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Bacterial adhesion to human epithelia via lectins constitutes a therapeutic opportunity to prevent infection. Specifically, BambL (the lectin from Burkholderia ambifaria) is implicated in cystic fibrosis, where lectin-mediated bacterial adhesion to fucosylated lung epithelia is suspected to play an important role. We employed structure-based virtual screening to identify inhibitors of BambL-saccharide interaction with potential therapeutic value. To enable such discovery, a virtual screening protocol was iteratively developed via 194 retrospective screening protocols against 4 bacterial lectins (BambL, BC2L-A, FimH, and LecA) with known ligands. Specific attention was given to the rigorous evaluation of retrospective screening, including calculation of analytical errors for enrichment metrics. The developed virtual screening workflow used crystallographic constraints, pharmacophore filters, and a final manual selection step. The protocol was applied to BambL, predicting 15 active compounds from virtual libraries of approximately 7 million compounds. Experimental validation using fluorescence polarization confirmed micromolar inhibitory activity for two compounds, which were further characterized by isothermal titration calorimetry and surface plasmon resonance. Subsequent testing against LecB from Pseudomonas aeruginosa demonstrated binding specificity of one of the hit compounds. This report demonstrates the utility of virtual screening protocols, integrating ligand-based pharmacophore filtering and structure-based constraints, in the search for bacterial lectin inhibitors.

Chronic infections with Pseudomonas aeruginosa are associated with the formation of bacterial biofilms. The tetrameric P. aeruginosa lectin LecA is a virulence factor and an anti-biofilm drug target. Increasing the overall binding affinity by multivalent presentation of binding epitopes can enhance the weak carbohydrate-ligand interactions. Low-nanomolar divalent LecA ligands/inhibitors with up to 260-fold valency-normalized potency boost and excellent selectivity over human galectin-1 were synthesized from d-galactose pentaacetate and benzaldehyde-based linkers in four linear steps.

Summary Carbohydrate-binding proteins from pathogenic bacteria and fungi have been shown to be implicated in various pathological processes, where they interact with glycans present on the surface of the host cells. These interactions are part of the initial processes of infection of the host and are very important to study at the atomic level. Here, we report the room temperature neutron structures of PLL lectin from Photorhabdus laumondii in its apo form and in complex with deuterated L-fucose, which is, to our knowledge, the first neutron structure of a carbohydrate-binding protein in complex with a fully deuterated carbohydrate ligand. A detailed structural analysis of the lectin-carbohydrate interactions provides information on the hydrogen bond network, the role of water molecules, and the extent of the CH-π stacking interactions between fucose and the aromatic amino acids in the binding site.

International audience; Pseudomonas aeruginosa is an opportunistic ESKAPE pathogen that produces two lectins, LecA and LecB, as part of its large arsenal of virulence factors. Both carbohydrate-binding proteins are central to the initial and later persistent infection processes, i.e. bacterial adhesion and biofilm formation. The biofilm matrix is a major resistance determinant and protects the bacteria against external threats such as the host immune system or antibiotic treatment. Therefore, the development of drugs against the P. aeruginosa biofilm is of particular interest to restore efficacy of antimicrobials. Carbohydrate-based inhibitors for LecA and LecB were previously shown to efficiently reduce biofilm formations. Here, we report a new approach for inhibiting LecA with synthetic molecules bridging the established carbohydrate-binding site and a central cavity located between two LecA protomers of the lectin tetramer. Inspired by in silico design, we synthesized various galactosidic LecA inhibitors with aromatic moities targeting this central pocket. These compounds reached low micromolar affinities, validated in different biophysical assays. Finally, X-ray diffraction analysis revealed the interactions of this compound class with LecA. This new mode of action paves the way to a novel route towards inhibition of P. aeruginosa biofilms.

All eukaryotic cells are covered with a dense layer of glycoconjugates, and the cell walls of bacteria are made of various polysaccharides, putting glycans in key locations for mediating protein-protein interactions at cell interfaces. Glycan function is therefore mainly defined as binding to other molecules, and lectins are proteins that specifically recognize and interact non-covalently with glycans. UniLectin was designed based on insight into the knowledge of lectins, their classification, and their biological role. This modular platform provides a curated and periodically updated classification of lectins along with a set of comparative and visualization tools, as well as structured results of screening comprehensive sequence datasets. UniLectin can be used to explore lectins, find precise information on glycan-protein interactions, and mine the results of predictive tools based on HMM profiles. This usage is illustrated here with two protocols. The first one highlights the fine-tuned role of the O blood group antigen in distinctive pathogen recognition, while the second compares the various bacterial lectin arsenals that clearly depend on living conditions of species even in the same genus. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Searching for the structural details of lectins binding the O blood group antigen Basic Protocol 2: Comparing the lectomes of related organisms in different environments.

SummaryThe opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.

