Dissertations / Theses on the topic 'Transglutaminase'

Conoscenze preesistenti: Le transglutaminasi (TGase) sono una classe di enzimi ampiamente diffusa tra gli organismi procarioti ed eucarioti. Gli enzimi di questa famiglia catalizzano modifiche post-traduzionali in molte proteine attraverso reazioni di trasferimento dell’acile, reazioni di deaminazione e di crosslinking (polimerizzazione) tra residui peptidici di lisina (accettore di acile) e glutammina (donatore di acile) intra- o inter-catena proteica. A causa della sua facilità di espressione e di purificazione, l’unica TGase ampiamente usata per le applicazioni industriali è la TGase microbica estratta da Streptomyces mobaraensis (MTGase). Oggigiorno la MTGase è disponibile in commercio ed è ampiamente usata nell’industria dei biopolimeri, in cosmetica, per applicazioni cliniche, nell’industria tessile della lana e soprattutto nell’industria alimentare. La sua abilità di catalizzare legami crociati in molti substrati proteici differenti è sempre più usata non solo per la produzione di salsicce, prosciutti e formaggi ma, molto recentemente, anche per la detossificazione della farina, come possibile terapia alternativa alla dieta senza glutine. Ne consegue che oggigiorno le applicazioni industriali della MTGase stiano aumentando, coinvolgendo sempre più settori e producendo una ricerca scientifica su questo argomento sempre più fervente, allo scopo di tentare di rispondere a specifiche esigenze industriali, come l’implementazione di sistemi di purificazione della MTGase più efficienti, la ricerca di fonti alternative di transglutaminasi microbica, e di fonti sicure di enzimi ricombinanti. Scopo del progetto di dottorato: lo scopo principale del progetto è l’identificazione di nuove forme di transglutaminasi microbica che possano diventare un’alternativa a quella attualmente in uso. È stata eseguita un’analisi approfondita delle sequenze note allo scopo di ottenere una classificazione delle TGase microbiche attraverso la loro similarità a forme note. Per selezionare le migliori candidate che possano essere forme attive in appropriate condizioni, le sequenze selezionate sono state soggette di modellamento molecolare e simulazioni molecolari. Per testare l’attività enzimatica, sono stati effettuati dei saggi sperimentali su una nuova forma trovata ed un’ulteriore nuova forma è stata espressa. Risultati: il presente lavoro propone in primo luogo un’analisi, ad oggi assente, dell’ampio panorama delle transglutaminasi microbiche, sviluppando la prima classificazione delle TGase microbiche basata sulle loro caratteristiche di sequenza e sulle loro specifiche strutture secondarie predette. Al fine di classificare ed analizzare le caratteristiche strutturali di tutte le sequenze annotate come aventi un TGase core, sono state utilizzate tecniche computazionali che coinvolgono analisi di sequenza, studi comparativi, costruzione di alberi filogenetici, modellamento per omologia e simulazioni di dinamica molecolare. Tramite questo approccio, è stata effettuata una classificazione preliminare di queste sequenze dividendole in cinque gruppi principali. Ogni gruppo è stato studiato dal punto di vista delle sequenze per analizzare la presenza di motifs specifici. Per tre di questi cinque gruppi, sono state studiate anche le strutture secondarie e, da questa analisi, sono state rilevate caratteristiche specifiche per ogni gruppo. Inoltre, due nuove forme di TGase microbica (mTGase) sono state studiate in dettaglio: K. albida mTGase e l’ipotetica mTGase da SaNDy (organismo non rivelato per possibilità di brevetto). Per la prima, in comparazione con la MTGase, sono state effettuate analisi della tasca relativa al sito attivo e simulazioni di dinamica molecolare. Per la seconda, invece, sono state utilizzate tecniche sperimentali per purificare l’ipotetico enzima al fine di testarne l’attività su substrati alimentari. Saggi sperimentali su entrambe le proteine sono ancora in corso, al fine di trovare le migliori condizioni di attività enzimatica e i migliori substrati di reazione. Le simulazioni di dinamica molecolare eseguite sulla mTGase di K. albida hanno suggerito alcune spiegazioni alla maggiore specificità di questo enzima rispetto alla MTGase, dimostrata sperimentalmente da Steffan e colleghi, ed alcune indicazioni per variare le condizioni di attività usate per testarla. Inoltre, l’analisi dei substrati ha permesso di trovare nuovi possibili substrati, sui quali l’enzima potrebbe essere impiegato ai fini della riduzione delle allergenicità. D’altro canto, l’enzima estratto da SaNDy, mostrando una più alta somiglianza con la MTGase, potrebbe essere meno selettivo della mTGase da K. albida nei confronti di specifici substrati, pertanto potrebbe essere possibile una sua applicazione anche su substrati gliadinici, tuttavia, per provare ciò, sono necessari ulteriori esperimenti. Note: il presente lavoro di dottorato è stato principalmente svolto presso il Laboratorio di Bioinformatica del CNR di Avellino sotto la supervisione del Dr. Facchiano, tuttavia, tutte le simulazioni di dinamica molecolare sono state eseguite presso il Dipartimento di Biochimica dell’Università di Zurigo, nel laboratorio di biologia strutturale e computazionale sotto la supervisione del Prof. A. Caflisch e del suo gruppo di ricerca (periodo di formazione all’estero obbligatorio). I saggi di attività sperimentale sul substrato gliadinico sono stati effettuati dal laboratorio di spettrometria di massa CeSMA-ProBio presso il CNR di Avellino; e l’ipotetica mTGase da SaNDy è stata invece clonata, espressa e purificata durante la collaborazione con il laboratorio di Molecular Sensing presso il CNR of Avellino.
Background: transglutaminases (TGase) are a class of enzymes widely spread in eukaryotic and prokaryotic organisms. Enzymes of this family catalyze post-translational modifications in many proteins by acyl transfer reactions, deamidation and crosslinking (polymerisation) between protein intra- or inter-chain glutamine (acyl donor) and lysine (acyl acceptor) peptide residues. Due to its facility of expression and purification, the only TGase enzyme widely used for industrial applications is the microbial TGase extracted from Streptomyces mobaraensis (MTGase). Nowadays the MTGase is commercially available and widely used in biopolymers industry, in cosmetics, in clinical applications, in wool textiles, and above all in the food processing industry. Its ability to catalyze crosslinks on many different protein substrates is increasingly used not only for sausage, ham and cheese production but, very recently, also for flour detoxification, as a possible alternative therapy to the gluten free diet. It follows that nowadays the industrial applications of MTGase have increased, covering more and more fields producing a very active scientific research about this topic aimed at attempt to meet specific industrial needs, as the implementation of more efficient system for MTGase production, the research of alternative sources of microbial TGase, and safe source of recombinant enzymes. Aims of the doctorate project: the main aim of the project is the identification of novel forms of microbial TGases that could become an alternative to that in use. A depth screening of known sequences has been performed, with the aim of obtaining a classification of microbial TGases for their similarity to known forms. To select the best candidates to be active forms under appropriate conditions, molecular modelling and molecular simulations have been performed on selected sequences. To test the enzymatic activity, experimental assays have been performed with a novel form, and another novel form has been expressed. Results: the present work proposes at first an analysis, lacking so far, of the wide microbial transglutaminase world, developing the first classification of the microbial TGase based on their sequence features and their specific predicted secondary structures. In order to classify and analyze the structural features of all the sequences annotated as having a TGase core computational techniques involving sequence analyses, comparative studies, building of phylogenetic trees, homology models and molecular dynamic simulations have been used. From this approach, a preliminary classification of these sequences was done by dividing them in five main groups. Each group has been investigated from the sequence point of view to analyze the presence of specific motifs. For three of this five groups, also the secondary structures have been investigated and, from this analysis, features specific for each group have been detected. Moreover, two novel forms of microbial TGase (mTGase) have been investigated in the detail: K. albida mTGase and the hypothetical mTGase from SaNDy (organism not disclosed for patent opportunity). Molecular dynamics simulations and active site pocket analyses have been performed for the first, in comparison with MTGase. For the second, instead, experimental technique has been used to purify the hypothetical enzyme in order to test it on food related substrates. Experimental assays on both the proteins are still ongoing, to find the best enzymatic activity conditions and the best substrates of reaction. The molecular dynamic simulations performed on K. albida mTGase have suggested some explanations to the higher specificity of this enzyme than MTGase, experimentally demonstrated by Steffen et colleague, and several indications to change the activity conditions used to test it. Moreover, the substrates screening has allowed to find novel possible substrates, on which this enzyme could be employed for the allergenicity reduction. On the other hand, the enzyme extracted from SaNDy, showing a higher similarity with MTGase, could be less selective than K. albida mTGase for specific substrates, so it could be possible its application also on the gliadin substrate, but to prove it further experiments are necessary. Note: the present PhD work has been mainly performed in the Bioinformatics Laboratory at the CNR of Avellino under Dr. Facchiano’s supervision, however all the MD simulations have been performed at the Biochemistry Department of the University of Zurich, in the computational and structural biology laboratory under the supervision of Prof. A. Caflisch and his research group (compulsory abroad training period). Experimental activity assays on gliadin substrate have been performed by the spectrometry mass CeSMA-ProBio lab at the CNR of Avellino; and the hypothetical mTGase from SaNDy was instead cloned, expressed and purified in collaboration with the Laboratory for Molecular Sensing at the CNR of Avellino.

Orientador: Hélia Harumi Sato
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A transglutaminase catalisa a formação de ligações cruzadas entre grupos ?-amino de resíduos de lisina e o grupo ?-carboxiamida de resíduos de glutamina de proteínas. Esta enzima pode ser usada para unir diferentes proteínas e melhorar suas propriedades funcionais. A transglutaminase obtida de micro-organismos por processos fermentativos apresenta vantagens em relação à enzima obtida de plantas e tecidos animais para as aplicações industriais. Neste trabalho, foram estudados os efeitos de diferentes subprodutos ou resíduos agroindustriais no meio de cultivo para a fermentação submersa de Streptomyces sp. CBMAI-837, visando o aumento da atividade e da produtividade da enzima. Entre os substratos proteicos avaliados, o uso de 2,5% (m:v) de farelo de algodão ou de 2,5% (m:v) de farelo de soja no meio de cultivo resultou no aumento da atividade enzimática de 1,2 U.mL-1 (214%) e 1,0 U.mL-1 (182%), respectivamente, em relação ao valor obtido pela fermentação do micro-organismo em meio de cultivo contendo 2,5% de farinha de soja (0,57 U.mL-1). Em relação às fontes de carbono principais avaliadas, a adição de 2,0% de glicerol ou de 12% de melaço de cana de açúcar permitiu o aumento da atividade de transglutaminase em 0,94 U.mL-1 (167%) e 0,88 U.mL-1 (157%), respectivamente. Foi verificado que a adição de 1% de quitina nativa no meio de cultivo favoreceu a produção da enzima elevando a atividade de transglutaminase em 181% em relação ao meio de cultivo sem quitina. Os efeitos da aplicação da TGase de Streptomyces sp. CBMAI-837 em massa de farinha de trigo mole e na fabricação de pães foram avaliados e os resultados foram comparados com a atuação de uma TGase comercial (BioBond) formulada para aplicação em cereais. De forma geral, as duas enzimas avaliadas apresentaram o mesmo efeito sobre a massa quanto ao aumento da resistência à extensão, a redução da máxima extensão da massa e a redução da pegajosidade da massa. Efeito antagônico foi observado na hidrofobicidade de superfície das proteínas da massa sendo que este parâmetro foi reduzido pela adição da TGase de Streptomyces sp. CBMAI-837 e foi elevado pela enzima comercial. A adição de TGase na preparação da massa de farinha de trigo mole resultou em aumento da massa molecular das proteínas, indicantivo da formação de ligações cruzadas entre proteínas. A aplicação da transglutaminase de Streptomyces sp. CBMAI-837 em massa de pão de farinha de trigo mole promoveu a redução do volume do pão em 9% e o aumento da firmeza em 32%. O aumento da quantidade de solvente adicionado na massa de 53% para 56% permitiu o aumento do volume dos pães, no entanto, com pouca diferença dos parâmetros de textura em relação ao controle para a TGase comercial BioBond e para a TGase de Streptomyces sp. CBMAI-837
Abstract: Transglutaminase catalyzes the cross-linking reaction between a ?-carboxyamide of a glutamine residue from a peptide bond and the ?-amino group of a lysine. TGase can bind different proteins and improve their functional properties. The microbial transglutaminase shows advantages over the enzyme extracted from plants and mammals. In the present study, the effect of different industrial wastes and byproducts in the culture medium during the submerged fermentation of Streptomyces sp. CBMAI-837 was studied aiming to increase the enzyme activity and yield. Amongst the substrates with high protein content, the use of 2,5% of cottonseed meal or 2,5% of soybean meal in the culture medium increased the transglutaminase activity to 1.2 U.mL-1 (214%) and 1.0 U.mL-1 (182%), respectively, as compared to the results obtained using 2,5% of soybean flour. With regard to the main carbon sources, both 2% glycerol and 12% sugar cane molasses increased the transglutaminase activity to 0.94 U.mL-1 (167%) and 0.88 U.mL-1 (157%), respectively. It was observed that the addition of 1% of chitin on culture medium increased the transglutaminase activity by 181% as compared to the results obtained without the addition of chitin. The effects of the TGase from Streptomyces sp. CBMAI - 837 on soft wheat flour dough and on the manufacture of bread were evaluated, and the results were compared with the performance of a commercial TGase (BioBond) formulated for specific applications in cereal products. In general, both enzymes had the same effect on the rheological properties of the doughs, increasing the resistance to extension, reducing the maximum extension and reducing stickiness of the dough. The surface hydrophobicity of the protein dough was reduced by the addition of TGase from Streptomyces sp. CBMAI 837 but increased by the addition of the commercial enzyme. In general the analysis of the protein structure indicated an agglomeration of the proteins causing an increase in molecular weight. The application of transglutaminase from Streptomyces sp. CBMAI-837 in the formulation of bread loaves decreased the bread volume by 9% and increased firmness by 32%. Increasing the amount of solvent added to the dough from 53% to 56% increased the volume of the loaves, but resulted in little difference in the texture profiles of the loaves made with the addition of the commercial BioBond and Streptomyces sp. CBMAI ¿ 837 TGases, in relation to the control
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos

