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Perdikoulis, Michael V. "Studies on the modular organization of human properdin and C1q of the complement pathway." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312551.
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Batista, Adelina Braga. "Potential fungicidal and insecticidal proteins present in seeds Dioclea megacarpa Rolfe." Universidade Federal do CearÃ, 2009. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=3789.
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CoordenaÃÃo de AperfeiÃoamento de NÃvel SuperiorDioclea megacarpa Rolfe is the correct synonym for D. relexa var. grandiflora. This species belongs to Fabaceae, the legume family. Previous studies, realized in our research group, showed the presence of active proteins for several phytopathogenic fungi in the seeds of this species, among of them Aspergillus niger. Thus, the present work was proposed with the objective of determining the bioactivity of protein(s) from D.megacarpa seeds against fungi, leading to its/their purification and partial characterization and further investigation of its/their action mechanism. Another approach of this work it was analyze the insecticidal potential of glucose/mannose-specific lectin, isolated from D.megacarpa seeds (Moreira et al., 1983), against the cowpea bruchid Callosobruchus maculatus. For this, seed flour was placed in contact with 0.15 M NaCl (1:5, w/w), followed by stirring for 3 h, filtration through a nylon cloth, re-extraction for 1 h, centrifugation at 11,500 x g, for 30 min, at 4 oC. The supernatant obtained, named total extract, showed antifungal activity against A. niger and presented several bioactive proteins, including lectin (129.27 UH/mgP, using trypsinized rabbit erythrocytes), trypsin inhibitor (18.91 mg de tripsina inibida/gF), urease (47.50 U/gF), toxin (LD50 119.60 mgP/Kg mice body weight ), chitinase (1.66 nKat/mgP) e β-1,3-glucanase (0.55 nKat/mgP). On the other hand, peroxidasic and proteolytic activities were not detected. For purification of antifungal principle, several chromatographies were performed on Sephadex G-50, Chitin and Resource Q, this last connected to an FPLC system. The purified antifungal protein, named Dm-PAF, with apparent molecular mass of 67-68 kDa (SDS-PAGE), did not show any haemagglutinating or chitinolytic activity and presented its NH2-terminal sequence blocked. Dm-PAF, at a very low concentration (0.015 ÂgP/ÂL), it was able to inhibit the growth of Saccharomyces cerevisiae and Candida tropicalis yeasts. The investigation of the antifungal action mechanism excluded the possibility of interaction between Dm-PAF and H+-ATPase pumps. In addition, the glucose/mannose-specific lectin, obtained from Sephadex G-50 column, exhibited a potent insecticidal activity against C. maculatus, interfering in important parameters related to life cycle of this insect. These data show to be the D. megacarpa seeds a rich source of biologically interesting proteins, possibly involved in the defense mechanism of plants
Dioclea megacarpa Rolfe à usada como sinonÃmia de D. relexa var. grandiflora, uma espÃcie pertencente à famÃlia Fabaceae (Leguminosae). Estudos prÃvios, realizados por nosso grupo de pesquisa, demonstraram a presenÃa em suas sementes de proteÃnas ativas contra fungos fitopatogÃnicos, dentre esses Aspergillus niger. Assim, o presente trabalho foi proposto no intuito de avaliar a bioatividade de proteÃnas de sementes de D. megacarpa contra fungos, conduzindo à sua purificaÃÃo e caracterizaÃÃo parcial, bem como à investigaÃÃo de seu mecanismo de aÃÃo. Outro objetivo deste trabalho foi examinar o potencial inseticida da lectina com especificidade por glucose-manose, isolada de sementes de D. megacarpa (Moreira et al., 1983), contra o bruquÃdeo do feijÃo-caupi Callosobruchus maculatus. Para tanto, farinha de sementes foi posta em contato com NaCl 0,15 M (1:5, p/v), seguida de agitaÃÃo contÃnua por 3 h, filtraÃÃo em pano de trama fina, re-extraÃÃo por 1 h e centrifugaÃÃo a 11.500 x g, 30 min, 4 oC. O sobrenadante obtido, denominado de extrato total, se mostrou ativo contra A. niger e apresentou vÃrias proteÃnas bioativas, compreendendo lectina (129,27 UH/mgP, com eritrÃcitos tripsinizados de coelho), inibidor de tripsina (18,91 mg de tripsina inibida/gF), urease (47,50 U/gF), toxina (DL50 119,60 mgP/Kg de peso corpÃreo de camundongo), quitinase (1,66 nKat/mgP) e β-1,3-glucanase (0,55 nKat/mgP). Por outro lado, atividades peroxidÃsica e proteolÃtica nÃo foram detectadas. Para purificaÃÃo da proteÃna antifÃngica, foram realizadas cromatografias em matrizes de Sephadex G-50, Quitina e Resource-Q, essa Ãltima acoplada ao sistema de FPLC. A proteÃna antifÃngica purificada de sementes de D. megacarpa, denominada de Dm-PAF, com massa molecular aparente de 67-68 kDa (PAGE-SDS), nÃo mostrou atividades hemaglutinante e quitinÃsica e apresentou sua seqÃÃncia NH2-terminal bloqueada. Dm-PAF, em concentraÃÃo baixÃssima (0,015 ÂgP/ÂL), se mostrou capaz de inibir o crescimento das leveduras Saccharomyces cerevisiae e Candida tropicalis, cuja investigaÃÃo do mecanismo de aÃÃo nÃo revelou envolvimento dessa proteÃna com bombas de H+-ATPase. Em adiÃÃo, a lectina ligante a glucose-manose, obtida na cromatografia em Sephadex G-50, mostrou potente atividade inseticida contra C. maculatus, interferindo em parÃmetros importantes relacionados ao ciclo de vida do inseto. Os dados apresentados mostram as sementes de D. megacarpa como uma rica fonte de proteÃnas interessantes, possivelmente envolvidas no mecanismo de defesa das plantas
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Does, Maria Petronella. "Chimeric proteins of stinging nettle lectin, chitinase and [beta]-1,3-glucanase." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2000. http://dare.uva.nl/document/55397.
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Sousa, Michelle Amelia De. "Investigation into the lectin component of Type II ribosome inactivating proteins." Thesis, University of Warwick, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264904.
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Baba, Kei'ichi. "LECTIN AND RELATED PROTEINS IN THE BARK OF Sophora japonica L." Kyoto University, 1990. http://hdl.handle.net/2433/78243.
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Reidy, Michael James. "Engineering of the RTB Lectin as a Carrier Platform for Proteins and Antigens." Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/26155.
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The major obstacle many promising drugs struggle to overcome is the barrier imposed by the outer cell membrane. In addition to technologies such as liposomes and cell-penetrating peptides, more attention is being given to the class of proteins known as lectins to deliver therapeutic and antigenic proteins to the interiors of cells. Lectins bind to but do not modify sugars, and provide an efficient route to endocytosis. The galactose/N-acetyl-galactosamine specific lectin ricin B-chain (RTB) is especially attractive in possibly fulfilling a carrier role due to its well-characterized endocytotic trafficking and its efficacy over a wide range of cell types. By producing RTB recombinantly in plants it is possible to create a fully active, non-toxic carrier that does not rely on the processing of large amounts of toxic material (e.g. castor bean). Payload molecules such as small molecules and proteins can be attached to RTB via chemical conjugation at primary amine groups, without the loss of lectin or uptake activities. The biotin/streptavidin interaction and direct genetic fusion of polypeptides also provide efficient mechanisms for the attachment of payload proteins to RTB. An immunoglobulin domain-based scaffolding mechanism bridges modified RTB and payload proteins when co-expressed in Agrobacterium-infiltrated plant leaves. Carrier and payload proteins expressed in plants and E. coli, respectively, and purified independently are not able to assemble into an efficient carrier/payload arrangement. These findings show that plant cells are able to correctly produce the two components of the carrier/payload system and assemble them into an efficient and flexible capture and carry technology.Ph. D.
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Down, Rachel Elizabeth. "Use of endogenous plant defensive proteins to confer resistance to aphids in crop plants." Thesis, Durham University, 1998. http://etheses.dur.ac.uk/4786/.
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A liquid artificial diet system, which was suitable for bioassay of added compounds, was developed for the glasshouse potato aphid, Aulacorthum solani. The diet supported normal growth and reproduction of this insect. Once established, the artificial diet bioassay system was used to test potential insecticidal activities of a variety of proteins found naturally occurring in plants. Effects on survival, development and fecundity were measured. The lectin found in snowdrop, Galanthus nivalis agglutinin (GNA) was found to significantly reduce the fecundity of A. solani, in terms of parthenogenetic nymph production, when administered in artificial diets at the 0.1% w/v level. No significant reductions in survival were found, although GNA administered in vitro did appear to slow the development of A. solani. Transgenic potato plants expressing GNA were used in a growth room trial to show that the reduction in fecundity with the in vitro trials could be reproduced in planta. Aphids feeding on the GNA-expressing potatoes had a significantly lower cumulative nymph production than those feeding on non- transformed plants. The transgenic plants had no effect on the survival of A. solani. The GNA-expressing plants were tested in a larger scale glasshouse trial and resulted in a significantly slower buildup of aphids when compared to control potatoes, thus confirming the results of the artificial diet bioassays and in planta growth room trials. Immunohistochemical studies were performed to detect the presence of GNA in the gut lumen of A. solani fed on artificial diet containing 0.1% w/v GNA; the lectin was observed to be selectively concentrated in the region of the epithelial membrane in the stomach, suggesting that binding to surface carbohydrates or glycoproteins was taking place. Binding to the gut surface has been suggested to mediate lectin toxicity in higher animals, and other insects. A synergistic effect was observed with transgenic potatoes expressing a double construct encoding GNA and bean chitinase (BCH); A. solani cumulative nymph production on these plants was significantly reduced compared to aphids feeding on control and GNA-only expressing plants. However, interestingly, BCH-only expressing plants did not significantly affect the fecundity of A. solani, although a slight reduction in nymph production was observed. On the basis of reports in the literature that suggested that chitin-binding lectins were toxic to insects, an attempt to isolate the gene encoding the chitin-binding stinging nettle lectin was made. RNA was extracted from nettle rhizomes and used to prepare a cDNA library. Successful library construction was verified. PGR methods and a primary screen of the library were used in an attempt to locate the gene.
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Toot, Amanda Lee. "Localization and characterization of C-type lectin-like family of proteins in Leptospira interrogans." [Ames, Iowa : Iowa State University], 2007.
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Aroso, Miguel Ângelo Mouta Martins. "Characterisation of ZG16p, a unique mammalian lectin from pancreatic zymogen granules." Doctoral thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/14099.
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Doutoramento em BioquímicaThe mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.
Os mecanismos de biogénese dos grânulos secretores e a secreção regulada das enzimas digestivas, nas células acinares do pâncreas, ainda não são totalmente compreendidos. Para esclarecer estes processos, que são de importância biológica e clínica (ex., pancreatite), é necessário um melhor conhecimento molecular dos componentes da membrana dos grânulos, as suas funções e interações. A aplicação da proteómica contribuiu largamente para a identificação de novas proteínas dos grânulos de zimogénio (ZG) mas ainda não foi acompanhada por uma melhor caracterização das suas funções. Este estudo teve como objectivos a) o isolamento e identificação de novas proteínas associadas à membrana dos ZG; b) a caracterização das propriedades bioquímicas e da função da lectina ZG16p, uma proteína associada a membrana dos ZG; c) explorar o potencial da ZG16p como uma nova ferramenta para marcar o compartimento endolisossomal. Inicialmente, efetuamos uma abordagem proteómica ao estudo das frações dos ZG, a qual nos levou à identificação de novas proteínas periféricas da ZGM com capacidade de se ligarem a proteoglicanos (Chymase e PpiB). Depois, começamos a desvendar as propriedades moleculares e (múltiplas) funções da lectina ZG16p. A ZG16p é uma proteína única nos mamíferos com capacidade de se ligar a glicanos e a proteoglicanos. Pela primeira vez, foi revelado que a ZG16p é extremamente resistente a proteases através do desenvolvimento de um ensaio de digestão com enterokinase. Adicionalmente, demonstrei que a ZG16p se liga a um complexo de elevado peso molecular (também resistente a proteases) e forma homodímeros muito estáveis. À luz destas descobertas, nós sugerimos que a ZG16p poderá actuar como um elo de ligação aos proteoglicanos, ajudando na formação e estabilização de uma rede/estrutura (matriz submembranar) ligada ao lúmen da ZGM, que desempenhará uma função importante durante a segregação e empacotamento dos zimogénios. A ZG16p poderá atuar como um elo de ligação entre a matriz e os zimogénios agregados devido à sua capacidade para formar dímeros. Adicionalmente, a resistência da ZG16p a protéases poderá ser de maior importância após a secreção, uma vez que é sabido que a ZG16p se liga a fungos patogénicos nos intestinos. Investiguei ainda, o papel dos domínios de ligação da ZG16p na sua segregação para os ZG em células AR42J, um modelo pancreático. A mutação pontual dos motivos de ligação a glicanos e a proteoglicanos não alterou a segregação da ZG16p para os ZG. Também demonstrei que quando a ZG16p se encontra no citoplasma liga-se ao compartimento endolisossomal. Como é sabido, a desregulação da autofagia devido ao funcionamento defeituoso dos lisossomas está associado à pancreatite, por isso iremos discutir o papel potencial da ZG16p nesta doença.