Glycolipids are present on the surfaces of all living cells and thereby represent targets for many protein receptors, such as lectins. Understanding the interactions between lectins and glycolipids is essential for investigating the functions of lectins and the dynamics of glycolipids in living membranes. This review focuses on lectins binding to the glycosphingolipid globotriaosylceramide (Gb3), an attractive host cell receptor, particularly for pathogens and pathogenic products. Shiga toxin (Stx), from Shigella dysenteriae or Escherichia coli, which is one of the most virulent bacterial toxins, binds and clusters Gb3, leading to local negative membrane curvature and the formation of tubular plasma membrane invaginations as the initial step for clathrin-independent endocytosis. After internalization, it is embracing the retrograde transport pathway. In comparison, the homotetrameric lectin LecA from Pseudomonas aeruginosa can also bind to Gb3, triggering the so-called lipid zipper mechanism, which results in membrane engulfment of the bacterium as an important step for its cellular uptake. Notably, both lectins bind to Gb3 but induce distinct plasma membrane domains and exploit mainly different transport pathways. Not only, several other Gb3-binding lectins have been described from bacterial origins, such as the adhesins SadP (from Streptococcus suis) and PapG (from E. coli), but also from animal, fungal, or plant origins. The variety of amino acid sequences and folds demonstrates the structural versatilities of Gb3-binding lectins and asks the question of the evolution of specificity and carbohydrate recognition in different kingdoms of life.

Because of the antimicrobial resistance crisis, lectins are considered novel drug targets. Pseudomonas aeruginosa utilizes LecA and LecB in the infection process. Inhibition of both lectins with carbohydrate‐derived molecules can reduce biofilm formation to restore antimicrobial susceptibility. Here, we focused on non‐carbohydrate inhibitors for LecA to explore new avenues for lectin inhibition. From a screening cascade we obtained one experimentally confirmed hit, a catechol, belonging to the well‐known PAINS compounds. Rigorous analyses validated electron‐deficient catechols as millimolar LecA inhibitors. The first co‐crystal structure of a non‐carbohydrate inhibitor in complex with a bacterial lectin clearly demonstrates the catechol mimicking the binding of natural glycosides with LecA. Importantly, catechol 3 is the first non‐carbohydrate lectin ligand that binds bacterial and mammalian calcium(II)‐binding lectins, giving rise to this fundamentally new class of glycomimetics., A screening yields the first non‐carbohydrate small molecules mimicking the interaction of carbohydrates in the binding sites of bacterial lectins. The catechols, known as PAINS, were carefully validated in numerous biophysical assays. A crystal structure in complex with Pseudomonas aeruginosa LecA and NMR analyses with mammalian C‐type lectin Langerin prove the catechol moiety as a general replacement motif for carbohydrates in calcium(II)‐binding lectins.

Fungal lectins are a large family of glycan-binding proteins, with no enzymatic activity. They play fundamental biological roles in the interactions of fungi with their environment and are found in many different species throughout the fungal kingdom. In particular, their contribution to defence against feeders has been emphasized and extracellular lectins may be involved in the recognition of bacteria, fungal competitors and specific host plants. Their carbohydrate specificities and quaternary structures vary widely, but evidence for an evolutionary relationship within the different classes of lectins is provided by the high degree of amino acid sequence identity shared by the different fungal lectins. The UniLectin3D database contains 194 3D structures of fungal lectins, of which 129 are characterized with their carbohydrate ligand. UniLectin3D lectin classes from all origins were used to construct 107 lectin motifs in 26 folding configurations and to screen 1,223 species deposited in the genomic portal MycoCosm of the Joint Genome Institute. The resulting 33 485 protein sequences of putative lectins are organized in MycoLec, a publicly available and searchable database. The characterization of the lectin candidates in fungal genomes is based on systematic statistics regarding potential carbohydrate ligands, protein lengths, signal peptides, relative motif positions and amino acid compositions of fungal lectins. These results shed light on the evolution of the lectin gene families.

Carbohydrate-binding proteins (lectins) are auspicious targets in drug discovery to combat antimicrobial resistance; however, its non-carbohydrate drug-like inhibitors are still spacious. Here, we present a druggable pocket in a β-propeller lectin BambL from Burkholderia ambifaria as a potential target for allosteric inhibitors. This site was identified employing 19 F NMR fragment screening and a computational pocket prediction algorithm SiteMap. The structure-activity relationship study revealed the most promising fragment with a dissociation constant of 0.3±0.1 mM and a ligand efficiency of 0.3 kcal mol-1 HA-1 that affected the orthosteric site. This effect was substantiated by site-directed mutagenesis in the orthosteric and secondary pockets. Future drug-discovery campaigns that aim to develop small molecule inhibitors can benefit from allosteric sites in lectins as a new therapeutic approach against antibiotic-resistant pathogens.