Orientador: Hélia Harumi Sato
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A isomaltulose e a trealulose são dissacarídeos isômeros estruturais, que podem ser obtidos a partir da sacarose utilizando-se glicosiltransferase bacteriana. Esses dissacarídeos são considerados açúcares alternativos de grande potencial para uso nas indústrias de alimentos e farmacêutica porque são hidrolisados e absorvidos mais lentamente e apresentam baixo potencial cariogênico comparado com a sacarose. Foi estudada a imobilização de células de Serratia plymuthica ATCC 15928, produtora de glicosiltransferase por gelificação iônica em gel alginato contendo transglutaminase (TG) e também a utilização de células livres para a conversão de sacarose em isomaltulose e trealulose. Utilizando-se células livres de Serratia plymuthica ATCC 15928 foi obtido 70% de conversão em isomaltulose e 8% de trealulose a 25°C por 10 bateladas de 15 minutos, a partir de solução de sacarose 30%. Entre as cinco amostras de alginato de sódio testadas, para a imobilização das células de S. plymuthica ATCC 15928 com e sem adição de TG, foram obtidos melhores resultados (médio de três bateladas) de conversão de sacarose (37,4% de isomaltulose) utilizando o alginato de sódio B, de alta viscosidade (14.000cP Sigma ¿ A 7128) em presença de TG. Nas condições estudadas (1,7% de alginato de sódio, 30% de massa celular úmida, solução de cloreto de sódio 0,2Mol/L, 2% de TG e 35% de sacarose) também houve maior facilidade de formação de grânulos uniformes. A presença de TG como agente de reticulação na matriz de imobilização melhorou a estabilidade de conversão por três bateladas onde observou-se resultado médio 27% maior com relação a matriz com o mesmo tipo de alginato (B) em ausência de TG. A composição da matriz de imobilização com adição de TG foi otimizada por metodologia de planejamento experimental, assim como a adição de gelatina como fonte de proteína adicional para promoção de ligações cruzadas catalisadas pela TG. Os melhores resultados de conversão de sacarose (solução 35%) em isomaltulose (72,66% de isomaltulose e 8% de trealulose em 4 bateladas de 24horas) foram obtidos utilizando-se matriz de polissacarídeo-proteína composto de 1,7% de alginato de sódio 14.000cP (Sigma®-A7128), 0,25mol/L de CaCl2, 0,5% de gelatina, 3,5% de TG e concentração de massa celular úmida superior a 35% (m:v). Verificou-se que a adição de ALMP na matriz de alginato de cálcio-gelatina-TG para imobilização de S. plymuthica, testada por planejamentos experimentais seqüenciais, não aumentou a estabilidade da taxa de conversão de sacarose em isomaltulose quando comparada com as células imobilizadas em matriz de alginato de cálcio-gelatina-TG. Em processo contínuo utilizando-se coluna empacotada com células de S. plymuthica imobilizadas em matriz otimizada e descrita acima, foi obtida taxa de conversão média de 64% de sacarose em isomaltulose durante 200 horas de processo, equivalente a 0,27g de isomaltulose/g de células imobilizadas/hora em coluna a 25°C e fluxo de substrato (35% de sacarose) 0,2mL/min
Abstract: The isomaltulose and trehalulose are disaccharides and structural isomers, which can be obtained from sucrose using bacterial glycosyltransferase. These disaccharide are considered alternative sugars with great potential for use in the food and pharmaceutical industries because they are hydrolyzed and absorbed more slowly and have a low cariogenic potential compared with sucrose. The conversion of sucrose to isomaltulose and trehalulose was estudied using immobilized and free cells of Serratia plymuthica ATCC 15928. The cells were immobilized by ionic gelation in alginate gel containing transglutaminase. Using free cells of Serratia plymuthica ATCC 15928 was obtained 70% isomaltulose conversion and 8% trehalulose conversion at 25° C in 10 batches of 15 minutes from a 30% sucrose solution. Among the five samples of sodium alginate tested for S. plymuthica ATCC 15928 cells immobilization, with or without the addition of TG, the best results (average of three batches) were obtained using sodium alginate B, high viscosity (14.000cP Sigma - A 7128) in the presence of TG, leading to 37.4% isomaltulose conversion from sucrose. In the studied conditions (1.7% sodium alginate, 30% wet cell mass solution of sodium chloride 0.2 Mol/L, 2% TG, 35% sucrose) was also easier to form uniform granules. The presence of TG as a crosslinking agent in the immobilization matrix improved the stability during three batches, resulting in an 27% higher average conversion with respect to a same type of alginate (B) matrix in absence of TG. Immobilization matrix compositions with addition of TG was optimized by experimental design methodology, as well as the addition of gelatin as a protein source for promoting additional crosslinking catalyzed by TG. The best results conversion of sucrose (35% solution) into isomaltulose (72.66% of isomaltulose and 8% of trehalulose in 4 batches of 24 hours) were obtained using proteinpolysaccharide matrix composed of 1.7% alginate 14.000cP sodium (Sigma® A7128), 0.25 Mol/L CaCl2, 0.5% gelatin, 3.5% TG, and wet cell mass concentration of 35% (w:v). It has been found that the addition of ALMP (amidated low methoxyl pectin) into the calcium alginate-gelatin-TG matrix for immobilization of S. plymuthica, tested by sequential experimental design, do not increase the stability of sucrose to isomaltulose conversions rate when compared with cells immobilized in calcium alginate -gelatin-TG matrix. In continuous process using a packed column with S. plymuthica cell's immobilized in the optimized matrix described above, it was obtained an average conversion rate of 64% sucrose to isomaltulose during a 200 hours process, equivalent to 0.27g isomaltulose per gram of immobilized cell per hour, in a column at 25° C and using flow substrate (35% sucrose) of 0.2 mL / min
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos

Coeliac disease (CD) is a gluten intolerance condition that results in the flattening of the villi, which line the bowel. It is the most common cause of malabsorption of food nutrients. This inability to absorb sufficient levels of nutrients causes many of the common symptoms experienced by CD patients. Some of the symptoms, which lead to an increase in mortality rate, include chronic diarrhea, fatigue, iron-deficient anemia and osteoporosis. People with CD have higher than normal levels of certain antibodies in their blood. Thus, the concentration of anti-transglutaminase antibody (anti-tTG) in human sera is an important analytical marker for the diagnosis of CD. An immunosensor is a type of biosensor that has an antigen or antibody fragment as its biological recognition component. The specificity of the molecular recognition of antigens by antibodies to form a stable complex is the basis of immunosensor technology. In this work, overoxidized polypyrrole (OvoxPpy) was electrosynthesized as a noval sensor platform on a glassy carbon electrode (GCE). The OvoxPpy was then doped with gold-nanoparticles (GNP) by electrodeposition using cyclic voltammetry to form GNP|OvoxPpy||GCE electrode system. Morphology and size of the GNP|OvoxPpy||GCE nanocomposite were determined using scanning electron microscopy. The electrochemical immunosensor for anti-tTG antibodies was prepared by immobilizing transglutaminase antigen (tTG-antigen) onto the GNP|OvoxPpy||GCE by drop coating and allowed to incubate for 2 hrs. The electrochemical characterization of the nanocomposite platform and immunosensor were studied by voltammetry and electrochemical impedance spectroscopy (EIS)...

2009/2010
Tissue transglutaminase (TG2) is a multifunctional enyzme involved in cell growth and differentiantion, receptor mediated endocytosis, cell adhesion and morphology, stabilization of extracellular matrix, membrane trafficking and structure/function, signal transduction, regulation of cytoskeleton and apoptosis. Multiple lines of evidence suggest an involvement of TG2 autoimmune diseases, cancer and in neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, Huntington's disease and Parkinson's disease. In all of the neurodegenerative diseases examined to date, TG2 activity is upregulated in selectively vulnerable brain regions, TG2 proteins are associated with inclusion bodies characteristic of the diseases, and prominent proteins in the inclusion bodies are modified by TG2 enzyme. It is important to identify TG2 substrates as they may offer an understanding of how the TG2-catalyzed post-translational modification has an impact on physiology and disease. Identification of these substrates may lead to novel drug targets and new diagnostic markers for several TG2-related diseases. A variety of different methods have been proposed for the identification of TG2 substrates. In this work we applied a new method for identification of TG2 substrates (interactors) by using a selection of cDNA phage display libraries followed by massive gene sequencing with 454 system. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential substrates and interactors, which were subsequently confirmed in functional assays. Within the identified clones, some had been previously described as interacting proteins (fibronectin, SMOC1, EIF4G2, MYO18A, GSTO2), while others were new. When compared to standard systems, such as microtiter ELISA, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction. We expect that this approach to library and selection analysis can also be extended to other methods traditionally used to study protein-protein interactions, as well as to the study of the selection of peptides and antibodies by phage display.
L'enzima transglutaminasi tissutale è un enzima multifunzionale. Questa proteina gioca un ruolo importante durante lo sviluppo, crescita e differenziamento cellulare, endocitosi mediata da recettore, adesione e morfologia cellulare, stabilizzazione della matrice extracellulare, traffico e struttura/funzione di membrana, trasduzione del segnale, regolazione del citoscheletro ed apoptosi. Molteplici evidenze indicano un coinvolgimento di TG2 in diverse patologie neurodegenerative, incluso il morbo di Alzheimer, la paralisi progressiva supranucleare, il morbo di Huntington e quello di Parkinson. In tutte le malattie neurodegenerative esaminate finora, l'attività della TG2 è aumentata in specifiche regioni cerebrali e le proteine sono associate in corpi d’inclusione caratteristici di tali patologie dove vengono modificate dall'enzima TG2. E’ importante identificare i substrati della TG2 per comprendere come le modifiche post-traduzionali introdotte da questo enzima siano coinvolte nella patogenesi delle suddette malattie. Molteplici metodiche sperimentali sono state proposte ai fini dell'identificazione dei substrati della TG2. In questo lavoro è stato applicato un nuovo metodo per l’identificazione dei substrati della TG2 (interattori), selezionando una libreria di cDNA espressa come phage display, seguito da un sequenziamento genico massivo utilizzando il sistema 454 Life Sciences. La classificazione e l’analisi di più di 120,000 sequenze di DNA ha permesso di identificare molti substrati e potenziali interattori, che sono stati successivamente confermati con le analisi funzionali. All’interno dei cloni identificati, alcuni erano già stati precedentemente descritti come proteine interagenti (interattori) (fibronectina, SMOC1, EIF4G1, MYO18A, GSTO2), mentre altri sono stati identificati come nuovi. Nella comparazione con i metodi standard, come, ad esempio, ELISA, il metodo qui descritto risulta enormemente più rapido e fornisce un numero molto maggiore di informazioni relative alle interazioni analizzate, permettendo quindi una migliore caratterizzazione di sistemi complessi. Ad esempio, nel caso della fibronectina, è stato possibile identificare i domini specifici coinvolti nell’interazione. Prevediamo che questo approccio per l’analisi e la selezione di librerie, possa essere applicato anche ad altri metodi tradizionalmente usati per lo studio di interazioni proteina- proteina, così come allo studio di selezioni di peptidi e anticorpi tramite la tecnica del phage display.
XXIII Ciclo
1979

Orientador: Hélia Harumi Sato
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Transglutaminase é uma enzima capaz de catalisar a formação de ligações cruzadas intra- e intermoleculares entre proteínas, peptídeos e aminas primárias por meio de ligações covalentes entre resíduos de lisina e glutamina. Desta forma, transglutaminase pode ser utilizada em diversos setores industriais para o desenvolvimento de novos produtos ou para a modificação de suas características. A linhagem B6 isolada de amostra de solo coletada na região do Estado de Minas Gerais foi identificada como tendo características morfológicas típicas de actinomicetos e pela análise da região 16S rRNA há colocou na subclasse Streptomyces próximo a linhagem Streptomyces angustmycinicus NBRC 3934T. A fim de aumentar a produção de transglutaminase (2,75 U/mL) pela linhagem Streptomyces sp. B6, o meio de fermentação foi submetido a processos de otimização. Como primeiro passo da otimização, o crescimento do micro-organismo e a produção da enzima foram estudados através de uma pré-seleção de fontes de carbono, nitrogênio e sais no meio de produção. Após as análises das diferentes fontes, um delineamento experimental do tipo Plackett-Burman foi utilizado para a seleção dos componentes do meio de cultivo que afetam a produção de transglutaminase. Os resultados do delineamento experimental indicaram que a produção de transglutaminase foi influenciada negativamente pela peptona bacteriológica e MgSO4.7H2O, positivamente pelo amido de batata, glicose, peptona de caseína e KH2PO4.7H2O e não foi influenciada pelo farelo de soja, considerando um nível de confiança de 95%. A concentração de amido de batata foi fixada no maior nível testado no planejamento Plackett-Burman devido à gelificação do meio de fermentação em concentrações maiores. Assim, os três fatores que influenciaram a produção de transglutaminase (glicose, peptona de caseína e KH2PO4.7H2O) foram otimizados para obter o máximo de produção da enzima utilizando delineamento composto central. Sob a condição otimizada, a qual continha 25 g/L de farinha de soja, 35 g/L de amido de batata, 5 g/L de glicose, 24,5 g/L de peptona de caseína e 8 g/L de KH2PO4.7H2O, a atividade enzimática atingiu 6,13 U/mL, apresentando 125% à mais de atividade em relação á obtida no meio antes da otimização. A transglutaminase microbiana produzida pela linhagem Streptomyces sp. B6 exibiu atividade ótima em 45°C e em pH de 6,5 e 11,0. A enzima manteve-se estável na faixa de pH 3,0-11,0 durante 60 minutos à 40°C durante 3 horas. A transglutaminase não foi inibida por Ca2+, Na+, Co2+, Mn2+, K+, Mg2+, Ba2+, EDTA, L-cisteína e glutationa na concentração de 5 mM, mas foi inibida na presença de Hg2+, Cu2+, Zn2+ e Fe2+ na concentração de 5mM. A linhagem Streptomyces sp. B6 é uma nova fonte de transglutaminase com características interessantes para aplicações biotecnológicas
Abstract: Transglutaminase is an enzyme capable of catalyzing the forming intra-and intermolecular cross-linking between proteins, peptides and primary amines by covalent bonds between lysine and glutamine residues. Thus, transglutaminase can be used in food processing industries to develop new products and modify their characteristics. The B6 strain was isolated from soil sample collected in the region state of Minas Gerais was identified as having morphological characteristics typical of the actinomycetes, and the 16S rRNA analysis placed it in the Streptomyces subclade, closely related to Streptomyces angustmycinicus NBRC 3934T. In order to increase the transglutaminase production (2.75 U/mL) from Streptomyces sp. B6 strain, the fermentation medium was subjected to optimization processes. In the first step of optimization, the micro-organism growth and enzyme production were studied through a pre-selection of carbon, nitrogen and salts sources in the culture medium. After analysis of different sources, the Plackett¿Burman experimental design was used for screening the components of the culture medium that affect the transglutaminase production. Results of the experiment indicated that production of transglutaminase was negatively influenced by bacteriological peptone and MgSO4.7H2O, positively influenced by potato starch, glucose, casein peptone and KH2PO4.7H2O and was not influenced by soybean meal, considering 95% of confidence level. The potato starch concentration was fixed at the highest level tested in Plackett¿Burman design due to gelation of the fermentation medium in higher concentrations. Thus, the three factors that influence the transglutaminase production (glucose, casein peptone and KH2PO4.7H2O concentrations) were optimized to obtain the maximum transglutaminase production using central composite design. Under the proposed optimized condition, which contained 25 g/L soybean meal, 35 g/L potato starch, 5 g/L glucose, 24.5 g/L casein peptone and 8 g/L KH2PO4.7H2O, the enzyme activity reached 6.13 U/mL, which was 125% more than the activity in relative obtained medium before optimization. The microbial transglutaminase produced by Streptomyces sp. B6 strain exhibited optimal activity at 45 oC and at pH 6.5 and 11.0. The enzyme remained stable in the pH range from 3.0 - 11.0 for 60 minutes and at 40 oC temperature for 3 hours. The transglutaminase was not inhibited by Ca2+, Na+, Co2+, Mn2+, K+, Mg2+, Ba2+, EDTA, L-cysteine and glutathione in concentration 5 mM, but was inhibited in the presence of Hg2+, Cu2+, Zn2+ and Fe2+ in concentration 5 mM. In conclusion, Streptomyces sp. B6 strain is a new source of transglutaminase with interesting features for biotechnological applications
Mestrado
Ciência de Alimentos
Mestre em Ciência de Alimentos