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Fraser, Stuart Tallis. "Lectin - carbohydrate interactions in lympho-haemopoiesis: a study of L-selectin, ligands of L-selectin and CD24 inthe rat." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1998. http://hub.hku.hk/bib/B31236844.
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Fraser, Stuart Tallis. "Lectin - carbohydrate interactions in lympho-haemopoiesis : a study of L-selectin, ligands of L-selectin and CD24 in the rat /." Hong Kong : University of Hong Kong, 1998. http://sunzi.lib.hku.hk/hkuto/record.jsp?B20667450.
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Selvaraj, Justin Antony Verfasser], and Peter [Akademischer Betreuer] [Kremsner. "Investigation of lectin complement proteins in urinary schistosomiasis and visceral leishmaniasis / Justin Antony Selvaraj ; Betreuer: Peter Kremsner." Tübingen : Universitätsbibliothek Tübingen, 2016. http://d-nb.info/1197694374/34.
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Selvaraj, Justin Antony [Verfasser], and Peter [Akademischer Betreuer] Kremsner. "Investigation of lectin complement proteins in urinary schistosomiasis and visceral leishmaniasis / Justin Antony Selvaraj ; Betreuer: Peter Kremsner." Tübingen : Universitätsbibliothek Tübingen, 2016. http://d-nb.info/1197694374/34.
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Grant, George. "Local (intestinal) and systemic responses of animals to ingested soyabean Glycine max proteins, antinutritional effects of lectin and trypsin inhibitors." Thesis, University of Aberdeen, 1988. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU010227.
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The poor growth of young rats fed fully supplemented diets containing raw soyabean appeared to be due to interference with local (intestinal) metabolism, resulting in apparently poor digestion and absorption of dietary nitrogen, coupled with changes in systemic intermediary metabolism, leaning to low overall retention of absorbed nitrogen and slightly incresed catabolism of body lipid. Low serum insulin concentrations and pancreas and small intestine enlargement were also evident. These changes were due to a number of anti-nutritional factors in soyabean: 1. Trypsin inhibitors (Kunitz + Bowman-Birk) depressed growth rate by reducing digestion and absorption of dietary nitrogen and interfering with retention of absorbed nitrogen. They also induced considerable enlargement of the pancreas. 2. The lectin inhibited growth primarily as a result of interference with retention of absorbed nitrogen. It also caused enlargement of the pancreas and of the small intestine. 3. Anti-nutritional factor/s, devoid of haemagglutinating or trypsin inhibitory activity, caused a loss of muscle and also possibly increased production and secretion of mucus in the small intestine. The poor growth of young soyabean-fed animals was thus due to the combined effects of these anti-nutritional factors. As the rats matured, the inhibitory effects of soyabean upon growth diminished and, after 16-24 weeks on the diet, were negligible. On the other hand, pancreas enlargement persisted upon long-term (up to 96 weeks) feeding with raw soyabean. Enlargement of the whole gastrointestinal tract was also evident upon prolonged soyabean feeding. With rats kept for more than 1 year on soyabean diet, there was apparently an increased incidence (approximately 15%) of pre-cancerous or cancerous changes in the pancreas. This amy have been due, in part, to a synergism between the lectin and trypsin inhibitors and unsaturated lipids. Aqueous heat-treatment greatly reduced but did not eliminate the anti-nutritional effects of soyabean. Pre-treatment of meal with hot aqueous ethanol was more effective.
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Silva, Andre Luis Coelho da. "Pulchellina: uma potente toxina vegetal inativadora de ribossomos - RIP tipo 2. estudos in vitro e in vivo." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-07012009-112409/.
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Pulchellina é uma proteína inativadora de ribossomo (RIP) isolada de sementes de Abrus pulchellus fragmento que codifica a cadeia A da pulchellina (PAC) foi clonado e inserido no vetor pGEX-5X para expressar a cadeia A recombinante (rPAC) como uma proteína de fusão em Escherichia coli. A análise da seqüência de aminoácidos mostrou que a rPAC apresenta uma alta identidade seqüencial (> 86%) com a cadeia A da abrina-c. A habilidade que a rPAC possui para depurinar rRNA em ribossomos de levedura também foi demonstrada em testes in vitro. Objetivando verificar a atividade tóxica do produto heterólogo, nós promovemos a associação in vitro da rPAC com a cadeia B recombinante da pulchellina (rPBC). Ambas as cadeias foram incubadas na presença de um sistema de redução/oxidação, originando um heterodímero ativo (rPAB). O rPAB apresentou uma massa molecular aparente de aproximadamente 60 kDa, similar a pulchellina nativa. As atividades tóxicas do rPAB e da pulchellina nativa foram comparadas através da injeção intraperitonial em camundongos, usando diferentes diluições de cada proteína. O rPAB foi capaz de matar 50% dos animais testados com doses de 45μg.kg-1. Nossos resultados mostraram que o heterodímero recombinante apresenta tanto toxicidade quanto um padrão conformacional similar a pulchellina nativa. Estudos usando cultura de tecidos também foram realizados com o objetivo de investigar a presença da pulchellina em calos obtidos a partir de sementes de A. pulchellus. Segmentos de cotilédones de sementes imaturas foram inoculados em meio MS suplementado com diferentes concentrações de auxina, citocinina e sacarose para promover a indução dos calos. A expressão da pulchellina nos calos foi monitorada através de RT-PCR e testes de atividade biológica. Os calos obtidos após 35 dias foram congelados, macerados e submetidos a extração de RNA total e proteínas. Um fragmento específico de DNA que codifica a cadeia A da pulchellina foi amplificado a partir do RNA total sugerindo a síntese da proteína nos calos. Isto foi confirmado no extrato bruto de calos, que mostrou atividade hemaglutinante contra sangue de coelho e uma alta toxicidade quando injetado via intraperitoneal em camundongos.O extrato bruto também foi submetido à cromatografia de afinidade em coluna de Sepharose-4B. A fração retida na coluna apresentou duas bandas protéicas quando analisadas em gel de poliacrinamida, sob condições desnaturantes, apresentando um padrão similar ao obtido com a pulchellina de semente.Pulchellin is a type 2 ribosome-inactivating protein (RIP) isolated from seeds of the Abrus pulchellus tenuiflorus plant. The DNA fiagment encoding Pulchellin A-chain (PAC) was cloned and inserted in pGEX-5X to express the recombinant pulchellin Achain (rPAC) as a fusion protein in Escherichin coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. Intending to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reducedloxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of about 60 D a similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection in mice using different dilutions. The rPAB was able to kill 50% of the tested mice with doses of 45μg.kg-1. Our results indicated that the recombinant heterodimer presented toxic activity and a conformational pattern similar to pulchellin. Studies using tissue cultures were also performed to investigate the presence of the pulchellin in callus established from seed explants of A. pulchellus. Cotyledon segments of immature seeds were inoculated in basal medium MS supplemented with different concentrations of auxin, citokinin and sucrose in order to determine the best callus induction. The pulchellin expression was monitored in callus cultures by RT-PCR and biological activity. The calli obtained aRer 35 days were freeze dried, macerated and submitted to extraction of total RNA and proteins. A specific DNA fragment codifying the A-chain pulchellin was amplified from callus RNA suggesting the synthesis of the protein. This was confirmed in the calli crude extract that showed haemagglutinating activity against rabbit blood cells and a high intraperitoneal toxicity to mice. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein, showed to be composed by two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to the results obtained with seeds pulchellin.
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Kruger, Sarah Jane, and n/a. "Characterisation of Proteins from Grevillea robusta and NMR Studies of the Serine Protease Inhibitor." Griffith University. School of Science, 2004. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040618.150708.
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Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.
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Carlsson, Jenny. "Interaction Studies in Complex Fluids with Optical Biosensors." Doctoral thesis, Linköpings universitet, Tillämpad Fysik, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-14694.
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In this thesis interactions in complex fluids, such as serum and meat juice, were analysed with optical biosensor techniques. Panels of lectins immobilised on gold surfaces were used for investigation of differences in protein glycosylation pattern in sera and meat juices between various species. The present panel was also used for investigation of global glycosylation changes of serum proteins in type 1 diabetes patients. Biorecognition was evaluated with null ellipsometry and scanning ellipsometry combined with multivariate data analysis techniques (MVDA). Principal component analysis (PCA) showed that the lectin panel enabled discrimination between sera from the different species as well as for the different meat juices. The results also indicate that there is a measurable global alteration in glycosylation pattern of serum proteins in type 1 diabetic patients compared to healthy subjects. Using an artificial neuronal net (ANN), it was also possible to correctly categorise unknown serum samples into their respective class or group. The analytical potential of combining information from lectin panels with multivariate data analysis was thereby demonstrated. Also, a sensitive and specific method based on surface plasmon resonance (SPR) for detection of insulin autoantibodies (IAA) in serum samples from individuals at high risk of developing type 1 diabetes (T1D) has been developed. When measuring trace molecules, such as autoantibodies, in undiluted sera with label-free techniques like SPR, non-specific adsorption of matrix proteins to the sensor surface is often a problem, since it causes a signal that masks the analyte response. The developed method is an indirect competitive immunoassay designed to overcome these problems. Today, IAA is mainly measured in radio immunoassays (RIAs), which are time consuming and require radioactively labelled antigen. With our SPR-based immunoassay the overall assay time is reduced by a factor of >100 (from 4 days to 50 min), while sensitivity is maintained at a level comparable to that offered by RIA. Finally, the assay was used in a screening study of newly diagnosed type 1 diabetes patients and non-diabetic subjects.
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Jiménez-Castells, Carmen 1982. "Capture and identification of carbohydrate-binding proteins by SPR and CREDEX-MS." Doctoral thesis, Universitat Pompeu Fabra, 2010. http://hdl.handle.net/10803/7237.
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Carbohydrate-binding proteins of non-immunological origin -lectins- have been recognized over the last decades as decisive players in numerous biological processes, ranging from cellcell communication, fertilization, pathogen-cell adhesion to metastasis. Consequently, there is an increasing interest in finding powerful and nanosized tools to screen for these molecules and to study their carbohydrate interactions in detail. Here, two complementary approaches are described to characterize lectin-carbohydrate interactions with high sensitivity, low sample consumption, and without the need for sample labelling: SPR and CREDEX-MS. In SPR, we have developed an approach where the sugar is immobilized onto a sensor surface through a tailor-made peptide module that allows (1) to capture the lectin, (2) to characterize the interaction through kinetic and thermodynamic parameters, and (3) to identify the interacted protein by mass spectrometry. In CREDEX-MS, based on proteolytic excision of proteincarbohydrate complexes and mass spectrometric analysis, the peptides comforming the carbohydrate binding domain are identified.Las lectinas (proteínas de origen no inmune capaces de reconocer azúcares) se han revelado en las últimas décadas como participantes cruciales en multitud de procesos biológicos, tales como la comunicación célula-célula, la fertilización, la adhesión del patógeno a la célula y la metástasis, entre muchos otros. Por lo tanto, existe un gran interés en el desarrollo de técnicas analíticas potentes para el estudio de las interacciones lectina-carbohidrato. En este trabajo, se describen dos aproximaciones complementarias mediante las cuales se pueden caracterizar las interacciones lectinas-azúcar con gran sensibilidad, poca utilización de muestra y sin la necesitad de ningún marcaje. En la técnica basada en resonancia de plasmón superficial (SPR), el azúcar es inmovilizado sobre una superficie a través de un módulo peptídico, lo cual permite (1) capturar la lectina, (2) caracterizar su interacción mediante parámetros cinéticos y termodinámicos y (3) identificar posteriormente la proteína mediante espectrometría de masas. Complementariamente, la técnica CREDEX-MS, basada en la excisión proteolítica del complejo proteína-azúcar y posterior análisis por espectrometría de masas, nos permite identificar los péptidos que forman parte del dominio de unión al azúcar.
