based on an alignment with the sequence of a putative flavonoid GT found in Citrus sinensis.

Plant secondary products such as flavonoids have a variety of roles in plants including UV protection, antifeedant activity, pollinator attraction, stress response, flavor, and many more. These compounds also have effects on human physiology. Glucosylation is an important modification of many flavonoids and other plant secondary products. In grapefruit, glucosylation is important in the synthesis of the bitter compound naringin and several flavonoid glucosyltransferase (GT) enzymes have been characterized from young grapefruit leaf tissue. To study structure and function of flavonoid GTs, it is necessary to isolate cDNA’s that can be cloned and manipulated. In prior work, the plant secondary product glucosyltransferase (PSPG) box was used to identify putative GT clones. We report on results from experiments to test the hypothesis that PGT clones 4 and 11 are plant secondary product GTs, specifically flavonoid GTs. Previously, PGT 4 was cloned into a bacterial expression system, however all protein was localized into inclusion bodies and GT activity could not be tested. For this work, recombinant PGT 4 and PGT 11 were transformed into yeast and the proteins expressed and screened for glucosyltransferase activity with a variety of flavonoid substrates including flavanones, flavones, and flavonols.

Glucosyltransferases (GTs) are enzymes which perform glucosylation reactions, which involve attaching a UDP-activated glucose molecule to acceptor molecules specifi c to the enzyme. The enzyme which our lab focuses its research on is a fl avonol-specifi c 3-OGT found in Citrus paradisi, or grapefruit (Cp3GT). This enzyme is part of the class of enzymes known as fl avonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Cp3GT in an attempt to discover the residues important for substrate and regiospecifi city. In this study, we are testing the basis of substrate septicity of Cp3GT. We hypothesize that incorporation of fi ve amino acids specifi c to Citrus sinensis GT (CsGT) into Cp3GT at 308th position may facilitate mCp3GT to use anthocyanidins as one of the substrates. We report our fi ndings thus far concerning the addition of specifi c residues to the Cp3GT’s amino acid sequence based on an alignment with the sequence of a putative fl avonoid GT found in Citrus sinensis.

Flavonoids are a class of plant metabolites with a C6-C3-C6 structure. They are responsible for a large range of biological functions including UV protection, pigmentation, and anti-microbial properties. Citrus paradisi, the grapefruit, contains a wide variety of flavonoids, including the target flavonols which are characterized by a hydroxyl group at the C3 position. A glucose molecule is added to flavonols by 3-Oglucosyltransferases (3-O-GTs). C. paradisi F3-O-GT only glucosylates flavonols; however, Vitis vinifera (grape) 3-O-GT can accept both flavonols and anthocyanidins. The two enzymes have some identity with one another but sequence alignment pinpointed several areas of non-homology. Homology modeling using the crystallized structure of the V. vinifera 3-GT revealed sites within the non-homologous areas that could influence the binding site most directly. The 382 site was of particular interest with arginine in C. paradisi changed to tryptophan in V. vinifera, a much bulkier and non-charged amino acid. Site-directed mutagenis was performed to form the R382W mutant line and transformed into yeast for expression after induction with methanol. Western blot was used to determine the optimal protein induction time, after which the cells were harvested and broken to extract the proteins. Isolation and purification of the protein in question allows for enzyme analysis. This is performed by measuring incorporation of radioactive glucose onto various substrates from each flavonoid class. High counts indicate that the enzyme is active upon the substrate while low counts indicate little to no activity. Characterization will also be performed by varying reaction conditions. Thus, the optimal pH, temperature, substrate quantity, enzyme quantity, and reaction duration can be determined for this specific mutant. These experiments will determine if the R382W mutation has a significant impact on the substrate specificity or reaction conditions for the enzyme. A change in activity to include other classes of flavonoids besides flavonols indicates that the mutation site has a direct impact on the conformation of the binding site. Failure of the mutation to change substrate specificity still provides valuable information for the structure and function of the enzyme. This has implications for engineering enzymes to perform specific functions.