Biology
Ph.D.
Angiogenesis is the formation of new blood vessels from the endothelium of pre-existing vasculature and is the main mechanism of vascularization during physiological processes such as embryonic development, growth, regeneration, and wound healing and pathological process of vascularizing tumors. The major source of these environmental signals is the interactions between the cell and the extracellular matrix (ECM). Cell adhesion molecules are found on the surface of all cells and play a role in cell-cell and cell-extracellular matrix interactions. Tissue transglutaminase is ubiquitously expressed in various tissue types, occurs in both an intracellular and extracellular form, and is highly expressed in endothelial cells and smooth muscle cells in the vasculature. Tissue transglutaminase is secreted from cells externally and binds to the cell surface or extracellular matrix. It has been implicated in the stabilization of the extracellular matrix and in cell-ECM interactions by cross-linking matrix proteins. In 1993, angiocidin was isolated from lung carcinoma extracts by affinity chromatography using a peptide fragment of thrombospondin-1 (TSP-1), a matrix protein that has been implicated in mechanisms of tumor progression. The recombinant protein was expressed and shown to be a potential inhibitor of angiogenesis but the mechanism of action was not characterized. Affinity chromatography showed that tissue transglutaminase binds to recombinant angiocidin. Our studies have shown: Angiocidin binds to tissue transglutaminase in situ and in vitro through different binding studies. Recombinant angiocidin was also a substrate for the enzymatic activity of tissue transglutaminase. Both endogenous and exogenous angiocidin were able to crosslink to themselves. This interaction with angiocidin inhibits transglutaminase function by inhibiting amine incorporation, crosslinking of extracellular matrix proteins, and the promotion of cell-matrix interactions. Additionally, the monomeric and crosslinked angiocidin have different biological activity. Polymeric angiocidin is more adhesive, inhibits, migration, and is resistant to proteolytic degradation. Our studies suggest that angiocidin inhibits angiogenesis by its ability to bind to tissue transglutaminase and crosslink to itself, thereby destabilizing the cell- extracellular matrix interactions through its interaction with tissue transglutaminase. These studies may help to understand the mechanism of recombinant angiocidin in inhibiting tumor vasculature, as well as understanding the significance of these post-translational modifications of endogenous and exogenous proteins in cellular biology.
Temple University--Theses

L’autofagia è un processo cellulare altamente conservato responsabile della degradazione di proteine ed organelli cellulari. L’autofagia avviene in maniera basale in condizioni normali, ma può essere indotta in risposta ad uno stress, come ad esempio deprivazione di nutrienti, ipossia, disfunzioni mitocondriali e infezioni. La transglutaminasi tissutale (TG2) si accumula, sia in vivo che in vitro, ad alti livelli in cellule in condizioni di stress. Su questa base, abbiamo deciso di studiare se la TG2 potesse giocare un ruolo nel processo autofagico. A questo proposito abbiamo utilizzato topi “knockout” per la TG2 e linee cellulari in cui l’enzima fosse sia assente o iper-espresso. La mancanza della TG2, sia in vivo che in vitro, provocava un evidente accumulo dell’isoforma LC3 II presente sugli autofagosomi, suggerendo una notevole induzione di autofagia. Al contrario, la formazione in queste stesse cellule di vescicole acide era molto scarsa, indicando un difetto nella maturazione finale degli autofagolisosomi. Infatti, trattando le cellule con NH4Cl, un inibitore dell’attività lisosomale, si aveva un marcato accumulo di LC3 II e di mitocondri danneggiati simile a quello osservato nelle cellule senza la TG2. Questi dati indicano un ruolo della TG2 nelle modificazioni post-traduzionali di proteine coinvolte nella maturazione degli autofagosomi accompagnati dall’accumulo di molti mitocondri danneggiati.
Autophagy is a highly conserved cellular process responsible for the degradation of long-lived proteins and organelles. Autophagy occurs at low levels under normal conditions, but it is enhanced in response to stress, e.g., nutrient deprivation, hypoxia, mitochondrial dysfunction and infection. “Tissue” transglutaminase (TG2) accumulates, both in vivo and in vitro, to high levels in cells under stressful conditions. Therefore, in this study, we investigated whether TG2 could also play a role in the autophagic process. To this end, we used TG2 knockout mice and cell lines in which the enzyme was either absent or overexpressed. The ablation of TG2 protein both in vivo and in vitro, resulted in an evident accumulation of microtubule-associated protein 1 light chain 3 cleaved isoform II (LC3 II) on pre-autophagic vesicles, suggesting a marked induction of autophagy. By contrast, the formation of the acidic vesicular organelles in the same cells was very limited, indicating an impairment of the final maturation of autophagolysosomes. In fact, the treatment of TG2 proficient cells with NH4Cl, to inhibit lysosomal activity, led to a marked accumulation of LC3 II and damaged mitochondria similar to what we observed in TG2-deficient cells. These data indicate a role for TG2-mediated post-translational modifications of proteins in the maturation of autophagosomes accompanied by the accumulation of many damaged mitochondria.

Site-specific modification of proteins by transglutaminase Transglutaminase (TGase; EC 2.3.2.13) catalyzes the reaction between the γ-amido group of a protein-bound Gln residue (–CONH2, the acceptor) and an amino group (–NH2, the donor) of an alkyl-amine (protein-CONH2 + H2N-ligand → protein-CONH-ligand + NH3). In the case of protein substrates, TGase causes an intra- and inter-molecular crosslinking of proteins by formation of an isopeptide bond involving the side chains of Gln and Lys residues. The acyl donor can be also a small amido-ligand mimicking the Gln residue, so that TGase allows a useful and interesting variability of substrates, thus leading to the modification of proteins at the level of Gln or Lys residues using appropriate substrate reagents. Recently, the microbial TGase from S. mobaraensis has attracted a strong interest for protein modification, considering its stability, high reactivity and small size. The X-ray structure of this TGase has been solved and shown to contain an active site given by a triad Cys-His-Asp in analogy to a protease. A striking result of recent studies is that reactions mediated by TGase can be site-specific with some proteins, sometimes leading to the modification of only one Gln residue among the many Gln residues of a protein substrate. On the other hand, there is only a moderate specificity for Lys residues. With the view to shed light into the molecular features dictating the site-specific reaction(s) of TGase, in this Thesis a number of TGase-mediated reactions have been studied using proteins of known structure and dynamics, as apomyoglobin (apoMb), egg-white lysozyme (LYS) and bovine pancreatic ribonuclease A (RNase). Amino- as well amido-ligands have been used in the TGase-mediated reactions, so that it was possible to analyse the specificity of modification of both Gln and Lys in the examined proteins. We have found an almost strict specificity of TGase-mediated reactions at the level Gln91 of apoMb, a residue embedded in the highly flexible or unfolded helix-F of the holo protein, as given by previous NMR measurements and limited proteolysis data. Also a Gln-mimicking ligand can be covalently linket by TGase at a Lys residue of the same chain region. Thus, we concluded that local enhanced flexibility or even fully local unfolding dictates the site-specific reaction with TGase. While RNase can be selectively modified by using an amido-ligand at the level of the ɛ-amino group of Lys1, a similar Lys1 of LYS was instead fully unreactive. It was possible to relate this finding to the flexibility and rigidity of the N-terminal region of RNase and LYS, respectively, on the basis of the crystallographically determined B-factor values (a measure of chain flexibility) of these two proteins. A nicked species of RNase with the single peptide bond Asn34-Leu35 cleaved (RNase Th1) and a LYS derivative with a single disulfide bridge reduced among the four of native LYS (LYSCM6, 127) were shown to be much more reactive in the TGase-mediated reactions than the parent intact proteins, in agreement with their enhanced flexibility or partial unfolding. Moreover, we could demonstrate that the sites or regions susceptible to TGase reactions are also prone to limited proteolysis phenomena, implying that both TGase and a protease require some local unfolding for a site-specific enzymatic reaction. Indeed, this in keeping with view that the biorecognition phenomenon is similar for both enzymes, considering also the fact that TGase acts as a reverse protease (amide synthesis instead of hydrolysis). An interesting outcome of these studies resides in the fact that we can envisage a novel enzymatic method of covalent coupling of an amino-polymer as poly(ethylene)glycol (PEG) to specific Gln residue(s) of proteins of pharmaceutical interest. Indeed, using TGase and an amino derivative of PEG (PEG-NH2), it was possible to prepare homogeneous PEGylated derivatives of apoMb, human growth hormone (hGH) and granulocyte colony-stimulating factor (G-CSF). Overall, we have interpreted our findings as indicating that the selective TGase-mediated reactions require a flexible or unfolded polypeptide substrate. Therefore, it is possible to predict the sites of TGase attack on a protein substrate, provided that its structure and dynamics are known. Considering the increasing relevance of PEGylated protein drugs and the high regulatory demands for their approval, it can be anticipated that the innovative methods for the site-specific PEGylation of proteins using TGase will be considered a useful advance in the methodologies used for protein modification.
Modifica sito-specifica di proteine con transglutaminasi La transglutaminasi (TGasi; EC 2.3.2.13) catalizza la reazione tra il gruppo γ-ammidico di un residuo di Gln (-CONH2, accettore) ed un gruppo amminico (-NH2, donatore) di una alchil-ammina (proteina-CONH2 + H2N-ligando → proteina-CONH-ligando + NH3). Con substrati proteici la TGasi determina una reticolazione intra- e inter-molecolare di proteine mediante la formazione di un legame isopeptidico tra le catene laterali dei residui di Gln e Lys. Il donatore acilico può essere anche un ammido-ligando in grado di mimare la catena laterale di Gln e, pertanto, la TGase consente una modifica di proteine a livello dei residui di Gln o Lys utilizzando opportuni reagenti. Recentemente, la TGase microbica da S. mobaraensis ha suscitato un notevole interesse per la modifica enzimatica di proteine, considerando la sua stabilità, elevata reattività e piccole dimensioni. La struttura ai raggi-X di questa TGase ha rivelato la presenza di un sito attivo costituito da una triade Cys-His-Asp in analogia al sito attivo di una proteasi. Recenti studi hanno dimostrato che le reazioni mediate da TGase possono essere sito-specifiche, portando talvolta alla modifica di un solo residuo Gln tra i molti residui Gln di un substrato proteico. D'altra parte, è stata accertata solo una moderata specificità per i residui di Lys. Con lo scopo di chiarire i motivi strutturali che determinano la specificità di azione della TGase, in questa Tesi sono state studiate una serie di reazioni TGase-mediate utilizzando proteine di struttura e dinamica note, come apomioglobina (apoMb), lisozima da bianco d’uovo (LYS) e ribonucleasi pancreatica bovina (RNase). Sono stati utilizzati sia ammino- che ammido-ligandi nelle reazioni con TGase ed in tal modo è stato possibile analizzare la specificità di modifica sia a livello di Gln che di Lys con le proteine esaminate. E’ stata accertata una specificità rigorosa delle reazioni TGase-mediate a livello di Gln91 di apoMb, un residuo localizzato nel segmento corrispondente all’elica F della proteina nativa e risultato molto flessibile o unfolded in apoMb da precedenti misure NMR e da dati di proteolisi limitata. Anche un ligando in grado di mimare il residuo di Gln può essere legato da TGase a livello di una Lys incorporata nello stesso segmento flessibile. Pertanto, è stato concluso che una elevata flessibilità locale o unfolding determina la reazione sito-specifica di TGase. Mentre con la RNase si può ottenere con TGase ed un ammido-ligando la specifica modifica a livello del gruppo ɛ-aminico di Lys1, la simile Lys1 del LYS non reagisce affatto in simili condizioni. E 'stato possibile mettere in relazione questo fatto con la flessibilità e la rigidità della regione N-terminale di RNase e LYS, rispettivamente, sulla base dei valori del fattore-B (correlato alla flessibilità della catena polipeptidica) di queste due proteine ottenuti da dati cristallografici. Un derivato di RNase con il singolo legame peptidico Asn34-Leu35 idrolizzato (RNase Th1) ed un derivato di LYS con un ponte disolfuro ridotto (LYSCM6, 127) sono risultati molto più reattivi nelle reazioni con TGase rispetto alle corrispondenti proteine native, in accordo con la loro maggiore flessibilità o parziale denaturazione. Inoltre, abbiamo potuto dimostrare che i siti o regioni suscettibili di reazioni con TGase subiscono anche fenomeni di proteolisi limitata, significando che sia la TGase che una proteasi necessitano di un local unfolding per determinare una reazione enzimatica sito-specifica. In effetti, questo è linea con il fatto che il bioriconoscimento del substrato è simile per entrambi gli enzimi, considerando il fatto che la TGase è una proteasi inversa (sintesi invece che idrolisi di una ammide). Un risultato interessante di questi studi risiede nel fatto che si apre la strada ad un nuovo metodo enzimatico di binding covalente di poli(etilene)glicole (PEG) a livello di specifici residui di Gln in proteine di interesse farmaceutico. Infatti, utilizzando TGase ed un ammino-derivato di PEG (PEG-NH2), è stato possibile preparare derivati peghilati omogenei di apoMb, ormone della crescita umano (hGH) e granulocyte colony-stimulating factor (G-CSF). In generale, i risultati ottenuti hanno indicato chiaramente che le reazioni selettive di TGase richiedono un substrato flessibile o unfolded. Pertanto, è possibile prevedere i siti di attacco di TGase su un substrato proteico, se sono note la sua struttura e dinamica. Considerando la crescente importanza dei farmaci proteici peghilati e le speciali richieste di omogeneità dettate dalle autorità regolatorie, si può prevedere che la peghilazione sito-specifica con TGase di proteine di interesse farmaceutico troverà utili applicazioni.

Transglutaminases (EC 2.3.2.13) are enzymes that catalyze an acyl transfer reaction between a &gamma
-carboxyamide group of a peptide bound glutaminyl residue (acyl donor) and a variety of primary amines (acyl acceptors), including the amino group lysine. Transglutaminase has a potential in obtaining proteins with novel properties, improving nutritional quality of foods with the addition of essential amino acids, preparing heat stable gels, developing rheological properties and mechanical strength of foods and reducing the applications of food additives. The aim of this study is to develop intracellular and extracellular microbial protransglutaminase (pro-MTG) producing recombinant Pichia pastoris strains by using genetic engineering techniques. In this context first,protransglutaminase gene (pro-mtg) from Streptomyces mobaraensis was amplified by PCR both for intracellular and extracellular constructs using proper primers then they were cloned into the pPICZ&alpha
-A expression vectors, separately. Both intracellular (pPICZ&alpha
A::pro-mtgintra) and extracellular (pPICZ&alpha
A::pro-mtgextra) constructs were prepared with strong alcohol oxidase 1 promoter which is induced by methanol. Pichia pastoris X33 cells were transfected by linear pPICZ&alpha
A::pro-mtgintra and pPICZ&alpha
A::pro-mtgextra, separately and plasmids were integrated into the Pichia pastoris X33 genome at AOX1 locus. After constructing the recombinant P. pastoris strains, batch shaker bioreactor experiments were performed for each recombinant cell and the best producing strains were selected according to Dot blot and SDS-PAGE analyses. The selected recombinant P. pastoris strains, carrying pPICZ&alpha
A::promtgextra gene and pPICZ&alpha
A::pro-mtgintra gene in their genome were named as E8 and I1, respectively. Afterwards, a controlled pilot scale bioreactor experiment in a working volume of 1 L was performed with E8 clone and produced pro-MTG was activated by Dispase I. The variations in the recombinant MTG activity, cell concentration, total protease activity, AOX activity and organic acid concentrations throughout the bioprocess were analyzed and specific growth rates, specific consumption rates and yield coefficients were calculated regarding to measured data. Maximum MTG activity was obtained as 4448 U L- 1 and the maximum cell concentration was measured as 74.1 g L-1 at t=36 h of the bioprocess. In this study, an active transglutaminase enzyme was produced extracellularly by P. pastoris for the first time and the third highest extracellular MTG activity was achieved with E8 clone.