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Pabst, Elisabeth Sophie [Verfasser], Jörg [Akademischer Betreuer] Durner, Jörg [Gutachter] Durner, and Erich [Gutachter] Glawischnig. "Arabidopsis thaliana legume lectin-like proteins at the interface between systemic acquired resistance and abiotic stress responses / Elisabeth Sophie Pabst ; Gutachter: Jörg Durner, Erich Glawischnig ; Betreuer: Jörg Durner." München : Universitätsbibliothek der TU München, 2018. http://d-nb.info/1161528547/34.
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Santos, Adriano dos [UNESP]. "Estudo da afinidade das proteínas rTgMIC1 e rTgMIC4 da Toxoplasma gondii com fetuína e asialofetuína utilizando técnica piezelétrica." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/92055.
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As proteínas de micronema TgMIC1 e TgMIC4 (TgMICs) da Toxoplasma gondii fazem parte de um complexo proteico localizado na superfície do parasita responsável pelo processo de adesão e invasão celular. Os objetivos desse trabalho foram estudar dispositivos piezelétricos contendo em cada um, uma das MICs recombinantes (rTgMIC1 ou rTgMIC4) e utilizá‐los na determinação das constantes de afinidade entre elas com a fetuína e asialofetuína, empregando o modelo da Isoterma de Langmuir. Os dispositivos foram desenvolvidos por meio a abordagem de monocamadas automontadas (SAM) mista de tióis, utilizando solução etanólica contendo 2,5 mM de ácido 11‐mercaptoundecanóico (11‐MUA) e 7,5 mM de 6‐mercapto‐1‐hexanol (C6OH). A formação da SAM, realizada em temperatura ambiente por 12 h, foi monitorada pela técnica de Microbalança a Cristal de Quartzo com Fator Dissipativo (QCM‐D) e voltametria cíclica utilizando o par redox [FeII(CN)6]4‐/ FeIII(CN)6]3‐. Os resultados obtidos por ambas as técnicas evidenciaram a formação de SAM rígida e de elevado grau de cobertura superficial após cinética lenta em que processos de adsorção e organização dos tióis ocorreram simultaneamente. Para a imobilização das rTgMICs, solução aquosa contendo 10 mM de EDC (N‐etil‐N‐(dimetilaminopropil) carbodiimida) e 20 mM de NHS (N‐hidroxisuccinimida) foi utilizada para a ativação dos grupos carboxílicos presentes na SAM por 2 h, e o processo acompanhado por QCM‐D apresentou resultados compatíveis com aqueles encontrados na literatura. Pela mesma técnica, foi possível verificar que ambas as rTgMICs se imobilizam sobre o cristal de quartzo após sua incubação com solução 0,15 mg/mL de cada rTgMIC em tampão Tris‐HCl contendo 200 mM de NaCl...
The Toxoplasma gondii micronemal proteins TgMIC1 and TgMIC4 (TgMICs) are part of a protein complex located on the surface of the parasite responsible for the process of cellular adhesion and invasion. The goal of the present work was to study a piezoelectric device containing the recombinant MICs (rTgMIC1 or rTgMIC4) and use it in determining the affinity constants between the MICs with fetuin and asialofetuin, employing the Langmuir isotherm model. The devices were developed using the approach of self‐assembled monolayer (SAM) in ethanolic solution containing 2.5 mM of 11‐mercaptoundecanoic acid (11‐MUA) and 7.5 mM of 6‐mercaptohexanol (C6OH). The SAM formation, held at room temperature for 12 h, was monitored by the Quartz Crystal Microbalance technique with Dissipative Factor (QCM‐D) and cyclic voltammetry using the redox couple [FeII(CN)6]4‐/FeIII(CN) 6]3‐. The results obtained from both techniques showed the formation of a rigid and high surface degree coverage SAM after a slow kinetic process in which adsorption and organization of the thiols occur simultaneously. For the immobilization of rTgMICs, aqueous solution containing 10 mM EDC N‐ethyl‐N‐(dimethylaminopropyl) carbodiimide and 20 mM NHS (N‐hydroxysuccinimide) was used for 2 h, and the process followed by QCM‐D was consistent with those found in the literature. By the same technique it was found that both rTgMICs are immobilized on the quartz crystal after incubation whit solution 0.15 mg/mL of each rTgMIC in Tris‐HCl containing 200 mM NaCl (pH 8.0) for 2 h. Unlike the functionalization of the quartz crystal rTgMIC4, remaining adsorption sites were observed in the process using the rTgMIC1, wherein the blocking step using 0.1% gelatin solution for 2 h was required. Throughout the QCM‐D technique it was possible... (Complete abstract click electronic access below)
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Santos, Adriano dos. "Estudo da afinidade das proteínas "rTgMIC1" e "rTgMIC4" da Toxoplasma gondii com fetuína e asialofetuína utilizando técnica piezelétrica /." Araraquara : [s.n.], 2012. http://hdl.handle.net/11449/92055.
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Orientador: Paulo Roberto BuenoBanca: Emanuel Carrilho
Banca: Maria Cristina Roque Antunes Barreira
Resumo: As proteínas de micronema TgMIC1 e TgMIC4 (TgMICs) da Toxoplasma gondii fazem parte de um complexo proteico localizado na superfície do parasita responsável pelo processo de adesão e invasão celular. Os objetivos desse trabalho foram estudar dispositivos piezelétricos contendo em cada um, uma das MICs recombinantes (rTgMIC1 ou rTgMIC4) e utilizá‐los na determinação das constantes de afinidade entre elas com a fetuína e asialofetuína, empregando o modelo da Isoterma de Langmuir. Os dispositivos foram desenvolvidos por meio a abordagem de monocamadas automontadas (SAM) mista de tióis, utilizando solução etanólica contendo 2,5 mM de ácido 11‐mercaptoundecanóico (11‐MUA) e 7,5 mM de 6‐mercapto‐1‐hexanol (C6OH). A formação da SAM, realizada em temperatura ambiente por 12 h, foi monitorada pela técnica de Microbalança a Cristal de Quartzo com Fator Dissipativo (QCM‐D) e voltametria cíclica utilizando o par redox [FeII(CN)6]4‐/ FeIII(CN)6]3‐. Os resultados obtidos por ambas as técnicas evidenciaram a formação de SAM rígida e de elevado grau de cobertura superficial após cinética lenta em que processos de adsorção e organização dos tióis ocorreram simultaneamente. Para a imobilização das rTgMICs, solução aquosa contendo 10 mM de EDC (N‐etil‐N‐(dimetilaminopropil) carbodiimida) e 20 mM de NHS (N‐hidroxisuccinimida) foi utilizada para a ativação dos grupos carboxílicos presentes na SAM por 2 h, e o processo acompanhado por QCM‐D apresentou resultados compatíveis com aqueles encontrados na literatura. Pela mesma técnica, foi possível verificar que ambas as rTgMICs se imobilizam sobre o cristal de quartzo após sua incubação com solução 0,15 mg/mL de cada rTgMIC em tampão Tris‐HCl contendo 200 mM de NaCl... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The Toxoplasma gondii micronemal proteins TgMIC1 and TgMIC4 (TgMICs) are part of a protein complex located on the surface of the parasite responsible for the process of cellular adhesion and invasion. The goal of the present work was to study a piezoelectric device containing the recombinant MICs (rTgMIC1 or rTgMIC4) and use it in determining the affinity constants between the MICs with fetuin and asialofetuin, employing the Langmuir isotherm model. The devices were developed using the approach of self‐assembled monolayer (SAM) in ethanolic solution containing 2.5 mM of 11‐mercaptoundecanoic acid (11‐MUA) and 7.5 mM of 6‐mercaptohexanol (C6OH). The SAM formation, held at room temperature for 12 h, was monitored by the Quartz Crystal Microbalance technique with Dissipative Factor (QCM‐D) and cyclic voltammetry using the redox couple [FeII(CN)6]4‐/FeIII(CN) 6]3‐. The results obtained from both techniques showed the formation of a rigid and high surface degree coverage SAM after a slow kinetic process in which adsorption and organization of the thiols occur simultaneously. For the immobilization of rTgMICs, aqueous solution containing 10 mM EDC N‐ethyl‐N‐(dimethylaminopropyl) carbodiimide and 20 mM NHS (N‐hydroxysuccinimide) was used for 2 h, and the process followed by QCM‐D was consistent with those found in the literature. By the same technique it was found that both rTgMICs are immobilized on the quartz crystal after incubation whit solution 0.15 mg/mL of each rTgMIC in Tris‐HCl containing 200 mM NaCl (pH 8.0) for 2 h. Unlike the functionalization of the quartz crystal rTgMIC4, remaining adsorption sites were observed in the process using the rTgMIC1, wherein the blocking step using 0.1% gelatin solution for 2 h was required. Throughout the QCM‐D technique it was possible... (Complete abstract click electronic access below)
Mestre
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Ngoepe, Mafora Gloria. "Heterologous expression of a Mukwa (pterocarpus angolensis ) seed lectin (Pal) gene in Escherichia coli, Saccharomyces cerevisiae and Yarrowia lipolytica and construction of Pal recombinant vector for expression in Aspergillus niger." Thesis, University of Limpopo (Turfloop Campus), 2011. http://hdl.handle.net/10386/857.
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Thesis (M.Sc. (Microbiology)) --University of Limpopo, 2011Pterocarpus angolensis seed lectin (PAL), a 28 kDa non glycosylated protein, was initially successfully cloned and expressed in E. coli for ease of high protein production. It was discovered, however, as in similar studies that the recombinant PAL yield in E. coli is low and localized intracellularly. This makes extraction even more difficult because most of the protein is lost either when the cell undergoes lysing or when there is incomplete extraction. As a result of the low yields in E. coli, expression vectors were constructed for pal expression in S. cerevisiae, Y. lipolytica and A. niger. Colony PCR of S. cerevisiae transformants confirmed the presence of pal gene whilst sequencing revealed a 66% homology to native PAL. Expression of recombinant PAL in S. cerevisiae, which was expected to be intracellular, was doubtfully unsuccessful since no signal was detected following Western blot analysis. A pBARMTE1-pal expression vector was successfully constructed and could be used for expression studies in Aspergillus niger, however, it was not used in this study. A pal gene whose codons were optimized for Y. lipolytica was synthesized and successfully cloned and expressed in Y. lipolytica. Gene sequence alignment of native pal and the codon optimized pal showed 81% homology whilst the amino acid alignment showed 100% homology. A 31 kDa, recombinant PAL was successfully expressed in Y. lipolytica. The recombinant PAL was approximately 3 kDa larger than native PAL. It was established that this is due to glycosylation of the recombinant PAL. This recombinant protein was found to be more thermostable than native PAL since it demonstrated haemagglutination activity after 10 minutes of exposure in a boiling water bath and only lost activity after 2 hours of exposure to boiling. This study succeeded in producing a more stable extracellular recombinant PAL which demonstrated biochemical activity that was largely similar to that of native PAL but only differed in carbohydrate specificity and haemagglutinating strengths.
Flemish Interuniversity Council (VLIR-UOS)-Own Initiative Project,the SARBIO- South African Regional Co-operation in Biochemistry, Molecular Biology and Biotechnology, the (CSIR) Council for Scientific and Industrial Research,the (NRF) National Research Foundation,(TBI) The Biovac Institute Foundation, and the (SIDA) Swedish International Agency
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Stroppa, Valter Luís Zuliani. "Influência de lecitina e PGPR no processo de microestruturação de chocolate amargo." [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/266882.
