No description available.

No description available.

Lectin affinity chromatography has proven to be a powerful method to separate oligosaccharides based on their stereochemical structures. This technique has not been used for the separation of glycolipids since mixtures of these compounds form micelles in aqueous solution. Since N-acetylgalactosamine (GalNAc) is commonly found in glycolipids, three GalNAc-specific lectins were selected to develop a lectin affinity chromatographic method for glycolipids. To circumvent the difficulty of working with micelles, the autoradiographic detection of ¹²⁵l-labeled lectins binding to glycolipids on thin-layer chromatograms was used to study the glycolipid-binding specificity of the lectins from Helix pomatia, Wisteria floribunda and Dolichos biflorus. All three lectins detected the Forssman glycolipid which has a terminal GalNAcα1-3 residue. The Helix pomatia and Wisteria floribunda lectins are also bound to glycolipids with GalNAcβ-linked residues. The interactions of these lectins with glycolipid derived, ³H-labeled oligosaccharides were also analyzed by affinity chromatography on agarose-immobilized lectins. Only the immobilized Helix pomatia lectin was able to specifically bind oligosaccharides with α-linked GalNAc residues. The Helix pomatia lectin was selected to develop an affinity chromatography system for the purification of intact glycolipids having terminal GalNAcα1-3 residues. This technique relies on the ability of the immobilized lectin to bind its oligosaccharide ligands in aqueous solutions of tetrahydrofuran (THF) which inhibits micelle formation and permits the separation of non-specifically bound glycolipids. Forssman glycolipid and a human blood group A-active hexaosylceramide were bound to the Helix pomatia column equilibrated in water/THF (5:95). After applying a step gradient of increasing water content (to 50% water), the specifically bound glycolipids were eluted when GalANc was included in the mobile phase. Using these chromatographic conditions, the Forssman glycolipid from the neutral lipid fraction of sheep erythrocyte stroma and the A-active glycolipids from a total extract of type A human erythrocytes were purified in the Helix pomatia column. The ability to purify human A-active glycolipids from total lipid extracts in a single chromatographic step with the Helix pomatia column was used to isolate A-active glycolipids present in erythrocytes from donors from a rare blood group B(A). The erythrocytes from B(A) subgroup of blood group B individuals, are weakly hemagglutinated by a murine monoclonal anti-A antibody although these erythrocytes should not express blood group A antigens. The Helix pomatia lectin was used to determine the presence and isolate A-active glycolipids from the neutral lipid fraction of erythrocytes from two blood group B(A) donors. However, A-active glycolipids were absent in the glycolipid extracts from erythrocytes from a third B(A) donor and plasma of all three B(A) donors as well as erythrocytes of blood group B and O donors. Based on the fact that only glycolipids and oligosaccharides with GalNAcα1-3 residues specifically bind to the Helix pomatia column, this lectin column was used to isolate the 'terminal products' of the biosynthetic pathway of the human blood group A glycolipids and glycopeptides from the human epidermoid carcinoma cell line A-431. The metabolically active A-431 cells were grown in the presence of ³H-labeled monosaccharide precursors and the Helix pomatia column was used to determine and compare the rate of incorporation of labeled precursors in the A-active glycoconjugates from these cells. / Ph. D.

No / The influence of surfactants on the stability of cyclodextrin (CD) Pickering emulsions is not well understood. In this study, we report two-way effects of Tween 80 and soybean lecithin (PL) on the long term stability of Pickering emulsions stabilized by the self-assembled microcrystals of alpha-CD and medium chain triglycerides (MCT). The CD emulsions in the absence and presence of Tween 80 or PL at different concentrations were prepared and characterized by the droplet size, viscosity, contact angle, interfacial tension and residual emulsion values. After adding Tween 80 and PL, similar effects on the size distribution and contact angle were observed. However, changes of viscosity and interfacial tension were significantly different and two-way effects on the stability were found: (i) synergistic enhancement by Tween 80; (ii) inhibition at low and enhancement at high concentrations by PL. The stability enhancement of Tween 80 was due to the interfacial tension decrease caused by the interaction of Tween 80 with CD at the o/w interface at lower concentrations, and significant viscosity increase caused by the Tween 80-CD assembly in the continuous phase. For PL at low concentrations, the replacement of alpha-CD/MCT by alpha-CD/PL particles at the o/w interface was observed, leading to inhibitory effects. High concentrations of PL resulted in an extremely low interfacial tension and stable emulsion. In conclusion, the extensive inclusion of surfactants by CD leads to their unique effects on the stability of CD emulsions, for which the changes of viscosity and interfacial tension caused by host-guest interactions play important roles.

Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

Uma nova lectina extraída da semente de Artocarpus integrifólia, denominada KM+ foi recentemente descrita. KM+ e haptotática para neutrófilos, promove a aglutinação de hemácias dos grupos A, B, 0, estimula a proliferação de linfócitos do baço de camundongos e liga-se em &#945 D-manose, &#945 metil manosidio e &#945 D-glicose. Esta lectina é composta por quatro monômeros, com peso molecular de 13.150 daltons cada, unidos por interações não covalentes. KM+ contem 1,8% de carboidratos e apresentou quatro isoformas com pontos isoelétricos entre 4,2 e 5,2. Este trabalho teve como objetivos estudar modificações estruturais de KM+ em função de parâmetros como temperatura, força iônica, pH, agentes desnaturantes, ligação com D-manose, monitoradas por dicroísmo circular (CD) e fluorescência. CD também foi utilizado para estimar o conteúdo de estrutura secundaria de KM+, utilizando-se dois programas descritos na literatura: SSE (Secondary Structure Estimation), que utiliza o método dos mínimos quadrados para a estimativa da estrutura secundaria e obtenção dos espectros básicos, baseados nos dados cristalográficos de proteínas de .estrutura resolvida; CCA (Convex Constraint Analisys) que utiliza o algoritmo simplex e a partir dos espectros de CD das proteínas de referencia calcula os espectros das componentes básicas. Para a estimativa das frações de estrutura secundária o segundo método utiliza o programa Lincomb. Os espectros de CD foram registrados no intervalo de 185 a 260 nm. O conteúdo em estrutura secundária, estimado pelo programa SSE foi: 0% de &#945-hélice, 41% de folha &#946, 27% de volta &#946 e 32,3 de estrutura desordenada; pelo programa CCA foi: 1% de &#945-hélice, 35% de folha &#946 anti-paralela, 21% de volta &#946 e/ou folha &#946 paralela, 15% de contribuições de aromáticos e/ou ligações dissulfeto, 28% de estrutura desordenada. Os desvios médios quadráticos para os programas SSE e CCA foram 12% e 1%, respectivamente. Portanto a lectina KM+ é principalmente constituída por estruturas tipo folha &#946 e tipo desordenada. A curva calculada pelo programa CCA foi mais bem estimada, pois tem o desvio médio quadrático 12 vezes menor que o do programa SSE. Este resultado, provavelmente ocorre devido aos seguintes fatores: (i) no programa CCA, o espectro da proteína a ser analisada e alinhado com os espectros das proteínas de referência, influenciando no calculo dos espectros básicos; (ii) maior número de proteínas com estrutura &#946 no grupo de referência do programa CCA. A estabilidade de KM+ em função da temperatura tem comportamento diferente em tampão sódio fosfato (PBS) daquele observado em água. Em PBS, quando a amostra esta a 70&#176C, a forma do espectro de CD mostrou-se consistente com um espectro de proteína desnaturada. Comumente, um espectro de proteína desnaturada caracteriza-se pela perda da estrutura secundaria predominante e aumento da estrutura desordenada. Em água, também a 70&#176C, na região da estrutura &#946 (216 nm) surge uma nova banda e na região da estrutura desordenada (195 nm) aparece uma banda com valores positivos mimetizando um espectro da estrutura &#945-hélice. Esta diferença de comportamento pode ser devida à força iônica. A desorganização promovida na molécula de KM+ por cloreto de guanidina foi típica de desnaturação. o máximo da emissão de fluorescência, da KM+ em PBS pH 7,2, foi a 328 nm, característico de resíduos de triptofano protegidos do solvente. Este máximo mudou para 340 nm em pH 10,5. Este resultado indica mudanças no ambiente químico do triptofano neste pH. O deslocamento para a região do vermelho indica, que em pH. os resíduos de triptofano estio em maior contato com o solvente. O número de sitios ligantes de D-manose J)a molécula de KM+, foi estimado pela supressão da fluorescência promovida