Lectins are non-covalent glycan-binding proteins mediating cellular interactions but their annotation in newly sequenced organisms is lacking. The limited size of functional domains and the low level of sequence similarity challenge usual bioinformatics tools. The identification of lectin domains in proteomes requires the manual curation of sequence alignments based on structural folds. A new lectin classification is proposed. It is built on three levels: (i) 35 lectin domain folds, (ii) 109 classes of lectins sharing at least 20% sequence similarity and (iii) 350 families of lectins sharing at least 70% sequence similarity. This information is compiled in the UniLectin platform that includes the previously described UniLectin3D database of curated lectin 3D structures. Since its first release, UniLectin3D has been updated with 485 additional 3D structures. The database is now complemented by two additional modules: PropLec containing predicted β-propeller lectins and LectomeXplore including predicted lectins from sequences of the NBCI-nr and UniProt for every curated lectin class. UniLectin is accessible at https://www.unilectin.eu/

Burkholderia cenocepacia is an opportunistic Gram‐negative bacterium that causes infections in patients suffering from chronic granulomatous diseases and cystic fibrosis. It displays significant morbidity and mortality due to extreme resistance to almost all clinically useful antibiotics. The bacterial lectin BC2L‐C expressed in B. cenocepacia is an interesting drug target involved in bacterial adhesion and subsequent deadly infection to the host. We solved the first high resolution crystal structure of the apo form of the lectin N‐terminal domain (BC2L‐C‐nt) and compared it with the ones complexed with carbohydrate ligands. Virtual screening of a small fragment library identified potential hits predicted to bind in the vicinity of the fucose binding site. A series of biophysical techniques and X‐ray crystallographic screening were employed to validate the interaction of the hits with the protein domain. The X‐ray structure of BC2L‐C‐nt complexed with one of the identified active fragments confirmed the ability of the site computationally identified to host drug‐like fragments. The fragment affinity could be determined by titration microcalorimetry. These structure‐based strategies further provide an opportunity to elaborate the fragments into high affinity anti‐adhesive glycomimetics, as therapeutic agents against B. cenocepacia., A new druggable site for anti‐adhesive therapy. We used fragment‐based virtual screening to explore the druggability of a region in the vicinity of the fucose binding site in a lectin from an opportunistic and highly drug‐resistant pathogen. The crystal structure of the target protein complexed with the lead fragment validated the existence of a secondary binding site, paving the way for the design of potent ligands to be employed in anti‐adhesive therapy.

Pathogenic micro-organisms utilize protein receptors in adhesion to host tissues, a process that in some cases relies on the interaction between lectin and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analyse by X-ray crystallography the structures of the lectin LecB from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 2% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven as especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we design general protocols which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.

International audience; Glycosaminoglycans (GAGs) are complex linear polysaccharides. GAG-DB is a curated database that classifies the three-dimensional features of the six mammalian GAGs (chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronan, and keratan sulfate) and their oligosaccharides complexed with proteins. The entries are structures of GAG and GAG-protein complexes determined by X-ray single-crystal diffraction methods, X-ray fiber diffractometry, solution NMR spectroscopy, and scattering data often associated with molecular modeling. We designed the database architecture and the navigation tools to query the database with the Protein Data Bank (PDB), UniProtKB, and GlyTouCan (universal glycan repository) identifiers. Special attention was devoted to the description of the bound glycan ligands using simple graphical representation and numerical format for cross-referencing to other databases in glycoscience and functional data. GAG-DB provides detailed information on GAGs, their bound protein ligands, and features their interactions using several open access applications. Binding covers interactions between monosaccharides and protein monosaccharide units and the evaluation of quaternary structure. GAG-DB is freely available.

Lectins, such as adhesins and toxins, are carbohydrate-binding proteins that recognise glycans of cells and their secretions. While mediation of microbe-microbe and microbe-host interactions by lectins has long been recognised in the lung and gut, little is known about those in the vagina, where such interactions are implicated in health and various disease states. These include sexually transmitted infections, cervical cancer and poor pregnancy outcomes such as preterm birth. In this study, the curated UniLectin3D database was used to establish a lectin classification based primarily on taxonomy and protein 3D structure. The resulting 109 lectin classes were characterised by specific Hidden Markov Model (HMM) profiles. Screening of microbial genomes in the UniProt and NCBI NR sequence databases resulted in identification of >100 000 predicted bacterial lectins available at unilectin.eu/bacteria. Screening of the complete genomes of 90 isolates from 21 vaginal bacterial species showed that the predicted lectomes (ensemble of predicted lectins) of Lactobacilli associated with vaginal health are substantially less diverse than those of pathogens and pathobionts. Both the number of predicted bacterial lectins, and their specificities for carbohydrates correlated with pathogenicity. This study provides new insights into potential mechanisms of commensal and pathogen colonisation of the reproductive tract that underpin health and disease states.

Two orthogonal, metal free click reactions, enabled to glycosylate ubiquitin and its mutant A28C forming two protein scaffolds with high affinity for BambL, a lectin from the human pathogen Burkholderia ambifaria. A new fucoside analogue, with high affinity with BambL, firstly synthetized and co-crystallized with the protein target, provided the insights for sugar determinants grafting onto ubiquitin. Three ubiquitin-based glycosides were thus assembled. Fuc-Ub, presented several copies of the fucoside analogue, with proper geometry for multivalent effect; Rha-A28C, displayed one thio-rhamnose, known for its ability to tuning the immunological response; finally, Fuc-Rha-A28C, included both multiple fucoside analogs and the rhamnose residue. Fuc-Ub and Fuc-Rha-A28C ligands proved high affinity for BambL and unprecedented immune modulatory properties towards macrophages activation., Metal free click reactions used to glycosylate ubiquitin and its mutant A28C afforded two protein scaffolds with high affinity for Burkholderia ambifaria lectin (BambL).

