Made available in DSpace on 2014-06-11T19:25:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-05-08Bitstream added on 2014-06-13T18:29:02Z : No. of bitstreams: 1 santos_a_me_araiq.pdf: 1458862 bytes, checksum: f7fe58d688854cba187d0f1e30f22ce7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As proteínas de micronema TgMIC1 e TgMIC4 (TgMICs) da Toxoplasma gondii fazem parte de um complexo proteico localizado na superfície do parasita responsável pelo processo de adesão e invasão celular. Os objetivos desse trabalho foram estudar dispositivos piezelétricos contendo em cada um, uma das MICs recombinantes (rTgMIC1 ou rTgMIC4) e utilizá‐los na determinação das constantes de afinidade entre elas com a fetuína e asialofetuína, empregando o modelo da Isoterma de Langmuir. Os dispositivos foram desenvolvidos por meio a abordagem de monocamadas automontadas (SAM) mista de tióis, utilizando solução etanólica contendo 2,5 mM de ácido 11‐mercaptoundecanóico (11‐MUA) e 7,5 mM de 6‐mercapto‐1‐hexanol (C6OH). A formação da SAM, realizada em temperatura ambiente por 12 h, foi monitorada pela técnica de Microbalança a Cristal de Quartzo com Fator Dissipativo (QCM‐D) e voltametria cíclica utilizando o par redox [FeII(CN)6]4‐/ FeIII(CN)6]3‐. Os resultados obtidos por ambas as técnicas evidenciaram a formação de SAM rígida e de elevado grau de cobertura superficial após cinética lenta em que processos de adsorção e organização dos tióis ocorreram simultaneamente. Para a imobilização das rTgMICs, solução aquosa contendo 10 mM de EDC (N‐etil‐N‐(dimetilaminopropil) carbodiimida) e 20 mM de NHS (N‐hidroxisuccinimida) foi utilizada para a ativação dos grupos carboxílicos presentes na SAM por 2 h, e o processo acompanhado por QCM‐D apresentou resultados compatíveis com aqueles encontrados na literatura. Pela mesma técnica, foi possível verificar que ambas as rTgMICs se imobilizam sobre o cristal de quartzo após sua incubação com solução 0,15 mg/mL de cada rTgMIC em tampão Tris‐HCl contendo 200 mM de NaCl... / The Toxoplasma gondii micronemal proteins TgMIC1 and TgMIC4 (TgMICs) are part of a protein complex located on the surface of the parasite responsible for the process of cellular adhesion and invasion. The goal of the present work was to study a piezoelectric device containing the recombinant MICs (rTgMIC1 or rTgMIC4) and use it in determining the affinity constants between the MICs with fetuin and asialofetuin, employing the Langmuir isotherm model. The devices were developed using the approach of self‐assembled monolayer (SAM) in ethanolic solution containing 2.5 mM of 11‐mercaptoundecanoic acid (11‐MUA) and 7.5 mM of 6‐mercaptohexanol (C6OH). The SAM formation, held at room temperature for 12 h, was monitored by the Quartz Crystal Microbalance technique with Dissipative Factor (QCM‐D) and cyclic voltammetry using the redox couple [FeII(CN)6]4‐/FeIII(CN) 6]3‐. The results obtained from both techniques showed the formation of a rigid and high surface degree coverage SAM after a slow kinetic process in which adsorption and organization of the thiols occur simultaneously. For the immobilization of rTgMICs, aqueous solution containing 10 mM EDC N‐ethyl‐N‐(dimethylaminopropyl) carbodiimide and 20 mM NHS (N‐hydroxysuccinimide) was used for 2 h, and the process followed by QCM‐D was consistent with those found in the literature. By the same technique it was found that both rTgMICs are immobilized on the quartz crystal after incubation whit solution 0.15 mg/mL of each rTgMIC in Tris‐HCl containing 200 mM NaCl (pH 8.0) for 2 h. Unlike the functionalization of the quartz crystal rTgMIC4, remaining adsorption sites were observed in the process using the rTgMIC1, wherein the blocking step using 0.1% gelatin solution for 2 h was required. Throughout the QCM‐D technique it was possible... (Complete abstract click electronic access below)

C-type lectin-like receptors (CTLRs) play critical roles in immunity and homeostasis by recognising a variety of microbial or endogenous ligands. Clecsf8 is a member of the Dectin-2 family of CTLRs. Clecsf8 shares important similarities with its relatives Mincle and Dectin-2, such as the lack of an integral signalling motif and a single, calcium dependent ligand binding domain. They were shown to associate with the FcRγ adaptor, which is essential for receptor surface expression and downstream signalling. Recent publications revealed an important role for Clecsf8 in anti-mycobacterial immunity. It was reported to recognise the mycobacterial cord factor (TDM), similar to the related CTLR Mincle, as well as a possible role in candidiasis. In this study, we characterised the underlying mechanism of Clecsf8 expression in a context of mycobacterial disease. The generation of novel anti-Clecsf8 monoclonal antibodies allowed us to characterise the expression of Clecsf8 in detail in homeostasis and inflammation in murine models in vivo and culture systems in vitro. We found Clecsf8 to be predominantly expressed on monocytes/macrophages and neutrophils within e. g. the peritoneal cavity, blood and bone marrow. Notably, Clecsf8 was expressed only weakly in the lung, but strongly upregulated in a pulmonary mycobacterial infection model. In vitro, Clecsf8 expression on elicited macrophages was strongly induced upon treatment with microbial stimuli in a Myd88- and Mincle dependent manner. Interestingly, surface expression of Clecsf8 in a murine fibroblast cell line was greatly enhanced by co-transfection of Mincle, but not another related CTLR, Dectin-2. Notably, we confirmed mycobacteria as a ligand of CLECSF8, but found no role for the receptor in Candida immunity. In conclusion, Clecsf8 is a myeloid expressed, mycobacterial receptor, showing significant interdependence with Mincle and is regulated through the Myd88 pathway.

