Architecture and Evolution of Blade Assembly in β-propeller Lectins

International audienceLectins with a β-propeller fold bind glycans on the cell surface through multivalent binding sites and appropriate directionality. These proteins are formed by repeats of short domains, raising questions about evolutionary duplication. However, these repeats are difficult to detect in translated genomes and seldom correctly annotated in sequence databases. To address these issues, we defined the blade signature of the five types of β-propellers using 3D-structural data. With these templates, we predicted 3887 β-propeller lectins in 1889 species and organised this new information in a searchable online database. The data reveals a widespread distribution of β-propeller lectins across species. Prediction also emphasises multiple architectures and led to uncover a novel β-propeller assembly scenario. This was confirmed by producing and characterizing a predicted protein coded in the genome of Kordia zhangzhouensis. The crystal structure shows a new intermediate in the evolution of β-propeller assembly and demonstrates the power of our tool

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, Le(Y) 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

Synergism of the Two Myb Domains of Tay1 Protein Results in High Affinity Binding to Telomeres

Double-stranded regions of the telomeres are recognized by proteins containing Myb-like domains conferring specificity toward telomeric repeats. Although biochemical and structural studies revealed basic molecular principles involved in DNA binding, relatively little is known about evolutionary pathways leading to various types of Myb domain-containing proteins in divergent species of eukaryotes. Recently we identified a novel type of telomere-binding protein YlTay1p from the yeast Yarrowia lipolytica containing two Myb domains (Myb1, Myb2) very similar to the Myb domain of mammalian TRF1 and TRF2. In this study we prepared mutant versions of YlTay1p lacking Myb1, Myb2, or both Myb domains and found that YlTay1p carrying either Myb domain exhibits preferential affinity to both Y. lipolytica (GGGTTAGTCA)n and human (TTAGGG)n telomeric sequences. Quantitative measurements of the protein binding to telomeric DNA revealed that the presence of both Myb domains is required for a high affinity of YlTay1p to either telomeric repeat. Additionally, we performed detailed thermodynamic analysis of the YlTay1p interaction with its cognate telomeric DNA, which is to our knowledge the first energetic description of a full-length telomeric-protein binding to DNA. Interestingly, when compared with human TRF1 and TRF2 proteins, YlTay1p exhibited higher affinity not only for Y. lipolytica telomeres but also for human telomeric sequences. The duplication of the Myb domain region in YlTay1p thus produces a synergistic effect on its affinity toward the cognate telomeric sequence, alleviating the need for homodimerization observed in TRF-like proteins possessing a single Myb domain

Structural insights into Aspergillus fumigatus lectin specificity: AFL binding sites are functionally non-equivalent

International audienceThe Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications

Heptabladed β‐propeller lectins PLL2 and PHL from Photorhabdus spp. recognize O ‐methylated sugars and influence the host immune system

O‐methylation is an unusual sugar modification with a function that is not fully understood. Given its occurrence and recognition by lectins involved in the immune response, methylated sugars were proposed to represent a conserved pathogen‐associated molecular pattern. We describe the interaction of O‐methylated saccharides with two β‐propeller lectins, the newly described PLL2 from the entomopathogenic bacterium Photorhabdus laumondii, and its homologue PHL from the related human pathogen Photorhabdus asymbiotica. The crystal structures of PLL2 and PHL revealed up to 10 out of 14 potential binding sites per protein subunit to be occupied with O‐methylated structures. The avidity effect strengthens the interaction by 4 orders of magnitude. PLL2 and PHL also interfere with the early immune response by modulating the production of reactive oxygen species and phenoloxidase activity. Since bacteria from Photorhabdus spp. have a complex life cycle involving pathogenicity towards different hosts, the involvement of PLL2 and PHL might contribute to the pathogen overcoming insect and human immune system defences in the early stages of infection

X-ray Structures and Thermodynamics of the Interaction of PA-IIL from Pseudomonas aeruginosa with Disaccharide Derivatives

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Structural basis for mannose recognition by alectin from opportunistic bacteria Burkholderia cenocepacia

International audienceChronic colonization of the lungs by opportunist bacteria such as Pseudomonas aeruginosa and members of the Burkholderia cepacia complex (Bcc) is the major cause of morbidity and mortality among cystic fibrosis (CF) patients. PA-IIL (lecB gene), a soluble lectin from P. aeruginosa, has been the subject of much interest because of its very strong affinity for fucose. Orthologs have been identified in the opportunist bacteria Ralstonia solanacearum, Chromobacterium violaceum and Burkholderia of Bcc. The genome of the J2315 strain of B. cenocepacia, responsible for epidemia in CF centers, contains three genes that code for proteins with PA-IIL domains. The shortest gene was cloned in Escherichia coli and pure recombinant protein, BclA, obtained. The presence of native BclA in B. cenocepacia extracts was checked by proteomics approach. The specificity of recombinant BclA was characterized using surface plasmon resonance showing a preference for mannosides and supported with glycan array experiments demonstrating a strict specificity for oligomannose-type N-glycan structures. The interaction thermodynamics of BclA with α-methyl-mannoside (αMeMan) demonstrates a dissociation constant (Kd) of 2.75 x 10-6 M. The X-ray crystal structure of the complex with αMeMan was determined at 1.7Å resolution. The lectin forms homodimers with one binding site per monomer, acting cooperatively with the second dimer site. Each monomer contains two calcium ions and one sugar ligand. Despite strong sequence similarity, the differences between BclA and PA-IIL in their specificity, binding site, and oligomerisation mode indicate that the proteins should have different roles in the bacteria

Structural insights into Aspergillus fumigatus

International audienceThe Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications

Glycan array results for AFL interaction with biologically important oligosaccharides.

<p>The bars show all of an oligosaccharide’s average relative binding above 10 % (see Methods). Immobilized Fuc response given for comparison. BG stands for blood group. Oligosaccharides with mixed epitopes marked with asterisk. Oligosaccharide structures are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083077#pone.0083077.s008" target="_blank">Table S2</a> and structures of top binders of each group are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083077#pone.0083077.s004" target="_blank">Figure S4</a>.</p

3D structure of AFL complexed with MeSeFuc.

<p>AFL monomer (chain A) overall architecture with individual blades and binding sites labelled (A) and side-view of AFL dimer (chains A and B) with intermonomer contacts shown (B). MeSeFuc ligands are shown as purple sticks. AFL protein sequence with secondary structure elements highlighted in colors according to panel A (C). </p