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes of nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interactions is not well understood. The significance of our research is in identifying cellular mechanisms of how LecB facilitates P. aeruginosa infection. We introduce LecB as a new member of the list of bacterial molecules that bind integrins and show that P. aeruginosa can move forward underneath attached epithelial cells by loosening cell-basement membrane attachment in a LecB-dependent manner., The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as a virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as a cross-linker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and causes rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin–laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and causes the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by cross-linking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild-type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results, we suggest that LecB has a role in disseminating bacteria along the cell-basement membrane interface.

Selection from dynamic combinatorial libraries (DCL) benefit from the dynamic nature of the library that can change constitution upon addition of a selection pressure, such as ligands binding to a protein. This technology has been predominantly used with small molecules interacting with each other through reversible covalent interaction. However, application of this technology in biomedical research and drug discovery has been limited by the reversibility of covalent exchange and the analytical deconvolution of small molecule fragments. Here we report a supramolecular approach based on the use of a constant short PNA tag to direct the combinatorial pairing of fragment. This PNA tag yields fast exchange kinetics, while still delivering the benefits of cooperativity, and provides favourable properties for analytical deconvolution by MALDI. A selection of > 6 000 assemblies of glycans (mono-, di-, tri-saccharides) targeting AFL, a lectin from pathogenic fungus, yielded a 95 nM assembly, nearly three orders of magnitude better affinity than the corresponding glycan alone (41 µM).

The search for new biomolecules requires a clear understanding of biosynthesis and degradation pathways. This view applies to most metabolites as well as other molecule types such as glycans whose repertoire is still poorly characterized. Lectins are proteins that recognize specifically and interact noncovalently with glycans. This particular class of proteins is considered as playing a major role in biology. Glycan-binding is based on multivalence, which gives lectins a unique capacity to interact with surface glycans and significantly contribute to cell-cell recognition and interactions. Lectins have been studied for many years using multiple technologies and part of the resulting information is available online in databases. Unfortunately, the connectivity of these databases with the most popular omics databases (genomics, proteomics, and glycomics) remains limited. Moreover, lectin diversity is extended and requires setting out a flexible classification that remains compatible with new sequences and 3D structures that are continuously released. We have designed UniLectin as a new insight into the knowledge of lectins, their classification, and their biological role. This platform encompasses UniLectin3D, a curated database of lectin 3D structures that follows a periodically updated classification, a set of comparative and visualizing tools and gradually released modules dedicated to specific lectins predicted in sequence databases. The second module is PropLec, focused on β-propeller lectin prediction in all species based on five distinct family profiles. This chapter describes how UniLectin can be used to explore the diversity of lectins, their 3D structures, and associated functional information as well as to perform reliable predictions of β-propeller lectins.

LecA/PA-IL (Pfam PF07828) is a soluble galactose-binding lectin from bacterium Pseudomonas aeruginosa. The lectin is specific for α-galactose present on glycosphingolipids of the globoside family and has therefore been proposed to play a role in cell adhesion and in internalization of bacteria in epithelial cells. The lectin has also direct toxic activity. Search for high-affinity inhibitors can be performed on the recombinant lectin, with use of surface plasmon resonance assays and structural studies.

LecB/PA-IIL (Pfam PF07472) from bacterium Pseudomonas aeruginosa is a fucose-binding lectin with unusual high affinity for glycans. The occurrence of LecB and related proteins is limited to few opportunistic bacterial species, some of them being responsible for severe infections in immune-compromised patients. This lectin is therefore of interest as a target for the design of anti-infectious compounds, but can also be used for research and biotechnology. LecB is a small protein that can be produced in good quantity in recombinant system and purified by affinity chromatography.

International audience; Lectins, and related receptors such as adhesins and toxins, are glycan-binding proteins from all origins that decipher the glycocode, i.e. the structural information encoded in the conformation of complex carbohydrates present on the surface of all cells. Lectins are still poorly classified and annotated, but since their functions are based on ligand recognition, their 3D-structures provide a solid foundation for characterization. UniLectin3D is a curated database that classifies lectins on origin and fold, with cross-links to literature, other databases in glycosciences and functional data such as known specificity. The database provides detailed information on lectins, their bound glycan ligands, and features their interactions using the Protein-Ligand Interaction Profiler (PLIP) server. Special care was devoted to the description of the bound glycan ligands with the use of simple graphical representation and numerical format for cross-linking to other databases in glyco-science. We conceived the design of the database architecture and the navigation tools to account for all organisms, as well as to search for oligosaccharide epitopes complexed within specified binding sites. UniLectin3D is accessible at https://www.unilectin. eu/unilectin3D.

Dinitroanilines are chemical compounds with high selectivity for plant cell &alpha, tubulin in which they promote microtubule depolymerization. They target &alpha, tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum, which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum. These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.

