A diverse array of proteins has evolved to detect and affect carbohydrate structures, thereby performing critical roles in important biological events. Carbohydrate recognition usually employs a high degree of precision, as discriminating between two carbohydrate structures can depend on a single hydrogen bond or the configuration of a hydroxyl group. My work has focused on the molecular recognition of carbohydrate antigens by two biologically important classes of carbohydrate-binding proteins: antibodies and lectins. Single crystal x-ray diffraction has been employed to study the IgG2a antibody LPT3-1 and the lectins Griffonia simplicifolia 1-A4 (GSI-A4) and Lathyrus odoratus lectin (LOdL). LPT3-1 targets the conserved inner core structure of lipooligosaccharide from Neisseria meningitidis, the leading cause of meningitis and septicaemia. Structural characterization of LPT3-1 with an inner core fragment demonstrates how this antibody achieves selective cross-reactivity to variants of the inner core and provides insight that could support the development of a broadly protective N. meningitidis vaccine. Legume lectin GSI-A4 displays specificity towards the terminal galactose and N-acetyl-D-galactosamine of carbohydrates, yet the closely related lectin GSI-B4 will only recognize a terminal galactose. The structures of GSI-A4 co-crystallized with two different carbohydrates reveals the mechanism by which GSI-A4 displays this cross-reactivity, which allows for specific recognition of two important tumour-associated carbohydrate antigens. LOdL is a member of the Mannose/Glucose legume lectin family that can recognize an array of clinically significant antigens including abnormal glycosylation patterns on gp120 of HIV. Characterization of LOdL in complex with glucose at high resolution provides a putative primary sequence and molecular level insight into the molecular recognition displayed by this lectin. Structural data indicates LOdL is cross-reactive with the related glucose epimer mannose, and would display a similar if not identical affinity for glucose and mannose, enabling cross-reactivity with oligosaccharides displaying a terminal mannose. The similarity in sequence and primary recognition between LOdL and Pisum sativum lectin (PSL) suggests that LOdL also shares oligosaccharide specificity with PSL and similarly could demonstrate anti-HIV activity. Overall, the structural characterization of these three carbohydrate-binding proteins reveals mechanisms by which antibodies and lectins can employ selective cross-reactivity to discriminate among clinically-relevant carbohydrate structures. / Graduate / mj3parker@gmail.com

A liquid artificial diet system, which was suitable for bioassay of added compounds, was developed for the glasshouse potato aphid, Aulacorthum solani. The diet supported normal growth and reproduction of this insect. Once established, the artificial diet bioassay system was used to test potential insecticidal activities of a variety of proteins found naturally occurring in plants. Effects on survival, development and fecundity were measured. The lectin found in snowdrop, Galanthus nivalis agglutinin (GNA) was found to significantly reduce the fecundity of A. solani, in terms of parthenogenetic nymph production, when administered in artificial diets at the 0.1% w/v level. No significant reductions in survival were found, although GNA administered in vitro did appear to slow the development of A. solani. Transgenic potato plants expressing GNA were used in a growth room trial to show that the reduction in fecundity with the in vitro trials could be reproduced in planta. Aphids feeding on the GNA-expressing potatoes had a significantly lower cumulative nymph production than those feeding on non- transformed plants. The transgenic plants had no effect on the survival of A. solani. The GNA-expressing plants were tested in a larger scale glasshouse trial and resulted in a significantly slower buildup of aphids when compared to control potatoes, thus confirming the results of the artificial diet bioassays and in planta growth room trials. Immunohistochemical studies were performed to detect the presence of GNA in the gut lumen of A. solani fed on artificial diet containing 0.1% w/v GNA; the lectin was observed to be selectively concentrated in the region of the epithelial membrane in the stomach, suggesting that binding to surface carbohydrates or glycoproteins was taking place. Binding to the gut surface has been suggested to mediate lectin toxicity in higher animals, and other insects. A synergistic effect was observed with transgenic potatoes expressing a double construct encoding GNA and bean chitinase (BCH); A. solani cumulative nymph production on these plants was significantly reduced compared to aphids feeding on control and GNA-only expressing plants. However, interestingly, BCH-only expressing plants did not significantly affect the fecundity of A. solani, although a slight reduction in nymph production was observed. On the basis of reports in the literature that suggested that chitin-binding lectins were toxic to insects, an attempt to isolate the gene encoding the chitin-binding stinging nettle lectin was made. RNA was extracted from nettle rhizomes and used to prepare a cDNA library. Successful library construction was verified. PGR methods and a primary screen of the library were used in an attempt to locate the gene.