Tissue transglutaminase (TG2) is a calcium-dependent enzyme that natively catalyses the formation of isopeptidic bonds between protein- or peptide-bound glutamine and lysine residues. Physiologically, it is ubiquitously expressed in tissues, with roles in cellular differentiation, extracellular matrix stabilisation, and apoptosis, among others. However, its unregulated activity has been associated with various pathologies including fibrosis, cancer and celiac disease. Since most pathologies are associated with an increased transamidation activity, efforts have been directed towards the development of TG2 inhibitors. In this context, the work described in this thesis is centred on reversible inhibitors, building on recent work done within the Keillor group in two directions, namely localisation and potency. In a localisation-driven approach, we developed a photolabile derivative of a known reversible inhibitor, in order to form a covalent bond with the enzyme and determine the inhibitor’s binding site. In tandem, we optimised a protocol for the expression of TG2 incorporating ArgΔ10 and LysΔ8, amino acids that are 13C- and 15N-labelled to provide a mass shift of 10 and 8 Da, respectively, compared to the corresponding unlabelled amino acids. This “heavy” TG2 was developed as a tool for reference in the analysis of the tryptic digest of labelled protein. In a potency-driven approach, based on the observation that previous trans cinnamoyl inhibitor scaffolds were susceptible to nucleophilic attack by glutathione, we developed a bis(triazole) scaffold with reduced electrophilicity. The preparation of a small library of compounds showed that this scaffold demonstrates a preference for electron-withdrawing substituents, such as nitro groups. Continuing in a potency-driven approach, and inspired by work done in the identification of glutathione-resistant scaffolds, we studied a new alkynyl scaffold. While still susceptible to glutathione addition, these compounds showed a marked improvement in potency, with the lead compound having an IC50 of 930 nM and being established as a competitive inhibitor with a Ki of 420 nM, our most potent reversible inhibitor to date. Furthermore, this scaffold also produced an inhibitor lacking nitro groups (to limit eventual cellular toxicity), but maintaining good potency, with an IC50 value of 3.03 μM.

This study investigated the distribution of tissue transglutaminase (TG2) antigen and activity in human umbilical vein endothelial cells (HUVEC) and peripheral blood derived monocytes.  Both cell types produced and secreted TG2 with an approximate 50:50 distribution between cell associated and secreted TG2.  TG2 antigen in HUVEC was principally distributed between cell lysates and extracellular matrix (ECM) samples, yet the activity of ECM samples was lower.  The  highest specific activity for both cell types was found in conditional medium.  Monocyte cell lysates activity was consistently below the level of detection, suggesting that TG2 was inactive.  TG2 antigen levels for both cell types were stable, both in quantity and distribution in the samples, in the presence of inflammatory type mediators.  However, TG2 activity could be modified by inflammatory mediators.  HUVEC treated with conditioned medium from monocytes and vice versa, both demonstrated stable antigen levels but the activity of secreted TG2 could be increased.  This indicated that some factor secreted by monocytes augments TG2 activity the extracellular environment.  TG2 secretion by the classical ER/Golgi route was investigated by synthesis of de novo radiolabelled TG2.  Localisation of de novo synthesised TG2, from cells and from in vitro translation of TG2 in Xenopus oocyte extracts, demonstrated that TG2 was not found associated with ER/Golgi membranes.  This indicated that TG2 is not a classically secreted protein and is therefore secreted from the cytosol by another mechanism.  Local TG2 substrates were identified from 2D gel electrophoresis of HUVEC samples treated +/- calcium in the presence/absence of a TG specific inhibitor.  These proteins were analysed by mass spectrometry.  The nine proteins identified were keratin, albumin, actin, tropomyosin, HSP27, TCP-1-α, RhoGAP1, eIF3 and DPM synthase.

Transglutaminase (TGase; protein-glutamine y-glutamyltransferase, E.C. 2.3.2.13) is an enzyme that catalyses the acyl transfer reaction by introducing covalent cross-links between proteins, peptides and various primary amines. Until recently, commercial TGase has been derived from mammalian origin. Calcium-dependent TGase extracted from guinea-pig liver and blood plasma have been investigated for the purpose of their application in the food industry. However, supply, complicated separation and purification procedures as well as the requirement for calcium have made it almost impossible to apply mammalian TGase in food processing on an industrial scale. Microbial transglutaminase (MTGase) was first purified from the culture filtrate of Streptoverticillium S-8112, a variant of Stv. mobaraense and subsequently the extracellular enzyme has been purified from the culture filtrate of other Streptoverticillium and Streptomyces species. This enzyme is easily obtained by microbial fermentation and has been found to have the ability to induce cross-linking and gelation of food proteins. In addition, MTGase does not require calcium for activation which is of great advantage for the food industry as many food-proteins are easily precipitated in the presence of Ca2+ thus rendering them less sensitive to the enzymatic reaction. A commercial source of MTGase derived from Stv. mobaraense is available, however the optimum temperature range of this enzyme is 50 to 55 degrees C. Important to the food industry is the requirement of catalytic activity at low temperatures so the need for a low temperature variant is desirable. This thesis explores the possibility of finding a bacterial source which retains MTGase activity at low temperatures and can be produced on an industrial scale. Thus provided the MTGase functions at the required temperature the reduced catalytic activity can be offset by using more enzyme. Psychrophilic, psychrotrophic and mesophilic bacteria were screened for the presence of a related TGase gene. A PCR strategy which amplified the region of the gene encoding the putative active site of MTGase was utilised for the selection and cloning of the gene. This successful screening strategy led to the cloning of the entire coding sequence of the mature form of MTGase from mesophilic actinomycetes including several Streptoverticillium species (Stv. mobaraense, Stv. griseocarneum, Stv. cinnamoneum ssp. cinnamoneum) and Streptomyces lavendulae and also from a previously unreported mesophilic bacteria, Stv. baldaccii. Structural relationships of the gene and protein were analysed by Southern and western blotting, respectively. MTGase derived from Stv. baldaccii was examined to determine the optimal growth conditions for maximum enzyme activity and whether this enzyme could function at low temperatures. Stv. baldaccii TGase exhibits characteristics of cold-adapted enzymes found in psychrophilic bacteria. Stv. baldaccii TGase has a lower temperature optimum, higher specific activity at low temperatures and thermal instability at moderately high temperatures. Industrial applications often require continuous large volumes of enzyme product. In this study a purification scheme was developed for the isolation of endogenous MTGase from the culture filtrate of Stv. baldnccii. However for commercial applications a recombinant source would overcome problems with supply, production time and complex and expensive growth requirements. MTGase gene encompassing the entire coding region for the protein from Stv. baldaccii was expressed in E. coli and produced an active enzyme. The recombinant MTGase shared similar immunological and enzymatic characteristics as the endogenous enzyme. The findings of this thesis are: (i) A PCR method was developed for selection and cloning of the gene based on the sequence encoding the mature active form and the putative active site encoding region of the TGase gene from Stv. mobaraense; (ii) The entire coding sequence of the mature form of MTGase from mesophilic Streptoverticillium species (Stv. mobaraense, Stv. griseocarneum, Stv. cinnamoneum ssp. cinnamoneum and Stv. baldaccii) and Streptomyces lavendulae were compared; (iii) Structural analysis of the protein by western blotting revealed that there is a related protein produced within the Streptoverticillium species with both the Pro-TGase and the active mature enzyme detected in the culture filtrate. Southern blot hybridisation revealed that MTGase produced within Streptoverticillium species is related by genomic organisation, with only one copy of the gene detected; (iv) Stv. baldaccii TGase production was optimised by a systematic analysis of growth conditions: TGase production was favoured by growth at low temperatures with maximum growth and enzyme activity occurring when cultured cells changed from exponential phase to stationary phase; (v) Stv. baldaccii TGase exhibits characteristics of cold-adapted enzymes found in psychrophilic bacteria with its low temperature optimum, higher specific activity at low temperatures and thermal instability at 55 degrees C; (vi) Comparison of the deduced amino acid sequences of the TGase gene cloned from Stv. mobaraense and Stv. baldaccii showed approximately 80 % identity. This difference in the protein sequence of the two MTGases may be responsible for the lower activity optima and heat instability of Stv. baldaccii TGase; (vii) The MTGase gene encompassing the entire coding region for the protein from Stv. baldaccii was expressed in E. coli. The recombinant MTGase showed immunological and enzymatic characteristics similar to the endogenous form of the enzyme and therefore the same purification conditions were applied. Taken together I have shown that MTGase from Stv. baldaccii has high specific activity at low temperatures and the enzyme is produced at comparable levels to Stv. mobaraense under a variety of conditions. The enzyme can be over-expressed in E. coli thus providing a convenient production pathway since E. coli media has been optimised as a result of many studies to minimise the cost of production. A recombinant source would overcome supply, reduce production time and produce the enzyme cheaply and in abundant amounts. This thesis provides detailed proof of concept for the development of a commercial, high activity, temperature desensitised enzyme for use in biofilm production and protein manipulation with potential application in the food and beverage industry.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Science, Environment, Engineering and Technology
Full Text

Orientador: Flavia Maria Netto
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O leite bovino contém proteínas que são consideradas antigênicas e capazes de induzir algum tipo de resposta imunológica, dentre elas destaca-se a ß-lactoglobulina (ß-Lg). A utilização da enzima transglutaminase (TG), que catalisa reações de ligação e-(?-glutamina)lisil inter ou intramoleculares, incorporação de aminas e desaminação tem demonstrado eficiência em alterar o potencial antigênico de alimentos. O presente trabalho teve como objetivo estudar o efeito da reação de polimerização induzida pela TG na atividade antigênica da ß-Lg e sua digestibilidade, na presença dos agentes redutores glutationa (GSH) e ácido ascórbico (AA). As modificações da ß-Lg com a TG na presença dos agentes redutores foram realizadas através de experimentos fatoriais 22 nos quais as variáveis independentes foram a relação enzima:substrato (E/S) (0 ¿ 51,6 U TG g -1 de proteína) e concentração dos agentes redutores, [GSH] (0 - 0,67 mmol/L) ou [AA] (0- 0,02 mol/L) e as variáveis dependentes foram grau de polimerização e concentração de IgE. A polimerização foi avaliada por eletroforese em gel de poliacrilamida (SDS-PAGE), e grau de polimerização (GP) através da determinação de lisinas livres. A avaliação da antigenicidade foi feita por ELISA, utilizando soro de camundongos BALB/c sensibilizados com ß-Lg na forma nativa. A resistência das amostras à ação da pepsina foi avaliada por eletroforese em gel de poliacrilamida na presença de SDS e tricina (SDSPAGE/Tricina). Os tratamentos realizados com TG e GSH apresentaram polímeros de alta massa molar (MM > 97,4 kDa), exceto para a condição do ensaio 5 (0,34 mmol/L GSH), que não utilizou TG. Os tratamentos com TG e AA também apresentaram polímeros de alta massa molar (MM > 97,4 kDa), exceto para a condição do ensaio 2 (44,1 U de TG g -1 e 0,003 mol/L de AA) e ensaio 5 (0,01 mol/L AA, que não utilizou TG). O GP foi maior para as amostras tratadas com TG na presença de agentes redutores, quando comparadas às amostras tratadas somente com TG ou agente redutor. Os resultados mostraram que a presença dos agentes redutores levou ao aumento da polimerização pela TG ao desnaturar a proteína levando à maior exposição dos aminoácidos lisina e glutamina. Não houve diminuição na antigenicidade da ß-Lg tratada com TG na presença de GSH ou AA, porém houve aumento da antigenicidade de algumas amostras obtidas sob algumas das condições dos ensaios realizados com os agentes redutores quando comparadas à ß-Lg. Os tratamentos realizados com TG na presença dos agentes redutores não aumentaram a digestibilidade da ß-Lg pela pepsina. Os digeridos das amostras obtidas sob as condições relativas aos níveis extremos do planejamento experimental (±1,41) e o ponto central, foram avaliados quanto à sua antigenicidade. Os resultados mostraram que não houve diminuição da antigenicidade da ß-Lg tratada com TG e GSH ou TG e AA., quando comparadas à ß-Lg. A polimerização com TG na presença de agentes redutores GSH e AA não foi capaz de modificar a antigenicidade das amostras digeridas, sugerindo que não houve modificações na proteína capazes de aumentar sua digestibilidade pela pepsina ou modificar regiões de epítopos
Abstract: Cow¿s milk contains proteins which are considered antigenic and able to lead to immune response, among them ß-lactoglobulin (ß-Lg) is the most important. The transglutaminase (TG) is the only enzyme commercially used that catalyses inter or intramolecular cross linking reaction in various proteins and demonstrates efficiency to alter the antigenic potential of food. The current work aimed at studying the effect of the reaction of induced polymerization by TG in presence of the reducing agents glutathione (GSH) as well as ascorbic acid (AA) in the antigenic activity of ß-Lg and its digestibility. The modifications in ß-Lg induced by TG in presence of reducing agents were carried out using factorial experiments 22, in which the independent variables were the enzyme:substrate ratio (E/S) (0 ¿ 51,6 U TG g -1) and concentration of reducing agents, [GSH] (0 - 0,67 mmol/L) or [AA] (0- 0,02 mol/L), and the dependent variables were the polymerization degree and concentration of IgE. Polymerization was assessed by electrophoresis in polyacrylamide gel (SDSPAGE), and the polymerization degree (PD) was determined by the free lysine content. The assessment of antigenicity was by ELISA assay, using serum of BALB/c mice sensitized with native ß-Lg. The resistance of the samples to the action of pepsin was evaluated by electrophoresis in polyacrylamide gel in presence of SDS and tricine (SDSPAGE/Tricine). The treatments using TG and GSH showed high molecular weight polymers (MM > 97,4kDa), except for a test condition 5 (0,34 mmol/L GSH), where TG was not used. The treatments with TG and AA also showed polymers of high molecular weight (MM > 97,4kDa), except for the test condition 2 (44,1 U TG g -1 and 0,003 mol/L of AA) and test 5 (0,01 mol/L AA, without TG). The PD was higher in the samples treated with TG in presence of reducing agents, when compared to samples treated only with TG or reducing agents. The results showed that the presence of reducing agents led to the increase of polymerization by TG since they denature the protein, leading to increased exposure of the amino acids glutamine and lysine, substrate for TG. The results showed that there was no reduction in the antigenicity of ß-Lg treated with TG in presence of GSH or AA, however an increase in the antigenicity of some modified samples was observed when compared to the native ß-Lg. . The treatments carried out with TG and reducing agents did not increase the digestibility of the ß-Lg by pepsin. The antigenicity of the digested from the samples obtained under the conditions of the extreme levels of experimental planning (±1,41) and at the central point were assessed. The results showed that after digestion with pepsin there was no decrease of the antigenicity of ß-Lg treated with TG in the presence of GSH or AA as compared to digested ß-Lg. Polymerization with TG in the presence of reducing agents was not able to alter the antigenicity of the samples, which suggests that there were no modifications in the protein that would be able to increase its digestibility by pepsin as well as the epitopes regions of the ß-Lg
Mestrado
Nutrição Experimental e Aplicada à Tecnologia de Alimentos
Mestre em Alimentos e Nutrição

