Biology subjects

Imberty, A.

Publications and source records attributed to Imberty, A..

11 recordsLinked to original sources

bioRxiv2026

Structural Basis for Human Milk Oligosaccharides Recognition by Evolutionarily Dominant Norovirus Genogroup II Genotypes

Human noroviruses are a major cause of acute gastroenteritis. Human milk oligosaccharides (HMOs) contain glycan motifs similar to histo-blood group antigens and may act as soluble decoys that reduce viral attachment. However, the molecular basis of HMO recognition by the P domain of the major capsid protein VP1 of different norovirus genotypes is not fully understood. Here, we combined ligand-based and protein-based NMR experiments with X-ray crystallography to study HMO binding to the P domain of the latent GII.2, dominant GII.4 and emerging GII.17 noroviruses genotypes. Screening of 12 HMOs by STD NMR showed binding of all fucosylated HMOs to GII.4 and GII.17 and none for non-fucosylated HMOs. In contrast, GII.2 showed little or no detectable binding under the same conditions. Seven crystal structures of GII.4 or GII.17 with complexed HMOs supported fucose anchoring via five conserved residues. The remaining sugar moieties formed genotype-specific secondary interactions with an extended, shallow and solvent exposed binding surface whose shape is dictated by divergent surface loops. GII.4 recognition is mainly driven by hydrogen bonds whereas GII.17 relied more strongly on water-mediated interactions. Chemical shift perturbation from NMR titrations (CSP NMR) of GII.4 corroborated the fucose interactions and gave dissociation constants of 20-33 mM, consistent with weak and rapidly exchanging interactions.

biochemistry↗

bioRxiv2023

Cell-free expression and characterization of multivalent rhamnose-binding lectins using biolayer interferometry

Lectins are important biological tools for binding glycans, but recombinant protein expression poses challenges for some lectin classes, limiting the pace of discovery and characterization. To discover and engineer lectins with new functions, workflows amenable to rapid expression and subsequent characterization are needed. Here, we present bacterial cell-free protein synthesis as a means for efficient, small-scale expression of multivalent, disulfide bond-rich, rhamnose-binding lectins. Furthermore, we demonstrate that the cell-free expressed lectins can be directly coupled with BLI analysis, either in solution or immobilized on the sensor, to measure interaction with carbohydrate ligands without purification. This workflow enables determination of lectin substrate specificity and estimation of binding affinity. Overall, we believe that this method will enable high-throughput expression, screening, and characterization of new and engineered multivalent lectins for applications in synthetic glycobiology.

synthetic biology↗

bioRxiv2022

Extending Janus lectins architecture: characterization and application to protocells

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being developed for producing tools with precise topology and specificity. We developed the concept of chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the {beta}-propeller domain from Ralstonia solanacearum and the {beta}-trefoil domain from fungus Marasmius oreades, is specific for fucose and -galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The synthetic protein binds to H1299 lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering.

synthetic biology↗

bioRxiv2022

Building artificial plant cell wall on lipid bilayer by assembling polysaccharides and engineered proteins

The cell wall constitutes a fundamental structural component of plant cells, providing them with mechanical resistance and flexibility. Mimicking that wall is a critical step in the conception of an experimental model of the plant cell. The assembly of cellulose/hemicellulose in the form of cellulose nanocrystals and xyloglucans as a representative model of the plant cell wall has already been mastered, however, those models lacked the pectin component. In this work, we used an engineered chimeric protein designed for bridging pectin to the cellulose/hemicellulose network, therefore achieving the assembly of complete cell wall mimics. We first engineered proteins, i.e. carbohydrate-binding module from Ruminococcus flavefaciens able to bind oligo-galactorunan, resulting in high-affinity polygalacturonan receptors with Kd in the micromolar range. A Janus protein, with cell wall gluing property, was then designed by assembling this CBM with a Ralstonia solanacearum lectin specific for fucosylated xyloglucans. The resulting supramolecular architecture is able to bind fucose-containing xyloglucans and homogalacturonan ensuring high affinity for both. A two-dimension assembly of an artificial plant cell wall was then built first on synthetic polymer and then on supported lipid bilayer. Such artificial cell wall can serve as a basis for the development of plant cell mechanical models and thus deepen the understanding of the principles underlying various aspects of plant cells and tissues. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC="FIGDIR/small/501355v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@18af594org.highwire.dtl.DTLVardef@ec3dorg.highwire.dtl.DTLVardef@95d44aorg.highwire.dtl.DTLVardef@a47bea_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

The lectin LecB induces patches with basolateral characteristics at the apical membrane to promote Pseudomonas aeruginosa host cell invasion