Orientador: Paulo Roberto Bueno / Banca: Emanuel Carrilho / Banca: Maria Cristina Roque Antunes Barreira / Resumo: As proteínas de micronema TgMIC1 e TgMIC4 (TgMICs) da Toxoplasma gondii fazem parte de um complexo proteico localizado na superfície do parasita responsável pelo processo de adesão e invasão celular. Os objetivos desse trabalho foram estudar dispositivos piezelétricos contendo em cada um, uma das MICs recombinantes (rTgMIC1 ou rTgMIC4) e utilizá‐los na determinação das constantes de afinidade entre elas com a fetuína e asialofetuína, empregando o modelo da Isoterma de Langmuir. Os dispositivos foram desenvolvidos por meio a abordagem de monocamadas automontadas (SAM) mista de tióis, utilizando solução etanólica contendo 2,5 mM de ácido 11‐mercaptoundecanóico (11‐MUA) e 7,5 mM de 6‐mercapto‐1‐hexanol (C6OH). A formação da SAM, realizada em temperatura ambiente por 12 h, foi monitorada pela técnica de Microbalança a Cristal de Quartzo com Fator Dissipativo (QCM‐D) e voltametria cíclica utilizando o par redox [FeII(CN)6]4‐/ FeIII(CN)6]3‐. Os resultados obtidos por ambas as técnicas evidenciaram a formação de SAM rígida e de elevado grau de cobertura superficial após cinética lenta em que processos de adsorção e organização dos tióis ocorreram simultaneamente. Para a imobilização das rTgMICs, solução aquosa contendo 10 mM de EDC (N‐etil‐N‐(dimetilaminopropil) carbodiimida) e 20 mM de NHS (N‐hidroxisuccinimida) foi utilizada para a ativação dos grupos carboxílicos presentes na SAM por 2 h, e o processo acompanhado por QCM‐D apresentou resultados compatíveis com aqueles encontrados na literatura. Pela mesma técnica, foi possível verificar que ambas as rTgMICs se imobilizam sobre o cristal de quartzo após sua incubação com solução 0,15 mg/mL de cada rTgMIC em tampão Tris‐HCl contendo 200 mM de NaCl... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Toxoplasma gondii micronemal proteins TgMIC1 and TgMIC4 (TgMICs) are part of a protein complex located on the surface of the parasite responsible for the process of cellular adhesion and invasion. The goal of the present work was to study a piezoelectric device containing the recombinant MICs (rTgMIC1 or rTgMIC4) and use it in determining the affinity constants between the MICs with fetuin and asialofetuin, employing the Langmuir isotherm model. The devices were developed using the approach of self‐assembled monolayer (SAM) in ethanolic solution containing 2.5 mM of 11‐mercaptoundecanoic acid (11‐MUA) and 7.5 mM of 6‐mercaptohexanol (C6OH). The SAM formation, held at room temperature for 12 h, was monitored by the Quartz Crystal Microbalance technique with Dissipative Factor (QCM‐D) and cyclic voltammetry using the redox couple [FeII(CN)6]4‐/FeIII(CN) 6]3‐. The results obtained from both techniques showed the formation of a rigid and high surface degree coverage SAM after a slow kinetic process in which adsorption and organization of the thiols occur simultaneously. For the immobilization of rTgMICs, aqueous solution containing 10 mM EDC N‐ethyl‐N‐(dimethylaminopropyl) carbodiimide and 20 mM NHS (N‐hydroxysuccinimide) was used for 2 h, and the process followed by QCM‐D was consistent with those found in the literature. By the same technique it was found that both rTgMICs are immobilized on the quartz crystal after incubation whit solution 0.15 mg/mL of each rTgMIC in Tris‐HCl containing 200 mM NaCl (pH 8.0) for 2 h. Unlike the functionalization of the quartz crystal rTgMIC4, remaining adsorption sites were observed in the process using the rTgMIC1, wherein the blocking step using 0.1% gelatin solution for 2 h was required. Throughout the QCM‐D technique it was possible... (Complete abstract click electronic access below) / Mestre

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / As lectinas consitem em uma classe diversificada de proteÃnas, capazes de reconhecer estruturas glicÃdicas de forma reversÃvel e com alta especificidade, no entanto sem alterar suas estruturas quÃmicas, participando de vÃrios processos celulares importantes. Dentre as diferentes famÃlias de lectinas, as isoladas a partir de leguminosas sÃo as mais extensivamente estudadas, havendo sido relatada a influÃncias dessas molÃculas sobre diversos processos patolÃgicos, incluindo a carcinogÃnese. NotÃveis propriedades antitumorais tÃm sido detectadas para algumas lectinas de leguminosas, resultantes da sua habilidade em induzir a morte celular ou a autofagia em cÃlulas cancerÃgenas, o que atraÃdo atenÃÃo para suas possiveis aplicaÃÃes biomÃdicas. AlÃm disso, algumas lectinas desse grupo especificas para galactose/N-acetil-D-galactosamina (Gal/GalNAc) tÃm se mostrado Ãteis como marcadores histoquÃmicos na pesquisa do cÃncer e a caracterizaÃÃo estrutural dessas lectinas em complexo com diferentes epÃtopos cancerÃgenos vem sendo realizada com sucesso. A lectina isolada a partir das sementes da leguminosa Vatairea macrocarpa (VML) à uma lectina bem caracterizada especÃfica para Gal/GalNAc capaz de reconhecer especificamente o antÃgeno Tn (GalNAc-α-O-Ser), naturalmente encontrado em O-mucinas presentes em diferentes tipos de cÃncer. As estruturas cristalogrÃficas para a VML em complexo com o antÃgeno Tn e GalNAc foram determinadas com resoluÃÃes de 1.4 e 1.7 Ã, respectivamente. A maioria das lectinas obtidas a partir de fontes naturais consiste em misturas de diferentes isoformas, uma caracterÃstica indesejada para aplicaÃÃes biomÃdicas. Com base nisso, uma construÃÃo recombinante para VML (rVML) foi expressa em Escherichia coli, sendo obtida de forma solÃvel em com alto rendimento. A estrutura cristalina para a rVML, bem como para seus complexos com o antÃgeno Tn, GalNAc e α-Lactose foram determinadas com resoluÃÃes de 1.7, 2.7, 2 e 1.8 Ã, respectivamente, apresentando a mesma estrutura geral e padrÃes de interaÃÃo que a lectina silvestre. Com o intuito de gerar um perfil comparativo entre a VML e outras lectinas de leguminosas capazes de reconhecer o antÃgeno Tn, foram realizadas anÃlises de docking molecular utilizando fragmentos de O-mucinas diferentemente decorados com o antÃgeno Tn. Esse perfil ressalta como alteraÃÃes sutis no elenco ou disposiÃÃo dos aminoÃcidos constituintes