Lectins are multivalent carbohydrate binding proteins that specifically recognise diverse sugar structures and mediate a variety of biological processes, such as cell-cell and host-pathogen interactions, serum glycoprotein turnover and innate immune responses. Lectins have received considerable attention in recent years on account of their properties leading to wide use in research and biomedical applications. Seeds of leguminous plants are mainly rich sources of lectins, but lectins are also found in all classes and families of organisms. Legume lectins have similar tertiary structures, but exhibit a large variety of quaternary structures. The carbohydrate binding site in them is made up of four loops, the first three of which are highly conserved in all legume lectins. The fourth loop, which is variable, is implicated in conferring specificity. Legume lectins which share the same monosaccharide specificity often exhibit markedly different oligosaccharide specificities. This thesis primarily concerns with structure solution and analysis of lectins from the legume and β-prism II fold families using X-ray crystallography. Apart from having the property of specifically and reversibly binding to carbohydrates, lectins are also interesting models to study sequence-structure relationships, especially of how minor change in the sequence may bring about major changes in oligomerization and binding. Chapter 1 gives an overview of different structural types of plant lectins and describes in detail, their carbohydrate binding features. The details of the various experimental procedures employed during the course of this research, are explained in Chapter 2. Chapter 3 describes the crystal structure of a β-prism II fold lectin (RVL), from Remusatia vivipara, an epiphytic plant of traditional medicinal value, and analysis of its binding properties. This lectin was established to have distinct binding properties and has nematicidal activity against a root-knot nematode with the localization site identified as the high-mannose displaying gut-lining in the nematode. The crystal structure of RVL revealed a new quaternary association of this homodimeric lectin, different from those of reported β-prism II lectins. Functional studies on RVL showed that it fails to bind to simple mannose moieties yet showed agglutination with rabbit blood cells (which have mannose moieties on the surface) and some high mannose containing glycoproteins like mucin and asialofetuin. Further, ELISA and glycan array experiments indicated that RVL has high affinity to N-glycans like trimannose pentasaccharide such as in gp120, a capsid glycoprotein of HIV virus, necessary in virus-association with the host cell. The structural basis for this N-glycan binding was revealed through structure analysis and molecular modelling, and it was demonstrated that there are two distinct binding sites per monomer, making RVL a truly multivalent lectin. Evolutionary phylogeny revealed the divergence in the β-prism II fold proteins with regards to the number of sugar-binding regions per domain, oligomerization and specificity. Chapter 4 deals with the structural studies on a galactose-specific legume lectin (DLL-II) from Dolichos lablab, a leguminous plant. The lectin was found to be a planar tetramer in the crystal structures of the native and ligand bound forms, as expected from our solution studies and phylogenetic analysis. The protein is a heterotetramer with subunits differing only in the presence or absence of a C-terminal helical region at the core of the tetramer. Due to the static disorder in all the crystals, the central helix could be oriented in either direction. Structure analysis of DLL-II proved to be an interesting endeavour as static disorder compounded with twinning in the crystal made the data processing and structure solution a challenging process. Subsequent structure and sequence alignments led to the identification of an adenine-binding pocket in the hydrophobic core of the tetramer. Based on this, DLL-II lectin was co-crystallized with adenine and the structure revealed the presence of adenine at the predicted binding site. Chapter 5 describes the identification and analysis of potential plant lectins/lectin-like domains in the genome of Oryza sativa, using bioinformatics approaches. This project was initiated to study the occurrence of legume-lectin like domains (a predominant dicot feature) in O. sativa, which is a monocot. Later, a large scale genome analysis for all types of lectin domains was carried out through exhaustive PSI-BLAST, profile matching by HMMer, CDD and MulPSSM. The final validation was carried out by assessing the carbohydrate binding potential of the domain by examining the sugar binding sites. The primary interest in undertaking this work was to find the occurrence of association of these domains with other domains as in protein receptor kinases, where lectin is the receptor domain. Though primarily initiated as a bioinformatics project, further structural characterization was attempted by cloning, expression and purification of some of the annotated lectin proteins using prokaryotic expression systems. The protein expression was attained in reasonable amounts for a few of the annotated legume lectin homologs, however purification is yet to be achieved as the expressed proteins are insoluble. A part of the results described in this thesis and the other related projects that the author was involved are reported in the following publications. 1) Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity Bhat GG, Shetty KN, Nagre NN, Neekhra VV, Lingaraju S, Bhat RS, Inamdar SR, Suguna K, Swamy BM. 2010. Glycoconjugate J. 27(3):309-320 2) Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin Thamotharan S, Karthikeyan T, Kulkarni KA, Shetty KN, Surolia A, Vijayan M & Suguna K. 2011. Acta Crystallographica D 67(3):218-227 3) Crystal structure of a β-prism II lectin from Remusatia vivipara Shetty KN, Bhat GG, Inamdar SR, Swamy BM, Suguna K. 2012. Glycobiology 22(1): 56-69. 4) Structure of a galactose binding lectin from Dolichos lablab Shetty KN, Lavanyalatha V, Rao RN, SivaKumar N & Suguna K (Under review) 5) Occurrence of lectin-like domains: Oryza sativa genome analysis. Shetty KN & Suguna K. (Manuscript in preparation)