Made available in DSpace on 2014-08-20T13:59:15Z (GMT). No. of bitstreams: 1 tese_marcia_vaz_ribeiro.pdf: 713659 bytes, checksum: 2dc33e63ce136dffc6ae1dfd420cfab3 (MD5) Previous issue date: 2011-10-31 / The medicinal Alternanthera (Amaranthaceae) species, such as A. philoxeroides, present a great variety of bioactive compounds, among which are the betacyanins, nitrogen pigments that belong to the betalains class. These compounds are widely used as additives for food and drugs, due to their antioxidant action and lack of toxicity, which have already been proven. Techniques have been developed in order to improve productivity and performance of this pigment, one of those is the use of in vitro elicitors or in vivo stressing agents. Both have an important role in the transduction process of signals that regulate the defense genes in plants, acting as stimulators of production or degradation of several primary and secondary metabolites. This work aimed to assess, in A. philoxeroides plants, the growth and production characteristics of betacyanin in in vitro plants; the levels of photosynthetic pigments, betacyanins, lipid peroxidation and antioxidant enzymes activity in in vivo plants under salt stress, and also to quantify the level of betacyanin and the 5GT-DBs gene expression in the biosynthetic route of this compound in in vitro plants submitted to elicitation by NaCl and by tyrosine. For this, three trials were conducted. In the first one, A. philoxeroides explants were inoculated in MS medium with increasing NaCl concentrations (0, 50, 100, 150, 200 and 250 mM) for 35 days. In the second one, plants from in vitro cultures were acclimatized in greenhouses and irrigated with a sodium chloride solution (0, 200 and 400 mM) for 30 days. The third trial had two essays, one composed of in vitro A. philoxeroides plants in a liquid MS medium in vermiculite substrate for 35 days. After this period, a NaCl solution (400 mM) was added to the medium and the shoots were collected after 0, 12, 24, 36 and 48 hours of exposure. In the second one, nodal segments were inoculated in MS medium with and without tyrosine (0 e 75 µM), and its aerial parts were collected after 35 days. In the growth analysis, reduction of the averages was observed for the following variables: height, number of shoots, number of sprouts and root number and length; for the plants that have grown in the sodium chloride medium. The highest concentrations of betacyanins were found in the stalks of plants from MS medium, with 50 mM of NaCl. After 30 days of in vivo cultivation, the levels of chlorophyll a, chlorophyll b and carotenoids decreased as the salt concentration increased, while the reason of chlorophyll a/b in plants submitted to a higher salt concentration presented a difference in comparison to the control. Higher levels of betacyanin were observed on stalks, when compared to the leaves, in the highest salt concentrations. On the leaves, there was a significant increase of lipid peroxidation and superoxide dismutase activity. On the roots, there was an increase of enzymes catalase and ascorbate peroxidase. Regarding the analysis of differential expression (qRT-PCR), it was possible to observe that from 12 to 24 hours of salt stress, the 5-GT gene expression firstly increased, then there was a decrease in 36 hours and a new increase in 48 hours. The 5-GT gene also showed increased expression as a response to tyrosine. It was possible to conclude that A. philoxeroides elicited in vitro with sodium chloride present a decrease of the assessed morphological parameters, but in low concentrations betacyanin synthesis is stimulated. Salt stress causes greater degradation in the photosynthetic pigments, increment of betacyanin synthesis in stalks and damage to the cell membranes of the leaves. The increase of antioxidant enzymes activity stimulated the protective system against oxidative stress on in vivo A. philoxeroides plants. It is suggested that in this species, the enzyme bethanidine 5-Oglucosyltransferase reaches its highest expression in 48 hours of exposure to salt elicitation and also when grown in a medium containing tyrosine. / As espécies medicinais do gênero Alternanthera (Amaranthaceae) como A. philoxeroides apresentam uma variedade de compostos bioativos, entre eles as betacianinas, que são pigmentos nitrogenados pertencentes à classe das betalaínas. Esses compostos são amplamente utilizados como aditivos de produtos alimentícios e medicamentos devido à sua comprovada ação antioxidante e ausência de toxicidade. Técnicas têm sido desenvolvidas para aprimorar a produtividade e o rendimento deste pigmento, sendo uma delas o uso de elicitores in vitro ou agentes estressantes in vivo. Ambos apresentam um importante papel no processo de transdução de sinais que regulam os genes de defesa nas plantas, agindo como estimuladores para a produção e ou degradação de diversos metabólitos, tanto primários quanto secundários. Assim, o objetivo do presente trabalho foi avaliar em plantas de A. philoxeroides, as características de crescimento e produção de betacianina em plantas cultivadas in vitro; os teores dos pigmentos fotossintéticos, betacianinas, peroxidação lipídica e atividade de enzimas antioxidantes em plantas cultivadas in vivo, sob estresse salino, além de, quantificar o teor de betacianina e a expressão do gene 5GT-DBs envolvido na rota biossintética, deste composto, em plantas in vitro submetidas à elicitação por NaCl e pelo aminoácido tirosina. Para isso, foram conduzidos três experimentos. No primeiro, explantes de A. philoxeroides foram inoculados em meio MS, com concentrações crescentes de NaCl (0, 50, 100, 150, 200 e 250 mM), durante 35 dias. No segundo, plantas provenientes da cultura in vitro foram aclimatizadas em casa de vegetação e irrigadas com solução de cloreto de sódio (0, 200 e 400 mM), por 30 dias. O terceiro experimento contou com dois ensaios, sendo o primeiro composto de plantas de A. philoxeroides cultivadas in vitro, em meio MS líquido, no substrato vermiculita, durante 35 dias. Após esse período, foi adicionada ao meio, solução de NaCl (400 mM) e coletada a parte aérea das plantas após 0, 12, 24, 36 e 48 horas de exposição ao sal, Já o segundo, segmentos nodais foram inoculados em meio MS, na presença e ausência de tirosina (0 e 75 µM), tendo sua parte aérea coletada após 35 dias de cultivo. Nas análises de crescimento observou-se redução das médias para as variáveis altura, número de gemas, número de brotos e no número e comprimento de raiz, nas plantas crescidas nos meios contendo cloreto de sódio. As maiores concentrações de betacianinas foram encontradas nos caules de plantas cultivadas em meio MS com 50 mM de NaCl. Após 30 dias de cultivo in vivo os teores de clorofilas a, clorofila b, e carotenóides decresceram à medida que aumentou a concentração de sal, enquanto a razão clorofila a/b das plantas submetidas à maior concentração de sal apresentou