do sÃtio de ligaÃÃo a carboidrato, que talvez nÃo influenciem a capacidade de ligaÃÃo a monossacarÃdeo, podem impactar diretamente a habilidade dessas lectinas em reconhecer antÃgenos em condiÃÃes naturais. Adicionalmente aos jà caracterizados efeitos biolÃgicos relatados para VML, a similaridade entre sua estrutura e perfis de interaÃÃes quando comparadas a outras lectinas comumente utilizadas como marcadores histoquÃmicos (e.g., VVLB4 e SBA), sugerem fortemente a possÃvel utilizaÃÃo da VML como uma nova ferramenta na pesquisa do cÃncer. Esse trabalho consiste no primeiro relato de estruturas cristalogrÃficas para uma lectina de leguminosa especÃfica para Gal/GalNAc da tribo Dalbergieae. / Lectins are a very diverse class of proteins able to bind specific sugar structures reversibly and with high specificity, but without enzymatically modifying them, triggering several important cellular processes. Among the different lectin families, legume lectins are the most thoroughly studied and have been widely reported to exhibit a number of links to many pathological processes, including carcinogenesis. The remarkable anti-tumor properties of some legume lectins, resulting from their ability to induce programmed cell death and/or autophagocytosis in cancer cells have attracted much attention for biomedical applications. Moreover, a few galactose/N-acetylgalactosamine (Gal/GalNAc)-binding lectins from this group have proven to be useful markers for cancer histochemistry, and the structural characterization of these lectins bound to specific cancer epitopes has been carried out successfully. The seed lectin isolated from the legume tree Vatairea macrocarpa (VML) is a well characterized Gal/GalNAc-binding protein able to specifically recognize naturally occurring O-mucins presenting the carcinoma epitope Tn antigen (GalNAc-α-O-Ser). The crystal structures of VML in complex with Tn antigen and GalNAc have been determined at the resolution of 1.4 and 1.7 Ã, respectively. Unfortunately, most of lectins obtained from natural sources consist in a mixture of forms, which is an undesired feature for biomedical applications. Thus, the recombinant form of VML (rVML) was expressed in Escherichia coli, being obtained soluble and wih high yielding. The crystal structure for rVML, as well as for the complex with Tn antigen, GalNAc and α-Lactose have been determined at resolutions of 1.7, 2.7, 2 and 1.8 Ã, respectively, presenting the same overall structure and binding patterns as the wild lectin. Molecular docking analysis of this new structure and other Tn-binding legume lectins to O-mucin fragments differently decorated with this antigen provides a comparative binding profile among these proteins, stressing that subtle alterations that may not influence monosaccharide binding can, nonetheless, directly impact the ability of these lectins to recognize naturally occurring antigens. In addition to the specific biological effects of VML, the structural and binding similarities between it and other lectins commonly used as histological markers (e.g., VVLB4 and SBA) strongly suggest that VML can be used as a new tool for cancer research. This is the first report of crystal structures of a Gal/GalNAc-binding legume lectin from the Dalbergieae tribe.

No description available.

by Wang He-Xiang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 161-179). / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / LIST OF CONTENTS --- p.v / LIST OF TABLES --- p.xi / LIST OF FIGURES --- p.xii / LIST OF ABBREVIATIONS --- p.xvi / Chapter CHAPTER 1. --- General Introduction --- p.1 / Chapter CHAPTER 2. --- Literature Review --- p.5 / Chapter 2.1. --- Lectins --- p.5 / Chapter 2.1.1. --- Aspects of lectins --- p.5 / Chapter 2.1.2. --- Isolation and purification of lectins --- p.8 / Chapter 2.1.3. --- Characteristics of lectins --- p.9 / Chapter 2.1.4. --- Effects of lectins on biological activities --- p.10 / Chapter 2.1.4.1. --- The role of lectins in plant defence --- p.11 / Chapter 2.1.4.2. --- The specificity of some legume lectins --- p.13 / Chapter 2.1.4.3. --- Some properties of animal lectins --- p.14 / Chapter 2.1.4.4. --- Hypotensive activity of the lectins --- p.18 / Chapter 2.1.4.5. --- Lectins in immunology --- p.20 / Chapter 2.2. --- Mushroom Lectins and Polysaccharides --- p.24 / Chapter 2.2.1. --- General aspects of mushroom lectins and polysaccharides --- p.24 / Chapter 2.2.2. --- Mushroom lectins --- p.25 / Chapter 2.2.2.1. --- Hericium erinaceum lectin --- p.26 / Chapter 2.2.2.2. --- Lactarius deterrimus lectin --- p.26 / Chapter 2.2.2.3. --- Laetiporus sulfureus lectin --- p.27 / Chapter 2.2.2.4. --- Grifola frondosa lectin --- p.28 / Chapter 2.2.2.5. --- Volvariella volvacea lectin --- p.28 / Chapter 2.2.2.6. --- Flammulina veltipes lectin --- p.29 / Chapter 2.2.2.7. --- Ischnoderma resinosum agglutinin --- p.31 / Chapter 2.2.2.8. --- Lectins from Agaricus spp --- p.31 / Chapter 2.2.3. --- Mushroom polysaccharides --- p.34 / Chapter 2.2.3.1. --- Lentinan --- p.35 / Chapter 2.2.3.2. --- "PSK (trade name, Krestin)" --- p.35 / Chapter 2.2.3.3. --- PSP (Polysaccharopeptide) --- p.37 / Chapter 2.2.3.4. --- PSPC (polysaccharide-peptide complex) --- p.38 / Chapter CHAPTER 3. --- Isolation and Characterization of Two Distinct Lectins from the Cultured Mycelium of the Edible Mushroom Tricholoma mongolicum --- p.44 / Chapter 3.1. --- Introduction --- p.44 / Chapter 3.2. --- Materials and Methods --- p.45 / Chapter 3.2.1. --- Strain and culture condition --- p.45 / Chapter 3.2.2. --- Extraction --- p.46 / Chapter 3.2.3. --- Purification --- p.46 / Chapter 3.2.4. --- Hemagglutination activity --- p.47 / Chapter 3.2.5. --- Test of hemagglutination inhibition by various carbohydrates --- p.47 / Chapter 3.2.6. --- MW estimation by gel filtration and SDS- PAGE --- p.48 / Chapter 3.2.7. --- Glycoprotein staining with PAS reagent --- p.49 / Chapter 3.2.8. --- Carbohydrate content --- p.49 / Chapter 3.2.9. --- Thermal stability --- p.49 / Chapter 3.2.10. --- pH stability --- p.49 / Chapter 3.2.11. --- Effect of cations --- p.50 / Chapter 3.2.12. --- Amino acid analysis --- p.50 / Chapter 3.2.13. --- Antiproliferative activity of lectins --- p.50 / Chapter 3.2.14. --- Statistics --- p.51 / Chapter 3.3. --- Results --- p.51 / Chapter 3.3.1. --- Extraction and purification --- p.51 / Chapter 3.3.2. --- General characteristics of lectins --- p.52 / Chapter 3.3.3. --- Antiproliferative activity of lectins --- p.54 / Chapter 3.4. --- Discussion --- p.55 / Chapter 3.5. --- Summary --- p.58 / Chapter CHAPTER 4. --- The Immunomodulatory and Antitumor Activities of Lectins from the Mushroom Tricholoma mongolicum --- p.79 / Chapter 4.1. --- Introduction --- p.79 / Chapter 4.2. --- Materials and Methods --- p.81 / Chapter 4.2.1. --- Lectins --- p.81 / Chapter 4.2.2. --- Animals --- p.81 / Chapter 4.2.3. --- Assay for antitumor activity --- p.81 / Chapter 4.2.4. --- Assessment of tumor growth and host survival after lectin treatment --- p.82 / Chapter 4.2.5. --- Mitogenic activity of lectins --- p.82 / Chapter 4.2.6. --- Production of nitrite ions in response to lectin treatment --- p.83 / Chapter 4.2.7. --- Preparation of concanavalin A-stimulated lymphokines --- p.84 / Chapter 4.2.8. --- Assay for macrophage activating factor --- p.85 / Chapter 4.2.9. --- Production of tumor necrosis factor (TNF) --- p.86 / Chapter 4.2.10. --- Bioassay for tumor necrosis factor --- p.86 / Chapter 4.2.11. --- Statistics --- p.87 / Chapter 4.3. --- Results --- p.87 / Chapter 4.3.1. --- Antitumor activity --- p.87 / Chapter 4.3.2. --- Assessment of tumor growth and host survival --- p.87 / Chapter 4.3.3. --- Mitogenic activity --- p.88 / Chapter 4.3.4. --- Production of nitrite ions --- p.89 / Chapter 4.3.5. --- Production of macrophage activating factor --- p.89 / Chapter 4.3.6. --- Tumor necrosis factor assay --- p.90 / Chapter 4.4. --- Discussion --- p.90 / Chapter 4.5. --- Summary --- p.94 / Chapter CHAPTER 5. --- Hypotensive and Vasorelaxing Activities of a Lectin (TML-1) from the Edible Mushroom Tricholoma mongolicum --- p.109 / Chapter 5.1. --- Introduction --- p.109 / Chapter 5.2. --- Materials and Methods --- p.111 / Chapter 5.2.1. --- Animals --- p.111 / Chapter 5.2.2. --- In vivo blood pressure measurement in rats --- p.112 / Chapter 5.2.3. --- Study employing blockade of autonomic ganglion transmission --- p.113 / Chapter 5.2.4. --- Study employing alpha-adrenergic blockade --- p.113 / Chapter 5.2.5. --- Study employing beta-adrenergic blockade --- p.114 / Chapter 5.2.6. --- Study employing cholinergic blockade --- p.114 / Chapter 5.2.7. --- Study employing histaminergic blockade --- p.114 / Chapter 5.2.8. --- Study employing inhibitor of the renin- angiotensin system --- p.115 / Chapter 5.2.9. --- Preparation of right atrium for in vitro studies --- p.115 / Chapter 5.2.10. --- Preparation of aorta for in vitro studies --- p.116 / Chapter 5.2.11. --- Adenosine receptor binding assays --- p.116 / Chapter 5.2.12. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.118 / Chapter 5.2.13. --- Statistics --- p.118 / Chapter 5.3. --- Results --- p.118 / Chapter 5.3.1. --- Blood pressure changes in vivo --- p.118 / Chapter 5.3.2. --- Pharmacological studies using receptor antagonists --- p.119 / Chapter 5.3.3. --- Adenosine receptor binding assay --- p.119 / Chapter 5.3.4. --- Effects on the right atrium in vitro --- p.120 / Chapter 5.3.5. --- Effect of TML-1 on vascular relaxation --- p.120 / Chapter 5.3.6. --- Effect of methylene blue on the hypotensive activity of TML-1 --- p.120 / Chapter 5.4. --- Discussion --- p.120 / Chapter 5.5. --- Summary --- p.123 / Chapter CHAPTER 6. --- A Polysaccharide-Peptide Complex with Immunoenhancing and Antitumor Activities from Cultured Mycelia of the Mushroom Tricholoma mongolicum --- p.134 / Chapter 6.1. --- Introduction --- p.134 / Chapter 6.2. --- Materials and Methods --- p.135 / Chapter 6.2.1. --- Extraction --- p.135 / Chapter 6.2.2. --- Purification --- p.135 / Chapter 6.2.3. --- PSP for purpose of comparison --- p.136 / Chapter 6.2.4. --- Polysaccharide and protein contents --- p.136 / Chapter 6.2.5. --- MW determination of F1 using gel filtration --- p.136 / Chapter 6.2.6. --- Animals --- p.136 / Chapter 6.2.7. --- Antiproliferative activity assay --- p.137 / Chapter 6.2.8. --- Mitogenic activity --- p.137 / Chapter 6.2.9. --- Production of nitrite ions --- p.138 / Chapter 6.2.10. --- Macrophage activating factor assay --- p.138 / Chapter 6.2.11. --- Antitumor activity assay --- p.139 / Chapter 6.2.12. --- Statistics --- p.139 / Chapter 6.3. --- Results --- p.140 / Chapter 6.3.1. --- Purification of polysaccharide-peptide complex --- p.140 / Chapter 6.3.2. --- Antiproliferative activity --- p.140 / Chapter 6.3.3. --- Mitogenic activity in vitro --- p.140 / Chapter 6.3.4. --- Molecular weight of Fl --- p.141 / Chapter 6.3.5. --- Mitogenic activity in vivo --- p.141 / Chapter 6.3.6. --- Production of nitrite ions --- p.141 / Chapter 6.3.7. --- Production of macrophage activating factor --- p.141 / Chapter 6.3.8. --- Antitumor activity in vivo --- p.142 / Chapter 6.4. --- Discussion --- p.142 / Chapter 6.5. --- Summary --- p.144 / GENERAL DISCUSSION --- p.155 / CONCLUSIONS --- p.158 / REFERENCES

Cheung, Hang Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 138-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction of Lectins --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Definition and History of Lectins --- p.1 / Chapter 1.1.2 --- More than Just Carbohydrate Binding --- p.2 / Chapter 1.1.3 --- Classification of Lectins --- p.3 / Chapter 1.2 --- Plant Lectins --- p.4 / Chapter 1.2.1 --- History of Plant Lectins --- p.4 / Chapter 1.2.2 --- Occurrence of Plant Lectins --- p.5 / Chapter 1.3 --- Physiological Roles of Plant Lectins --- p.6 / Chapter 1.3.1 --- Lectins as Storage Proteins --- p.6 / Chapter 1.3.2 --- Lectins as Defense Proteins --- p.7 / Chapter 1.3.3 --- Lectins as mediator in symbiosis with bacteria --- p.8 / Chapter 1.4 --- Biological Activities of Plant Lectins --- p.9 / Chapter 1.4.1 --- Immunomodulatory Activity --- p.9 / Chapter 1.4.2 --- Lectins and Cancer --- p.10 / Chapter 1.4.3 --- A ntiviral A ctivity --- p.12 / Chapter 1.5 --- Lectins in Glycomic Study --- p.14 / Chapter 1.5.1 --- Background --- p.14 / Chapter 1.5.2 --- Glyco-catch method --- p.15 / Chapter 1.5.3 --- Lectin Blot Analysis --- p.16 / Chapter 1.6 --- Aim of current study --- p.17 / Chapter Chapter 2: --- Purification and Characterization of a Lectin from Musa acuminata --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Purification Scheme --- p.20 / Chapter 2.2.2 --- Assay of Hemagglutinating A ctivity --- p.21 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.22 / Chapter 2.