| dc.description.abstract | Decades long studies on plant lectins carried out in this laboratory have contributed substantially to glycobiology and helped in the initiation and development of macromolecular crystallography in India. Subsequently the studies were extended to microbial lectins. It was realised that no lectin from archea has been thoroughly characterised. A genomic bioinformatic search led to the identification of several lectins from archea (Chapter 1).
An archeal lectin from Methanococcus voltae A3, christened Mevo lectin, has been cloned, expressed and purified. Crystallographic and solution studies of Mevo lectin and its complexes, the first effort of its kind on an archeal lectin, reveal a structure similar to β-prism I fold lectins from plant and animal sources, but with a quaternary association involving a ring structure with seven-fold symmetry. Each subunit in the heptamer carries one sugar binding site on the first Greek Key motif. The oligomeric interface is primarily made up of a parallel β-sheet involving a strand of Greek Key I of one subunit and Greek Key ΙΙΙ from a neighboring subunit. The crystal structures of the complexes of the lectin with mannose, αMan(1,2)αMan, αMan(1,3)αMan, a mannotriose and a mannopentose revealed a primary binding site similar to that found in other mannose specific β-prism I fold lectins. The complex with αMan(1,3)αMan provides an interesting case in which a few subunits have the reducing end at the primary binding site, while the majority have the nonreducing end at the primary binding site. The structures of complexes involving the trisaccharide and the pentasaccharide exhibit cross-linking among heptameric molecules. The observed arrangements may be relevant to the multivalency of the lectin. Phylogenetic analysis of amino acid sequences indicates that Mevo lectin is closer to β-prism I fold animal lectins than with those of plant origin. The results presented here reinforce the conclusion regarding the existence of lectins in all three domains of life. It would also appear that lectins evolved to the present form before the three domains diverged (Chapter 2).
Mannose-binding lectins can specifically recognize and bind complex glycan structures on pathogens and have potential as antiviral and antibacterial agents. Mevo lectin has specificity toward terminal α1,2 linked manno-oligosaccharides. Mycobacterium tuberculosis (M. tuberculosis) expresses mannosylated structures including lipoarabinomannan (ManLAM) on its surface and exploits C-type lectins to gain entry into the host cells. ManLAM structure has mannose capping with terminal αMan(1,2)αMan residues and is important for recognition by innate immune cells. Here, we aim to address the specificity of Mevo lectin toward high-mannose type glycans with terminal αMan(1,2)αMan residues and its effect on M. tuberculosis internalization by macrophages. Isothermal titration calorimetry (ITC) studies demonstrated that Mevo lectin shows preferential binding toward manno-oligosaccharides with terminal αMan(1,2)αMan structures and showed a strong affinity for ManLAM, whereas it binds weakly to Mycobacterium smegmatis lipoarabinomannan, which displays relatively fewer and shorter mannosyl caps. Crystal structure of Mevo lectin complexed with a Man7D1 revealed the multivalent cross-linking interaction, which explains avidity-based high-affinity for these ligands when compared to previously studied manno-oligosaccharides lacking the specific termini. Functional studies suggest that M. tuberculosis internalization by the macrophage was impaired by binding of Mevo lectin to ManLAM present on the surface of M. tuberculosis. Selectivity shown by Mevo lectin toward glycans with terminal αMan(1,2)αMan structures, and its ability to compromise the internalization of M. tuberculosis in vitro, underscore the potential utility of Mevo lectin as a research tool to study host-pathogen interactions (Chapter 3).
As mentioned earlier, Mevo lectin belongs to a highly conserved β-prism I fold lectin family and contains a single carbohydrate binding motif (132GXXXD136) on Greek Key I. Structural studies on complexes of mannose and mannose containing sugars with the lectin established that both Asp 134 and Asp 136 (part of conserved carbohydrate binding motif), are involved in the binding to carbohydrates. Asp 134 plays an important role in determining the specificity and affinity towards manno-oligosaccharides with αMan(1,2)αMan at the non-reducing end. To further elucidate the mechanism of the carbohydrate binding by Mevo lectin, two single mutants (D134A and D136A) and one double-mutant (D134/136A) were generated by site-directed mutagenesis. Analytical gel filtration results showed that all three mutants exhibited similar oligomeric state as the native lectin. X-ray crystallographic studies of Mevo lectin mutants revealed no major structural differences between the native lectin and the mutants. Binding analysis of the mutants by ITC indicated that Asp 136 is indispensable for the carbohydrate binding of Mevo lectin, whereas the D134A mutant retained its monosaccharide binding with reduced affinity compared to the native lectin. However, binding to manno-oligosaccharides having αMan(1,2)αMan at the non-reducing ends, such as mannotetrose, mannoheptose and ManLAM reduced very substantially. As expected, D134/136A double-mutant completely lost its carbohydrate-binding activity. These results suggest that Asp 136 is irreplaceable and Asp 134 plays an important role in determining the specificity and affinity of Mevo lectin to manno-oligosaccharides with terminal αMan(1,2)αMan residues and ManLAM (Chapter 4). It would appear that jacalin-like lectins or lectin domains with an aspartyl residue at the position corresponding to 134 are likely to interfere with uptake of M. tuberculosis by macrophages. | en_US |