An extensive investigation into and charactaisation of factors influencing transgene expression following introduction of the transgoie into tobacco via Agrobacterium- mediated transformation was carried out. Characterisation of material supplied at the outset of this project revealed that this material was unacceptable for further analysis. It was thus deemed necessary to obtain large populations of transgenic tobacco heterogenous for levels of transgene expression. Characterisation of these populations (CaMV-lecA and ssRubisco-lecA plants) showed that all plants fell into one of four segregation classes based on segregation of the kanamycin-resistance selectable marker. Results showed that the majority of regenerants contained multiple nptII-containing inserts, while the presence of one or two such inserts was also found, albeit at a much lower frequency. Segregation analysis based on detection of the lecA transgene agreed, in the majority of cases, with these results. However, in a few cases it was found that data obtained from both segregation analyses did not agree, with the presence of a single lecA-containing transgene being detected in plants shown to contain two copies of the nptII-contaning transgene. This result indicates the occurrence of T-DNA rearrangement either within the tobacco genome or during T-DNA transfer and integration. Southern blot analyses allowed a detailed characterisation of T-DNA structure, copy number and number of integration sites to be undertaken. Results from these analyses revealed a higher frequency of T-DNA rearrangement within plants containing multiple inserts. However, such rearrangements did not correlate with a significant reduction in levels of transgene expression since all detected rearrangements were found to occur at or towards the left hand border of the T-DNA, that border distant to the lecA transgene. Plants containing more than one T-DNA were also frequently found to contain these T-DNAs arranged as an inverted repeat at a single locus although no significant relationship between copy number and the presence of such structures was found. Correlating transgene expression levels, as determined by radioimmunoassay-based quantitation of lectin protein in tissues of transgenic plants, with T-DNA copy number, organisation and structure revealed no significant relationship. It is thus feasible to conclude that the major contributory factor influencing levels of transgene expression is the location of T-DNA integration within the plant genome. Subsequent work concerned with investigating the nature of those integration site-specific factors i.e. 'position effect' indicated a possible role for methylation-induced modulation of gene expression. Results presented in this thesis provide an insight into the fate of transgenes following introduction into the plant genome and clearly demonstrate the importance of further exploring the molecular mechanisms underlying transgene expression variability.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17598号 / 工博第3757号 / 新制||工||1573(附属図書館) / 30364 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 森 泰生, 教授 跡見 晴幸 / 学位規則第4条第1項該当

Reniform nematode, Rotylenchulus reniformis is a semi-endoparasitic nematode infecting over 300 plant species including important fiber crops like cotton. Introgression of reniform nematode resistance from a distantly-related resistant species Gossypium longicalyx into cultivated upland species Gossypium hirsutum has been a challenge. An approach towards achieving nematode resistance in crop plants has been to identify candidate parasitism genes expressed in the nematode facilitating infection of host plant species, and silencing the same through reverse genetic approaches like RNAi. A cDNA library constructed from the sedentary female stage of reniform nematode revealed an EST coding for C-type lectins and occurring in high frequency. Identification and characterization of C-type lectins in reniform nematode is important in understanding the immune system of nematode and in planning strategies for the development of reniform nematode resistant cotton varieties. A total of 11 C-type lectin gene family members were identified across six life stages of reniform nematode, with each member expected to play a significant role in the development and parasitic establishment with the host plant. Conserved sites characteristic of C-type lectins found in other organisms have been identified in the C-type lectin genes in reniform