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Orientadores: Lireny Aparecida Guaraldo Gonçalves, Priscila EfraimDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: Chocolates são produtos compostos por uma fase lipídica com predominância de manteiga de cacau, e que contem liquor, açúcar emulsificantes e eventualmente leite em pó (ou derivados lácteos) e aromas. As características físicas dos chocolates como brilho, dureza, fusão a temperaturas da boca e estabilidade térmica são conseqüências da estruturação cristalina induzida na manteiga de cacau. Os triacilgliceróis presentes na manteiga de cacau podem-se organizar em até seis formas polimórficas sendo que apenas duas são estáveis e geram produtos de melhor qualidade. A adição de emulsificantes ao chocolate visa melhorar o comportamento reológico durante o processamento diminuindo as tensões interfaciais entre a fase gordurosa e as partículas sólidas. Os emulsificantes promovem o recobrimento das partículas sólidas com a fase oleosa lubrificando as interfaces que, desta maneira, poderão servir de elementos ativos para a nucleação. Este trabalho avaliou o efeito da adição dos dois emulsificantes mais utilizados na indústria, lecitina de soja e PGPR (poliglicerol poliricinoleato), no processo de estruturação cristalina de chocolate amargo. Foram produzidos chocolates com diferentes teores de emulsificantes conforme um planejamento fatorial completo 2² rotacionado. O nível de lecitina variou de 0,08 a 0,92% e o de PGPR de 0,02 a 0,58%. Para caracterizar esta influência foram utilizadas técnicas de reometria (determinação das propriedades de escoamento), ressonância magnética nuclear (acompanhamento da cinética de cristalização), Índice de temperagem (quantificação da pré-cristalização), e ensaios de ruptura (avaliação da resistência estrutural). Os valores de viscosidade plástica de Casson do chocolate medidos a 40ºC variaram de 1,4 a 5,9 Pa.s enquanto que o limite de escoamento variou de zero a 34Pa. Os parâmetros cinéticos avaliados de isotermas de cristalização a 15ºC ajustadas ao Modelo de Avrami resultaram no expoente n variando de 2,598 a 2,956 e o parâmetro k entre 5,15.10-6 e 2,85.10-5 min-n para os diferentes teores de emulsificantes. Estes efeitos foram associados à capacidade da lecitina em aumentar o volume cristalino e ao potencial nucleador do PGPR
Abstract: Chocolates are confectionary products composed by a lipid phase based mainly on cocoa butter that contains cocoa mass, sugar, emulsifiers and eventually milk solids and aromas. Physical characteristics of chocolates like gloss, hardness, melting at mouth temperature and thermal stability result from the crystalline structuration accomplished by the fat phase. The triacylglycerols in cocoa butter can be organized in up to six different polymorphs but only two of them are stable and therefore yield better quality products. The addition of emulsifiers to chocolates intends to improve the rheological behavior during processing by the reduction of the interfacial tension between the fat phase and the solid particles. The emulsifiers promote the coating of the solid particles by an oily layer, lubricating the interfaces that then can serve as active elements for nucleation. This work examines the effects of the addition of the two most used emulsifiers in chocolate, namely soy lecithin and PGPR (polyglycerol polyricinolate) in the crystalline structuration of chocolates. Chocolates with different contents of the two emulsifiers were produced, following a 22 complete factorial rotational design. The lecithin level varied from 0.08 to 0.92% and the PRPG content from 0.02 to 0.58%. The influence of the additives was characterized by rheometry (determination of the flowing parameters), nuclear magnetic resonance (monitoring the crystallization kinetics), Temperindex (pre-crystallization level quantification), and snap test (structural resistance). The values of Casson's plastic viscosity of the chocolates, measured at 40ºC, varied from 1.4 to 5.9 Pa.s while the yield value ranged between zero to 34 Pa. The kinetic parameters evaluated from crystallization isotherms at 15ºC modeled by the Avrami equation resulted in the exponent n varying from 2.598 to 2.956 and k parameters between 5.15 10-5 min-n and 2.85 10-5 min-n when the different amount of emulsifiers were used. These effects are associated to the ability of lecithin to enlarge the crystal volume and to the nucleating potential of PGPR
Mestrado
Engenharia de Processos
Mestre em Engenharia Química
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Campana, Patricia Targon. "Desnaturação e reenovelamento da frutalina, uma lectina ligante de D galactose." Universidade de São Paulo, 1998. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-03092008-154638/.
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Os estudos sobre o mecanismo de enovelamento das proteínas é o resultado de um estudo intenso utilizando métodos bioquímicos, biofísicos e teóricos. \"In vitro\", o estado inicial deste estudo é a proteína desnaturada. Neste trabalho, temos estudado o reenovelamento, após desnaturação térmica, de uma glicoproteína denominada frutalina; da família das lectinas. A característica principal desta classe de proteínas é sua habilidade para interagir com carboidratos e, portanto, combinar-se com glicocomponentes da superfície da célula, induzindo suas propriedades biológicas. A frutalina é uma lectina tetramérica extraída das sementes de Artocarpus incisa. Ela é ligante de D-galactose e o espectro de CD (dicroísmo circular) de sua estrutura nativa foi identificado como sendo dominado por folhas ?. A desnaturação térmica e as etapas do reenovelamento foram monitoradas por espectroscopia de CD, fluorescência e também pela perda da atividade hemaglutinante. As condições de desnaturação utilizadas foram aquecimento à 60°C por 30 a 60 minutos, dependendo do tempo de estocagem (a -18°C) da proteína na forma nativa. Os resultados indicaram que o reenovelamento é promovido por um processo de congelamento na presença de PBS contendo 0,l M de D-galactose seguida por centrífugoconcentração em Centriprep 3. A hemaglutinação positiva ocorreu tanto para a fração nativa quanto para a fração reenovelada. O reenovelamento da frutalina desnaturada também ocorreu com PBS contendo 0,1 M de solução de D-glicose. Quando a forma desnaturada foi concentrada antes do congelamento em PBS sem D-galactose ou em PBS contendo xilose, o reenovelamento não ocorreu. Estes resultados mostraram que o reenovelamento da frutalina foi dependente da ligação com a D-galactose ou D-glicose, bem como a importância do congelamento para obter a forma biologicamente ativa. A análise da estrutura secundária utilizando o programa CCA forneceu um resultado importante: para a forma nativa da frutalina obtivemos 85% de folhas ? paralelas e antiparalelas, incluindo voltas ?, enquanto que para a forma reenovelada obtivemos 73%, mostrando que a estrutura reenovelada, a nível secundário, se aproximou satisfatoriamente da nativa, concordando com os resultados obtidos nos testes de hemaglutinação.Our current understanding of the protein folding mechanism is the result of intense study employing biophysical, biochemical and theoretical methods. \"In vitro\", the initial state of the protein in this puzzle is its unfolded form. In the present work we have studied the refolding, after thermal denaturation, of the glycoprotein frutalin, a member of the lectin class. The main characteristic of these proteins is their ability to interact with carbohydrates and thus combine with glycocomponents of the cell surface, leading to their biological properties. Frutalin is a tetrameric lectin extracted from the seeds of Artocarpus incisa. It is D-galactose specific and its native CD spectrum was identified as being dominated by ? -sheet. The thermal unfolding and refolding steps were measured by CD and fluorescence spectroscopies together with the loss of hemagglutinating activity. The unfolding conditions used were 60°C for 30 to 60 minutes, depending on the protein storage time. The results indicate that refolding is promoted by the freezing process in the presence of 0,1 M D-galactose-PBS followed by three-fold concentration in a Centriprep 3. Positive hemagglutination occurred for both the native and refolded forms. Refolding of denatured frutalin also occurred with PBS containing 0,1 M D-glucose. When the unfolded form was concentrated before freezing in PBS without D-galactose or in PBS containing xylose, refolding did not occur. These results show that the refolding of frutalin is dependent on the binding of D-galactose or D-glucose, and demonstrate the importance of freezing in order to obtain the biologically activity form. An analysis of secondary structure using the CCA program showed an important result: the native form, presented 85% ? -sheet/ ?-turns, while in the refolded form, this content fell to 73%. These results show that the refolded form is very similar to the native protein, which is in agreement with the hemagglutination results.
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Campana, Patricia Targon. "Desnaturação e renovelamento de lectinas oligoméricas ligantes de D-galactose: estudos no equilíbrio termodinâmico." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-11062008-101210/.
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O estudo de enovelamento de proteínas tem sido um problema de fundamental importância em biofísica e biologia molecular. Neste trabalho, estudamos os processos de desnaturação e renovelamento das lectinas jacalina e frutalina. Estas lectinas são tetraméricas, apresentam alta homologia estrutural, porém diferem em atividade biológica, sendo a frutalina mais potente. Apesar desta homologia, estas lectinas diferem também nos processos de desnaturação e renovelamento como função da temperatura e o comportamento frente ao desnaturante químico hidrocloreto de guanidina (GndHCl). Ambas proteídas foram desnaturadas pela ação de GndHCl e suas curvas de desnaturação medidas por espectroscopia de fluorescência e CD. As medidas de fluorescência da frutalina deram valores de estabilidade conformacional de 17,12 kJ/mol e 12,34 kJ/mol, na presença e na ausência de D-Galactose, enquanto a jacalina forneceu valores de 8,12 kJ/mol para a transição NI e 5,61 kJ/mol para a transição IU em PBS. Os valores na presença de açúcar foram similares. NOs estudos da frutalina foram separadas as formas nativa, desnaturada, renovelada e uma forma molecularmente distinta chamada mal-enovelada, por cromatografia de exclusão molecular. Estas formas foram analisadas por atividades hemaglutinante e espectroscopias de CD e fluorescência. Todos os resultados obtidos confirmaram a ocorrência do renovelamento de ambas lectinas e que os monômeros renovelados, depois de alcançarem sua estrutura tridimensional, se associam espontaneamente para a formação dos tetrâmeros.Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of jacalin and frutalin. They are tetrameric lectins that present structural homology, buti jacalin shows a more marked biological activity than the latter. These proteins, despite their homology, have different unfolding/refolding behaviors as function of temperature and chemical agents. Both proteins were unfolded induced by guanidine hydrochlroide and their dnaturation curves mesuared by fluorescence emission and CD. Fluorescence measurments of frutalin gave values of conformational stability of 17.12 kJ/mol and 12.32 kJ/mol, in the presence and absence of D-Galactose, while jacaline gave values of 8.12 kJ/mol for NI transition and 5.61 kJ/mol for IU transition in PBS. In sugar presence the values are similar. In the frutalin studies were separeted the native, unfolded, refolded and a distinct molecular form denoted misfolded, by Size Exclusion Chromatography. These forms were analyzed for hemagglutination activity, CD and fluorescence spectroscopy. All the results obtained confirmed the successful refolding of the both lectins and the refolded monomers, after adopting their native three-dimensional structures, spontaneously assembled to form tetramers.
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Koshi, Yoichiro. "Development of New Chemical Methods toward Lectin Engineering." 京都大学 (Kyoto University), 2008. http://hdl.handle.net/2433/57283.
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Kyoto University (京都大学)0048
新制・課程博士
博士(工学)
甲第13851号
工博第2955号
新制||工||1436(附属図書館)
26067
UT51-2008-C767
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 濵地 格, 教授 森 泰生, 教授 白川 昌宏
学位規則第4条第1項該当
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Gomes, Pâmela Oliveira Martins. "Imobilização de Galectina-1 e Galectina-1 fusionada com Maltose Binding Protein (MBP-Gal-1) sobre superfície eletropolimerizada com [N-(3-Pirrol-1-il-propil)-4,4\'-bipiridina] (PPB) para a construção de um biossensor de lactose." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/59/59138/tde-11102018-100311/.
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As galectinas são proteínas que se ligam a -galactosídeos por meio do domínio de reconhecimento de carboidratos (CRD do inglês, Carbohydrate Recognition Domain) e que participam de vários processos de reconhecimento celular, sinalização, adesão e destinação intracelular de proteínas recém-sintetizadas. A primeira galectina, Galectina-1 (Gal-1), foi identificada em 1976 e possui um papel importante na progressão e proliferação tumoral, angiogênese, resistência a drogas e processos inflamatórios. Assim, é interessante a construção de dispositivos com Galectinas imobilizadas, preservando o CRD para o estudo de mecanismos e/ou detecção destas doenças. Neste trabalho a produção e caracterização de uma proteína recombinante fusionada, a MBP-Gal-1, foi descrita. A proposta do projeto foi pautada na hipótese de que a fusão da MBP à Gal-1 seria uma excelente estratégia para imobilização orientada da proteína de interesse, (Gal-1), sobre eletrodos modificados com filme polimérico, auxiliando na preservação da atividade da biomolécula imobilizada para posterior desenvolvimento de biossensor. A MBP-Gal-1 foi purificada utilizando 2 colunas com resinas diferentes: sepharose/lactose e amilose onde foi possível comprovar a atividade/preservação dos sítios ativos da Gal-1 e MBP, respectivamente. A proteína fusionada teve seu estado oligomérico estimado e seu raio hidrodinâmico determinado pela técnica Espalhamento Dinâmico da Luz sendo que a mesma se encontrava na forma monomérica com raio hidrodinâmico de 4 nm ± 1,26. A massa molecular de 57,834 kDa para a MBP-Gal-1 foi obtida através da técnica de Espectrometria de Massas MALDI-TOF/TOF. O PPB, material polimérico empregado na modificação dos eletrodos de carbono vítreo e ouro, foi sintetizado e teve sua estrutura confirmada pela técnica de Ressonância Magnética Nuclear; este material foi utilizado para a realização dos ensaios de Espectroscopia de Impedância Eletroquímica (EIE) e Ressonância de Plásmons de Superfície (SPR) para a construção do biossensor. Os ensaios EIE utilizando eletrodo de carbono vítreo modificado com PPB mostraram a importância da imobilização orientada da sonda (MBP-Gal-1) para garantir a preservação da atividade biológica da mesma, uma vez que os resultados relativos ao aumento da Resistência à Transferência de Carga (Rtc), após adição do alvo (lactose), foram da ordem de 80% a mais para a proteína fusionada MBP-Gal-1 quando comparados à proteína nativa Gal-1. Os ensaios de SPR revelaram maior SPR efetiva para a MBP-Gal-1 imobilizada sobre superfície de eletrodo Au-SPR modificado com PPB o qual apresentou bom desempenho na detecção de lactose.Galectins are proteins that bind to -galactosides by the Carbohydrate Recognition Domain (CRD) and participate in various processes of cell recognition, signaling, adhesion and intracellular destination of newly synthesized proteins. The first galectin, Galectin-1 (Gal-1), was identified in 1976 and plays an important role in tumor progression and proliferation, angiogenesis, drug resistance and inflammatory processes. Thus, it is interesting to desing devices with immobilized Galectins, preserving its CRD for the study of mechanisms and /or detection of such diseases. In this work the production and characterization of a fused recombinant protein, MBP-Gal-1, has been described. The project goal was based on the hypothesis that the fusion of the MBP to Gal-1 would be an excellent strategy for oriented immobilization of the protein of interest, (Gal-1), onto PPB- modified electrodes, promoting the preservation of the biomolecule activity immobilized for further development of a biosensor. MBP-Gal-1 was purified using 2 columns with different resins: sepharose/lactose and amylose and it was possible to prove the activity/preservation of both CRDs from Gal-1 and MBP, respectively. Dynamic Light Scattering showed that MBP-Gal-1 was in a monomeric form and with a hydrodynamic radius of 4 nm ± 1,26. The molecular mass of 57.834 kDa for MBP-Gal-1 was obtained by the technique of MALDI-TOF/TOF Mass Spectrometry. The PPB, a polymeric material used in the modification of glassy carbon and gold electrodes, was synthesized and its structure was confirmed by the Nuclear Magnetic Resonance (NMR); this material was used to carry out the Electrochemical Impedance Spectroscopy (EIS) and Surface Plasmon Resonance (SPR) tests for the construction of the biosensor. EIS assays using PPB-modified glassy carbon electrode showed the importance of probe-immobilization (MBP-Gal-1) to ensure the preservation of the biological activity of the protein, since the results related to the increase in Resistance of Charge Transfer (Rct), after addition of the target (lactose), were 80% higher for the fused protein MBP-Gal-1 when compared to the Gal-1 native-form protein. The SPR assays revealed a greater effective SPR for MBP-Gal-1 immobilized onto PPB-modified Au-SPR electrode surface which showed good performance in the detection of lactose.
