CV-IIL: New lectin from Chromobacterium violaceum
| Authors | |
|---|---|
| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | Chemica 43S |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | lectin; pathogen; crystal; Chromobacterium |
| Description | Bacterium Chromobacterium violaceum, a gram-negative saprophyte from soil and water, is usually considered non-pathogenic to human. However, infections in animals, including human, can be quite varied, ranging from mild diarrhoea to septicaemia leading to a rapid death. This bacterium has been found to be highly abundant in the water and borders of the Negro river, a major component of the Brazilian Amazon. It produces the violacein pigment, which exhibits an antimicrobial activity particularly against soil amoebae and trypanosomes. Because of its pharmaceutical interest, C. violaceum genome has been fully sequenced by the Brazilian National Genome Project Consortium [1]. The genome contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, probably involved in the occasional but often fatal cases of human C. violaceum infection. Homology search in the C. violaceum genome revealed that gene cv1741 displays homology with gene lecB from human pathogen Pseudomonas aeruginosa. Product of the gene lecB is the fucose-binding lectin PA-IIL that can play a crucial role in adhesion and specific recognition of a host by the pathogen and contributes to its virulence [2]. Similar properties of chromobacterial protein CV-IIL, the gene product of cv1741, could be expected. The recombinant CV-IIL protein has been prepared for structural and biochemical characterisation. The cv1741 gene was cloned into pET25b vector, and the protein expressed in E. coli TUNER (DE3) cells has been purified by affinity chromatography on Mannose-agarose. Purification yielded 25 mg of pure protein CV-IIL per litre of cultivation media and MS analysis confirmed purity and molecular mass of the obtained product. Competitive binding assays using ELLA methodology showed that CV-IIL displays high affinity towards L-fucose and D-mannose. Crystals of CV-IIL/fucose and CV-IIL/mannose complexes have been grown using PEG precipitants. Diffraction data have been measured at ESRF and structures have been solved at 1.1 Ĺ resolution. Analysis of the binding sites allows to rationalize the high affinity of the lectin for monosaccharides.. |
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