This thesis is concerned with the first ever X-ray crystallographic and complimentary solution studies on mycobacterial lectins. Lectins, described as multivalent carbohydrate binding proteins of non-immune origin, are found in all kingdoms of life. As explained in the introductory chapter, those from plants and animals are the best characterized in terms of structure and function. Although not that extensive, important studies have been carried out on viral, fungal and parasite lectins as well. Bacterial lectins studied so far can be classified in to fimbrial, surface and secretory (or toxic). Applications of lectins include blood typing, cell separation and purification of glycoconjugates, mitogenic stimulation of lymphocytes, mapping of neuronal pathways and drug targeting and delivery. The work reported in the thesis lies at the intersection of two major long range programs in this laboratory, one on lectins and the other on mycobacterial proteins. Three putative lectins Rv1419 and Rv2813 from M. tuberculosis and MSMEG_3662 from M. smegmatis were chosen for exploratory studies on the basis of preliminary genomic searches. Exploratory studies on Rv1419, Rv2813 and MSMEG_3662 are described in the second chapter. MSMEG_3662 contains two domains, a LysM domain and a lectin domain (MSL) connected by a long polypeptide chain. The two M. tuberculosis proteins, full length MSMEG_3662 and MSL were cloned, expressed, purified and characterized. Rv2813 did not show any appreciable agglutination activity. It showed ATPase activity. Clearly the protein was not a lectin. Rv1419, full length MSMEG_3662 and MSL exhibited lectin characteristics. Among them, Rv1419 and MSL could be crystallized. Preliminary X-ray diffraction studies on them were carried out. Rv1419 could be successfully expressed only once. However, that was enough for the determination of crystal structure and the glycan array analysis of the lectin (Chapter 3). The monomeric lectin has a β-trefoil fold. It has high affinity for LacNAc and its Neu5Ac derivatives. Modeling studies using complexes of homologous structures, led to the identification of two carbohydrate binding sites on the lectins. Sequence comparisons of Rv1419 with homologous proteins with known structures and phylogenetic analysis involving them provide interesting insights into the relationship among trefoil lectins from different sources. X-ray crystal structure analysis of MSL and its complexes with mannose and methyl-α-mannose, the first comprehensive effort of its kind on a mycobacterial lectin, reveals a structure very similar to β-prism II fold lectins from plant sources, but with extensive unprecedented domain swapping in dimer formation (Chapter 4). The two subunits in a dimer often show small differences in structure, but the two domains, not always related by 2-fold symmetry, have the same structure. Each domain carries three sugar-binding sites, similar to those in plant lectins, one on each Greek key motif. The occurrence of β-prism II fold lectins in bacteria, with characteristics similar to those from plants, indicates that this family of lectins is of ancient origin and had evolved into a mature system before bacteria and plants diverged. In plants, the number of binding sites per domain varies between one and three, whereas the number is two in the recently reported lectin domains from Pseudomonas putida and Pseudomonas aeruginosa. An analysis of the sequences of the lectins and the lectin domains shows that the level of sequence similarity among the three Greek keys in each domain has a correlation with the number of binding sites in it. Furthermore, sequence conservation among the lectins from different species is the highest for that Greek key which carries a binding site in all of them. Thus, it would appear that carbohydrate binding influences the course of the evolution of the lectin. LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides is underexplored. Binding of a full length MSMEG_3662 containing LysM and lectin (MSL) domains to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration (Chapter 5). This investigation demonstrates the presence of two binding sites of non-identical affinities per dimeric MSL-LysM molecule. Affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in MSL-LysM, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the present case two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of MSL-LysM for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far. A part of the work presented in this thesis has been reported in the following publications: • Patra D, Mishra P, Surolia A, Vijayan M. 2014. Structure, interactions and evolutionary implications of a domain-swapped lectin dimer from Mycobacterium smegmatis. Glycobiology, 24:956-965. • Patra D, Sharma A, Chandran D, Vijayan M. 2011. Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 67:596-599. • Patra D, Srikalaivani R, Misra A, Singh DD, Selvaraj M, Vijayan M. 2010. Cloning, expression, purification, crystallization and preliminary X-ray studies of a secreted lectin (Rv1419) from Mycobacterium tuberculosis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 66:1662-1665.