International audience; Pathogens frequently rely on lectins for adhesion and cellular entry into the host. Since these interactions typically result from multimeric binding of lectins to cell surface glycans, novel therapeutic strategies are being developed with the use of glycomimetics as competitors of such interactions. Herein we study the benefit of nucleic acid-based oligomeric assemblies with PNA-fucose conjugates. We demonstrate that the interactions of a lectin with epithelial cells can be inhibited with conjugates that do not form stable assemblies in solution but benefit from the cooperativity of ligand-protein interactions and PNA hybridization to achieve high affinity. A dynamic dimeric assembly fully blocked the binding of the fucose-binding lectin BambL of Burkholderia ambifaria, a pathogenic bacterium, to epithelial cells with an efficiency of more than 700 fold compared to L-fucose.

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N-acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X-ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969-975. © 2016 Wiley Periodicals, Inc.

The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as crosslinker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and caused rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin - laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and caused the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by crosslinking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results we suggest that LecB has a role in disseminating bacteria along the cell - basement membrane interface.ImportancePseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes for nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interaction is not well understood. The significance of our research is in identifying cellular mechanisms how LecB facilitates P. aeruginosa infection: We introduce LecB as new member to the list of bacterial molecules that bind integrins and show that P. aeruginosa can efficiently move forward underneath attached epithelial cells by loosening cell - basement membrane attachment in a LecB-dependent manner.

International audience; Burkholderia pseudomallei and Chromobacterium violaceum are bacteria of tropical and subtropical soil and water that occasionally cause fatal infections in humans and animals. Microbial lectins mediate the adhesion of organisms to host cells, which is the first phase in the development of infection. Here we report the discovery of two novel lectins from the above-mentioned bacteria-BP39L and CV39L. The crystal structures revealed that the lectins possess a seven-bladed -propeller fold. Functional studies conducted on a series of oligo-and polysaccharides confirmed the preference of BP39L for mannosylated saccharides and CV39L for rather more complex polysaccharides with a monosaccharide preference for β-L-fucose. The presented data indicate that the proteins belong to a currently unknown family of lectins.

International audience; Evidence of the mediation of glycan molecules in the interaction between viruses and their hosts is accumulating and is now partially reflected in several online databases. Bioinformatics provides convenient and efficient means of searching, visualizing, comparing, and sometimes predicting, interactions in numerous and diverse molecular biology applications related to the-omics fields. As viromics is gaining momentum, bioinformatics support is increasingly needed. We propose a survey of the current resources for searching, visualizing, comparing, and possibly predicting host-virus interactions that integrate the presence and role of glycans. To the best of our knowledge, we have mapped the specialized and general-purpose databases with the appropriate focus. With an illustration of their potential usage, we also discuss the strong and weak points of the current bioinformatics landscape in the context of understanding viral infection and the immune response to it.

International audience; Bacterial lectins are typically multivalent and bind noncovalently to specific carbohydrates on host tissues to facilitate bacterial adhesion. Here, we analyzed the effects of two fucose-binding lectins, BambL from Burkholderia ambifaria and LecB from Pseudomonas aeruginosa, on specific signaling pathways in B cells. We found that these bacterial lectins induced B cell activation, which, in vitro, was dependent on the cell surface expression of the B cell antigen receptor (BCR) and its coreceptor CD19, as well as on spleen tyrosine kinase (Syk) activity. The resulting release of intracellular Ca 2+ was followed by an increase in the cell surface abundance of the activation marker CD8, increased cytokine secretion, and subsequent cell death, replicating all of the events that are observed in vitro upon canonical and antigen-mediated B cell activation. Moreover, injection of BambL in mice resulted in a substantial, BCR-independent loss of B cells in the bone marrow with simultaneous, transient enlargement of the spleen (splenomegaly), as well as an increase in the numbers of splenic B cells and myeloid cells. Together, these data suggest that bacterial lectins can initiate polyclonal activation of B cells through their sole capacity to bind to fucose.

International audience; Lectins 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 tools

International audience; Multivalent structures displaying different instead of similar sugar units, namely heteroglycoclusters (hGCs), are stimulating the efforts of glycochemists for developing compounds with new biological properties. Here we report a four-step strategy to synthesize hexadecavalent hGCs displaying eight copies of αFuc and βGal. These compounds were tested for the binding to lectins LecA and LecB from Pseudomonas aeruginosa. While parent fucosylated (19) and galactosylated (20) homoclusters present nanomolar affinity with LecB and LecA, respectively, we observed that hGCs combining these sugars (11 and 13) maintain their binding potency with both lectins despite the presence of an unspecific sugar. The added multivalency is therefore not a barrier for efficient recognition by bacterial receptors and it opens the route for adding different sugars that can be selected for their immunomodulatory properties.