nematode for binding of Ca+2 and mannose. The highest level of expression of C-type lectins was observed in the sedentary female stage indicating it to be possibly the most sensitive stage to microbial infection and so a likely stage to target for its management. The site of secretion of C-type lectins in the sedentary female stage could be identified by in situ hybridization as the hypodermal region of the exposed posterior body region which is not inserted into the host root tissue. Phylogenetic analyses of C-type lectin domains of various nematode groups placed the plant-parasitic nematodes in one group indicating the possibility of co-evolution and probably carrying out a similar function aiding in the establishment of parasitism with host plants. Our findings now extend the spectrum of known nematode C-type lectin genes and suggest that lectin activity might be a more general feature of parasitism which could be explored in better understanding the interactions occurring at the host-nematode and nematode-pathogen interfaces.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Leguminosae is recognized by the large amount of isolated and characterized lectins, especially seeds. In this group stand out proteins extracted from species belonging to subtribe Diocleinae, the number of studies in different areas of knowledge. Lectins can be defined as proteins or glycoproteins which are not originated from a body's immune response and have the ability to recognize and bind reversibly to mono or oligosaccharides particular without, however, altering their chemical structures. Different works in our group are structurally haracterizing these proteins as well as elucidating some possible biotechnological applications for these molecules. In this sense, the objective of this study was to isolate and characterize lectins of different species of Leguminosae of Diocleinae subtribe and test them for toxicity to Artemia sp. naupilos, The effect on the smooth muscle of blood vessels and the detention lectin matrix agarose previously activated with cyanogenic bromide (CNBr). Were isolated and characterized the lectin Dioclea sclerocarpa, Dioclea lasiocarpa and Dioclea lasiophylla. Were also made circular dichroism studies on lectin Dioclea sclerocarpa and Dioclea lasiocarpa. The lectin Dioclea lasiophylla was tested against Artemia sp. order to assess their toxicity and was also immobilized on agarose matrix. We evaluated the effect of lectin Dioclea lasiocarpa in the smooth muscle of blood vessels. The knowledge gained from the three scientific articles published in this thesis is a major breakthrough in this promising field of study that has been continuously growing for biotechnological applications. / A famÃlia Leguminosae à reconhecida pela grande quantidade de lectinas isoladas e caracterizadas, especialmente de sementes. Neste grupo se destacam as proteÃnas extraÃdas de espÃcies pertencentes a subtribo Diocleinae, pela quantidade de estudos em diferentes Ãreas do conhecimento. Lectinas podem ser definidas como proteÃnas ou glicoproteÃnas que nÃo sÃo originadas a partir de uma resposta imunolÃgica do organismo e possuem a capacidade de reconhecer e se ligar reversivelmente a mono ou oligossacarÃdeos especÃficos sem, no entanto, alterar suas estruturas quÃmicas. Diferentes trabalhos no nosso grupo vÃm caracterizando estruturalmente essas proteÃnas, bem como elucidando algumas possÃveis aplicaÃÃes biotecnolÃgicas para essas molÃculas. Neste sentido, o objetivo deste trabalho foi isolar e caracterizar lectinas de diferentes espÃcies de Leguminosae da subtribo Diocleinae e testa-las com relaÃÃo à toxicidade para naupilos de Artemia sp., o efeito na musculatura lisa de vasos sanguÃneos e a imobilizaÃÃo de lectina em matriz de agarose previamente ativada com brometo cianogÃnico (CNBr). Foram isoladas e caracterizadas as lectinas de Dioclea sclerocarpa, Dioclea lasiocarpa e Dioclea lasiophylla. TambÃm foram feitos estudos de dicroÃsmo circular na lectina de Dioclea sclerocarpa e Dioclea lasiocarpa. A lectina de Dioclea lasiophylla foi testada contra Artemia sp.de forma a avaliar sua toxicidade e tambÃm foi imobilizada em matriz de agarose. Foi avaliado o efeito da lectina de Dioclea lasiocarpa na musculatura lisa de vasos sanguineos. O conhecimento acumulado a partir dos trÃs artigos cientÃficos publicados nesta tese constitui um grande avanÃo no neste campo promissor de estudo que vem em contÃnuo crescimento para aplicaÃÃo biotecnolÃgica.