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Bhatt, Veer Sandeep. "Non-lectin type Protein-carbohydrate Interactions: A Structural Perspective." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306858684.
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Pesquero, Naira Canevarolo. "Estudo da equivalência entre a lectina artin M obtida a partir da semente da jaca e a sua forma recombinante na afinidade por glicanas /." Araraquara : [s.n.], 2010. http://hdl.handle.net/11449/87992.
Full textAbstract:
Orientador: Paulo Roberto BuenoBanca: Marcelo Mulato
Banca: Maria Cristina Roque Antunes Barreira
Resumo: No presente trabalho foi avaliada a equivalência entre as formas nativa e recombinante da lectina Artin M utilizando como ligante a peroxidase de raiz forte (HRP) por meio da técnica de microbalança a cristal de quartzo. Para tal foi preparado um biossensor por meio da imobilização da lectina, tanto nativa como recombinante, na superfície do cristal de quartzo piezelétrico. A imobilização das lectinas foi realizada por meio da construção de uma monocamada auto organizada utilizando dois alcanotióis, ácido 11-mercaptoundecanóico e o 2-mercaptoetanol. Para o acoplamento das proteínas foram utilizados N-etil-N- (dimetilaminopropil) carbodiimida (EDC) e N-hidoxisuccinimida (NHS) que formam com os grupamentos carboxílicos um intermediário reativo. Após a preparação do biossensor foi utilizado um sistema de injeção em fluxo acoplado à microbalança de cristal a quartzo para o estudo da interação lectina-glicoconjugado. Desta forma, as interações da Artin M nativa e recombinante com a glicoproteína peroxidase de raiz forte foram estudadas por meio da determinação das suas constantes de afinidade aparente e de associação cinética, sendo que foram encontrados os valores de constante de afinidade aparente (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP, e os valores de constante cinética (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP. Os valores das constantes de interação obtidos evidenciaram a equivalência entre ambas as formas da lectina Artin M. Neste trabalho também foi determinada a constante de associação cinética da interação entre a lectina Artin M recombinante e linhagens celulares de leucemia mielóide aguda humana (NB4, K562 e U937), no intuito de melhor entender a diferença na citotoxicidade observada da Artin M sobre estas células ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In the present work was evaluated the equivalence between the native and recombinant forms of Artin M using horseradish peroxidase as ligand by means the quartz crystal microbalance technique. In this way, a biosensor was prepared immobilizing the lectin, native and recombinant forms, on piezoelectric quartz crystal surface. Lectins immobilization was realized constructing self assembled monolayers using the alkanethiols 11-mercaptoundecanoic acid and 2-mercaptoethanol. To the binding of proteins was used N-ethyl-N-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), which form with carboxylic groups a reactive intermediary. After biosensor preparation was utilized a flow injection system coupled to quartz crystal microbalance to study the lectin-glycoconjugated interaction. Thus the native Artin M and recombinant Artin M interaction with horseradish peroxidase glycoprotein were studied by determining its apparent affinity constant and association kinetic constants. The values obtained to apparent affinity constant were (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions, and the values obtained to association kinetic constant were (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions. These constant values evidence the equivalence between native and recombinant forms of Artin M lectin. During this work were also determined the association kinetic constant of the interaction between recombinant Artin M and leukemic lineages from human acute myeloid leukemia (NB4, K562 and U937), with the purpose of a better understanding in the different cytotoxic effect of Artin M on these cells. In this way the values of association kinetic constant obtained was (0,3 ± 0,1) x 10-7 , (0,9 ± 0,1) x 10-7 e (2,7 ± 0,3) x 10-7 mL cel -1 to the interactions between Artin M and NB4, K562 and U937, respectively
Mestre
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Arruk, Viviana Galimberti. "Avaliação do sistema complemento e produção de anticorpos de pacientes HIV negativos com neurocriptococose." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5144/tde-11012012-092626/.
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Cryptococcus sp é um fungo saprófita, cosmopolita, que causa micose sistêmica, geralmente, subaguda ou crônica, conhecida, sobretudo, por sua localização meníngea, após aquisição da infecção por via respiratória Embora seja ubíquo, a criptococose ocorre predominantemente em indivíduos imunodeficientes e podendo ocorrer, também, em indivíduos imunocompetentes. Os estudos experimentais e em humanos avaliando a ativação do sistema complemento e a produção de anticorpos específicos mostram que a resposta inata e de anticorpos são importantes para a delimitação do processo infeccioso por Cryptococcus sp, como também, a administração de anticorpos monoclonais podem induzir uma resposta eficaz na disseminação da doença. O sistema complemento contribui para a defesa do organismo contra o Cryptococcus sp de diferentes maneiras: secretando opsoninas e fatores quimiotáticos e colaborando com a ação dos anticorpos específicos, aumentando a interação entre a imunidade inata e adquirida. Os anticorpos antiglicuroxilomanana (GXM) possuem numerosas atividades biológicas: a) opsonização para fagocitose, b) ativação da via clássica do complemento resultando na deposição precoce de fragmentos de C3 no fungo, c) supressão do excesso de acúmulo de C3 pela via alternativa; d) facilitação do clareamento do GXM do soro in vivo, resultando no maior acúmulo de GXM nos tecidos ricos em células do sistema fagocítico mononuclear; e) proteção em modelos murinos da criptococose e f) facilitação de vários aspectos da imunidade celular ao Cryptococcus sp. O objetivo desse estudo foi avaliar a resposta humoral ao GXM e às proteínas da parede celular (Ag S) avaliando a atividade do sistema complemento como também a produção de anticorpos específicos em amostras séricas de adultos com e sem neurocriptococose. Foram coletadas 106 amostras de soro e divididas em 3 grupos: grupo 1- 21 indivíduos com neurocriptococose e baixa exposição a levedura, grupo 2- foi composto por 23 indivíduos saudáveis com alta exposição ao fungo e HIV negativos, granjeiros da cidade de Jumirim localizada a 164 km de São Paulo, na região de Sorocaba e, o grupo 3- 60 indivíduos saudáveis, HIV negativos e com baixa exposição ao Cryptococcus sp. Dois pacientes foram excluídos do estudo por apresentarem tumores (timona e câncer de pulmão). O sistema complemento foi avaliado por ensaio hemolítico (CH 50 e AP 50) e, a dosagem da proteína ligadora de manose (MBL) foi feita por ELISA. Os valores de CH 50 estiveram dentro da normalidade em 17/21, 13/23, 59/60 indivíduos dos grupos 1, 2 e 3 respectivamente. A média dos valores de CH 50 foi diferente significativamente entre o três grupos (P < 0,0001). O grupo 2 mostrou níveis reduzidos significantes em comparação aos dois outros grupos. Os valores de AP 50 estiveram dentro da normalidade em 11/21; 21/23 e 60/60 indivíduos dos grupos 1, 2 e 3 respectivamente. Houve diferença nos valores de AP 50 (P = 0,0005) e apenas um paciente do grupo 1 apresentou valores indetectáveis desta via. Houve diferença significante na dosagem de MBL entre os três grupos (P = 0,0277). Anticorpos IgG anti-GXM foram quantificados por ELISA e expressos por densidade óptica (DO). IgG anti GXM foi detectado em todos os grupos com diferença significante entre eles (P= 0,0127). As médias de IgG anti- GXM (DO) foram: 1.191 (0,49 a 1.217) no grupo 1, 1.572 (0,815 a 2.479) no grupo 2 e 0,965 (0,321 a 1.295) no grupo 3. Dois indivíduos assintomáticos do grupo 2 tiveram títulos de GXM detectáveis (1/256 e 1/32). Quatro pacientes com neurocriptococose faleceram (19%) e seus resultados mostravam: CH 50 normal, 2/4 tinham valores de AP 50 baixo (12 UI/mL) e indetectável; 3/4 tinham altos níveis de MBL e apenas um tinha baixa DO de IgG anti-GXM. Baseado em nosso estudo, podemos concluir que a resposta humoral (sistema complemento e anticorpos) não é suficiente para explicar a susceptibilidade a neurocriptococose, porém a alta e constante exposição ao Cryptococcus sp pode prevenir o desenvolvimento de doença, ou seja, a constante e intensa exposição ao fungo induz a produção de anticorpos que previnem a doença clínica mas não a infecção. Por outro lado fatores genéticos que determinam as concentrações de MBL podem influenciar na susceptibilidade a neurocriptococose. Os anticorpos contribuem para o clearence de GXM, entretanto as concentrações séricas não se correlacionam com resistência à doençaCryptococcus sp is a fungal pathogen with a worldwide distribution. Although it is ubiquitous in the environment, cryptococcal disease occurs predominantly in immunocompromised hosts and can also occur in apparently immunocompetent individuals. The innate immunity is of special relevance for the antifungal reaction, as it allows an immediate reaction and recognizes a broad variety of fungal pathogens. The host immune response is a major determinant of the outcome of cryptococcal infection; however, the antibodies response is poorly understood. In addition, most of the studies are experimental and there is restricted knowledge concerning the human immune response. Complement system has soluble factors, restrictive regulator proteins and cellular receptors involved in defense mechanism. Glucuroxylomannan (GXM) monoclonal antibodies (MAbs) have numerous biological activities: a) opsonization for phagocytosis, b) activation of the classical complement pathway leading to early deposition of C3 fragments on the yeast, c) suppression overall accumulation of C3 via the alternative pathway; d) clearance facilitation of GXM from serum in vivo, leading to increased accumulation of GXM in tissues rich in mononuclear phagocyte system; e) protection in murine models of cryptococcosis and f) facilitation of various aspects of cellular immunity to Cryptococcus sp. The goal of our study was to evaluate if the antibody response to GXM and cell wall proteins regarding specific antibodies as well as complement system in sera of immunocompetent adults with and without neurocryptococcosis. The aim of our research was to evaluate classical and alternative complement system pathway, to quantify mannose-binding lectin (MBL) as well antibody response to GXM and cell wall proteins (AgS) regarding specific antibodies in sera of immunocompetent adults with and without neurocryptococcosis. One hundred and six samples were collected and classified in 3 groups: group 1- 21 individuals with neurocryptococcosis and low exposure to the yeast; group 2- was composed by 23 healthy individuals, chicken farmings from Jurumirim, a town 164 km to São Paulo, and with high exposure to Cryptoccocus spp and HIV negative. The third group included 60 healthy HIV negative individuals with presumed low exposure to Cryptococcus. Two patients were excluded by report of previous malignancies (timoma and pulmonary cancer). The complement system was evaluated by hemolytic assay and ELISA to MBL. CH 50 and AP 50 values were within the normal range in 17/21; 13/23; 59/60 patients in groups 1, 2 and 3 respectivelly. Mean CH 50 values were significantly different among the three groups (P < 0,0001). Group 2 showed significantly reduced levels in comparison with groups 1 and 3. AP 50 values were within the normal range in 11/21; 21/23; 60/60 patients in groups 1, 2 and 3 respectivelly. There was difference in the AP 50 values (P=0,0005) and one no activation of this pathway in group 1. There was significant difference in MBL among the groups (P = 0,0277). GXM antibodies IgG was measured by ELISA and expressed as optical density (OD). GXM- IgG was detected in all the groups with significant difference among them (P = 0,0127). The means of IgG anti-GXM (OD) were: 1.191 (range 0,49 to 1.217) in group 1, 1.572 (range 0,815 to 2.479) in group 2 and 0,965 (range 0,321 to 1.295) in the group 3. Two of the group 2 individuals had low GXM titers (1/256 and 1/32) and no symptoms. Four patients (4/21; 19%) with neurocryptococcosis died and the results showed: normal classical pathway activation, 2/4 had low (12 UI/mL) or undetectable alternative pathway values ; 3/4 had high MBL concentrations and only one had low OD for IgG anti-GXM. In conclusion, our results suggest that constant and high exposure to Cryptococcus sp can prevent the development of cryptococcosis, i.e. constant and intensive fungal exposition induces protective antibodies to clinical disease but not to the infection. In the other side, genetic factors which determine MBL concentrations could influence the susceptibility to neurocryptococcosis. The antibodies contribute to GXM clearance, however, the concentrations did not correlate with the resistance to the disease
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Carding, S. R. "Immunochemical studies of mammalian beta-galactoside ?-binding lectins." Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.352601.