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.22 / Chapter 2.2.5 --- Protein Concentration Determination --- p.22 / Chapter 2.2.6 --- N-terminal amino acid sequence analysis --- p.22 / Chapter 2.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.23 / Chapter 2.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.23 / Chapter 2.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.24 / Chapter 2.2.10 --- Assay of Nitric Oxide Production by Murine Peritoneal Macrophages --- p.25 / Chapter 2.2.11 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.25 / Chapter 2.2.12 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.26 / Chapter 2.2.13 --- RNA Extraction --- p.27 / Chapter 2.2.14 --- Reverse Transcription: First Strand cDNA Synthesis --- p.28 / Chapter 2.2.15 --- Polymerasae Chain Reaction (PCR) --- p.28 / Chapter 2.3 --- Results --- p.32 / Chapter 2.4 --- Discussion --- p.46 / Chapter Chapter 3: --- Purification and Characterization of a Lectin from Gymnocladus chinensis Baill. --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Material and Methods --- p.50 / Chapter 3.2.1 --- Purification Scheme --- p.50 / Chapter 3.2.2 --- Assay of Hemaggl utinating Activity --- p.51 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.51 / Chapter 3.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.51 / Chapter 3.2.5 --- Protein Concentration Determination --- p.51 / Chapter 3.2.6 --- N-terminal amino acid sequence analysis --- p.52 / Chapter 3.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.52 / Chapter 3.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.52 / Chapter 3.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.52 / Chapter 3.2.10 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.52 / Chapter 3.2.11 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.53 / Chapter 3.2.12 --- Assay of Anti-fungal Activity --- p.53 / Chapter 3.3 --- Results --- p.56 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4: --- Introduction to Protease Inhibitors --- p.70 / Chapter 4.1 --- General Introduction --- p.70 / Chapter 4.2 --- Serine Protease Inhibitors --- p.71 / Chapter 4.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.73 / Chapter 4.2.2 --- Bowman-Birk Type Serine Protease Inhibitors (BBI) --- p.74 / Chapter 4.2.3 --- Squash Type Serine Protease Inhibitors --- p.75 / Chapter 4.3 --- Roles of Pis in Plants --- p.76 / Chapter 4.3.1 --- Pis as a defense protein --- p.76 / Chapter 4.3.2 --- Pis in seed germination --- p.78 / Chapter 4.4 --- Applications of Protease Inhibitors --- p.79 / Chapter 4.4.1 --- Pis in Cancer Prevention --- p.79 / Chapter 4.4.2 --- Pis in Crop Protection --- p.81 / Chapter 4.5 --- Aim of Current Study --- p.83 / Chapter Chapter 5: --- Isolation and Characterization of a Trypsin Inhibitor from the seeds of Lens culinaris --- p.84 / Chapter 5.1 --- Introduction --- p.84 / Chapter 5.2 --- Materials and Methods --- p.86 / Chapter 5.2.1 --- Purification Scheme --- p.86 / Chapter 5.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.87 / Chapter 5.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.88 / Chapter 5.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.88 / Chapter 5.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.88 / Chapter 5.2.6 --- Protein Concentration Determination --- p.89 / Chapter 5.2.7 --- N-terminal amino acid sequence analysis --- p.89 / Chapter 5.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.89 / Chapter 5.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.90 / Chapter 5.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.90 / Chapter 5.2.11 --- Assay of Anti-fungal Activity --- p.90 / Chapter 5.3 --- Results --- p.93 / Chapter 5.4 --- Discussion --- p.103 / Chapter Chapter 6: --- Isolation and Characterization of trypsin inhibitors trom the seeds of Vigna mungo (L.) Hepper --- p.106 / Chapter 6.1 --- Introduction --- p.106 / Chapter 6.2 --- Materials and Methods --- p.107 / Chapter 6.2.1 --- Purification Scheme --- p.107 / Chapter 6.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.109 / Chapter 6.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.109 / Chapter 6.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.109 / Chapter 6.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.109 / Chapter 6.2.6 --- Protein Concentration Determination --- p.109 / Chapter 6.2.7 --- N-terminal amino acid sequence analysis --- p.110 / Chapter 6.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.110 / Chapter 6.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.110 / Chapter 6.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.110 / Chapter 6.2.11 --- Assay of Anti-fungal Activity --- p.110 / Chapter 6.3 --- Results --- p.113 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7: --- General Discussion --- p.135 / References --- p.138

Leung, Ho Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 136-146). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.II / ACKNOWLEDGEMENT --- p.III / ABSTRACT --- p.IV / CHINESE ABSTRACT --- p.VI / TABLE OF CONTENT --- p.VII / OVERVIEW OF THIS PROJECT --- p.1 / Chapter SECTION 1: --- Purification and Characterization of hemagglutinins from French bean and mottled kidney bean / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.2 / Chapter 1.2 --- Physiological functions of plant lectins --- p.6 / Chapter 1.3 --- Physiological functions of animal lectins --- p.9 / Chapter 1.4 --- Biological functions of lectins --- p.12 / Chapter 1.5 --- Clinical and research applications of lectins --- p.16 / Chapter 1.6 --- Legume lectins --- p.17 / Chapter 1.7 --- Isolation and purification of lectins --- p.19 / Chapter 1.8 --- Objectives of the present study --- p.21 / Chapter Chapter 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Chemicals --- p.22 / Chapter 2.2 --- Assay of hemagglutinating