L'insuffisance cardiaque est une cause majeure de morbidité et de mortalité dans les pays industrialisés. Ce syndrome est le résultat de nombreuses maladies cardiaques qui induisent dans un premier temps un remodelage adaptatif du coeur : 1 'hypertrophie ventriculaire gauche(HVG). Cette HVG est associée à une réorganisation de la matrice extracellulaire (MEC)nécessaire dans la phase aigüe du remodelage mais délétère à long terme. Comme d'autres gènes, celui de la transglutaminase 2 (TG2) se retrouve surexprimé dans le cadre d'une HVG. La TG2 est une protéine multifonctiounelle très complexe qui peut agir comme transglutaminase (liaison entre acides aminés), GTPase (petite protéine G des récepteurs a-adrénergiques), protéine isomérase-disulfide et kinase. La régulation de ces fonctions est liée à sa structure tridimensionnelle qui est déterminée par son environnement en fonction des concentrations de calcium et de GTP. Zhang et ses collaborateurs ont démontré que de la surexpression de la TG2 entrainait une HVG (Zhang et al. , 2003). Par conséquent, l'inhibition de la TG2 pourrait présenter un intérêt thérapeutique dans le cadre d'une HVG. Notre étude s'intéresse donc aux conséquences de l'absence de la TG2 sur le remodelage précoce et tardif du coeur. Pour cela, des souris invalidées pour le gène de la TG2 totalement(TG2 KO) ou spécifiquement dans le cardiomyocyte (TG2 CRE +) ont subi une constriction de l'aorte thoracique (TAC) pour induire une HVG par surcharge de pression. Les souris TG2KO développent une HVG plus importante dès 7 jours par rapport à leurs contrôles suite à une TAC. Cette hypertrophie est associée à une atteinte de la fonction cardiaque avec une défaillance systolique et diastolique observée par échocardiographie et cathétérisme ventriculaire gauche. Paradoxalement, la fonction contractile et la transitoire calcique des cardiomyocytes issus des TG2 KO, ne sont pas impactées suite à la TAC. De plus, lorsque laTG2 est invalidée uniquement dans le cardiomyocyte (TG2 CRE +), il n'y a plus de différence d'hypertrophie et de fonction suite à une TAC. Ces résultats semblent corncider avec la localisation de la TG2 dans le milieu extracellulaire révélée par immunohistochimie. Par conséquent, k rôle de la TG2 dans la MEC semble prévaloir sur son rôle dans le cardiomyocyte. D'autant plus qu'une augmentation de la fibrose avec une surexpression des collagènes 1 et 3 a été observée dans les coeurs des TG2 KO après TAC. Ainsi, ces souris présentent un coeur plus fibrotique et une mauvaise adaptation à la surcharge de pression sans une atteinte de la contractilité des cardiomyocytes. En conclusion, la TG2 est fortement présente au sem de la MEC et non dans le cardiomyocyte. Son rôle dans la stabilisation des composants de la MEC (collagènes, fibronectine ) et l'adhésion cellulaire (via les intégrines) apparait comme crucial dans l'adaptation du coeur à une surcharge de pression. Enfin, la surexpression de la TG2 dans l'insuffisance cardiaque n'est peut-être pas une cause mais une conséquence de cet état pathologique. Toutefois, l'utilisation d'un inhibiteur spécifique de la TG2 peut avoir un intérêt thérapeutique pour contrôler ces activités dans la MEC et dans le développement de la fibrose
Heart failure is a major cause of death and morbidity in industrialized countries. This syndrome result from severa! heart diseases that initially induce an adaptive heart remodeling: Jeft ventricular hypertrophy (LVH). LVH is associated with an extracellular matrix (ECM)reorganization which is necessary in the acute phase of remodeling but long-term deleterious. Like others, gene of transglutaminase 2 (TG2) is found overexpressed in LVH. TG2 is very complex multifunctional protein that can act as transglutaminase (bond between aminoacids), GTPase (small G protein of the a-adrenergic receptors), protein disulfideisomerase-and kinase. Regulation of these functions depends on its three-dimensional structure which is determined by its environment according to the calcium and GTP concentrations. Zhang and colleagues demonstrated that TG2 overexpression was able to cause LVH. Therefore, inhibition of TG2 may offer a therapeutic value for heart failure. This study focuses on the consequences of the Jack of TG2 on the earl y and Jate heart remodeling. Mice invalidated completely for the TG2 gene (TG2 KO) or specifically in cardiomyocytes (TG2 CRE +) were submitted to a thoracic aortic constriction (TAC) to induce LHV by pressure overload. TG2 KO mice develop greater LHV at 7 and 28 days compared to their controls. This hypertrophy is associated with a heart functional defect characterized by asystolic and diastolic dysfunction measured by echocardiography and left ventricular pressure catheter. However, contractile function and calcium transient of isolated cardiomyocytesderived from TGi KO, are not modified after TAC. In addition, when TG2 is disabled on! y in the cardiomyocyte (TG2 CRE + ), its makes no difference for L VH and function after TAC. These results seem to be in agreement with the TG2 Jocalization in the extracellular medium revealed by immunohistochemistry. Therefore, the role of TG2 in the MEC seems to prevail over his role within the cardiomyocyte. Moreover, an increased fibrosis content (associated to an increased expression of collagen 1 and 3 genes) was observed in the hearts of TG2 KOafter TAC. Thus, these mice exhibit fibrotic hearts that impact the myocardium adaptation to pressure overload without a cardiomyocyte contractile dysfunction. In conclusion, TG2 is strongly present in ECM and not in the cardiomyocyte. IT's role in the stabilization of ECM components (collagen, fibronectin. . . ) and cell adhesion (via integrins) appears to be crucialfor the cardiac adaptation to increased pressure overload. Finally, TG2 overexpression in heart failure may not be a cause but a consequence of this disease. However, the use of as pecifie TG2 inhibitor may be tested in order to validate these results in our TAC model but also in others experimental models of HF, such as myocardial infarction

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE
The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA

Orientador: Hélia Harumi Sato
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A transglutaminase microbiana (MTGase; EC 2.3.2.13) é uma enzima capaz de catalisar a formação de ligações covalentes cruzadas entre proteínas, peptídeos e várias aminas primárias através da reação de acil transferência entre resíduos de glutamina e lisina. A incorporação de ligações covalentes cruzadas entre proteínas por ação da transglutaminase vem sendo empregada pela indústria alimentícia para modificar principalmente a textura, a viscosidade e a capacidade de formação de gel de alimentos. Este trabalho teve como principal objetivo estudar a estabilidade térmica, o efeito de inibidores e ativadores, a concentração e a purificação da transglutaminase microbiana produzida pela linhagem Streptomyces sp. CBMAI 837. No estudo da estabilidade térmica da enzima verificou-se que a transglutaminase de Streptomyces sp. CBMAI 837 é uma enzima termossensível, estável em temperaturas abaixo de 40°C e rapidamente inativada acima de 50°C. Parâmetros cinéticos e termodinâmicos da desnaturação térmica da enzima foram determinados para as seis temperaturas estudadas. Os tempos de meia-vida da enzima a 55 e 60°C foram estimados em 3,5 e 1,9 minutos, respectivamente. A influência de alguns compostos no aumento da estabilidade térmica da enzima foi investigada, sendo verificado que a adição de EDTA e KCl na concentração de 1% e de cisteína e glutationa na concentração de 0,1% aumentaram a estabilidade térmica da transglutaminase durante incubação a 45°C por 30 minutos. O efeito de compostos como etanol, ativadores e inibidores enzimáticos na atividade da MTGase de Streptomyces sp. CBMAI 837 foi estudado. O etanol na concentração de 10% (v:v) apresentou pouco efeito na atividade enzimática, enquanto que concentrações acima de 40% (v:v) provocaram rápida inativação da enzima. A MTGase foi ativada na presença de EDTA e cisteína e inativada na presença de iodoacetamida e ácido cloromercuribenzóico, sugerindo que esta é uma enzima cálcio independente com um resíduo de cisteína no sítio ativo. No estudo da concentração da MTGase foram avaliados diferentes métodos, sendo verificado que a precipitação com sulfato de amônio a 80% de saturação foi o método mais efetivo, possibilitando a concentração do sobrenadante de cultivo cerca de 4,5 vezes com rendimento de 142%. A aplicação da preparação enzimática bruta concentrada de MTGase de Streptomyces sp. CBMAI 837 em proteína texturizada de soja apresentou efeito similar ao da enzima comercial Activa® TG-BP quando aplicada nas mesmas condições. Na purificação da MTGase de Streptomyces sp. CBMAI 837 em coluna de afinidade Blue Sepharose CL-6B foram separadas 3 frações com atividade de transglutaminase (TG-BS1, TG-BS2 e TG-BS3), indicando a presença de isoenzimas. A massa molecular da MTGase presente nas frações purificadas TG-BS2 e TG-BS3 foi estimada em cerca de 35 KDa por SDS-PAGE. As três frações obtidas foram caracterizadas quanto ao pH ótimo de atividade enzimática. Foi observado que a fração parcialmente purificada TG-BS1 apresentou atividade ótima em pH 10,0 e um segundo pico de atividade em pH 6,0, enquanto as frações purificadas TG-BS2 e TG-BS3 apresentaram pH ótimo de atividade em pH 6,5 e também um segundo pico de atividade em pH 10,0
Abstract: The microbial transglutaminase (MTGase, EC 2.3.2.13) is an enzyme capable of catalyzing the formation of covalent cross-links among proteins, peptides and various primary amines by reaction of acyl transfer between glutamine and lysine residues. The incorporation of covalent cross-links between proteins by the action of transglutaminase has been used by the food industry to modify especially the texture, viscosity and gel forming ability of foods. This work aimed to study the thermal stability, effect of inhibitors and activators, concentration and purification of microbial transglutaminase produced by strain Streptomyces sp. CBMAI 837. It was observed in the study of thermal stability of the enzyme that the transglutaminase from Streptomyces sp. CBMAI 837 is a thermosensitive enzyme, stable at temperatures below 40°C and rapidly inactivated above 50°C. Kinetic and thermodynamic parameters of thermal denaturation of the enzyme were determined for six temperatures. It was estimated that the half-life times of the enzyme at 55 and 60°C were 3.5 and 1.9 minutes, respectively. The influence of compounds to increase the thermal stability of the enzyme was investigated, and it was found that the addition of EDTA and KCl at a concentration of 1% and cysteine and glutathione at a concentration of 0.1% increased the thermal stability of transglutaminase during the incubation at 45°C for 30 minutes. The effect of compounds such as ethanol, activators and inhibitors on enzymatic activity of MTGase from Streptomyces sp. CBMAI 837 was studied. The ethanol concentration 10% (v/v) had little effect on enzyme activity, while concentrations above 40% (v/v) resulted in rapid inactivation of the enzyme. The MTGase was activated in the presence of EDTA and cysteine and inactivated in the presence of iodoacetamide and chloromercuribenzoic acid, suggesting that this is a calcium independent enzyme with a cysteine residue at the active site. Different methods were evaluated in the study of the concentration of MTGase, confirming that the precipitation with ammonium sulfate at 80% saturation of the culture supernatant was the method more effective, being concentrated this enzyme about 4.5 fold with a yield of 142%. The application of crude enzyme preparation of MTGase from Streptomyces sp. CBMAI 837 concentrated by ammonium sulfate in texturized soy protein showed a similar effect to the one of commercial enzyme Activa® TG-BP when applied under the same conditions. Three fractions with transglutaminase activity (TG-BS1, TG-BS2 e TG-BS3) were separated in the purification of MTGase from Streptomyces sp. CBMAI 837 in column affinity Blue Sepharose CL-6B, indicating the presence of isoenzymes. The molecular mass of MTGase present in the purified fractions TG-BS2 e TG-BS3 was estimated at about 35 KDa by SDS-PAGE. The three fractions were characterized by optimum pH of enzymatic activity. It was observed that the partially purified fraction TG-BS1 showed optimal activity at pH 10.0 and a second peak of activity at pH 6.0, while the purified fractions TG-BS2 e TG-BS3 showed optimum pH activity at pH 6.5 and also a second peak of activity at pH 10.0
Mestrado
Mestre em Ciência de Alimentos

Transglutaminases are enzymes that stabilize extracellular matrices by catalyzing the formation of protease-resistant isopeptide crosslinks between and within their substrate proteins. Two transglutaminase isoforms, tissue transglutaminase (tTG) and factor XIIIa (FXIIIa), are expressed in skeletal tissues. Tissue transglutaminase has been previously localized in bone to osteoblasts, the osteoid layer and the pericellular matrix of osteocytes. Factor XIIIa has been localized to mineralizing chondrocytes, however, its expression in osteoblasts has not been reported yet. In order to understand the role of transglutaminases in hard tissue formation, we have investigated tTG and FXIIIa expression and activity during osteoblast differentiation and mineralization using the well-characterized MC3T3-E1 preosteoblast cell line. We show that tTG and FXIIIa are expressed at all time points examined in the cell cultures, and that their expression and activity does not appear to be significantly changed by the differentiation or mineralization state of the cells. We show that tTG localizes homogeneously throughout the cells and the extracellular matrix, whereas FXIIIa distribution is polarized to one side of the cells at earlier time points, but becomes incorporated into the matrix produced by differentiating cells in mature cultures. These expression profiles suggest a dual role, initially in cell adhesion and attachment and, later, in protein assembly, where the transglutaminases likely stabilize the matrix and assist in the formation of an extracellular matrix competent for subsequent mineralization. The identification and characterization of two transglutaminase isoforms in osteoblasts suggest a potentially overlapping roles for these two enzymes in the MC3T3-E1 cell culture system.

Tissue-type transglutaminase (tTG) is the most ubiquitously distributed of its enzyme family, displaying a wide range of substrates. It is a multifunctional enzyme which acts as a GTP binding protein, a stabilizer of extracellular matrix proteins, and as an effector enzyme in the apoptotic cell death pathway. Recent evidence to suggest that tTG is inactive within the healthy cell has raised questions as to the exact conditions necessary for intracellular and extracellular activity of the enzyme. To investigate the role of tTG in extracellular and intracellular processes, three model cell lines displaying various levels of intracellular tTG via sense and antisense transfection with the human cDNA for tTG were examined for the effects of altered intracellular tTG levels on cell morphology. The two fibroblast cell lines examined (Met B and Swiss 3T3) did not display any alterations in cell morphology with vastly increased intracellular tTG. The endothelial cell line ECV304 however, showed a reduction in cell spreading with reduced intracellular tTG, concommitant with collapse of the microtubule network, an effect duplicated by inactivation of extracellular tTG using a monoclonal antibody (CUB7402) to neutralize its activity. Reduced cell spreading was accompanied by the loss of polymerization of extracellular fibronectin and the loss of incorporation of a biotinylated tTG substrate into high molecular weight extracellular matrix material. Using a specially adapted in-vitro technique (the FN-cell ELSA), cell surfacerelated tTG activity was found to be reduced in tTG antisense clones of ECV304 cells, and increased in tTG sense clones of Swiss 3T3 cells. This activity could also be neutralized by pre-incubating cells with the anti-tTG monoclonal antibody CUB7402. This assay also revealed that the majority of extracellular tTG activity detected using standard techniques was present due to leakage resulting from cell manipulation such as trypsinization from culture surfaces and mechanical shear forces, or activation of the pre-existing matrix-bound enzyme through trypsin treatment, however a portion of the extracellular activity was attributable to the slow externalisation of the enzyme from the intact cell. An examination of intracellular tTG crosslinking events was made using an electropermeabilization metod, where it was observed that tTG became activated t form SDS-insoluble "shells" in the presence of high intracellular Ca2+ These shells contained cytoskeletal elements and probably also some trapped genomic DNA, implying a function for these shells as cell-sized "scars" preventing loss of cell contents upon mechanical rupture of the cells. A similar tTG activation was observed to occur in mechanically injured cells in-vitro and in-vivo, this time involving a re-distribution of tTG activity to the extracellular matrix from the intracellular compartment. During fibrosis of rat kidneys induced by subtotal nephrectomy (Snx), tTG and s(y-glutamyl)lysine were observed by immunofluorescence microscopy to be increased in renal tubule cells. With increasing tissue damage, c(Y-glutamyl)lysine was also observed in the extracellular renal interstitial space, suggesting a role for tTG in the stabilization of extracellular matrix following tissue damage. The release of tTG into the extracellular space may be due to a lack of sufficient clearance of apoptotic bodies resulting in secondary necrosis and subsequent release of the enzyme.