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Vemulapalli, Tracy H. "Genetic and Immunological Analyses of a Brucella abortus Protein Exhibiting Lectin-like Properties." Thesis, Virginia Tech, 2000. http://hdl.handle.net/10919/31194.
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Brucella abortus is a facultative, intracellular zoonotic pathogen, which can cause undulant fever in humans and abortion in cattle. Despite all of the progress in brucellosis research, there are still many unanswered questions regarding the molecular mechanisms involved in the pathogenesis of Brucella infections. To better understand the Brucella antigens involved in virulence and/or immunity, genetic and immunologic characterization of a 16 kDa protein of B. abortus was performed. Using PCR methods, the gene encoding the 16 kDa protein was cloned and sequenced. PCR and Southern blot analysis revealed that the gene is conserved among the 6 nomen species of Brucella. Overexpression of this protein in B. abortus vaccine strain RB51 was achieved using Brucella groE and sodC promoters as well as its own promoter. Protection and clearance studies were performed in mice to determine the role of this protein in Brucella immunity and pathogenesis. Inoculation with either strain RB51 overexpressing the 16 kDa protein or a DNA vaccine encoding the 16 kDa protein gene failed to provide significant protection. No difference was noted between the splenic clearance of B. abortus strain 2308 and its recombinant overexpressing the 16 kDa protein. A mutant of strain 2308 (2308D16) was created by disrupting the 16 kDa proteinâ s gene with a chloramphenicol resistance cassette. Western blot analysis indicated that the O antigen profile of strain 2308D16 differed from that of strain 2308. Mice cleared strain 2308D16 faster than strain 2308 indicating the potential attenuation of the disruption mutant. Purified 16 kDa protein was obtained by overexpressing it in E. coli via the pRSET expression system. Western blotting results initially identified this protein as an immunoglobulin-binding protein. Hemagglutination assay revealed that the 16 kDa protein exhibits lectin-like properties. Preliminary studies using hemagglutination inhibition identified mannose as a possible sugar to which the 16 kDa protein can interact. The lectin-like properties exhibited by the 16 kDa protein appears to influence smooth lipopolysaccharide production, and thereby may be involved in virulence.Master of Science
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Carrière, Violaine. "La proteine mr60 : une lectine oligomerique intracellulaire." Orléans, 1999. http://www.theses.fr/1999ORLE2006.
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La mr60 est une proteine membranaire, intracellulaire, qui a ete mise en evidence comme etant une lectine specifique du mannose. Mr60 est identique a ergic-53, une proteine navette, identifiee comme un marqueur du compartiment intermediaire. La proteine mr60/ergic-53 endogene est presente sous forme d'oligomeres (des dimeres et des hexameres). Dans une premiere partie nous avons tente de caracteriser a l'aide de glycosynhtons fluorescents le motif oligomannosidique le plus specifiquement reconnu par la lectine. Nous avons ensuite, a l'aide de proteines recombinantes, etudie la relation existant entre l'oligomerisation de la proteine et sa fonction lectine. La proteine totale fixe le mannose et est oligomerique : elle forme des dimeres et des hexameres. Deux proteines tronquees dans les parties transmembranaire et cytosolique ont ete preparees et caracterisees. La plus longue, qui contient la cysteine 466 proche de l'extremite c-terminale de la proteine recombinante mais pas la cysteine 475, fixe les mannosides et forme des dimeres mais pas d'hexameres. La plus courte, qui ne possede pas les deux cysteines 466 et 475, ne fixe pas les mannosides et ne forme pas de dimeres stables ou de formes oligomeriques plus importantes. Cette etude montre que le domaine luminal de mr60/ergic-53 contient un element qui permet la stabilisation de la proteine sous forme oligomerique et qu'une proteine tronquee doit posseder au moins une des deux cysteines proche du domaine transmembranaire pour s'oligomeriser et etre capable de fixer un ose. Nous avons egalement prepare, en utilisant l'immunisation genique, des antisera monospecifiques contre la proteine totale et contre une proteine tronquee.
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Neto, Antonio EufrÃsio Vieira. "CaracterizaÃÃo estrutural da frutalina, uma lectina α-D-Galactose ligante de sementes de Artocarpus incisa e anÃlise das suas bases moleculares de ligaÃÃo a D-galactose." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15539.
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CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel SuperiorAs lectinas sÃo proteÃnas que contÃm pelo menos um domÃnio nÃo catalÃtico que lhes permite reconhecer seletivamente e se ligar de uma forma reversÃvel a glicanos especÃficos. A Frutalina à uma lectina obtida a partir das sementes de Artocarpus incisa, conhecida popularmente como fruta-pÃo. O isolamento foi realizado por cromatografia de afinidade em coluna de Agarose-D-Galactose e sua caracterizaÃÃo demonstrou que a Frutalina à uma glicoproteÃna, principalmente α-D-galactose ligante, mas que tambÃm reconhece α-D-Manose. Possui 2,1% de carboidratos e apresenta, em seu perfil de SDS-PAGE, duas bandas protÃicas com massas moleculares aparentes de 12 e 15 kDa, sendo uma proteÃna oligomÃrica, encontrando-se como tetrÃmero apenas em pH alcalino, com massa molecular aparente de 60 kDa. Massas diversas em torno de 16 kDa foram observadas nos espectros deconvoluÃdos em espectrometria de massas, o que corrobora a presenÃa de isoformas. Este trabalho mostra a cristalizaÃÃo e anÃlises dos dados obtidos por difraÃÃo de raios-x para determinaÃÃo da estrutura tridimensional desta lectina, e para isso foram realizados ensaios de cristalizaÃÃo da Frutalina isolada a partir das sementes maduras, na presenÃa do ligante (α-D-galactose) e na sua forma apo (sem ligantes). Os cristais de Frutalina cresceram principalmente em poÃos de pH 8,5 contendo PEG como precipitante e etileno-glicol e os melhores cristais apareceram apÃs uma semana de maturaÃÃo, sendo difratados a uma resoluÃÃo mÃxima de 1,81 Ã. A melhor soluÃÃo para o grupo espacial, considerando eixos e planos de simetria, foi obtida para o grupo espacial I2 com a obtenÃÃo de um Rfactor de 38,6% e LLG de 19,9. A estrutura da Frutalina apresenta, em cada unidade monomÃrica, um β prisma simÃtrico, com trÃs grupos de 4 folhas beta, cada. O sÃtio de reconhecimento a carboidratos, à semelhante ao da Jacalina, e envolve o N-terminal da cadeia α, demonstrando, na regiÃo, um enovelamento caracterÃstico de lectinas da famÃlia Moraceae. O sÃtio de ligaÃÃo da Frutalina consiste numa cavidade prÃxima ao N-terminal da cadeia α, formada por quatro resÃduos-chave: Gly25, Tyr146, Trp147 e Asp149. As bases de interaÃÃo com o ligante sÃo relacionadas ao nÃmero de interaÃÃes, que ocorrem entre a hidroxila do C1 e o resÃduo Tyr146, a hidroxila do C3 e o resÃduo Gly25, a hidroxila do C4 e os resÃduos de Gly25 e Asp149 e a hidroxila do carbono 6 aos resÃduos Tyr146, Trp147 e Asp149. Algumas hidroxilas da α-D-Galactose tambÃm utilizam de interaÃÃes com molÃculas de Ãgua estruturais, para buscar estabilidade no sÃtio de reconhecimento a carboidratos. O grande nÃmero de interaÃÃes corrobora com a grande afinidade que a Frutalina tÃm a galactose e à sua grande capacidade de aglutinaÃÃo, alÃm de proporcionar uma anÃlise das dimensÃes da lectina em relaÃÃo ao ligante, onde se visualiza que o sitio de ligaÃÃo à muito maior que o aÃÃcar, o que pode justificar a preferÃncia que a Frutalina costuma apresentar por glicoconjugados de maior massa molecular, proporcionando maior encaixe, e maior nÃmero de interaÃÃes quÃmicas entre um glicoconjugado maior.
Lectins are proteins containing at least one non-catalytic domain that allows them to recognize and selectively bind a reversible specific glycans. Frutalin is a lectin obtained from Artocarpus incisa seeds, popularly known as breadfruit. The isolation was performed by affinity chromatography on column of Agarose-D-Galactose and their characterization shows that frutalin is a glycoprotein mainly α-D-galactose ligand, because it also recognizes epimers of α-D-mannose. It has 2.1% of carbohydrates and presents, in its SDS-PAGE profile, two protein bands with apparent molecular masses of 12 and 15 kDa, with an oligomeric protein, lying as tetramer only in alkaline pH, with apparent molecular mass of 60 kDa. Several masses around 16 kDa was observed in deconvoluted spectra in Mass Spectrometry, which confirms the presence of isoforms. This work shows the crystallization and analysis of data obtained by x-ray diffraction to determine the three-dimensional structure of this lectin, and that were performed crystallization trials of frutalin isolated from the mature seeds in the presence of ligand (D-galactose) and the way apo (no binders). The frutalin crystals have grown primarily in wells of pH 8.5 containing PEG as precipitant and ethylene glycol and the best crystals appeared after one week of maturation being diffracted to a maximum resolution of 1.81 Ã. The best solution, for the space group, considering axes and planes of symmetry, has been obtained for the I2 space group, with the construction of an Rfactor of 38.6% and LLG = 19.9. The structure of frutalin presents in each monomeric unit, a symmetrical β-prism with three groups of four beta strands each. The carbohydrate recognition site is similar to the jacalin, and involves the N-terminus of the α chain, showing, in the region, a characteristic folding of the Moraceae family. The frutalin binding site cavity is near the N-terminus of the α chain formed by four key residues Gly25, Tyr146, Asp149, and Trp147. The bases of interaction with the binder are related to the number of interactions occurring between the C1 hydroxyl and Tyr146 residue, C3 hydroxyl and Gly25 residue, C4 hydroxyl and Asp149/Gly25 residues, and C6 hydroxyl and Tyr146/Trp147/Asp149 residues. Some hydroxyls of α-D-galactose also utilize interactions called structural waters, to seek stability in the carbohydrate recognition site. The large number of interactions agrees with the high affinity that frutalin has with galactose and its great capacity for agglutination, in addition to providing an analysis of the dimensions of the lectin in relation to the binder, which may justify the preference that frutalin tends to present by higher molecular weight glycoconjugates, and that happens due to the most fitting, and the greatest number of chemical interactions among a larger glycoconjugate.
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Martin, Andrew. "Glycosylated green fluorescent protein for carbohydrate binding protein analysis." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/glycosylated-green-fluorescent-protein-for-carbohydrate-binding-protein-analysis(9ddae46e-b4d7-4c08-8240-94b9b804ac68).html.
