Nikola Kostlánová

The Fucose-Binding Lectin from Ralstonia Solanacearum: A New Type of Beta-Propeller Architecture Formed by Oligomerization and Interacting with Fucoside, Fucosyllactose, and Plant Xyloglucan.

Plant pathogens, like animal ones, use protein-carbohydrate interactions in their strategy for host recognition, attachment, and invasion. The bacterium Ralstonia solanacearum, which is distributed worldwide and causes lethal wilt in many agricultural crops, was shown to produce a potent l-fucose-binding lectin, R. solanacearum lectin, a small protein of 90 amino acids with a tandem repeat in its amino acid sequence. In the present study, surface plasmon resonance experiments conducted on a series of oligosaccharides show a preference for binding to αFuc1-2Gal and αFuc1-6Gal epitopes. Titration microcalorimetry demonstrates the presence of two binding sites per monomer and an unusually high affinity of the lectin for αFuc1-2Gal-containing oligosaccharides (KD = 2.5 × 10-7 m for 2-fucosyllactose). R. solanacearum lectin has been crystallized with a methyl derivative of fucose and with the highest affinity ligand, 2-fucosyllactose. X-ray crystal structures, the one with α-methyl-fucoside being at ultrahigh resolution, reveal that each monomer consists of two small four-stranded anti-parallel β-sheets. Trimerization through a 3-fold or pseudo-3-fold axis generates a six-bladed β-propeller architecture, very similar to that previously described for the fungal lectin of Aleuria aurantia. This is the first report of a β-propeller formed by oligomerization and not by sequential domains. Each monomer presents two fucose binding sites, resulting in six symmetrically arranged sugar binding sites for the β-propeller. Crystals were also obtained for a mutated lectin complexed with a fragment of xyloglucan, a fucosylated polysaccharide from the primary cell wall of plants, which may be the biological target of the lectin.

A New Ralstonia Solanacearum High-Affinity Mannose- Binding Lectin Rs-Iil Structurally Resembling the Pseudomonas Aeruginosa Fucose-Specific Lectin Pa- Iil.

The plant pathogen Ralstonia solanacearum produces two lectins, each with different affinity to fucose. We described previously the properties and sequence of the first lectin, RSL (subunit Mr 9.9 kDa), which is related to fungal lectins (Sudakevitz, D., Imberty, A., and Gilboa‐Garber, N., 2002, J Biochem 132: 353–358). The present communication reports the discovery of the second one, RS‐IIL (subunit Mr 11.6 kDa), a tetrameric lectin, with high sequence similarity to the fucose‐binding lectin PA‐IIL of Pseudomonas aeruginosa. RS‐IIL recognizes fucose but displays much higher affinity to mannose and fructose, which is opposite to the preference spectrum of PA‐IIL. Determination of the crystal structure of RS‐IIL complexed with a mannose derivative demonstrates a tetrameric structure very similar to the recently solved PA‐IIL structure (Mitchell, E., et al., 2002, Nature Struct Biol 9: 918–921). Each monomer contains two close calcium cations that mediate the binding of the monosaccharide and explain the outstandingly high affinity to the monosaccharide ligand. The binding loop of the cations is fully conserved in RS‐IIL and PA‐IIL, whereas the preference for mannose versus fucose can be attributed to the change of a three‐amino‐acid sequence in the ‘specificity loop’.

A soluble fucose-specific lectin from Aspergillus fumigatus conidia - Structure, specificity and possible role in fungal pathogenicity

Aspergillus fumigatus is an important allergen and opportunistic pathogen. Similarly to many other pathogens, it is able to produce lectins that may be involved in the host-pathogen interaction. We focused on the lectin AFL, which was prepared in recombinant form and characterized. Its binding properties were studied using hemagglutination and glycan array analysis. We determined the specificity of the lectin towards l-fucose and fucosylated oligosaccharides, including α1-6 linked core-fucose, which is an important marker for cancerogenesis. Other biologically relevant saccharides such as sialic acid, d-mannose or d-galactose were not bound. Blood group epitopes of the ABH and Lewis systems were recognized, LeY being the preferred ligand among others. To provide a correlation between the observed functional characteristics and structural basis, AFL was crystallized in a complex with methyl-α,l-selenofucoside and its structure was solved using the SAD method. Six binding sites, each with different compositions, were identified per monomer and significant differences from the homologous AAL lectin were found. Structure-derived peptides were utilized to prepare anti-AFL polyclonal antibodies, which suggested the presence of AFL on the Aspergillus’ conidia, confirming its expression in vivo. Stimulation of human bronchial cells by AFL led to IL-8 production in a dose-dependent manner. AFL thus probably contributes to the inflammatory response observed upon the exposure of a patient to A. fumigatus. The combination of affinity to human epithelial epitopes, production by conidia and pro-inflammatory activity is remarkable and shows that AFL might be an important virulence factor involved in an early stage of A. fumigatus infection.