International audience; The approach developed here offers a straightforward and efficient access to β-C-glycosyl barbiturate ligands, spanning from glycomimetics to multivalent C-neoglycoconjugates, with the aim of deciphering structural parameters impacting the binding to pathogenic lectins. We reinvestigated the Knoevenagel condensation of barbituratic acid on protecting-group free carbohydrates and successfully designed sodium and 5,5-disubstituted N,N-dimethyl barbiturate forms of D-galactose, L-fucose, melibiose, 2′-fucosyllactose, and maltose and evaluated their binding affinity by isothermal titration calorimetry with LecA (galactose-binding lectin) and LecB (fucose-binding lectin) from Pseudomonas aeruginosa and RSL (fucose-binding lectin) from Ralstonia solanacearum. The barbiturate ring was shown to be detrimental for binding to LecA (K D in mM range) and even more to LecB (noninteraction) while RSL is much more tolerant especially in the presence of an aromatic group (K D in μM range). However, distancing the barbiturate ring from the recognition carbohydrate residue by using oligosaccharides increased affinity up to low micromolar range. Extension of our convenient synthetic approach led in two steps to melibiose-based C-glycosyl barbiturate cluster and C-glycosyl barbiturate glycopolymers exhibiting a dramatic enhancement of binding avidity for LecA.

Multivalency plays a major role in biological processes and particularly in the relationship between pathogenic microorganisms and their host that involves protein–glycan recognition. These interactions occur during the first steps of infection, for specific recognition between host and bacteria, but also at different stages of the immune response. The search for high-affinity ligands for studying such interactions involves the combination of carbohydrate head groups with different scaffolds and linkers generating multivalent glycocompounds with controlled spatial and topology parameters. By interfering with pathogen adhesion, such glycocompounds including glycopolymers, glycoclusters, glycodendrimers and glyconanoparticles have the potential to improve or replace antibiotic treatments that are now subverted by resistance. Multivalent glycoconjugates have also been used for stimulating the innate and adaptive immune systems, for example with carbohydrate-based vaccines. Bacteria present on their surfaces natural multivalent glycoconjugates such as lipopolysaccharides and S-layers that can also be exploited or targeted in anti-infectious strategies., The authors acknowledge the support from EU COST Actions CM1102 and COST BM1003. In addition, the work reviewed has been supported by the sources listed in the original publications cited and by institutions mentioned in the affiliation list., The authors also thank the Unit of Information Resources for Research of the Spanish National Research Council (URICI-CSIC) for the co-financing of this publication in Open Access.

International audience; The design of multivalent glycoconjugates has been largely studied using a variety of core scaffolds. Cyclotriveratrylenes (CTV) have been scarcely used in this context. The synthesis of the tri- and hexa-propargylated CTV cores allowed the prepn. of the corresponding glycoclusters with galactose or fucose residues through azide-alkyne "click" chem. Their interactions with bacterial lectins (LecA, LecB and BambL) was confirmed and studied by titrn. microcalorimetry. Adsorption onto carbon nanotubes was then attempted based on the hydrophobic CTV cores. Their incorporation into field effect transistor (FET) devices was accomplished but the data collected largely suggested a poor interaction of CTV-based glycoclusters with the carbon nanotubes leading to non-specific detection of the lectins.

CBMs (carbohydrate-binding modules) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the CBM40 family, coded from a section of the gene NanI from Clostridium perfringens . Glycan arrays revealed its preference towards α(2,3)-linked sialosides, which was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)-sialyl-lactose with a K d of ∼30 μM, the highest affinity value for this class of proteins. Inspired by lectins' structure and their arrangement as multimeric proteins, we have engineered a dimeric form of the CBM, and using SPR (surface plasmon resonance) we have observed 6–11-fold binding increases due to the avidity affect. The structures of the CBM, resolved by X-ray crystallography, in complex with α(2,3)- or α(2,6)-sialyl-lactose explain its binding specificity and unusually strong binding. * CBM, : carbohydrate-binding module; CFG, : Consortium for Functional Glycomics; GlcNAc, : N -acetylglucosamine; HBS-T, : Hepes-buffered saline with Tween 20; ITC, : isothermal titration calorimetry; LFA, : Limax flavus agglutinin; MAA, : Maackia amurensis agglutinin; MIC, : minimal inhibition concentration; MME, : monomethyl ether; Neu5Ac, : N -acetylneuraminic acid; PSL-I, : Polyporus squamosus lectin I; SiaOPr, : Neu5Ac derivative with a propargyl aglycon; 3′-SL, : 3′-sialyl-lactose; 6′-SL, : 6′-sialyl-lactose; 3′-SLN, : 3′-sialyl-lactosamine; 6′-SLN, : 6′-sialyl-lactosamine; SNA-I, : Sambucus nigra agglutinin I; SPR, : surface plasmon resonance; TSA, : thermal shift assay; WGA, : wheatgerm agglutinin