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The interactions of glycoconjugates with carbohydrate binding proteins are responsible for a wide range of recognition events in vivo; including immune response, cell adhesion and signal transduction. Glycoconjugates have already found many medicinal uses as therapeutic and diagnostic agents, but their full potential is yet to be realised. Access to a variety of homogeneously glycosylated glycoproteins is essential for the study of these important carbohydrate binding events. This requires the chemical synthesis and attachment of biologically relevant glycans to unglycosylated protein scaffolds in a site selective manner. Here we describe the use of a range of glycosyl iodoacetamides to glycosylate proteins selectively via their cysteine residues. We have chosen the green fluorescent protein mutant GFPuv for use as a protein scaffold due its known tolerance of two cysteine mutations (E6C and I229C) and the previous successful derivatisation of these cysteines with iodoacetamides.1 The inherent fluorescence of GFPuv also makes it a useful candidate for fluorescence based binding assays or cell labelling studies.16 active, mutants of GFPuv were created using a mixture of site directed mutagenesis and DNA shuffling (including one mutant containing six reactive cysteine residues). This was achieved by producing random combinations of two synthetic variants of GFPuv, one of which contained 33 surface cysteines. 94 bacterial colonies expressing active GFPuv were then sequenced and the new chimeric genes analysed. Four monosaccharides and one trisaccharide (N-glycan core mimic) suitable for the chemical glycosylation via cysteines were synthesised and successfully used to create a selection of homogeneous neoglycoproteins. These neoglycoproteins were demonstrated to interact differently with different lectins (including ConA, GNL and Jacalin) in a qualitative fluorescence based assay. Interactions were shown to vary with glycan structure, position of glycosylation sites and the number of glycosylation sites.
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Sun, Yedi. "Novel Functionalized Lectins Engineered by Affinity-Guided DMAP Chemistry." 京都大学 (Kyoto University), 2013. http://hdl.handle.net/2433/174964.
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Tan, Guosheng. "Raman spectroscopy studies of the influence of cAMP on the structure of the cAMP receptor protein (CRP)." Diss., Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/27687.
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Djabayan-Djibeyan, Pablo. "A comparison of lectins in green Venezuelan marine algae." Thesis, University of Portsmouth, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343338.
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Pesquero, Naira Canevarolo [UNESP]. "Estudo da equivalência entre a lectina artin M obtida a partir da semente da jaca e a sua forma recombinante na afinidade por glicanas." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/87992.
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No presente trabalho foi avaliada a equivalência entre as formas nativa e recombinante da lectina Artin M utilizando como ligante a peroxidase de raiz forte (HRP) por meio da técnica de microbalança a cristal de quartzo. Para tal foi preparado um biossensor por meio da imobilização da lectina, tanto nativa como recombinante, na superfície do cristal de quartzo piezelétrico. A imobilização das lectinas foi realizada por meio da construção de uma monocamada auto organizada utilizando dois alcanotióis, ácido 11-mercaptoundecanóico e o 2-mercaptoetanol. Para o acoplamento das proteínas foram utilizados N-etil-N- (dimetilaminopropil) carbodiimida (EDC) e N-hidoxisuccinimida (NHS) que formam com os grupamentos carboxílicos um intermediário reativo. Após a preparação do biossensor foi utilizado um sistema de injeção em fluxo acoplado à microbalança de cristal a quartzo para o estudo da interação lectina-glicoconjugado. Desta forma, as interações da Artin M nativa e recombinante com a glicoproteína peroxidase de raiz forte foram estudadas por meio da determinação das suas constantes de afinidade aparente e de associação cinética, sendo que foram encontrados os valores de constante de afinidade aparente (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP, e os valores de constante cinética (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 para as interações jArtinM-HRP e rArtinM-HRP. Os valores das constantes de interação obtidos evidenciaram a equivalência entre ambas as formas da lectina Artin M. Neste trabalho também foi determinada a constante de associação cinética da interação entre a lectina Artin M recombinante e linhagens celulares de leucemia mielóide aguda humana (NB4, K562 e U937), no intuito de melhor entender a diferença na citotoxicidade observada da Artin M sobre estas células...
In the present work was evaluated the equivalence between the native and recombinant forms of Artin M using horseradish peroxidase as ligand by means the quartz crystal microbalance technique. In this way, a biosensor was prepared immobilizing the lectin, native and recombinant forms, on piezoelectric quartz crystal surface. Lectins immobilization was realized constructing self assembled monolayers using the alkanethiols 11-mercaptoundecanoic acid and 2-mercaptoethanol. To the binding of proteins was used N-ethyl-N-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS), which form with carboxylic groups a reactive intermediary. After biosensor preparation was utilized a flow injection system coupled to quartz crystal microbalance to study the lectin-glycoconjugated interaction. Thus the native Artin M and recombinant Artin M interaction with horseradish peroxidase glycoprotein were studied by determining its apparent affinity constant and association kinetic constants. The values obtained to apparent affinity constant were (4,6 ± 0,9) x 103 e (2,6 ± 0,5) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions, and the values obtained to association kinetic constant were (7 ± 3) x 103 e (7 ± 2) x 103 L mol -1 to jArtinM-HRP e rArtinM-HRP interactions. These constant values evidence the equivalence between native and recombinant forms of Artin M lectin. During this work were also determined the association kinetic constant of the interaction between recombinant Artin M and leukemic lineages from human acute myeloid leukemia (NB4, K562 and U937), with the purpose of a better understanding in the different cytotoxic effect of Artin M on these cells. In this way the values of association kinetic constant obtained was (0,3 ± 0,1) x 10-7 , (0,9 ± 0,1) x 10-7 e (2,7 ± 0,3) x 10-7 mL cel -1 to the interactions between Artin M and NB4, K562 and U937, respectively
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Zelensky, Alex N., and Alex Zelensky@anu edu au. "In silico analysis of C-type lectin domains structure and properties." The Australian National University. The John Curtin School of Medical Research, 2005. http://thesis.anu.edu.au./public/adt-ANU20050318.185314.
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Members of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. This thesis presents the results of several computational and experimental studies of the CTLD structure, function and evolution.¶
Core structural properties of the CTLD fold were explored in a comparative analysis of the 37 distinct CTLD structures available publicly, which demonstrate significant structural conservation despite low or undetectable sequence similarity. Pairwise structural alignments of all CTLD structures were created with three different methods (DALI, CE and LOCK) and analysed manually and using a computational algorithm developed for this purpose. The analysis revealed a set of conserved positions and interactions, which were classified based on their role in CTLD structure maintenance.¶
The CTLD family is large and diverse. To organize and annotate the several thousand of known CTLD-containing protein sequences and integrate the information on their evolution, structure and function a local database and a web-based interface to it were developed. The software is written in Perl, is based on bioperl, bioperl-db and Apache::ASP modules, and can be used for collaborative annotation of any collection of phylogenetically related sequences.¶
Several studies of CTLD genomics were performed. In one such study, carried out in collaboration with the RIKEN structural genomics centre, CTLD sequences from the Caenorhabditis elegans genome were identified and clustered into groups based on similarity. The most representative members of the groups were then selected, which if characterized structurally would tell most about the C. elegans CTLDs and provide templates for homology modelling of all C. elegans CTLD structures.¶
In the other whole-genome study, the CTLD family in the puffer fish Fugu rubripes was analysed using the draft genome sequence. This work extended and complemented three genome-level surveys on human, C. elegans and D. melanogaster reported previously. The study showed that the CTLD repertoire of Fugu rubripes is very similar to that of mammals, although several interesting differences exist, and that Fugu CTLD-encoding genes are selectively duplicated in a manner suggesting an ancient large-scale duplication event. Another important finding was the identification of several new CTLDcps, which had mammalian orthologues not recognized previously.¶
CBCP, a novel CTLD-containing protein highly conserved between fish and mammals with previously unknown domain architecture, was predicted in the Fugu study based solely on ab initio gene models from the Fugu locus and cross-species genomic DNA alignments. To test if the prediction was correct, a full-length cDNA of the mouse CBCP was cloned, its tissue distribution characterized and untranslated regions determined by RACE. The full-length mCBCP transcript is 10 kb long, encodes a protein of 2172 amino acids and confirms the original prediction. The presence of a large N-terminal NG2 domain makes CBCP a member of a small but very interesting family of Metazoan proteins.
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Chagas, Kélem de Nardi. ""Avaliação do gene estrutural da proteína de ligação à lectina (MBL) e sua relação com a transmissão materno-fetal do HIV"." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/5/5144/tde-16112005-114946/.
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Avaliou-se a expressão do gene mbl2 em 79 crianças e suas mães HIV positivas com o objetivo de avaliar a sua influência na transmissão vertical. Os pacientes divididos em dois grupos: crianças HIV positivas e suas mães (n=18) e crianças HIV negativas e suas mães (n=61) foram avaliados pelo CH50 e AP50 (ensaios hemolíticos), dosagem e avaliação funcional da MBL, ativação da cascata terminal do complemento (ELISA) e o gene mbl2 (PCR). Os resultados não mostraram diferença significante entre os níveis séricos, atividade funcional e o gene da MBL entre os grupos, excluindo a sua influência sobre a transmissão materno-fetal do HIVIt was evaluated the mbl2 gene expression in 79 children and their HIV positive mothers with the aim to evaluate its influence on mother-to-child HIV. The patients were divided in two groups: HIV positive children and their mothers (n=18) and HIV negative children and their mothers (n=61) were evaluated by CH50 and AP50 (hemolytic assays); levels and functional MBL and terminal complement cascade (ELISA) and mbl2 gene (PCR). The results didn't show significant difference amons serum levels, functional activities and MBL gene between the groups, excluding the influence in the mother-to child HIV transmission.
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Raemaekers, Romaan J. M. "Expression of functional plant lectins in heterologous systems." Thesis, Durham University, 2000. http://etheses.dur.ac.uk/4621/.
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The mannose-binding lectin from snowdrop (Galanthus nivalis agglutinin; GNA) was produced in Escherichia coli and purified as a functional protein after denturation/renaturation. Incorporation of the four extra C-terminal residues recently revealed from X-ray crystallographic data demonstrated that these residues increase binding to the glycoprotein carboxypeptidase Y. However, no differences in activities were observed in haemagglutination assays when compared to native GNA and toxicity towards rice brown planthopper (Nilaparvata lugens', BPH) in artificial diet bioassays was unaltered. Site-directed mutagenesis of the carbohydrate-binding site of GNA provided evidence of a direct correlation between the binding potential of GNA to BPH gut glycoprotein 'receptors' and the toxicity levels of GNA towards BPH nymphs. Functional recombinant plant lectins GNA and PHA (Phaseolus vulgaris agglutinin) were expressed in Pichia pastoris using native signal peptides or the Saccharomyces a-factor prepro-sequence to direct secretion. The a-factor prepro-sequence was inefficiently processed unless Glu-Ala repeats were added at the C-terminal end. In the latter case, removal of the Glu-Ala repeats was itself inefficient leading to recombinant lectins with heterogenous N-termini. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed and fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs in Pichia. A fusion protein containing both GNA and GFP (GNA-GFP) was expressed in Pichia pastoris. Simultaneous dual activities (i.e. carbohydrate binding and fluorescence) of recombinant GNA-GFP were demonstrated. Partial cleavage in the linker region resulted in co-purification of GNA which increased the binding activity of the fusion protein. Selective binding of GNA-GFP to haemocytes in the haemolymph of Lacanobia oleracea was observed, both in vitro and when the protein was fed to insects in diet.
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Clarke, Christopher B. A. "An investigation into the role of water in protein-carbohydrate complexation." Thesis, University of Birmingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368787.
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Prasad, Alpana. "Immune function and structural analysis of recombinant bovine conglutinin and human lung surfactant protein-D." Thesis, University of Oxford, 2000. http://ora.ox.ac.uk/objects/uuid:f9a5ae66-4ed0-4bdf-90eb-c873ca44147d.