Human tissue transglutaminase (TG2) catalyses transamidation and deamidation reactions through a nucleophilic cysteine residue (CYS277). TG2 activity was found to increase in celiac disease, cystic fibrosis, neurodegenerative disorders and cancer. For this, TG2 has received much focus as a target for drug discovery and many inhibitors have been designed and tested. The most important of these have an electrophilic warhead that reacts covalently with CYS277 resulting in an irreversible inhibition of TG2. The work presented in this thesis aimed at the development of computational methods that could aid in the design and testing of potential TG2 inhibitors. 3-D models of TG2 active site were developed starting from published X-ray crystal structures by means of docking experiments with known irreversible inhibitors followed by molecular dynamics (MD) simulations. The models were validated by additional docking runs and MD simulations involving a larger set of compounds with a range of activities against TG2. The models performed reasonably well in the validation process and were, therefore, chosen as active site models of TG2. No straightforward correlation could be found to rank the compounds based on their activities. This was the rationale for the next stage of the work, where the mechanism of inhibition of TG2 by two classes of inhibitors was studied. The covalent-bond-forming events for the inhibitors bearing acrylamide warheads were followed by applying quantum mechanics/molecular mechanics (QM/MM) umbrella sampling MD simulations to the reaction. The produced activation energies correlated well with the biological activities for the inhibitors and a mechanism with an oxyanion intermediate was proposed. The mechanism of inhibition by compounds having sulfonium ion warheads was investigated using reaction path experiments, where a transition state was first identified and verified and was used as a starting point for the reaction path. The activation energies again produced a reasonable correlation with biological activity and an SN2 mechanism was suggested for this inhibition. On a different level, two allosteric inhibitors proposed in the literature were docked into an allosteric site in TG2 predicted by a collaborator from the University of Strathclyde, and docking complexes were subjected to accelerated MD (aMD) to inspect whether the binding would induce significant conformational changes in TG2. The binding of one inhibitor in the predicted site caused bending in TG2 structure that could be a starting event for complete TG2 inactivation. The other inhibitor seemed to produce a similar effect when bound to the original GDP binding site. An even more profound conformational change was reported due to the binding of GDP in its original binding site. aMD, for the simulation times used (400-1000 nanoseconds), was able to represent some large conformational changes in TG2 brought about by the binding of allosteric inhibitors. To sum up, the work presented in this thesis was successful in applying various computational approaches to the analysis of inhibition of TG2 with irreversible and allosteric inhibitors.

Orientador: Flavia Maria Netto
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O leite bovino contém várias proteínas que são consideradas antigênicas e capazes de induzir resposta imune, dentre elas, a ß-lactoglubulina (ß-Lg) é uma das mais antigênicas. A enzima transglutaminase (TG) é a única enzima utilizada comercialmente que catalisa reação de ligação cruzada inter ou intramolecular em diversas proteínas, formando polímeros de alta massa molar. Até o momento, pouco se conhece sobre a modificação dos epítopos antigênicos na estrutura das proteínas do soro de leite modificada ou polimerizada pela enzima TG. O presente trabalho teve como objetivo estudar o efeito da reação de polimerização induzida pela TG na atividade antigênica da ß-Lg. A modificação da ß-Lg com a TG foi realizada em dois experimentos: (1) a ß-Lg foi tratada termicamente (80 °C/60 min) em diferentes concentrações (3, 5 e 7%) e polimerizada com TG (5, 10, 25 e 50 U/g de proteína) e (2) a ß-Lg (3, 5 e 7%) foi polimerizada com TG 25 U/g de proteína na presença de agente redutor Cys nas concentrações 0,05, 0,1, 0,25 e 0,4 mol/L. A caracterização da ß-Lg polimerizada foi realizada por eletroforese (SDS-PAGE) e cromatografia líquida de alta eficiência de exclusão molecular (CLAE-EM). A antigenicidade da proteína foi avaliada por métodos imunoquímicos, utilizando soro de camundongos BALB/c e técnicas de Immunoblotting e ELISA. Quando a ß-Lg 3% foi tratada termicamente e polimerizada com TG, 40% da ß-Lg permaneceu nas formas monomérica e dimérica. Já as amostras tratadas com maior concentração de proteína (5% e 7%) apresentaram de 70% a 87% de material com massa molar (MM) > 100 kDa, sendo que ß-Lg 7% polimerizada com 25U TG/g foi a que apresentou maior grau de polimerização. Na presença de Cys, observou-se maior percentual de produtos com MM > 100 kDa que o obtido com o tratamento térmico. Nas amostras ß-Lg 5% e ß-Lg 7%, a presença da Cys possivelmente aumentou a susceptibilidade da ß-Lg à polimerização. As amostras selecionadas para avaliação da antigenicidade foram ß-Lg nativa, ß-Lg 7% tratada termicamente e polimerizada com 25U TG/g de proteína (ß-Lg 7% TT 25TG) e ß-Lg 7% polimerizada com 25 U/g de proteína na presença de Cys 0,25 mol/L (ß-Lg 7% 0,25Cys TG). Os resultados mostraram que os animais sensibilizados com ß-Lg polimerizada na presença de agente redutor Cys apresentaram níveis séricos de IgE e IgG menores, comparados aos grupos imunizados com ß-Lg nativa e ß-Lg tratada termicamente e polimerizada com TG, sugerindo que a polimerização na presença de agente redutor Cys pode reduzir o potencial antigênico da proteína ao modificar .e/ou ocultar regiões de epítopos na proteína
Abstract: The bovine milk contains several proteins that are considered antigenic and capable of inducing immune responses, among them ß-lactoglubulin (ß-Lg) is one of the most antigenic protein. The enzyme transglutaminase (TG) is the only enzyme commercially used that catalyzes reaction of crosslinking inter or intramolecular in several proteins, forming polymer of high molecular mass. Up to now, little is known about modification of epitopes of whey proteins by the TG. The present study examines the effect of the polymerization reaction induced by TG on the antigenic activity of ß-Lg. The ß-Lg was modified by TG in two experiments: (1) ß-Lg was heat treated (80 °C/60 min) at different concentrations (3, 5 and 7%) and modified with TG (5, 10 , 25 and 50U / g protein), and (2) ß-Lg (3, 5 and 7%) was modified with TG 25U / g of protein in the presence of reducing agent Cys ( 0.05, 0.1, 0.25, 0.4 mol/L). The characterization of the modified ß-Lg was performed by electrophoresis (SDSPAGE) and high performance liquid chromatography molecular exclusion (HPLCMS). The antigenicity of the protein was measured by Immunoblotting and ELISA, using serum from BALB/c mices. When ß-Lg 3% was heat treated and modified by TG, 40% of the ß-Lg remained as monomeric and dimeric forms. However, the samples treated at higher protein concentration (5% and 7%) showed 70% to 87% of material with molecular weigh > 100 kDa. ß-Lg 7% modified with 25U TG / g presented the greatest degree of polymerization. In the presence of Cys, higher percentage of products with MW > 100 kDa was obtained than with the previous heat treatment. The presence of Cys increased the susceptibility of ß-Lg for polymerization, especially those treated at protein 5 and 7% concentrations. The samples selected for evaluation of the antigenicity were native ß-Lg, 7% ß-Lg treated with 25U TG / g of protein (ß-Lg 7% TT 25TG) and 7% ß-Lg treated with 25U TG / g protein in the presence of Cys 0.25 mol / L (ß-Lg 7% 0.25 Cys TG). The results showed that animals sensitized with ß-Lg in the presence of reducing the agent Cys had lower levels of IgG and IgE, compared to the groups immunized with native ß-Lg and ß-Lg treated with high temperature and modified with TG, suggesting that the polymerization in the presence of reducing agent Cys can modify or hide epitopes and reduce the potential antigenic of the protein
Mestrado
Nutrição Experimental e Aplicada à Tecnologia de Alimentos
Mestre em Alimentos e Nutrição

La transglutaminase tissulaire (TG2) est une enzyme de la famille des transglutaminases (EC 2.3.2.13) qui est exprimée de manière ubiquitaire chez les mammifères. Cette enzyme catalyse la formation d'une liaison amide intra- ou intermoléculaire entre un résidu glutamine et un résidu lysine. Ce processus biologique conduit à la modification post-traductionnelle des protéines. Un nombre croissant de publications associe la surexpression de cette enzyme et la déréglementation de son activité, avec un certain nombre de pathologiques humaines telles que les maladies neurodégénératives (maladie d’Alzheimer, maladie de Huntington, maladie de Parkinson), la fibrose tissulaire, certains cancers et la maladie cœliaque. Le développement d'inhibiteurs puissants et sélectifs de la TG2 est primordial pour identifier soit des outils pharmacologiques pour comprendre les processus biologiques dépendant de cette enzyme ou soit des candidats médicaments pour traiter les pathologies liées à la surexpression de la TG2. La majorité des composés inhibiteurs synthétisés jusqu'à présent agissent en bloquant de manière irréversible la réaction de transamidification de la TG2 en ciblant spécifiquement la cystéine 277 présente dans le site actif de la TG2.L’objectif de ce travail a été d’identifier et de sélectionner des molécules de faible poids moléculaire inhibant de façon sélective et puissante l’activité de transamidification de la TG2. Nous présenterons l’optimisation de deux séries originales de composés (synthèse, études de relation de structure-activité) comportant un noyau aromatique central de type naphtalénique ou indolique et une fonction acrylamide comme accepteur de Michael pour piéger la fonction thiol de la cystéine 277. Un certain nombre de composés synthétisés montre une inhibition nanomolaire de la TG2 (IC50 = 1.7-6 nM) avec un excellent profil de sélectivité vis-à-vis de TG1, TG6 et FXIIIa (IC50 > 10 µM). Ces inhibiteurs inhibent efficacement la TG2 dans des extraits de tissus et de cellules. Aucune toxicité apparente n’a été observée pour des concentrations inférieures à 10 µM d’inhibiteur sur les lignées vSMCs et SH-SY5Y. Les valeurs de KI, kinact et kinact/KI ont été également déterminés sur deux inhibiteurs sélectionnés (23b et 78f) pour leurs activités biologiques. La formation d’une liaison covalente entre la cystéine 277 de la TG2 et ces deux inhibiteurs a été prouvée par digestion trypsique suivie d’une analyse LC-MS/MS
Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme of the mammalian transglutaminase (TG) family which catalyzes the formation of an intra- or inter-molecular isopeptide bond between a glutamine and a lysine, leading to the post-translational modification of proteins. An increasing number of literature has associated the over-expression of this enzyme, and the deregulation of its activity, with a number of human physio-pathological states like neurodegenerative disorders (Alzheimer’s disease, Huntington’s disease, Parkinson’s disease), tissue fibrosis, certain cancers, and celiac disease. The development of potent and selective TG2 inhibitors has become primordial to reach either a pharmacological probe, to understand the biological processes that depend on this enzyme, or a drug candidate, to treat the pathologies related with its overexpression. The majority of the inhibitory compounds synthesized so far act by irreversibly blocking the transamidation reaction of TG2. These TG2 inhibitors specifically target the cysteine 277 present in the TG2 active site. The aim of this work was the identification and selection of new potent and selective small molecules to inhibit the TG2 transamidation activity. We present the optimization of two new series of compounds (synthesis, structure-activity relationship studies) bearing naphthalene or indole aromatic rings as the central backbone structure. Both series present an acrylamide group as the Michael acceptor in order to react with the thiol group of cysteine 277. Several of the synthesized compounds showed a nanomolar inhibition over TG2 (1.7-6 nM) with an excellent selectivity profile over TG1, TG6 and FXIIIa (IC50 > 10 µM). These inhibitors showed high specificity on inhibiting TG2 in tissue and cell extracts. No apparent toxicity up to 10 µM was observed in vSMCs and SH-SY5Y cell lines. Their KI, kinact et kinact/KI were also determined on two selected inhibitors (23b and 78f) for their biological activities. The formation of a covalent bond between the cysteine 277 of TG2 and these two inhibitors was proven by tryptic digestion followed by LC-MS/MS analysis

Nous avons etudie un ensemble de maladies neurodegeneratives associees a des proteines contenant une sequence polyglutaminique excessivement longue, la plus frequente etant la choree de huntington. Le gain de fonction apporte par l'expansion polyglutaminique conduit a la formation d'agregats letaux pour les neurones. Pour expliquer cette agregation, nous avons postule que la transglutaminase pourrait catalyser la formation de polymeres stabilises par des liaisons (-glutamine) lysine entre les polyglutamines et des residus lysine de proteines neuronales. Nous avons montre que des peptides synthetiques contenant des motifs polyglutaminiques sont, in vitro, d'excellents substrats de la transglutaminase, et que leur reactivite augmente avec le nombre de residus glutamine. Ces peptides forment, in vitro, des agregats insolubles en presence de proteines cerebrales. Nous avons ensuite etudie les proprietes de substrat de l'huntingtine, proteine associee a la choree de huntington. Nous avons observe que l'huntingtine mutee est incorporee dans des polymeres de hauts poids moleculaires dans les tissus affectes, alors qu'elle reste monomerique dans les tissus sains. Nous avons montre que l'huntingtine est, in vitro, un substrat de la transglutaminase et que la constante de vitesse de la reaction augmente avec la longueur du motif polyglutaminique. La transglutaminase peut selectivement incorporer la forme mutee de l'huntingtine dans des polymeres de tres hauts poids moleculaires, reproduisant, in vitro, ce que l'on observe dans les tissus affectes. Les inhibiteurs de la transglutaminase bloquent la polymerisation de l'huntingtine. Comme la transglutaminase neuronale pourrait etre activee par les flux calciques accompagnant la conduction de l'influx nerveux, nous pensons que la formation de polymeres sous l'action de l'enzyme constitue l'etape initiale de la maladie.