activity --- p.24 / Chapter 2.3 --- Purification protocol --- p.26 / Chapter 2.4 --- Assay of saccharide inhibition of hemagglutination --- p.28 / Chapter 2.5 --- Assay of pH stability --- p.28 / Chapter 2.6 --- Molecular mass determination and N-terminal sequence determination --- p.28 / Chapter 2.7 --- Assay of mitogenic activity --- p.29 / Chapter 2.8 --- Assay of antiproliferative activity --- p.30 / Chapter 2.9 --- Assay for antifungal activity --- p.30 / Chapter 2.10 --- Assay of HIV-1 reverse transcriptase inhibitory activity --- p.31 / Chapter 2.11 --- Assay of stability towards trypsin and chymotrypsin --- p.31 / Chapter 2.12 --- Assay of nitric oxide production --- p.32 / Chapter 2.13 --- Assay ofHIV-1 integrase --- p.32 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification scheme --- p.35 / Chapter 3.2 --- Size determination and N-terminal sequencing --- p.36 / Chapter 3.3 --- Temperature stability assay --- p.37 / Chapter 3.4 --- pH stability assay --- p.37 / Chapter 3.5 --- Saccharides inhibition of hemagglutination --- p.37 / Chapter 3.6 --- Stability towards Trypsin and Chymotrypsin --- p.38 / Chapter 3.7 --- Anti-proliferative activity --- p.38 / Chapter 3.8 --- HTV-1 reverse transcriptase inhibition --- p.39 / Chapter 3.9 --- Mitogenic activity --- p.39 / Chapter 3.10 --- Nitric oxide production --- p.39 / Chapter 3.11 --- HIV-1 integrase --- p.39 / Chapter 3.12 --- Defensin --- p.40 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification scheme --- p.68 / Chapter 4.2 --- Sequence comparison --- p.69 / Chapter 4.3 --- Physical Stability of the hemagglutinins --- p.70 / Chapter 4.4 --- Protease Stability --- p.71 / Chapter 4.5 --- Sugar Specificity Assay --- p.72 / Chapter 4.6 --- Anti-proliferative Aactivity toward Cancer Cells --- p.73 / Chapter 4.7 --- HTV-1 reverse trancriptase and H̐ơþV integrase inhibition --- p.74 / Chapter 4.8 --- Mitogenic activity --- p.75 / Chapter 4.9 --- Antifungal protein --- p.76 / Chapter Chapter 5 --- CONCLUSION --- p.78 / Chapter SECTION 2: --- Purification and Characterization of flammulolysin from mushroom Flαmmulinα velutipes / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- General Introduction --- p.79 / Chapter 1.2 --- Mechanisms of hemolysis --- p.80 / Chapter 1.3 --- Biological role of hemolysins --- p.80 / Chapter 1.4 --- Mushroom hemolysin --- p.82 / Chapter 1.5 --- Applications of hemolysins --- p.83 / Chapter 1.6 --- Objectives of the present study --- p.83 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.84 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification and sequence determination --- p.90 / Chapter 3.2 --- Effect of sugars and salts on hemolysin --- p.90 / Chapter 3.3 --- Effect of Temperature and pH on hemolysin --- p.91 / Chapter 3.4 --- Effect of Proteases on hemolysin --- p.91 / Chapter 3.5 --- Effect of osmotic protection on hemolysin --- p.91 / Chapter 3.6 --- Effect of hemolysin on tumor cells --- p.91 / Chapter 3.7 --- Effect of hemolysin on spleen cells --- p.92 / Chapter 3.8 --- Effect of hemolysin on bacterial growth --- p.92 / Chapter 3.9 --- Effect of hemolysin on fungal growth --- p.92 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification and sequence comparison of hemolysin --- p.103 / Chapter 4.2 --- Sugar and Salts inhibition --- p.104 / Chapter 4.3 --- Temperature stability --- p.105 / Chapter 4.4 --- pH stability --- p.106 / Chapter 4.5 --- Protease stability --- p.106 / Chapter 4.6 --- Osmotic Protection --- p.106 / Chapter 4.7 --- Anti-tumour activity of the hemolysin --- p.107 / Chapter 4.8 --- Anti-fungal activity --- p.108 / Chapter Chapter 5 --- CONCLUSION --- p.109 / Chapter SECTION 3: --- Purification and Characterization of antifungal peptide from buckwheat seeds Fagopyrum esculentum / Chapter Chapter 1 --- INTRODUCTION / Chapter 1.1 --- Plant antiftmgal proteins --- p.110 / Chapter 1.2 --- Classification of antifungal proteins --- p.110 / Chapter 1.3 --- Distribution of antifungal proteins in plants --- p.111 / Chapter 1.4 --- Mechanisms of antifungal activity --- p.111 / Chapter 1.5 --- Future Perspectives of Antifungal proteins --- p.112 / Chapter 1.6 --- Antifungal peptide from Buckwheat --- p.112 / Chapter 1 .7 --- Objectives of the present study --- p.113 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.114 / Chapter Chapter 3 --- EXPERIMENTAL RESULTS / Chapter 3.1 --- Purification and sequence determination --- p.118 / Chapter 3.2 --- Effect on anti-fungal activity --- p.118 / Chapter 3.3 --- Effect of temperature and pH on antifungal activity --- p.118 / Chapter 3.4 --- Effect of the antifungal peptide on tumor cells --- p.119 / Chapter 3.5 --- Effect of antifungal peptide on HIV-1 Reverse transcriptase Activity --- p.119 / Chapter 3.6 --- Effect of antifungal peptide on spleen cells and NO Production --- p.119 / Chapter Chapter 4 --- DISCUSSION / Chapter 4.1 --- Purification scheme and N-terminal sequence --- p.130 / Chapter 4.2 --- Antifungal Activity --- p.131 / Chapter 4.3 --- Physical stability --- p.131 / Chapter 4.4 --- Anti-proliferative activity toward cancer cells --- p.131 / Chapter 4.5 --- HTV-1 Reverse Transcriptase Inhibitory activity --- p.132 / Chapter 4.6 --- Mitogenic activity and nitric oxide production --- p.132 / Chapter Chapter 5 --- CONCLUSION --- p.133 / OVERALL CONCLUSION --- p.134 / REFERENCES --- p.136