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Recognition of sugar moieties on the surface of microorganisms is one of the ways the body distinguishes potential pathogens from self-cells. The sugarbinding proteins, lectins, mediate this recognition role of the first line of defence against infections, preceding the antibody-mediated (adaptive) immune response. Collectins are calcium-dependent carbohydrate-binding proteins that have been implicated in innate immunity. Bovine conglutinin (BC) and lung surfactant protein-D (SP-D), belong to the family of 'collectins' which are characterised by four domains: an N-terminal cysteine-rich region, a collagenlike region linked with the carbohydrate recognition domain (CRD) via an ahelical neck region. BC and SP-D show remarkable similarity in their amino acid sequence (79% identity), function and biological characteristics. They have been shown to mediate microbial clearance either by directly binding to bacteria leading to phagocytosis or interacting with complement system components. The present study aims to elucidate the biological function of these proteins more precisely. Recombinant fragments (r) of BC and SP-D consisting of their CRDs and neck regions have been cloned in pET-21a and pMal-c2 vectors respectively, for expression in Escherichia coli. Recombinant conglutinin was expressed in BL21(DE3)pLysS and isolated by a denaturation-renaturing procedure. Binding of rBC(N/CRD) to mannan and complement component, iC3b, was assessed in real-time by BIAcore. The dissociation constants were calculated by Scatchard analysis. The carbohydrate structures present on the surface of the microorganisms play an important role in mediating the interactions with the immune cells. The recombinant molecules showed calcium-dependent binding to lipopolysaccharides (LPS) from gram-negative bacteria Pseudomonas aeruginosa, Klebsiella pnuemonia and Salmonella typhosa, which was inhibited in presence of sugars. rBC(N/CRD) also bound to whole bacteria as assessed by ELISA and retained its capacity to recognise various complement system components and the carbohydrate moieties on the surface of various pathogenic microorganisms. The recombinant protein retained its ability to bind various sugar residues, although with lower affinity than that of the native molecule. rBC(N/CRD) is able to bind and aggregate bacteria and cause agglutination of bacterial cell suspensions. A novel model has been used to describe the interactions of the collectins at the molecular level based on specificity of carbohydrate-recognition by the collectins. The pyocin mutant strain 1291 series of Neisseria gonorrhoeae has sequential deletions of the terminal sugars in their lipooligosaccharides (LOS). Conglutinin showed a preferential high affinity binding to 1291a mutant that expresses GlcNAc as the terminal hexose, in comparison to other mutants. This provides a unique system to understand the specific cell-surface interactions in relevance to a particular lectin. Further elucidation of the function of CRD and neck region at a structural level is in progress, using X-ray crystallography. Since the submission of the thesis, the structure of the monomeric CRD has been solved, which revealed a remarkable similarity to the SP-D and MBL structure. Trials are underway to get the structure of the trimeric CRDs. These studies aim to provide a better understanding of the collectinpathogen interaction at the biological and structural levels. The ultimate aim is to determine if the recombinant forms of these proteins can be used therapeutically to enhance the uptake and killing of pathogens.
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BERTHIER, LAURENT. "Contribution a l'etude des proteines des mouts et des vins de champagne : purification d'une lectine vegetale et d'une proteine levurienne." Reims, 1999. http://www.theses.fr/1999REIMS014.
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Parmi les composes decrits comme ayant une action stabilisante sur la mousse des boissons effervescentes, les glycoproteines semblent etre particulierement impliquees. Celles-ci sont presentes dans les mouts et vins de champagne a de faibles teneurs. Le fractionnement par chromatographie d'echange d'ions n'autorise pas leur purification, a cause de leurs caracteristiques intrinseques. C'est pourquoi, nous nous sommes orientes vers la recherche d'activites biologiques, souvent mises a profit dans une strategie de purification. Les vegetaux et les levures contiennent un grand nombre de lectines dont la plupart sont glycosylees. Apres avoir mis en evidence l'existence d'activites erythroagglutinantes parmi les proteines de la baie de raisin (cepage chardonnay), il nous a paru opportun d'entreprendre la purification de cette lectine. Une pre-purification consiste en un fractionnement des proteines totales sur colonne de concanavaline a ; la fraction retenue est ensuite rechromatographiee sur colonne de q-sepharose. Parmi les composes inhibiteurs de l'activite biologique testes, seuls les para-nitrophenyls-glycosides presentent une affinite marquee. Les fractions presentant une reponse positive a l'agglutination des erythrocytes trypsinises ou desialyles sont rassemblees puis soumises a une colonne d'affinite de para-aminophenyl--d-glucoside-sepharose. L'elution est effectuee par changement de tampon mettant en jeu la presence d'aminoacides protonables au sein du site non-catalytique. A partir de 200 mg de lyophilisat de macromolecules isolees par ultrafiltration, 325 g de lectine ont pu etre ainsi isoles. Une analyse en electrophorese sds-page revele la presence d'une seule bande d'une masse moleculaire d'environ 13000. Une chromatographie de gel filtration indique que la lectine purifiee est monomerique. Trois bandes sont visualisees apres focalisation isoelectrique a des pl respectivement egaux a 4,1 ; 4,4 et 4,9 correspondant vraisemblablement a trois isoformes. Cette lectine ne semble pas etre conservee durant la vinification. Une deuxieme proteine, presentant la meme activite biologique, a pu etre isolee d'un vin champenois par chromatographie d'affinite suivie d'une ief preparative. Une chromatographie preparative sur sephacryl s200 termine la purification. La masse moleculaire determine par chromatographie (20100) ou par sds-page (19000) revele sa structure monomerique. Nous montrons par immunologie et analyse des sucres l'origine levurienne de cette proteine riche en mannose.
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Haine-Joubert, Raymonde. "Contribution à l'etude des lectines solubles : isolement et caractérisation d'une lectine soluble spécifique des résidus béta-d-galactosyles à partir d'extraits de cerveau de mammifères." Paris 13, 1987. http://www.theses.fr/1987PA132001.
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L'effet de différents extraits de cerveau de mammifères contenant des activités lectines sur des cultures de cerveau de souris embryonnaires (e 14,5) et sur des cellules de cerveau de souris dissociées et maintenues en suspension (stades e 14,5, e17,5, p0, p4, p14, p23). Nous avons exploré les propriètés agglutinantes et agrégeantes de ces extraits et montre leur interaction spécifique avec des structures saccharidiques en particulier avec les residus de type béta -d-galactosyle. Ces molécules sont ubiquitaires, leur taux est régulé par le développement. Les propriétés physico-chimiques, la spécificité saccharidique et les différentes caracteristiques rattachent ces molecules aux galaptines, lectines solubles decrites pour d'autres tissus et d'autres especes. Une deuxieme activite lectine, specifique de l'heparine est extraite du cerveau en même temps que la galaptine. La caractérisation de la galaptine à partir d'extraits de cerveau de rat et de boeuf montre qu'il s'agit d'un dimère des sous unites >ou= 14 500 daltons pour le boeuf et de >ou= 16 000 daltons pour le rat. L'analyse de la composition en acides aminés fait ressortir un taux élevé de glycine et la richesse en acides aminés acides. L'isolement de la galaptine a permis de confirmer que certains effets des extraits bruts étaient bien liés à la présence de cette lectine dans les extraits
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Bulteau, François. "Ciblage in vivo des tumeurs via l'antigène Tn : Développement d'un cluster de Macrophage Galactose Lectine Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide." Thesis, Université Grenoble Alpes, 2020. http://www.theses.fr/2020GRALV048.
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L’ensemble des cellules, qu’elles soient procaryotes ou eucaryotes, est doté d’une couche de glycosylation externe riche et diversifiée, composant la face dominante immédiate en relation à leur environnement. Elles résultent de processus enzymatiques complexes liant les sucres entre eux et sur des protéines ou lipides. Des variations du « glycome » peuvent apparaître dans certaines pathologies. Les cancers sont les pathologies les plus fréquentes présentant des anomalies de ces glycosylations. Ces altérations sont quasi systématiques à la surface des cellules cancéreuses. Parmi celles-ci, l’antigène Thomsen-nouveau (Tn), un N-acétylgalactosamine (GalNAc) sur une sérine ou une thréonine, est fortement exprimé dans 90% des carcinomes mammaires ainsi que dans les cancers de la vessie, du col de l’utérus, de l’ovaire, du colon, de l’estomac et de la prostate. L’omniprésence de l’antigène Tn dans de nombreux cancers, associés à son absence dans les cellules saines, en fait une cible de choix pour la thérapie ciblée ou des vaccins synthétiques antitumoraux. Aucun anticorps ciblant l’antigène Tn n’est à ce jour disponible du fait de la difficulté à développer un anticorps avec une telle spécificité. Ainsi, nous nous sommes intéressés à une autre stratégie de ciblage, basée sur l’utilisation d’une molécule capable de reconnaître l’antigène Tn. Les lectines de type C sont une famille de protéines capables de se lier spécifiquement et de façon réversible à certains glucides, en présence de calcium. La macrophage galactose lectine (MGL) est une lectine de type C ayant une affinité très importante pour le GalNac et ses dérivés comme l’antigène Tn. Ce travail a consisté, dans un premier temps, à l’utilisation d’une forme recombinante soluble de la MGL pour valider le potentiel de cet outil pour le ciblage des cellules cancéreuses. Les différentes expériences, in vitro et in vivo, impliquant la MGL, ont démontré la capacité de cette dernière à cibler spécifiquement les tumeurs humaines via l’antigène Tn. La partie extracellulaire de la MGL est de ce fait un très bon candidat de vecteur pour le diagnostic et l’imagerie de tumeurs humaines et potentiellement pour l’administration de médicaments. Dans un deuxième temps, diverses stratégies de développement d’un outil bifonctionnel exploitant cette lectine ont été exploré. Le but était de créer une plateforme peptidique fonctionnalisable d’une part avec plusieurs domaines lectines, afin de contrôler l’affinité de reconnaissance, et d’autre part des groupements fonctionnels variable selon l’application recherché (diagnostique, thérapeutique, ...). Les différentes stratégies de couplage employées nous ont permis d’accrocher plusieurs CRD de lectine sur un support peptidique, cela en conservant l’état tridimensionnel et fonctionnel des protéines. Les caractérisations effectuées démontrent une importante augmentation de l’affinité directement fonction du nombre de lectine ajouté sur la plateforme. Ce travail ouvre la voie vers de nouveaux systèmes de ciblage des sucres modulable à façonAll cells, whether prokaryotic or eukaryotic, have a rich and diversified external glycosylation layer, forming the immediate dominant face in relation to their environment. They result from complex enzymatic processes linking sugars to each other and to proteins or lipids. Variations of the "glycome" can appear in certain pathologies. Cancers are the most frequent pathologies with abnormalities in these glycosylations. These alterations are almost systematic on the surface of cancer cells. Among them, the Thomsen-new antigen (Tn), an N-acetylgalactosamine (GalNAc) on a serine or threonine, is strongly expressed in 90% of mammary carcinomas as well as in cancers of the bladder, cervix, ovary, colon, stomach and prostate. The ubiquitous presence of the Tn antigen in many cancers, combined with its absence in healthy cells, makes it a target of choice for targeted therapy or synthetic anti-tumor vaccines. No antibody targeting the Tn antigen is currently available because of the difficulty in developing an antibody with such specificity. Thus, we were interested in an alternative targeting strategy, based on the use of a molecule capable of recognizing the Tn antigen. C-Type lectins are a family of proteins capable of specifically and reversibly binding to certain carbohydrates in the presence of calcium. Macrophage galactose lectin (MGL) is a C-type lectin with a high affinity for GalNac and its derivatives such as the Tn antigen. This work consisted, initially, in the use of a soluble recombinant form of MGL to validate the potential of this tool for the targeting of cancer cells. The different experiments, in vitro and in vivo, involving MGL, demonstrated the latter's ability to specifically target human tumors via the Tn antigen. The extracellular portion of MGL is therefore a very good vector candidate for the diagnosis and imaging of human tumors and potentially for drug delivery. In a second step, various strategies for the development of a bifunctional tool exploiting this lectin were explored. The goal was to create a peptide platform that could be functionalized on one hand with several lectin domains, in order to control recognition affinity, and on the other hand with functional groups that could be variable according to the application (diagnostic, therapeutic, ...). The different coupling strategies employed allowed us to attach several lectin CRDs to a peptide support, while preserving the three-dimensional and functional state of the proteins. The characterizations carried out show a significant increase in affinity directly related to the number of lectins added to the platform. This work paves the way to new customizable sugar-targeting systems
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Škeřík, Jan. "Analýza vybraných obsahových látek v extraktu z bezových větviček." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2016. http://www.nusl.cz/ntk/nusl-240544.
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My diploma thesis deals with the optimization and verification of conditions for separation of proteins, which are contained in the twigs of elder (Sambucus nigra L.) using the technique RP-HPLC. The measurements were made with the use of a HPLC system with a UV-VIS detector. Column Zorbax 300SB-C18 300 4,6 x 250 mm and particles the size of 5 microns were used. The theoretical part describes the attributes and usage of elder and especially its proteins. The basic characteristic of the used HPLC technique is introduced. The possibilities of how to identify proteins are described. The practical part demonstrates the individual steps of optimization of the HPLC method applied to the mixture of standard proteins. The application of this method in the real sample is also reported. The final part of the diploma thesis is focused on the comparison of analysed samples taken in different seasons.
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Wright, Lisa Michelle. "Biochemical and x-ray crystallographic studies of monocot lectins in their native and ligated states." Thesis, Liverpool John Moores University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263954.
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Magnusson, Alessandra Selinger. "Papel de peptídeos bioativos presentes no veneno de Lonomia obliqua sobre a angiogênese." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2016. http://hdl.handle.net/10183/158220.
Full textAbstract:
A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais.The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.