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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Com base na necessidade de obter novas formulaÃÃes mais eficientes e diante das propriedades apresentadas pelas molÃculas oriundas de algas marinhas, acredita-se que estas possam ser eficazes no processo de cicatrizaÃÃo. A lectina da alga marinha vermelha Pterocladiella capillacea (PcL) e os polissacarÃdeos da alga vermelha Solieria filiformis (SfP) inicialmente foram analisados em ensaio de toxicidade. PcL foi aplicada no ensaio do edema de pata seguido da dosagem de mieloperoxidase (MPO). Avaliou-se o efeito da lectina da alga Pterocladiella capillacea (PcL) e os polissacarÃdeos das algas Solieria filiformis (SfP) na cicatrizaÃÃo de feridas induzidas em ratos. Ambas as molÃculas foram submetidas a ensaios microbiolÃgicos e analisadas quanto ao efeito no processo de cicatrizaÃÃo em feridas limpas induzidas no dorso de ratos. SfP foi utilizado como um possÃvel veÃculo para a administraÃÃo de PcL e comparado ao Carbopol 940 (C). Os gÃis (0,9%) foram submetidos a anÃlise reolÃgica e entÃo aplicados nas lesÃes durante um perÃodo de tratamento de 10 (dez) dias, utilizando kollagenase como controle. O processo de cicatrizaÃÃo foi avaliado quanto ao tamanho das feridas, dosagem de MPO e anÃlise histolÃgica. PcL e SfP nÃo demonstraram toxicidade quanto aos parÃmetros de peso corpÃreo, ÃrgÃos e dosagens bioquÃmicas. Entretanto a anÃlise histolÃgica mostrou pequenas alteraÃÃes no fÃgado e rim. PcL (1, 3 e 9 mg/kg, i.v.) reduziu o edema induzido por carragenana e quando administrada com seu inibidor mucina nÃo foi possÃvel verificar a reduÃÃo do edema o qual foi confirmado pela dosagem de MPO. As duas molÃculas foram aplicadas em ensaios microbiolÃgicos e nÃo inibiram o crescimento de nenhum micro-organismo testado, os quais tambÃm nÃo foram capazes de utilizar SfP como fonte de carbono. A anÃlise reolÃgica mostrou que os SfP utilizados na formulaÃÃo dos gÃis (PcL+SfP e SfP) apresentaram a caracterÃstica de um pseudoplÃstico. A anÃlise macroscÃpica das feridas mostrou uma reduÃÃo da Ãrea da lesÃo nos animais tratados com PcL+SfP e PcL+C (53,5 e 60%, respectivamente) no sexto dia de administraÃÃo. Na anÃlise histolÃgica, nÃo foi observado infiltrado inflamatÃrio acentuado nos tecidos obtidos atà o 4 dia da administraÃÃo dos gÃis (PcL+SfP e PcL+C) e Kollagenase (controle positivo). No 6 dia, os animais nÃo tratados e os tratados apenas com SfP mostraram infiltrado inflamatÃrio. A dosagem de MPO demonstrou reduÃÃo no processo inflamatÃrio nas amostras contendo PcL, cujo resultado corrobora com a anÃlise histolÃgica. Em conclusÃo, PcL auxiliou no reparo de feridas, sugerindo seu uso futuro como uma possÃvel ferramenta para o tratamento de lesÃes. O papel biolÃgico e farmacolÃgico das lectinas e polissacarÃdeos de algas marinhas faz parte de uma Ãrea de estudos ainda pouco explorada, onde muito conhecimento deverà ser investido visto que estas biomolÃculas podem ser promissoras para a indÃstria farmacÃutica. / Based on the need for new formulations that are more efficient and on the properties provided by molecules derived from seaweed, it is believed that these can be effective in healing process. The lectin from the red seaweed Pterocladiella capillacea (PcL) and the polysaccharides of red algae Solieria filiformis (SfP) were initially analyzed in toxicity testing. PcL was applied to the paw edema test followed by measurement of myeloperoxidase (MPO). We evaluated the effect of the seaweed Pterocladiella capillacea lectin (PcL) and algal polysaccharides Solieria filiformis (SfP) in healing wounds in rats induced. Both molecules were submitted to microbiological tests and assayed for the effect on wound healing in wounds clean induced on the back of rats. SfP was used as a possible vehicle for the administration of PcL and compared to Carbopol 940 (C). The gels (0.9%) were analyzed rheological and