Carbohydrate-protein interactions are responsible for several biological functions. While these interactions maintain high levels of specificities, the binding strength of individual carbohydrate-protein recognitions are weak, with dissociation constants (Kd) ~10-3-10-6 M. In order to increase the binding strengths meaningful to physiological functions, multivalent, clustered patches of carbohydrate ligands are required. Synthetic glycoclusters contribute in a significant manner to understand the fine details of the weak carbohydrate-protein interactions. The extent of clustering of the ligands, spatial, topological orientations and the nature of the scaffolds are prominent issues to address the carbohydrate-protein interactions in general. Chapter 1 of the Thesis presents a summary of the synthetic cluster glycosides, mechanisms and energetics of their interactions with lectins. The presence of several ligands within the molecular scaffold is not sufficient, rather there exists a critical demand on the spatial disposition of the individual ligands in the multivalent ligand system to achieve enhanced binding affinities. In order to assess the multivalent effects, influence of linkers and the spatial disposition of the ligands, a systematic study was undertaken, involving a series of the most minimal of the multivalent sugar ligand system, namely, the bivalent sugar ligands. In a programme, it was desired to study the bivalent and monovalent sugar ligand-lectin interactions in a two-dimensional membrane model system. An appropriate model system was the Langmuir monolayer formations of the sugar ligands and their recognitions of the lectins at the interface. A series of bivalent and monovalent glycolipids were thus designed and synthesized. Molecular structure of the ligands utilized to study the lectins binding behavior at the air-water interface are presented in Figure 1. The sugar density dependent lectin binding at the air-water interface caused by the glycolipids was studied in detail. Prior to lectin binding studies, the monolayer behavior of the glycolipids (GL), non-sugars (NS) and their mixtures were assessed. It was observed that the apparent molecular areas of the mixed monolayers increased with increasing percentage of the glycolipid in the mixed monolayer. Interactions of the glycolipid mixed monolayers with lectin were assessed at a constant surface pressure of 10 mN/m. The adsorption kinetics of the lectin concanavalin A (Con A) with the mixed monolayers was monitored by the surface area variation (ΔA) as a function of time. The detailed studies showed a maximum increase in ΔA of 10% of the bivalent glycolipids in the mixed monolayer and a ΔA of 20% of the monovalent glycolipids (Figure 2). With both bivalent and monovalent glycolipids, change in the area per molecule had decreased progressively with higher percentage of the glycolipids in the monolayers. On the other hand, with ethylene glycol spacers, the relative responses and the amount of bound lectin increased. Figure 2. Ligand-lectin interactions at the air-water interface as a function of the percentage of (a) bivalent glycolipids and (b) monovalent glycolipids in the mixed monolayers. To verify the specificity of these interactions, the mannopyranoside non-specific lectin, namely, wheat germ agglutinin (WGA) was tested and there were no deviations in the ΔA for various ratios of the sugar–non-sugar mixed monolayers. The study established that (i) maximal binding of the lectin to the bivalent glycolipids occurred at lower sugar densities at the interface than that for the monovalent glycolipids and (ii) the surface presenting sparsely populated sugar residues are efficient for a lectin binding. Chapter 2 presents the details of synthesis and ligand-lectin interactions at the air-water interface, relevant in the two-dimensional membrane model system. A study of the multivalent effects originating through glycolipid micelles and their lectin interactions was undertaken in another programme. The kinetic studies of the glycolipid micelles-lectin