P. aeruginosa causes a substantial number of nosocomial infections and is the leading cause of death of cystic fibrosis patients. This Gram-negative bacterium is highly resistant against antibiotics and further protects itself by forming a biofilm. Moreover, a high genomic variability among clinical isolates complicates therapy. Its lectin LecB is a virulence factor and necessary for adhesion and biofilm formation. We analyzed the sequence of LecB variants in a library of clinical isolates and demonstrate that it can serve as a marker for strain family classification. LecB from the highly virulent model strain PA14 presents 13% sequence divergence with LecB from the well characterized PAO1 strain. These differences might result in differing ligand binding specificities and ultimately in reduced efficacy of drugs directed towards LecB. Despite several amino acid variations at the carbohydrate binding site, glycan array analysis showed a comparable binding pattern for both variants. A common high affinity ligand could be identified and after its chemoenzymatic synthesis verified in a competitive binding assay: an N-glycan presenting two blood group O epitopes (H-type 2 antigen). Molecular modeling of the complex suggests a bivalent interaction of the ligand with the LecB tetramer by bridging two separate binding sites. This binding rationalizes the strong avidity (35 nM) of LecBPA14 to this human fucosylated N-glycan. Biochemical evaluation of a panel of glycan ligands revealed that LecBPA14 demonstrated higher glycan affinity compared to LecBPAO1 including the extraordinarily potent affinity of 70 nM towards the monovalent human antigen Lewisa. The structural basis of this unusual high affinity ligand binding for lectins was rationalized by solving the protein crystal structures of LecBPA14 with several glycans.

Cell adhesion is a crucial feature of all multicellular organisms, as it allows cells to organise themselves into tissues to carry out specific functions. Here, we present a mimetic approach that uses multivalent lectins with opposing binding sites to crosslink glycan-functionalised giant unilamellar vesicles. The crosslinking process drives the progression from contact puncta into elongated protocellular junctions, which form the vesicles into polygonal clusters resembling tissues. Due to their carbohydrate specificity, different lectins can be engaged in parallel with both natural and synthetic glycoconjugates to generate complex interfaces with distinct lectin domains. In addition, the formation of protocellular junctions can be combined with adhesion to a functionalised support by other ligand-receptor interactions to render increased stability against fluid flow. Furthermore, we consider that adhesion is a complex process of attraction and repulsion by doping the vesicles with a PEG-modified lipid, and demonstrate a dose-dependent decrease of lectin binding and formation of protocellular junctions. We suggest that the engineering of prototissues through lectin-glycan interactions is an important step towards synthetic minimal tissues and in designing artificial systems to reconstruct the fundamental functions of biology.

The double-faced Janus lectin, designed by assembling sialic acid and fucose-specific lectin, organize multivalent heteroglyco compounds in mulitlayered material, and glycosylated protocells in prototissues., We engineered the first chimeric, bispecific lectin, with two rationally oriented and distinct recognition surfaces. This lectin, coined Janus lectin in allusion to the two-faced roman god, is able to bind independently to both fucosylated and sialylated glycoconjugates. The multivalent presentation of binding sites on each face of the Janus lectin is very efficient, resulting in avidities in the low nanomolar range for both fucosylated and sialylated surfaces. Moreover, novel heterovalent, bifunctional glycoclusters were synthetized that match the topology of the Janus lectin. Based on these tools, we constructed organized and controlled supramolecular architectures by assembling Janus lectin and glycocompound layer-by-layer. Furthermore, the Janus lectin was employed as biomolecular linker to organize protocells made from giant unilamellar vesicles of different nature, to more complex prototissues. In summary, tailor-made Janus lectins open wide possibilities for creating biomimetic matrices or artificial tissues.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate-based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug-like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d-mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure–activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure-based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half-lives in the 5–20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti-infectives against this multidrug-resistant pathogen. published

International audience; Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing 'pathoblockers' preventing biofilm formation and virulence. Here, we describe for the first time a covalent lectin inhibitor specific to a carbohydrate binding site. In addition we report its application in the LecA-specific in vitro imaging of biofilms formed by P. aeruginosa.

Burkholderia ambifaria is an opportunistic respiratory pathogen belonging to the Burkholderia cepacia complex, a collection of species responsible for the rapidly fatal cepacia syndrome in cystic fibrosis patients. A fucose-binding lectin identified in the B. ambifaria genome, BambL, is able to adhere to lung tissue, and may play a role in respiratory infection. X-ray crystallography has revealed the bound complex structures for four fucosylated human blood group epitopes (blood group B, H type 1, H type 2, and Lex determinants). The present study employed computational approaches, including docking and molecular dynamics (MD), to extend the structural analysis of BambL-oligosaccharide complexes to include four additional blood group saccharides (A, Lea, Leb, and Ley) and a library of blood-group-related carbohydrates. Carbohydrate recognition is dominated by interactions with fucose via a hydrogen-bonding network involving Arg15, Glu26, Ala38, and Trp79 and a stacking interaction with Trp74. Additional hydrogen bonds to non-fucose residues are formed with Asp30, Tyr35, Thr36, and Trp74. BambL recognition is dominated by interactions with fucose, but also features interactions with other parts of the ligands that may modulate specificity or affinity. The detailed computational characterization of the BambL carbohydrate-binding site provides guidelines for the future design of lectin inhibitors.