then applied to the lesions during a treatment period of 10 days, using kollagenase  as control. The healing process was evaluated on the size of the wounds, levels of MPO and histological analysis. The molecule SfP and PcL is not toxic for the parameters of body weight, organ and biochemical measurements. However, the histological analysis showed minor changes in liver and kidney. PcL (1, 3 and 9 mg / kg, i.v) reduced the edema induced by carrageenan and its inhibitor when administered with mucin was not possible to check the reduction of edema which was confirmed by measurement of MPO. The two molecules were used in microbiological assays and not inhibit growth of any microorganism tested and unable to use SfP as carbon source. The rheological analysis showed that the SfP used in the formulation of the gels (PcL+SfP and SfP) had the characteristic of a pseudoplastic. Macroscopic analysis of wounds showed a reduction in lesion area in the animals treated with PCL, PCL+SfP, PCL+C (53.5 and 60% respectively) on the sixth day of administration. In histological analysis, there was no severe inflammatory infiltrate in the tissues obtained until 4th day of administration of the gels (PcL and PcL+SfP, PcL+C) and Kollagenase (positive control). On day 6, the untreated animals and those treated only with SfP showed inflammatory infiltrate. The measurement of MPO showed a reduction in the inflammatory process in the samples containing PcL, whose results corroborate the histological analysis. In conclusion, PcL aid in wound repair, suggesting its use as a possible future tool for the treatment of lesions. The biological and pharmacological role of lectins and polysaccharides of seaweed is part of a study area little explored, where a lot of knowledge should be invested since these biomolecules can be promising for the pharmaceutical industry.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2010 / A 29 kDa lectin that shows specificity for galactose was isolated from Lonchocarpus capassa seeds by a combination of ammonium sulphate precipitation and affinity chromatography on a galactose-sepharose column. The 29 kDa lectin subunit co-purified with a 45 kDa subunit. The N-terminal sequence of the 29 kDa subunit showed homology to other legume lectins while that of 45 kDa subunit was capped. A 360 bp fragment was amplified using degenerate primers designed from internal protein sequences of the 29 kDa subunit and a 5´ RACE system primer. The cDNA fragment was cloned into pTz57R/Tvector and transformed into E. coli. The partial amino acid sequence of the lectin subunit was deduced from the nucleotide sequence of the clone. The 360 bp fragment consisted of 342 bp sequence coding for the start codon, leader sequence, N-Terminal sequence and sequences of the 79 amino acids from N-terminus. Comparison of the deduced amino acid sequence with other legume lectins showed regions of sequence homology with precursor sequences of Robinia pseudoacacia Bark lectin, a non seed lectin from Pisum sativum (pea), and the galactose specific peanut agglutinin (PNA) from Arachis hypogaea. Alignment of these sequences showed conserved regions including the metal binding sites found in all legume lectins. The 5´ end DNA sequence was used to design locus-specific primers which were used with genome walking cassette primers in an attempt to amplify the full L. capassa lectin gene. The cassette primers were designed from restriction enzyme sites on the cassette. Of all the restriction enzymes on the cassette Hind III and the L. capassa gene-specific primers amplified 288 bp of the 342 bp sequence already obtained from sequencing of the cDNA sequence with minor amino acid differences. Although the full lectin sequence was not obtained the study confirmed the presence of a galactose-specific lectin in L. capassa seeds.

"May 2003" / Bibliography: leaves 72-87. / iii, 87 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2003