interactions were conducted with the aid of surface plasmon resonance (SPR) technique. Prior to the SPR studies, the critical micellar concentration (CMC), aggregation number and the hydrodynamic diameter of each glycolipid (GL-1 to GL-6, Figure 1) micelles were determined. The glycolipid micelles were used as the analytes on a Con A immobilized surface. The sensorgrams obtained for the interaction of the various glycolipid micelles with Con A are presented in Figure 3. Figure 3. Sensorgrams obtained for the binding of various glycolipids micelles to a Con A immobilized surface, at a constant glycolipid concentration of 250 µM. The kinetic studies of the interactions were performed and the analysis showed that the bivalent analyte model provided a better fitting for the interaction sensorgrams. The analysis revealed that the ka1/kd1 values remained largely uniform for all the glycolipids, whereas the ka2/kd2 values were about two orders of magnitude larger for the bivalent glycolipid (GL-4 to GL-6) micelle-lectin interactions than for the monovalent series (GL-1 to GL-3) (Table 1). From the SPR studies, it emerged that the additional sugar unit in the bivalent glycolipid micelles provided a favorable complexation between the sugar ligand and the lectin. Further, the glycolipid micelles mediated layer-by-layer Con A multilayer formation was also studied by SPR and atomic force microscopy (AFM) methods. Chapter 3 provides the SPR studies of glycolipid micelles-lectin interactions. A study of the monomolecular recognitions of the mono- and bivalent sugar ligands 1-8 (Figure 4) to a lectin was undertaken subsequently. The kinetic studies of the bivalent vs monovalent ligands during lectin binding were conducted by employing the SPR technique, for which the sugar ligands 1-6 were used as the analytes on a lectin coated sensor surface. Figure 4. Structures of the mono- and bivalent sugar ligands 1-8 and the NS derivative. The following observations were made from the SPR analysis. (i) Within the mono- and bivalent series, the response units increased in the series 1–3 and 4–6; (ii) the equilibrium responses were attained within 105 seconds in the monovalent ligands and (iii) the association response gradually increased for the bivalent ligands 5 and 6 and reached an equilibrium after ~3 min. An important outcome of the kinetic studies was the identification of ka and kd for the monomolecular interactions, that were distinctly different for the bivalent ligands. Specifically, the ka was significantly faster and kd was slower for bivalent sugar ligands, in comparison to the monovalent sugar ligands (Table 2). Table 2. SPR derived kinetic parameters for the interactions of sugar ligand to a Con A immobilized surface at 25 oC. Isothermal titration calorimetry (ITC) studies were also conducted, in order to correlate the functional valencies and the thermodynamic parameters. The studies were conducted at ligand concentrations much below their CMCs. The general observations from the ITC studies were that the binding site saturations were slower for the monovalent sugar ligands, in comparison to the bivalent sugar ligands. It was observed that the binding affinities of bivalent ligands 5 and 6 enhanced ~5 times higher than the monovalent ligands 2 and 3 (Table 3). The effective linker length, which allowed the sugar ligands to be functionally active, was determined to be ~15 Å and this separation was necessary for the intermolecular cross-linking formation. The dynamic light scattering (DLS) study of the bivalent ligands 5 or 6-lectin complexes showed the presence of intermolecular cross-linked complexes that existed in solution from the initial stages of the binding process. Upon realizing the nanometric diameters of the sugar ligand-lectin complex, an attempt was undertaken to visualize the complexes by transmission electron micoscopy (TEM). In TEM, 4-Con A complex exhibited particle sizes in the range of 5-10 nm, matching nearly the size of the lectin alone. On the other hand, 5–Con A and 6–Con A complexes provided sizes varying between 20¬150 nm. These particle sizes corresponded to similar aggregate sizes derived from the DLS studies. Chaper 4 describes the kinetic, thermodynamic, DLS and TEM studies of sugar ligand-lectin intearctions. Table 3. Binding stoichiometries and thermodynamic parameters of the sugar ligand-Con A interactions at 25 oC.a Ligand n Ka (x 10 -4) ΔG ΔH TΔS 1 0.91 9.14 ( ± 0.75) -6.76 -3.39 3.37 2 1.01 5.76 (± 0.80) -6.49 -3.98 2.51 3 1.09 7.06 (± 1.23) -6.61 - 3.01 3.60 4 1.10 5.75 (± 0.27) -6.49 - 6.39 0.10 5 0.50 20.6 (± 1.7) -7.59 - 12.80 -5.21 6 0.47 37. 