Orientador: Flavia Maria Neto
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Estudos indicam que cerca de 2,5% dos recém-nascidos sofrem reações de hipersensibilidade ao leite bovino. Os principais componentes alergênicos do soro do leite bovino são as proteínas ß-Lactoglobulina (ß-Lg) e a-Lactoalbumina (a-La). A enzima transglutaminase (TG) tem sido utilizada para modificar as proteínas do soro do leite, podendo reduzir o seu potencial antigênico. O objetivo desse trabalho foi estudar o efeito do pH e relação enzima substrato (E:S) na polimerização do Isolado proteico do soro do leite (IPS) com a TG em diferentes condições utilizando a Metodologia de Superfície de Resposta, e avaliar a redução do potencial antigênico das proteínas pela suscetibilidade dos produtos obtidos à digestão com pepsina. O estudo da polimerização do IPS foi realizado por meio de experimentos fatoriais 22, nos quais as variáveis independentes foram a relação enzima:substrato (E:S) (15,7 ¿ 56,9 U TG /g de proteína) e pH (5,0 ¿ 8,4). A variável dependente foi a polimerização das amostras avaliada pela concentração relativa das proteínas ß-Lg ([ß-Lg]) e a-La ([a-La]) após a reação de polimerização, medida por densitometria do gel. Para o estudo da polimerização, foram realizados dois DCCR(Delineamento Composto Central Rotacional): DCCR 1 no qual foi utilizado o IPS sem qualquer tratamento e DCCR 2 no qual foi utilizado o IPS tratado termicamente. Para condição do DCCR 2, foi realizado um experimento preliminar afim de obter as melhores condições de tempo e temperatura de polimerização pela TG. A caracterização das amostras de IPS polimerizado foi realizada por eletroforese (SDS-PAGE). As amostras que apresentaram a menor [ß-Lg] foram empregadas para o estudo de resistência à pepsina, foram utilizados dois modelos de simulação da digestão gástrica: o adulto (182 U de pepsina/g de proteína, pH 2,0) e o infantil (23 U de pepsina/g de proteína, pH 4,0) seguida por caracterização por eletroforese (SDS-PAGE). A avaliação in vitro da antigenicidade dos digeridos gástricos foi realizada por ELISA, utilizando soro de camundongos BALB/c sensibilizados com ß-Lg na forma nativa. A polimerização do IPS pela TG foi mais eficiente quando a proteína foi previamente desnaturada por tratamento térmico. No DCCR 1 ocorreu maior polimerização da a-La do que da ß-Lg, indicando que esta proteína reage facilmente com a TG, mesmo sem tratamento térmico prévio. A digestão in vitro do IPS foi mais eficiente nas condições fisiológicas simulando o modelo adulto do que o infantil. Em ambos os modelos, a amostra tratada termicamente e polimerizada com TG (IPS/TT-TG) foi mais susceptível à pepsina e também foi a que apresentou a menor resposta frente IgE anti- ß-Lg. A diminuição moderada do potencial alergênico após os tratamentos realizados sugerem que houve modificação e ou ocultação de epítopos da estrutura da proteína
Abstract: Studies indicate that about 2.5% of newborns suffer from hypersensitivity reactions to cow¿s milk. The main allergenic components of bovine whey proteins are ß-lactoglobulin (ß-Lg) and a-lactalbumin (a-La). The enzyme transglutaminase (TG) has been used for modifying whey proteins, and may reduce their antigenic potential. The present work aimed at studying the effect of pH and enzyme substrate (E:S) on the polymerization of the IPS with TG under different conditions using Response Surface Methodology, and evaluate the reduction of potential of the antigenic proteins using the susceptibility of products to pepsin digestion. The study of the IPS polymerization was performed by factorial experiments 22, in which the independent variables were enzyme: substrate ratio (E: S) (15.7 to 56.9 U TG / g of protein (U g-1) ) and pH (5.0 - 8.4). The dependent variable was polymerization of the samples evaluated by the relative concentration of the ß-Lg ([ß-Lg]) and a-La ([La-a]) after the polymerization reaction, evaluated by densitometry of the gel. To study the polymerization, two CRCD (Central Rotatable Composite Design) were performed: CRCD 1 in which untreated WPI was used and CRCD 2 in which WPI denatured by heat treatment was used. The characterization of the samples was performed by SDS-PAGE. The evaluation of the polymerization was achieved by the relative concentration of the proteins ß-Lg ([ß-Lg]) and a-La (([a-La]) after polymerization, determined by densitometry. The samples with the lowest [ß-Lg] were chosen for the study of resistance to pepsin using two simulation models of gastric digestion, the adult (182 U pepsin / g of protein and pH 2.0) and infant (23 U pepsin / g of protein, pH 4.0). The resistance of the proteins to the action of pepsin was evaluated by SDS-PAGE. Evaluation of the antigenicity of the samples before and after gastric digestion was performed by ELISA using sera from BALB/c mice sensitized with ß-Lg in its native form. Between the two designs carried out for the polymerization of WPI by TG, the one in which the WPI has previously been denatured by heat treatment was more effective. The in vitro digestion of WPI was more efficient under conditions simulating the physiological adult model than the infant model. In both models the sample which was heat treated and subsequently polymerized by TG was more susceptible to pepsin, and showed the lowest anti-IgE response against ß-Lg, indicating that the allergenic potential was decreased after treatment. These results suggested that there was a modified and/or hidden of the epitopes
Mestrado
Nutrição Experimental e Aplicada à Tecnologia de Alimentos
Mestra em Alimentos e Nutrição

Orientador: Helia Harumi Sato
Dissertação (mestrado) - Universidade Estadual de Campinas. Faculdade de Engenharia de Alimentos
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Resumo: A transglutaminase (TGase, EC 2.3.2.13) é uma enzima que catalisa a formação de ligações cruzadas entre resíduos de glutamina e aminas livres de proteínas formando agregados protéicos. Foi estudado o aumento da escala de produção da transglutaminase de Streptomyces sp. P20, de frascos Erlenmeyer agitados de 50 mL contendo 15 mL de meio de cultivo para fermentador de bancada de 6 L com volume útil de 3 L. Para a fermentação em frascos agitados foi utilizado o meio de cultivo otimizado por MACEDO et al. (2007) e para a fermentação do microrganismo em reator de bancada foi utilizado esse útimo meio de cultivo modificado, composto de extrato de 2,5% de farinha de soja, 2% de amido de batata, 1% de peptona, 0,1% de glicose, 04% de KH2PO4 e 0,2% de MgSO4, ajustado para pH 7,0. Foi estudado, através de delineamento fatorial, os efeitos da temperatura, agitação e aeração na fermentação da linhagem de Streptomyces sp. P20 no reator de bancada e a produção de transglutaminase. O aumento da escala de 200 vezes proporcionou uma elevação na atividade enzimática de 7,2 vezes, e o tempo para obtenção desta atividade foi reduzido de 120 horas para 42 horas de fermentação, utilizando dois estágios de temperatura. A atividade máxima obtida em reator de bancada foi de 2,05 U/mL de caldo fermentado. No estudo da produção da transglutaminase de Streptomyces sp. P20, por fermentação em meio semi-sólido, utilizando-se como substratos farinha de feijão branco e farinha de amendoim, foram obtidos, respectivamente, atividade de transglutaminase iguais a 0,88 U/g de substrato seco e 0,77 U/g de substrato seco. A caracterização bioquica da transglutaminase bruta de Streptomyces sp. P20 foi estudada, bem como a aplicação da enzima em carne bovina e proteína texturizada de soja. Os valores de temperatura ótima e pH ótimo desta enzima foram avaliados, sendo verificado que a temperatura ótima de atividade foi de 35°C e o pH 6,5, sendo encontrada uma segunda faixa de pH ótimo em 9,0, o que pode indicar a presença de isoenzimas. A MTGase mostrou-se estável na faixa de pH 6,0 ¿ 9,0, e at·a temperatura de 35°C em pH 6,0 por 30 minutos. O aminoácido L-cisteína aumentou a estabilidade térmica da MTGase de Streptomyces sp. P20. O efeito de íons metálicos, do EDTA, L-cisteína, L-glutationa e PEG 6000 na atividade da MTGase foram avaliados. Os íons Hg2+, Cu2+, Zn2+ e Mn2+, inativaram a enzima, sugerindo a presença de grupos tiol no seu sítio ativo. O íon Mg2+ aumentou a atividade da MTGase cerca de 100%. A aplicação da MTGase de Streptomyces sp. P20 em carne bovina e em prote?a texturizada de soja foi eficiente quando comparada a aplicação da enzima comercial Activa TG-BP aplicada nas mesmas condições
Abstract: Transglutaminase (TGase, EC 2.3.3.13) is an enzyme that catalyzes cross-linking between the glutamine and free amine residues of proteins leading to the formation of protein aggregates. The scale up of the production of transglutaminase by Streptomyces sp. P20 from shaken flasks to a bench fermenter was studied. Initially, the effect of temperature and agitation on the microorganism was studied in 50 mL conical flasks containing 15 mL of culture medium previously optimized by MACEDO et al. (2007). The production in 250 mL conical flasks containing 50 mL of the same culture medium was then studied. For fermentation in the bench reactor the same medium was used, modified, containing extract of 2.5% powdered soy, 2% potato starch, 1% peptone, 0.1% glucose, 0.4% KH2PO4 and 0.2% MgSO4, pH 7.0. A factorial design was used to study the effects of temperature, agitation and aeration on the fermentation of the strain Streptomyces sp. P20 in a 6L bench fermenter. The x200 scale up led to a x7.2 enhancement in enzymatic activity, and the fermentation time decreased from 120 h to 42 h using two temperature (from 34°C during the first 24 hours to 26°C for the remaining time) and agitation (from 350 rpm during the first 24 hours to 150 rpm for the remaining time) shifts. The maximum transglutaminase activity obtained was 2.05 U/mL. In the solid state fermentation study using semi-solid media containing white bean flour and peanut flour, transglutaminase activities of 0.88 U/mg dried substrate and 0.77 U/mg dried substrate, respectively, were obtained. The biochemical characterization of the crude transglutaminase obtained from Streptomyces sp. P20 and its application in beef and texturized soy protein were studied. The optimum temperature and pH values for MTGase activity were investigated, exhibiting optimum activity at 35°C and at both pH 6.5 and pH 9.0, probably due to the presence of an isoenzyme. The enzyme was stable in the pH range from 6.0 ¿ 9.0, and at temperatures of up to 35?C it was stable for 30 minutes at pH 6.0. The amino acid Lcysteine enhanced the thermal stability of the MTGase from Streptomyces sp. P20. The effects of metal ions, EDTA, L-cysteine, L-glutathione and PEG 6000 on the activity of MTGase were investigated. The ions Hg2+, Cu2+, Zn2+ and Mn2+ inhibited enzyme activity, suggesting the presence of a thiol group at the active site of the enzyme. The ion Mg2+ increased MTGase activity by 100%. The application of MTGase from Streptomyces sp. P20 in beef and texturized soy protein was efficient when compared to the application of commercial Activa TG-BP under the same conditions
Mestrado
Mestre em Ciência de Alimentos

Les anticorps monoclonaux anti-tumoraux sont produits en cellules eucaryotes. Pour des raisons de temps et de cout, peu de candidats sont sélectionnés après des tests in vitro pour être produits à large échelle et testés in vivo. Pour tester plus d’anticorps plus rapidement, nous souhaitons produire des fragments variables simple chaine (scFv) chez E. coli. et les coupler à un fragment constant Fc par chimie click pour reconstituer des mimes d’immunoglobulines naturelles. Cette production indépendante des fragments est aussi un outil modulaire permettant de combiner rapidement un grand nombre scFv et de Fc différents.La chimie click est basée sur une réaction spécifique et de haut rendement entre un azide et un cyclooctyne. Les fragments ont donc été fonctionnalisés sur des résidus spécifiques (tags) par des composés chimiques pour introduire ces fonctions à leur extrémité. La première étape a consisté à introduire des tags en C-terminal du scFv anti-HER2 4D5 et en N-terminal du Fc d’IgG1 humaine. Les scFv ont été produits en cytoplasme d’E. coli à hauteur d’au moins 100 mg/L puis oxydés in vitro au sulfate de cuivre. Le fragment Fc a été produit classiquement en cellules humaines. Cinq réactions chimiques ou enzymatiques ont été optimisées et comparées en termes de spécificité et de rendement. La conjugaison d’une amine sur un tag glutamine catalysée par la transglutaminase microbienne a donné les meilleurs résultats. Le scFv a ainsi été dérivé par l’azadibenzocyclooctyne et le Fc par l’azide à hauteur de 60-70%. Lorsqu’ils sont mélangés, ces fragments forment un (scFv)2-Fc et un scFv-Fc avec des rendements globals respectifs de 10-20% et 20-30% après optimisation.Les mélanges scFv + Fc après réaction de chimie click se fixent de la même façon que le (scFv)2-Fc eucaryote au récepteur HER2. Il reste désormais à montrer que leur capacité à inhiber la prolifération d’une lignée exprimant ce récepteur est similaire. L’objectif final est d’obtenir une inhibition de croissance tumorale similaire sur des xénogreffes
Anti-tumoral monoclonal antibodies are currently produced in eukaryotic cells. For cost and time reasons, a limited number of potential candidates are selected after in vitro tests. They are produced at large scale and then tested in vivo. To test more antibodies and more rapidly, we chose to produce single chain variable fragments (scFv) in bacteria, and to couple them to the eukaryotic constant fragment (Fc) thanks to click chemistry to reconstitute immunoglobulin-like compounds. For a given cost, this enables to produce and test in vivo a larger number of clones. This independent production of fragments is also a flexible tool allowing the combination of different Fc isotypes/allotypes with different scFvs.Click chemistry is based on a specific and high-yield reaction between and azide and a cyclooctyne. Therefore, antibody fragments were functionalised on specific residues (tags) by chemical linker so that each part will contain one of these chemical moieties at their extremity. The first step consisted in introducing tags into the anti-HER2 scFv 4D5 C-terminus and human IgG1 Fc N-termini sequences. The scFvs were produced with yields higher than 100 mg/L in the E. coli cytoplasm and in vitro oxidized with copper sulfate. The Fc fragment was classically produced in human cells. Five chemical or enzymatical reactions were optimised and compared in terms of specificity and yield. The coupling between an amine and a glutamine tag catalysed by microbial transglutaminase gave the best results. The scFv fragment was thus functionalised with an azadibenzocyclooctyne and the Fc fragment with an azide at 60-70%. When mixed together, these fragments formed a (scFv)2-Fc and a scFv-Fc with global yields respectively of 10-20% and 20-30% after optimisation.After the click reaction, the scFv + Fc mix binds to the HER2 receptor on the same way as the eukaryotic (scFv)2-Fc in terms of HER2-binding and proliferation inhibition capacity. Now, it must be demonstrated that their proliferation inhibition of a HER2-positive cell line is similar. The final aim is to get a similar tumour growth inhibition on murine xenografts

La néphropathie à IgA (IgAN), est une maladie glomérulaire chronique primitive et principale cause d'insuffisance rénale dans le monde. Les causes et les facteurs aboutissant aux dépôts des complexes d'IgA1 sont inconnus. La forme soluble du récepteur (CD89s) complexée aux IgA joue un rôle clé dans la pathogenèse de cette maladie. Actuellement, aucun traitement spécifique n'est disponible et les options thérapeutiques sont limitées. La compréhension des mécanismes de la formation de ces complexes permettra d'envisager de nouvelles approches thérapeutiques. Dans cette perspective la première partie de cette thèse, met en évidence l'implication d'une protéine essentielle au développement de la N-IgA, la TG2, dans la régulation du clivage du CD89, et cela par la répression de la sérine phosphatase PP2A et l'activation de la métalloprotéase matricielle MMP-9. Dans les monocytes de patients l'expression diminuée de PP2A est associée à une tendance à l'augmentation de TG2, et inversement corrélée avec l'augmentation des complexes IgA1-CD89s. Afin de cibler ces complexes pathogéniques, un essai préclinique a été réalisé avec une protéase recombinante d'origine bactérienne clivant spécifiquement les IgA1 (IgA1-P). Les résultats ont formellement démontré la spécificité et l'efficacité de la protéase dans la réduction des complexes circulants et des dépôts d'IgA1 dans le modèle humanisé de N-IgA, associée à une diminution des marqueurs de l'inflammation et de l'hématurie. Les résultats ont mis en évidence le rôle de la dérégulation de l'axe TG2-PP2A-MMP-9 dans la formation des complexes IgA1-CD89s lors de la N-IgA, ainsi que l'efficacité de l'IgA1-P à éliminer ces complexes. Ces travaux suggèrent en plus du potentiel thérapeutique promoteur de l'IgA1-P, trois éventuelles cibles thérapeutiques envisageables pour la N-IgA
IgA nephropathy (IgAN) is a mesangial proliferative primary glomerulonephritis and a major cause of end-stage renal disease. Causes and factors leading to mesangial IgA1 deposition are unknown. The soluble form of the receptor (sCD89) complexed with IgA plays a key role in the pathogenesis of the disease. There is currently no specific treatment available and the therapeutic options are limited. A better comprehension of the mechanisms regulating the formation of IgA1-sCD89 complexes will unveil new strategies for targeted therapies. In this perspective, the first part of this thesis highlights the implication of the transglutaminase 2 (TG2), a protein essential for the development of IgAN, in the regulation of CD89 cleavage, in a mechanism involving the repression of the serine phosphatase PP2A and the activation of the matrix metalloproteinase MMP-9. While a trend towards TG2 increase is observed, PP2A expression is reduced in monocytes obtained from IgAN patients compared to controls, and inversely correlates with the levels of circulating hIgA1-sCD89 complexes. In order to target these pathogenic complexes, a preclinical assay has been performed with a recombinant protease, a bacterial protein that selectively cleaves human IgA1 (IgA1-P). Results formally demonstrate the specificity and the efficacy of the IgA1-P in the reduction of circulating complexes and mesangial IgA1 deposition in a humanized mouse model of IgAN, associated with a reduction in inflammation and hematuria. Concluding, the results presented in this thesis show a role for the TG2-PP2A-MMP-9 axis in the dysregulated formation of IgA1-sCD89 complexes during IgAN development, as well as the effectiveness of IgA1-P in the elimination of these complexes. In addition to the potential therapeutic use of IgA1-P, this work suggests the TG2-PP2A-MMP-9 axis as a new therapeutic candidate for IgAN treatment