pelo D-manose. Esta estimativa foi baseada na suposição de que todos os sítios ligantes de D-manose estivessem próximos aos resíduos de triptofano. A relação encontrada foi de 2 moles de D-manose/mol de KM+ / Recently a new lectin, KM+, isolated from Artocarpus integrifolia seeds was described. KM+ induces neutrophil migration, agglutination of human red blood cells, proliferation of mouse spleen cells and binding with monosacharides D-mannose, D-glicose and &#945-metil mannoside. This glycoprotein is composed of four monomers, assembled by non covalent bonds, has 500 aminoacids residues/mol, with a Molecular Weight of 52,000 Daltons and 1.8% of carbohydrates [27]. In this work structural changes of KM+ was studied as a function of temperature, pH, chemical denaturing agents as well as the binding with D-mannose. These changes were monitored by circular dichroism (CD) and fluorimetry. Circular Dichroism (CD) spectroscopy was used for the analysis of the secondary structure of KM+ in solution due do its capacity to indicate the presence and to estimate the proportion of &#945-helix, &#946-sheet, &#946-turn and unordered conformations. This measurent can be regarded as a function of the relative orientation of the chromophores responsible for their chiroptical activity. CD spectroscopy is also one of the methods of choice for monitorization of conformational changes in proteins as a function of solvents, pH, temperature, ionic strength and specific or non specific binding. Two programs which are in use for estimation of secondary structure: SSE, using the linear least squares method and CCA, using the simplex method, were evaluated in the present work. SSE uses a set of proteins with known X-ray data as the basis for evaluation while CCA uses only pure proteins experimental CD spectra. Fluorescence spectroscopy is very useful to monitore of protein conformational changes in solution due to the presence of intrinsic fluorophores. Fluorescence Measurements were performed at 25&#176C. Samples were excited at 280 nm and the emission was monitored in the range 290-450 nm. The maximum emission as a function of pH was at pH 7.0. The wavelength for maximum emission changed from 328 nm at pH 7.0 to 340 nm at pH 10.5. CD spectra were recorded over the range of 185 up to 260 nm. The Secondary structure content estimated by SSE program was: 0% &#945-helix, 41% &#946-sheet, 26% &#946-turn and 32% random with RMS of 12% and CCA program was: 1% &#945-helix, 35% antiparallel &#946-sheet, 21% &#946-turn and/or parallel B-sheet, 28% random, 15% aromatics contributions and dissulfide linkages with RMS of 1%. The fractions of secondary structure obtained when using CCA program were more consistent than those of SSE program. The simulation by CCA program was better probably due to its desconvolution of the spectral contribution of the common secondary structures using experimental CD curves of proteins. The stability of KM+, in PBS, as a function of temperature changes above 55&#176C but only at 70&#176C the shape of the CD spectrum is consistent with the loss of the native ordered secondary structure that should accompany protein unfolding. CD spectra of KM+ in water showed conformational changes as a function of temperature was not consistent with denaturated proteins. The unfolding of KM+ by GdnCl and SDS resulted in CD spectroscopic changes: consistent with the increased random structure and disappearance of beta sheet. Using the two denaturing agents together GdnCl and temperature, the denaturation was observed at lower decreased both GdnCl concentration and at lower temperature. The estimation of the number of binding sites for D-mannose was obtained through the fluorescence intensity decrease due to a quenching effect of D-mannose and showed that the stoichiometry of binding was 2 moles of D-mannoseimol of lectin