International audience; Chronic colonization of lungs by opportunist bacteria is the major cause of mortality for cystic fibrosis patients. Among these pathogens, Burkholderia cenocepacia is responsible for cepacia syndrome, a deadly exacerbation of infection that is the main cause of poor outcomes of lung transplantation. This bacterium contains three soluble carbohydrate‐binding proteins, including the B. cenocepacia lectin A (BC2L‐A), which is proposed to bind to oligomannose‐type N‐glycan structures to adhere to host tissues. In this work, several mannosylated glycoclusters and glycodendrimers with valencies ranging from four to 24 were prepared and their interactions with BC2L‐A were thermodynamically characterized by isothermal titration calorimetry. The results show that a 24‐valent structure binds to BC2L‐A at nanomolar concentration, which makes this compound the highest affinity monodisperse ligand for this lectin.

Glycosylation is a group of post-translational modifications that displays a large variety of structures and are implicated in a plethora of biological processes. Therefore, studying glycosylation requires different technical approaches and reliable tools, lectins being part of them. Here, we describe the use of the recombinant mushroom lectin PVL to discriminate O-GlcNAcylation, a modification consisting in the attachment of a single N-acetylglucosamine residue to proteins confined within the cytosolic, nuclear and mitochondrial compartments. Recombinant PVL (Psathyrella velutina lectin) (rPVL) displays significantly stronger affinity for GlcNAc over Neu5Ac residues as verified by thermal shift assays and surface plasmon resonance experiments, being therefore an excellent alternative to WGA (wheat germ agglutinin). Labeling of rPVL with biotin or HRP (horseradish peroxidase) allows its useful and efficient utilization by western blot. The staining of whole cell lysates with labeled-rPVL was dramatically decreased in response to O-GlcNAc transferase knockdown and seen to increase after pharmacological blockade of O-GlcNAcase. Also, HRP-rPVL seemed to be more sensitive than the anti-O-GlcNAc antibody RL2. Thus, rPVL is a potent new tool to selectively detect O-GlcNAcylated proteins. 27;2

Readily accessible, low-valency glycoclusters based on a triazine core bearing D-galactose and L-fucose epitopes are able to inhibit biofilm formation by Pseudomonas aeruginosa. These multivalent ligands are simple to synthesize, are highly soluble, and can be either homofunctional or heterofunctional. The galactose-decorated cluster shows good affinity for Pseudomonas aeruginosa lectin lecA. They are convenient biological probes for investigating the roles of lecA and lecB in biofilm formation.

Identification of selective and synthetically tractable ligands to glycan-binding proteins is important in glycoscience. Carbohydrate arrays have had a tremendous impact on profiling glycan-binding proteins and as analytical tools. We report a highly miniaturized synthetic format to access nucleic-acid-encoded hetero-glycoconjugate libraries with an unprecedented diversity in the combinations of glycans, linkers, and capping groups. Novel information about plant and bacterial lectin specificity was obtained by microarray profiling, and we show that a ligand identified on the array can be converted to a high-affinity soluble ligand by straightforward chemistry.

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA-IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA-IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra-galactosylated glycocluster and the PA-IL lectin for high concentration of lectins (2.5 μM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one-dimensional elongated aggregates to two-dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction. Copyright © 2013 John Wiley & Sons, Ltd.

The galactose-specific lectin LecA from Pseudomonas aeruginosa is a target for the development of new anti-infectious compounds. Sugar based molecules with anti-adhesive properties present great potential in the fight against bacterial infection and biofilm formation. LecA is specific for oligosaccharides with terminal α-galactoside residues and displays strong affinity for melibiose (αGal1-6Glc) with a Kd of 38.8 µM. The crystal structure of LecA/melibiose complex shows classical calcium-bridged binding of αGal in the primary binding site but also revealed a secondary sugar binding site with glucose bound. This sugar binding site is in close proximity to the galactose binding one, is independent of calcium and mainly involves interactions with a symmetry-related protein. This discovery would help to the design of new potent inhibitors targeting both binding sites.

Following an iterative oxime ligation procedure, cyclopeptide (R) and lysine-based dendron (D) were combined in all possible arrangements and successively functionalized with α-fucose and β-fucose to provide a new series of hexadecavalent glycosylated scaffolds (i.e., scaffolds RD16, RR16, DR16, and DD16). These compounds and smaller analogs (tetra- and hexavalent scaffolds R4 and R6) were used to evaluate the influence of the ligand valency and architecture, and of the anomer configuration in the binding to the αFuc-specific lectin LecB from Pseudomonas aeruginosa . Competitive enzyme-linked lectin assays (ELLA) revealed that only the RD16 architecture displaying αFuc (9A) reaches strong binding improvement (IC50 of 0.6 nM) over αMeFuc, and increases the α-selectivity of LecB. Dissociation constant of 28 nM was measured by isothermal titration micorcalorimetry (ITC) for 9A, which represents the highest affinity ligand ever reported for LecB. ITC and molecular modeling suggested that the high affinity observed might be due to an aggregative chelate binding involving four sugar head groups and two lectins. Interestingly, unprecedented binding effects were observed with β-fucosylated conjugates, albeit being less active than the corresponding ligands of the αFuc series. In particular, the more flexible lysine-based dendritic structures (15B and 18B) showed a slight inhibitory enhancement in comparison with those having cyclopeptide core.

Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the β-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics.

A family of fifteen glycoclusters based on a cyclic oligo-(1→6)-β-D-glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme-linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub-micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.