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas
A doença celíaca, caracterizada pela atrofia das vilosidades intestinais, afeta cerca de 1% da população e manifesta-se em indivíduos geneticamente predispostos. É uma doença autoimune e o seu principal agente desencadeador é o glúten, mais propiamente a gliadina, que sofre uma digestão incompleta, originando peptídeos de gliadina. Estes sofrem uma desaminação, pela ação da enzima denominada transglutaminase tecidular, tornando-os peptídeos imunogénicos, que serão responsáveis pela ativação do sistema imunitário e, consequentemente, pelos danos tecidulares no epitélio intestinal. A sua apresentação clínica pode variar desde manifestações gastrointestinais, como diarreia e distensão abdominal, até manifestações extra-intestinais, tais como anemia, baixa estatura e osteoporose. Os diversos quadros clínicos possíveis dificultam, por vezes, o seu diagnóstico. No que diz respeito ao tratamento, o único disponível para esta patologia é o recurso a uma dieta sem glúten. No entanto, atualmente encontram-se a ser investigadas novas abordagens terapêuticas, que serão essenciais para a melhoria da qualidade de vida destes doentes. Novas variantes modificadas de trigo com proteínas menos imunogénicas, a degradação enzimática dos peptídeos de gliadina através de suplementos orais, a diminuição da absorção intestinal de glúten, os inibidores da permeabilidade intestinal e da tTG, o bloqueio dos complexos HLA que fazem o reconhecimento dos peptídeos de gliadina desaminados, a inibição da migração das células T, a modulação da inflamação, os inibidores das MMPs e a vacinação são algumas das terapias alternativas que se encontram em estudo e que serão revistas. Com esta monografia, pretende-se rever o estado da arte, enfatizando a sua etiopatogenia, os sinais clínicos, diagnóstico e o tratamento utilizado atualmente, bem como as perspetivas futuras nesta área. Para a realização deste projeto, procedeu-se à pesquisa de artigos relevantes, recorrendo-se, para tal, à base de dados, como PubMed, ScientDirect, b-on, entre outras. Celiac disease, characterized by intestinal villous atrophy, affects about 1% of the population and manifests in genetically predisposed individuals. It is an autoimmune disease and its main trigger agent is gluten, gliadin more specifically, which undergoes incomplete digestion resulting in gliadin peptides. These undergo a deamination, by an enzyme, tissue transglutaminase, making immunogenic peptides, which are responsible for the activation of the immune system and, consequently, the tissue damage in the intestinal epithelium. The clinical presentation may vary from gastrointestinal manifestations, including diarrhea and abdominal distention, to extra-intestinal manifestations to such as anemia, failure to thrive and osteoporosis. The several possible clinical evaluations make the diagnostic more difficult. In regard to treatment, the only available for this disease is the use of a gluten-free diet. However, alternative therapeutic approaches are currently being investigated, which are essential for improving the quality of life of these patients. Variants of wheat modified with proteins less immunogenic, enzymatic degradation of gliadin peptides by oral supplements, decreased intestinal absorption of gluten, inhibitors of intestinal permeability and tTG, blocking HLA complexes which make the recognition of deamidated gliadin peptides, inhibition of T cell migration, modulation of inflammation, inhibitors of MMPs and immunization are some alternative therapies that are under study and which will be reviewed With this monograph we intended to review the pathogenesis, clinical signs, diagnosis and treatment currently used in CD, as well as the future prospects in this area. For this project, we proceeded to search for relevant articles, resorting to the database such as PubMed, ScientDirect, b-on, among others.

Les hydrogels sont constitués de réseaux élastiques de polymères hydrophiles qui emprisonnent une grande quantité d’eau. La nature des liens, physique ou chimique, qui existent entre les chaînes du réseau définit en partie leurs propriétés mécaniques. Un enjeu actuel important pour les applications envisagées en médecine régénérative est le renforcement mécanique des gels qu’on peut obtenir par mélange des deux types de liens afin de profiter des avantages de leurs réseaux respectifs. La gélatine, un biopolymère, est un bon système modèle pour répondre à cet objectif, car elle offre différentes voies de réticulation : physique, chimique et mixte. Cette thèse présente dans un premier temps une étude extensive de la formation d’hydrogels chimiques de gélatine à l’aide d’une transglutaminase microbienne. Etonnamment ces gels sont turbides, contrairement aux gels physiques qui sont transparents. Nous avons mené une caractérisation détaillée de la cinétique de réticulation et de la turbidité dans ces systèmes et nous avons expliqué ce phénomène par un mécanisme d’auto- mplification des inhomogénéités de concentration de polymères. Nous avons identifié les paramètres permettant de contrôler le niveau de turbidité et même de la supprimer en utilisant une gélatine plus chargée. Dans un deuxième temps, nous avons développé de façon contrôlée des systèmes mixtes transparents, où des liens chimiques et physiques sont introduits successivement. Nous avons montré que même une très faible quantité de liaisons covalentes permet d’obtenir un gel mixte ultra-résistant à la coupure. Nous avons ainsi mis au point une gamme de systèmes modèles qui ouvrent de nombreuses perspectives pour étudier quantitativement la relation entre propriétés structurelles et mécanisme de renforcement mécanique des gels
Hydrogels are elastic networks of hydrophilic polymers which trap a large amount of water. The nature of the cross-links, physical or chemical, between the network chains, determines in part their mechanical properties. An important current challenge for the regenerative medicine applications is the mechanical toughening of gels which can be obtained by mixing both types of bonds in order to take advantage of their respective networks. Gelatin, a biopolymer, is a good model system to meet this objective, as it offers different crosslinking routes: physical, chemical and mixed. This thesis presents firstly an extensive study of chemically crosslinked gelatin hydrogels with a microbial transglutaminase. Surprisingly these gels are turbid unlike physical ones that are transparent. We conduct a detailed characterization of the reaction kinetics and turbidity in these systems and explain this phenomenon by a self-amplification mechanism of polymer concentration inhomogeneities. We identify the parameters which control the level of turbidity and are even able to suppress it with the use of a more charged gelatin. In a second step we have developed, in a controlled way, transparent mixed systems, where chemical and physical bonds are successively introduced. We show that even a very small amount of covalent bonds is sufficient to make a mixed gel ultra-resistant to cutting. Theses model systems offer many perspectives to study quantitatively the relationship between structural properties and toughening mechanism of gels

Bone formation is an osteoblast-mediated process that is controlled by systemic factors such as hormones, growth factors and local cues that arise from the extracellular matrix (ECM). Bone ECM is elaborated by osteoblasts and therefore they can control their own activity. The ultimate goal of bone matrix formation is to elaborate an extracellular network, consisting mainly of fibronectin and collagen type I, that is capable of mineralizing and forming a strong tissue with appropriate tensile and elastic properties. This thesis describes studies that link transglutaminases (TGs), the protein cross-linking enzymes to type I collagen matrix deposition, osteoblast differentiation and bone formation. Findings here show that MC3T3-E1 osteoblasts require TG-activity for differentiation and proper production of collagenous matrices. We also show that osteoblasts produce two transglutaminase enzymes, transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), which are both expressed during osteoblast differentiation. The work further defines the roles of the two TGs in osteoblasts and shows that FXIIIA is the main TG-enzyme with transamidating activity in osteoblasts' ECM. Production of FXIIIA is induced during osteoblast differentiation and is externalized to the cell surface, then secreted to the ECM. TG2 was mainly found on the cell surface of osteoblasts with no transamidating activity; however, it is co-localized with FXIIIA on the cell surface. Studies conducted with chemical inhibitors, TG-substrates and activity probes suggest that TG-activity is required for osteoblast differentiation at three different levels. First, by positively affecting microtubule dynamics, delivery and fusion of secretory vesicles carrying cellular collagen type I to the plasma membrane. Second, by promoting fibronectin matrix deposition and collagen type I secretion. And third, by stabilizing the interaction between fibronectin and collagen type I in the ECM. Furthermore, we demonstrated that tubulin and fibronectin are candidate substrates for FXIIIA in osteoblasts. In summary, our studies are the first to describe FXIIIA transglutaminase expression in osteoblasts in vitro and in vivo, and first to link it to collagen secretion and osteoblast differentiation. Furthermore, these studies were the first to suggest a role for cellular FXIIIA in microtubule dynamics. We conclude that transglutaminase activity arising from FXIIIA can regulate osteoblast differentiation affecting extracellular matrix deposition.
La formation et le développement de l'os est un processus complexe dirigé par les ostéoblastes. Contrôlés par des hormones systémiques, des cytokines et d'autres facteurs locaux, les ostéoblastes sécrètent et assemblent la matrice extracellulaire (MEC) des tissus osseux. L'aboutissement de ce processus sera la génération d'un réseau extracellulaire constitué notamment de la fibronectine et du collagene de type I qui va se minéraliser en formant le tissu dur de l'os avec d'excellent propriétés mécaniques. Cette thèse présente des études liées aux transglutaminases (TGs) – une classe des enzymes responsables de la polymérisation (cross-linking) des protéines et d'autres composée biomacromoléculaires - en relation avec le collagène de type I secrété pendant l'élaboration de la MEC, la différentiation des ostéoblastes et l'élaboration du tissu osseux. Les principaux résultats de ces études portent sur l'observation que l'activité polymérisante de la TG est un facteur crucial pour la différentiation des cellules ostéoblastiques MC3T3-E1 et pour la production normale de la matrice collagénique. Un résultat essentiel de la présente recherche porte sur la découverte que les ostéoblastes synthétisent deux types d'enzymes TG, i.e. la transglutaminase 2 (TG2) et le facteur XIIIA (FXIIIA), qui sont tous les deux secrétés pendant la différentiation des ostéoblastes. Les résultats suivants éclaircirent les rôles des deux enzymes TG (TG2 et FXIIIA) dans l'activité des ostéoblastes en montrant que c'est le FXIIIA qui est l'enzyme TG dominante avec une activité de transamidation importante dans la MEC des ostéoblastes. FXIIIA est produit pendant la différentiation des ostéoblastes en s'externalisant vers la surface des cellules pour être par la suite sécrété dans la MEC. L'enzyme TG2 a été localisé seulement à la surface des cellules osteoblastiques. Même si le TG2 a été trouvé colocalisé avec le FXIIIA à la surface des cellules, aucune activité de transamidation n'est identifiée pour le TG2. Des études comportant des inhibiteurs chimiques, de substrats TG et de sondes d'activité TG suggèrent que l'activité TG est nécessaire pour la différentiation des ostéoblastes sur trois plans distincts, à savoir : (i) par une action bénéfique sur la dynamique des microtubules, l'acheminement et la fusion des vésicules sécrétoires qui transportent le collagène I cellulaire vers la membrane plasmatique; (ii) par l'accélération du dépôt de la matrice de fibronectine et la sécrétion du collagène de type I; (iii) par la stabilisation de l'interaction de la fibronectine avec le collagène I dans la MEC. De plus, nous avons démontré que la tubuline and la fibronectine ce sont de candidats substrat pour le facteur FXIIIA dans les ostéoblastes. En résumé, notre recherche décrit pour la première fois l'expression de l'enzyme transglutaminase FXIIIA dans les ostéoblastes, tant in vitro qu'in vivo, en corrélant l'expression du FXIIIA à la sécrétion et la différentiation des ostéoblastes. De plus, notre étude est la première en attribuant un rôle au facteur FXIIIA relative à la dynamique des microtubules. On conclut de notre étude que l'activité transglutaminase du facteur FXIIIA exerce une influence décisive dans les processus de différentiation des ostéoblastes avec un effet régulateur crucial à la sécrétion et au dépôt de la matrice extracellulaire.

Transglutaminase 2 (TGase 2) is a multifunctional calcium dependent enzyme that catalyzes protein modifications. TGase 2 is essential in neuronal cell differentiation and it has been reported that certain organophosphates are able to inhibit this process, and the organophosphate phenyl saligenin compound also disrupts TGase 2 activity. It has also been shown that the organophosphates chlorpyrifos (CPF) and chlorpyrifos oxon (CPFO), which cause developmental neurotoxicity, provoke several changes in differentiating rat C6 glioma cells at different levels. The aims of this thesis were to analyse the effects of CPF and CPFO on the TGases present in differentiating rat C6 glioma cells, to develop a new method for the purification of TGase 2 from guinea pig liver, to study possible direct interactions between TGase 2 and esterase inhibitors and to analyze a possible pathway for the externalisation of TGase 2. In the presence of sodium butyrate, rat C6 glial cells differentiated into an astrocyte phenotype. Differentiation of the cells was associated with an increase in the activity, protein levels and gene expression of TGase 2. Differentiation in the presence of CPF or CPFO generated an increase in the activity of TGase 2, a decrease in its levels of gene expression but had no effect on the protein levels. These effects could be associated with a direct interaction between the organophosphates and TGase 2. Chromatographic methods were developed to purify TGase 2 from guinea pig liver and the most effective one was a combination of ion exchange chromatography, protamine sulfate precipitation and hydrophobic interaction chromatography (HIC). The level of purity and yield obtained were superior to that of previously published methods. Furthermore, the final step of HIC could be applied directly to commercially available TGase 2 for the production of a highly purified TGase 2 sample. When TGase 2 purified in this manner was assayed in the presence of CPF and CPFO, enzyme activity was observed to increase significantly, suggesting a direct interaction with TGase 2. By contrast, phenyl saligenin phosphate was found to inhibit TGase activity in vitro, which suggests a direct effect that may involve a different binding site and/or mechanism to CPF or CPFO. The aspartyl protease inhibitor pepstatin A was also able to inhibit directly TGase activity in vitro. The final part of the project involved a short study of the potential association of TGase 2 with exosomes, in order to determine whether the latter might present a means of externalization of this enzyme. Exosomes purified from mouse N2a neuroblastoma cells were found to contain TGase 2, but its localization within the vesicles remains unclear.