Depuis longtemps les réactifs isothiocyanates (ITCs) sont largement utilisés dans le domaine de la bioconjugaison. Ces électrophiles forts réagissent avec les cystéines et les lysines des protéines pour former une liaison stable. Cette réactivité click permet de réaliser des marquages sélectifs ainsi que des fonctionnalisations de protéines. Cependant, les ITCs ne sont pas faciles à synthétiser et à isoler et leur stabilité ne permet pas une conservation optimale.Le but de ce projet est de développer le système enzymatique myrosinase-glucosinolate (MG) comme outil de conjugaison capable de former un ITC in situ. Le tandem MG est un mécanisme de défense des plantes de l’ordre des Brassicales bien connu. Dans ce système biochimique, la myrosinase opère comme thioglucosidase, en hydrolysant les glucosinolates (GLs), pour générer des ITCs. L’avantage de ce tandem enzymatique est de produire les ITCs à partir de précurseurs solubles dans l’eau, non toxiques, sous conditions douces.Afin d’explorer cet outil enzymatique, deux types de GLs non naturels ont été conçus. En raison de l’importance de l’interaction lectine-mannose dans les mécanismes d’adhésion bactérienne, nous avons conçu une petite librairie de GLs intégrant un mannoside. De cette façon il est possible d’étudier des lectines bactérienne (FimH). Le second type de glucosinolate est caractérisé par une deuxième fonction chimique, permettant de réaliser des réactions orthogonales.Le système MG a été évalué dans plusieurs approches de bioconjugaison telles que le marquage sélectif d’une lectine, la synthèse de néoglycoprotéines et la fonctionnalisation de nanoparticules. / Since many decades, Isothiocyanate (ITCs) reagents are widely used in bioconjugation approaches to create a stable bond onto the lysin and cysteine residues of proteins and peptides. Thanks to this click reaction it is possible to achieve a selective ligation or a high functionalization of proteins. On the others hand, isothiocyanates are not easy to prepare, to isolate, to store and most of the time insoluble in water.The aim of this work is to explore the myrosinase-glucosinolate (MG) enzymatic tandem as a ligation system, in order to release ITCs in-situ. The MG tandem is a well-known mechanism of defense in plants of the order Brassicales. In this biochemical system, myrosinase acts as a thioglucosidase, hydrolyzing glucosinolates (GLs) to liberate transient species that spontaneously form ITCs. The advantage of this enzymatic tandem is to generate ITCs from a stable non-toxic GLs precursor, soluble in water, using mild conditions.In order to develop this enzymatic tool, two kind of unnatural GLs were synthetized. Due to the important role of the mannoside-lectins interaction in the bacterial adhesion mechanism, at first, we designed a small library of GL bearing mannosides, in order to target and study the bacterial lectin FimH. The second kind of GL possess a chemical function allowing to study orthogonal reactions. After, the ability of the myrosinase to hydrolyze those unnatural GLs was investigated, as well their chemical reactivity.Then, the performances of the MG system was studied in different approaches of bioconjugation, such as: the synthesis of neoglycoproteins (NGPs), the site selective labeling of a lectin and the functionalization of gold nanoparticles. The feasibility of these strategies confirmed that the MG system can be used as an enzymatic tool in some bioconjugation approaches.

This thesis concerns the development of surface ligation techniques for the preparation of carbohydrate biosensors. Several methodologies were developed based on efficient photochemical insertion reactions which quickly functionalize polymeric materials, with either carbohydrates or functional groups such as alkynes or alkenes. The alkyne/alkene surfaces were then treated with carbohydrate azides or thiols and reacted under chemoselective Cu-catalyzed azide-alkyne cycloaddition (CuAAC) or photo-radical thiol-ene/yne click chemistry, thus creating a range of carbohydrate biosensor surfaces under ambient conditions. The methodologies were evaluated by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) flow through instrumentations with recurring injections of a range of lectins, allowing for real-time analysis of the surface interactions. The developed methods were proven robust and versatile, and the generated carbohydrate biosensors showed high specificities and good capacities for lectin binding.  The methods were then used to investigate how varying the glycan linker length and/or a sulfur-linkage affect the subsequent protein binding. The survey was further explored by investigating the impact of sulfur in glycosidic linkages on protein binding, through competition assays with various O/S-linked disaccharides in solution interactions with lectins. / QC 20120309

The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.