Starting with the structure analysis of ConA in the 70s, the crystal structures of hundreds of different lectins and their carbohydrate complexes have been determined. Lectins, multivalent carbohydrate-binding proteins which specifically bind different sugar structures, have received considerable attention in recent times on account of the realization of the importance of protein−sugar interactions, especially at the cell surface, in biological recognition. They occur in plants, animals, fungi, bacteria and viruses. Plant lectins constitute about 40% of the lectins of known structure. They can be classified into five structural groups, each characterized by a specific fold. Among them, legume lectins constitute the most extensively investigated group. Peanut lectin is a legume lectin which has been studied thoroughly in this laboratory. These studies have provided a wealth of structural and functional information. However, some gaps still exist in our understanding of the structure, interactions and multivalency of peanut lectin. The work presented here addresses these gaps. The hanging drop method was used for crystallizing PNA and its complexes. Intensity data were collected on Mar Research imaging plates mounted on Rigaku RU-200 or ULTRAX-18 X-ray generators. The Oxford cryosystem was used when collecting data at low temperature. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUCATE of CCP4 suite of programs. The molecular replacement program AMoRe was used for structure solutions. Structure refinements were carried out using the CNS software package and REFMAC of CCP4. Model building was done using the molecular graphics program FRODO. INSIGHT II, ALIGN, CONTACT and PROCHECK of CCP4 were used for the analysis and validation of the refined structure. Dynamic light scattering experiments were carried out using a Dyanpro Molecular Sizing Instrument, and the collected data were analyzed using Dynamic V6 software. Until recently, it has been possible to grow crystals of peanut lectin only when complexed with sugar ligands. It has now been possible to grow them at acidic pH in the presence of oligopeptides corresponding to a loop in the lectin molecule. Crystals have also been prepared in the presence of the peptides as well as lactose. Low pH crystal forms of the lectin−lactose complex similar to those obtained at neutral pH could also been grown. Thus, crystals of peanut lectin grown in different environmental conditions, at two pHs with and without sugars bound to the lectin, are now available. They have been used to explore the plasticity and hydration of the molecule. A detailed comparison among different structures shows that the lectin molecule is sturdy and the effect of changes in pH, ligand-binding and environment on it is small. The region involving the curved front β-sheet and loops around the second hydrophobic core is comparatively rigid. The back β-sheet involved in quaternary association, which exhibits considerable variability, is substantially flexible. So is the sugar-binding region. The numbers of invariant water molecules in the hydration shell are small and they are mainly involved in metal coordination or in stabilizing rare structural features. Small, consistent movements occur in the combining site on sugar-binding, although the site is essentially preformed. Crystal structures of peanut lectin complexed with Galβ1-3Gal, methyl-T-antigen, Galβ1-6GalNAc, Galα1-3Gal and Galα1-6Glc and that of a crystal grown in the presence of Galα1-3Galβ1-4Gal have been determined using data collected at 100 K. Use of water bridges as a strategy for generating carbohydrate specificity was earlier deduced from the complexes of the lectin with lactose (Galβ1-4Glc) and T-antigen (Galβ1- 3GalNAc). This has been confirmed through the analysis of the complexes with Galβ1-3Gal and methyl-T-antigen (Galβ1-3GalNAc-α-OMe). A detailed analysis of lectin−sugar interactions in the complexes shows that they are more extensive when β-anomer is involved in the linkage. As expected, the second sugar residue is ill defined when the linkage is 1-6. There are more than two-dozen water molecules, which occur in the hydration shells of all structures determined at resolutions better than 2.5 Å. Most of them are involved in stabilizing the structure, particularly loops. Water molecules involved in lectin−sugar interactions are also substantially conserved. The lectin molecule is robust and does not appear to be affected by change in temperature. Multivalency is believed to be important in the activity of lectins, although definitive structural studies on it have been few and far between. A study has been carried out on the complexation of tetravalent peanut lectin with a synthetic compound containing two terminal lactose moieties, using a combination of crystallography, dynamic light scattering and modelling. Light scattering indicates the formation of an apparent dimeric species and also larger aggregates of the tetrameric lectin in the presence of the bivalent ligand. The crystals of presumably crosslinked lectin molecules could be obtained. They diffract very poorly, but the X-ray data from them are good enough to define the positions of the lectin molecules. Extensive modelling on possible crosslinking modes of protein molecules by the ligand indicated that systematic crosslinking could lead to crystalline arrays. The studies also provided a rationale for the crosslinking in the observed crystal structure. The results obtained provide further insights into the general problem of multivalency in lectins. They indicate that crosslinking involving multivalent lectins and multivalent carbohydrates is likely to lead to an ensemble of a finite number of distinct periodic arrays rather than a unique array. PNA is among the most thoroughly studied lectins. Its structure demonstrated that open structures without point group symmetry cannot be ruled out for oligomeric proteins. It also contributed to the identification of legume lectins as a family of proteins in which small alterations in essentially the same tertiary structure lead to large changes in the quaternary association. Among other things, studies on PNA−sugar complexes led to the identification of water bridges as a strategy for generating carbohydrate specificity in addition to providing detailed information on PNA−sugar interactions. The work reported here significantly added to the information on this important lectin provided by earlier studies. On the basis of a detailed examination of structures of crystals grown under different environmental conditions, the relatively rigid and flexible regions of the molecule could be delineated. The picture that emerges is that of a robust protein with a substantially preformed combining site. The work also added to the information on the dependence of protein−sugar interactions on the different glycosidic linkages in disaccharides. The investigations reported here also provided further insights into the multivalency of peanut lectin.