4 (± 2. 4) -7.61 -11.54 -3.93 7 1.03 0.86 (± 0.06) -5.36 -7.9 -2.62 8 1.05 2.48 (± 0.12) -5.99 -6.3 -0.32 MeαMan 1.04 0.79 (± 0.04) -5.27 -7.83 -2.56 Ka is in the unit of M-1; ΔG, ΔH and TΔS are in the units of kcal mol-1. Errors in ΔG are ~1-4%. Errors in ΔH are in the range of 1-8%. Errors in TΔS are in the range of 1-6 %. A study was undertaken further to assess the kinetic interactions of the tumor-associated T-antigen with a lectin. Synthesis of amine-tethered T-antigen and lactose derivatives (Figure 5) were accomplished and an assessment of their kinetic interactions with lectin peanut agglutinin (PNA) was conducted. Figure 5. Structures of the amine-tethered T-antigen, lactose and mannose derivatives. The lectin PNA was used as the analyte onto the sugar ligand immobilized surfaces. It was found that the interaction with T-antigen showed higher response units than the lactose derivative (Figure 6). The kinetic studies of PNA with immobilized T-antigen and the lactose derivatives demonstrated that the binding followed a bivalent analyte model of the interaction. The T-antigen derivative interacted with the lectin and relatively faster association (ka) and a slower dissociation (kd) were observed, in comparison to the lactose derivative. The ratio of second binding kinetic constants (ka2/kd2) was observed higher than the first binding kinetic constants (ka1/kd1). Further, the ITC studies were conducted, in order to provide the thermodynamic parameters governing the lectin-T-antigen interactions. The combined approach of SPR and ITC studies showed that the T-antigen derivative exhibited a higher binding affinity to PNA than the lactose derivative. Chapter 5 presents synthesis of the T-antigen and lactose derivatives and studies of their lectin interactions. In summary, the thesis provides a detailed insight into the kinetic and thermodynamic parameters of the bivalent sugar ligand-lectin interactions, in comparison to the monovalent sugar ligands. Langmuir monolayer formation of the sugar ligands and the assessment of their lectin binding at the air-water interface demonstrated that the surface presenting sparsely populated sugar residues are efficient for a lectin binding. The kinetic studies of various glycolipid micelles-lectin interactions showed that the additional sugar unit in the bivalent glycolipid micelles provided a favorable complexation between the sugar ligand and the lectin. The detailed monomolecular kinetic studies showed that the bivalent sugar ligands underwent a faster association (kon) and a slower dissociation (koff) of the ligand-lectin complexes. The ITC studies on sugar ligand-lectin interactions led to identify not only the thermodynamic parameters, but also the influence of the hydrophobic alkyl units and the linker moieties. The DLS and TEM characterizations of sugar ligand-lectin complexes showed the status of the complexation, sizes and the morphologies. The studies were extended further to tumor associated T-antigen-lectin interactions. Overall, the Thesis establishes the most minimal multivalent sugar ligands, namely, the bivalent sugar ligand-letin interactions. The studies presented in the Thesis should be useful to design multivalent sugar ligands for highly avid lectin interactions and also to raise possibilities for the construction of defined lectin oligomers, facilitated through the multivalent sugar ligand-lectin cross-linking interactions.

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