The opportunistic bacterium Pseudomonas aeruginosa can infect mucosal tissues of the human body. To persist at the mucosal barrier, this highly adaptable pathogen has evolved many strategies, including invasion of host cells. Here, we show that the P. aeruginosa lectin LecB binds and cross-links fucosylated receptors at the apical plasma membrane of epithelial cells. This triggers a signaling cascade via Src kinases and PI3K leading to the formation of patches enriched with the basolateral marker PIP3 at the apical plasma membrane. This identifies LecB as causative bacterial factor for activating this well-known host cell response that is elicited upon apical binding of P. aeruginosa. Downstream of PI3K, Rac1 is activated to cause actin rearrangement and the outgrowth of protrusions at the apical plasma membrane. LecB-triggered PI3K activation also results in aberrant recruitment of caveolin-1 to the apical domain. In addition, we reveal a positive feedback loop between PI3K activation and apical caveolin-1 recruitment, which provides a mechanistic explanation for the previously observed implication of caveolin-1 in P. aeruginosa host cell invasion. Interestingly, LecB treatment also reversibly removes primary cilia. To directly prove the role of LecB for bacterial uptake, we coated bacteria-sized beads with LecB, which drastically enhanced their endocytosis. Furthermore, LecB deletion and LecB inhibition with L-fucose diminished the invasion efficiency of P. aeruginosa bacteria. Taken together, our study identifies LecB as missing link that can explain how PI3K signaling and caveolin-1 recruitment are triggered to facilitate invasion of epithelial cells from the apical side by P. aeruginosa. ImportanceAn intriguing feature of the bacterium P. aeruginosa is its ability to colonize highly diverse niches. P. aeruginosa can, besides biofilm formation, also enter and proliferate within epithelial host cells. Moreover, research during recent years has shown that P. aeruginosa possesses many different mechanisms to invade host cells. In this study we identify LecB as novel invasion factor. In particular, we show that LecB activates PI3K signaling, which is connected via a positive feedback loop to apical caveolin-1 recruitment, and leads to actin rearrangement at the apical plasma membrane. This provides a unifying explanation for the previously reported implication of PI3K and caveolin-1 in P. aeruginosa host cell invasion. In addition, our study adds a further function to the remarkable repertoire of the lectin LecB, which is all brought about by the capability of LecB to recognize fucosylated glycans on many different niche-specific host cell receptors.

microbiology↗

bioRxiv2022

A pore-forming β-trefoil lectin with specificity for the tumor-related glycosphingolipid Gb3

Lectins are efficient multivalent glycan receptors, deciphering the glyco-code on cell surfaces. The {beta}-trefoil fold, characterized by three lobe-shaped repeats, is adopted by several classes of lectins, often associated with other domains having enzymatic or toxic activity. Based on the UniLectin3D database classification, the sequence signature of trefoil lobes was defined and used to predict 44714 lectins from 4497 species. Among them, SaroL-1 from the lower eukaryote Salpingoeca rosetta was predicted to contain both {beta}-trefoil and aerolysin-like pore-forming domain. Recombinant SaroL-1 binds to galactose and derivatives, with a stronger affinity for cancer-related -galactosylated epitopes such as glycosphingolipid Gb3 embedded in giant unilamellar vesicles or cell membranes. Crystal structures in complex with Gb3 trisaccharide and GalNAc show similarity with pore-forming toxins. Recognition of the Gal epitope on glycolipids was necessary for hemolysis of rabbit erythrocytes and toxicity on cancer cells through carbohydrate-dependent pore-formation.

biochemistry↗

bioRxiv2021

Neutron crystallography reveals novel mechanisms used by Pseudomonas aeruginosa for host-cell binding

The 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.

biochemistry↗

bioRxiv2021

A Comprehensive Phylogenetic and Bioinformatics Survey of Lectins in the Fungal kingdom

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.

bioinformatics↗

bioRxiv2020

Proteome-wide prediction of bacterial carbohydrate-binding proteins as a tool for understanding commensal and pathogen colonisation of the vaginal microbiome

Bacteria use protein receptors called lectins to anchor to specific host surface sugars. The role of lectins in the vaginal microbiome, and their involvement in reproductive tract pathophysiology is poorly defined. Here we establish a classification system based on taxonomy and protein 3D structure to identify 109 lectin classes. Hidden Markov Model (HMM) profiles for each class were used to search bacterial genomes, resulting in the prediction of >100 000 bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species showed that potential pathogens produce a larger variety of lectins than commensals indicating increased glycan-binding potential. 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.

bioinformatics↗

bioRxiv2019

The Pseudomonas aeruginosa lectin LecB causes integrin internalization to facilitate crawling of bacteria underneath host cells

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 {beta}1-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 {beta}1-integrins and also of complete 3{beta}1-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.

microbiology↗

bioRxiv2019

Induction of Rare Conformation of Oligosaccharide by Binding to Calcium-dependent Bacterial Lectin: X-ray Crystallography and Modelling Study

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.

biophysics↗