The journey of structural studies on lectins, starting with ConA in the 70s, has crossed many milestones. Lectins, multivalent carbohydrate-binding proteins of non-immune origin, specifically bind diverse sugar structures. They have received considerable attention in recent times on account of the realization of the importance of protein-sugar interactions, especially at the cell surface, in biological recognition. They occur in plants, animals, fungi, bacteria and viruses. Plant lectins constitute about 40% of the lectins of known structure. They can be classified into five structural groups, each characterized by a specific fold. Among them, legume lectins constitute the most extensively investigated group. Basic Winged bean lectin (WBAI) is a glycosylated, homodimeric, legume lectin with Mr 58000. The structure of WBAI complexed with methyl-a-galactose, determined earlier in this laboratory, provided information about the oligomeric state and the carbohydrate specificity of the lectin in terms of lectin-monosaccharide interactions. The present work was initiated to understand the carbohydrate specificity of the lectin, especially at the oligosaccharide level, with special reference to its blood group specificity. The hanging drop method was used for crystallizing WBAI and its complexes. Intensity data were collected on Mar Research imaging plates mounted on Rigaku RU-200 or ULTRAX-18 X-ray generators. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUNCATE of CCP4 suite of programs. The molecular replacement program AMoRe was used for structure solution. Structure refinement was carried out using the CNS software package. Model building was done using the molecular graphics program O. INSIGHT II, ALIGN, CONTACT and PROCHECK of CCP4 were used for the analysis and validation of the refined structures. WBAI exhibits differential affinity for different monosaccharide derivatives of galactose. In order to elucidate the structural basis for this differential affinity, the crystal structures of the complexes of basic winged bean lectin with galactose, 2-methoxygalactose, N-acetylgalactosamine and methyl-a-N-acetylgalactosamine have been determined. Lectin-sugar interactions involve four hydrogen bonds and a stacking interaction in all of them. In addition, a N-H O hydrogen bond involving the hydroxyl group substituted at C2 exists in the galactose and 2-methoxygalactose complexes. The additional hydrophobic interaction, involving the methyl group, in the latter leads to the higher affinity of the methyl derivative. In the lectin - N- acetylgalactosamine complex the N-H O hydrogen bond is lost, but a compensatory hydrogen bond involving the oxygen atom of the acetamido group is formed. In addition, the CH3 moiety of the acetamido group is involved in hydrophobic interactions. Consequently, the 2-methyl and the acetamido derivatives of galactose have nearly the same affinity for the lectin. The methyl group, a-linked to the galactose, takes part in additional hydrophobic interactions. Therefore, methyl-a- N-acetylgalactosamine has higher affinity than N-acetylgalactosamine to the lectin. The structures of basic winged bean lectin-sugar complexes provide a framework for examining the relative affinity of galactose and galactosamine for the lectins that bind to them. The complexes also lead to a structural explanation for the blood group specificity of basic winged bean lectin, in terms of its monosaccharide specificity. The Tn-determinant (GalNAc-a-O-Ser/Thr) is a human specific tumor associated carbohydrate antigen. Having epithelial origin, it is expressed in many carcinogenic tumors including breast, prostate, lung and pancreatic cancers. The crystal structure of WBAI in complex with GalNAc-a-O-Ser (Tn-antigen) has been elucidated, in view of its relevance to diagnosis and prognosis of various human cancers. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the formation of the complex of the lectin with Tn-antigen. Modelling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein. WBAI binds A-blood group substance with higher affinity and B-blood group substance with lesser affinity. It does not bind the O substance. The crystal structures of the lectin, complexed with A -reactive and B - reactive di and tri saccharides, have been determined. In addition, the complexes of the lectin with fucosylated A- and B-trisaccharides and with a variant of the A-trisaccharide have been modelled. These structures and models provide valuable insights into the structural basis of blood group specificities. All the four carbohydrate binding loops of the lectin contribute to the primary combining site while the loop of variable length contributes to the secondary binding site. In a significant advance to the current understanding, the interactions at the secondary binding site also contribute substantially, albeit in a subtle manner, to determine the blood group specificity. Compared to the interactions of the B- trisaccharide with the lectin, the third sugar residue of the A -reactive trisaccharide forms an additional hydrogen bond with a lysine residue in the variable loop. In the former, the formation of such a hydrogen bond is prevented by a shift in the orientation of the third sugar resulting from an internal hydrogen bond in it. The formation of this bond is also facilitated by an interaction dependent change in the rotamer conformation of the lysyl residue of the variable loop. Thus, the difference in the interactions at the secondary site is generated by coordinated movements in the ligand as well as the protein. A comparison of the crystal structure and the model of the complex involving the variant of the A-trisaccharide results in the delineation of the relative contributions of the interactions at the primary and the secondary sites in determining blood group specificity. At the disaccharide level, WBAI exhibits higher affinity for á1-3 linked Gal/GalNAc containing oligosaccharides, compared to that of other á linked oligosaccharides. With an objective of understanding the preferential binding of WBAI for á 1-3 linked Gal/GalNAc containing oligosaccharides, crystal structure of the complexes of the lectin with Galá1-4Gal, Galá1-4GalâEt and Galá1-6Gal have been determined. The reducing sugar of the disaccharides with linkages other than á1-3 binds to the lectin through a water bridge whereas the same sugar moiety with á 1-3 linkage makes direct interactions with the loop L4 of the protein. The modelling study on the complex of the lectin with Galá1-2Gal further upholds this observation. Different structures involving WBAI, reported earlier and presented here, were used to investigate the plasticity of the lectin. The front curved â-sheet, which nestles the metal binding region and on which the carbohydrate binding loops are perched, is relatively rigid. On the contrary, the flat back â-sheet, involved in the quaternary association in legume lectins, is flexible. This flexibility is probably necessary to account for the variation in quaternary structure. With the results presented in this thesis, 14 crystal structures of WBAI, in the free form and in complex with different sugars, have been reported, all from this laboratory. It is now, perhaps, appropriate to examine the new information and insights gained from these investigations, on the structure and function of the lectin. Earlier X-ray studies of WBAI contributed substantially in establishing that legume lectins are a family of proteins in which small alterations in essentially the same tertiary structure lead to large alterations in quaternary association. Structural studies on WBAI, particularly those reported here, also contributed to the elucidation of the nuances of carbohydrate recognition by lectins. A comparative study of the available structures also revealed the flexible and rigid regions of the protein. The study of the influence of covalently linked sugars on the structure of Erythrina corallodendron lectin (ECorL), a homolog of WBAI, is the content of appendix of the thesis. The three-dimensional structure of the recombinant form of Erythrina Corallodendron lectin(rECorL) complexed with lactose, has been elucidated by X-ray crystallography. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites(Asn17). These changes take place in such a way that hydrogen bonds with the neighbouring protein molecules in rECorL compensate those made by the glycan with the protein in ECorl. contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.