Quantifying Siglec-sialylated ligand interactions: a versatile 19F-T2 CPMG filtered competitive NMR displacement assay.

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are integral cell surface proteins crucial for the regulation of immune responses and the maintenance of immune tolerance through interactions with sialic acids. Siglecs recognize sialic acid moieties, usually found at the end of N-glycan and O-glycan chains. However, the different Siglecs prefer diverse presentations of the recognized sialic acid, depending on the type of glycosidic linkage used to link to the contiguous Gal/GalNAc or sialic acid moieties. This fact, together with possible O- or N-substitutions at the recognized glycan epitope significantly influences their roles in various immune-related processes. Understanding the molecular details of Siglec-sialoglycan interactions is essential for unraveling their specificities and for the development of new molecules targeting these receptors. While traditional biophysical methods like isothermal titration calorimetry (ITC) have been utilized to measure binding between lectins and glycans, contemporary techniques such as surface plasmon resonance (SPR), microscale thermophoresis (MST), and biolayer interferometry (BLI) offer improved throughput. However, these methodologies require chemical modification and immobilization of at least one binding partner, which can interfere the recognition between the lectin and the ligand. Since Siglecs display a large range of dissociation constants, depending on the (bio)chemical nature of the interacting partner, a general and robust method that could monitor and quantify binding would be highly welcomed. Herein, we propose the application of an NMR-based a competitive displacement assay, grounded on 19F T2-relaxation NMR and on the design, synthesis, and use of a strategic spy molecule, to assess and quantify sialoside ligand binding to Siglecs. We show that the use of this specific approach allows the quantification of Siglec binding for natural and modified sialosides, multivalent sialosides, and sialylated glycoproteins in solution, which differ in binding affinities in more than two orders of magnitude, thus providing invaluable insights into sialoglycan-mediated interactions.

Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds.

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.

Plant-based production of a protective vaccine antigen against the bovine parasitic nematode Ostertagia ostertagi.

The development of effective recombinant vaccines against parasitic nematodes has been challenging and so far mostly unsuccessful. This has also been the case for Ostertagia ostertagi, an economically important abomasal nematode in cattle, applying recombinant versions of the protective native activation-associated secreted proteins (ASP). To gain insight in key elements required to trigger a protective immune response, the protein structure and N-glycosylation of the native ASP and a non-protective Pichia pastoris recombinant ASP were compared. Both antigens had a highly comparable protein structure, but different N-glycan composition. After mimicking the native ASP N-glycosylation via the expression in Nicotiana benthamiana plants, immunisation of calves with these plant-produced recombinants resulted in a significant reduction of 39% in parasite egg output, comparable to the protective efficacy of the native antigen. This study provides a valuable workflow for the development of recombinant vaccines against other parasitic nematodes.

A Versatile Urea Type Linker for Functionalizing Natural Glycans and Its Validation in Glycan Arrays.

The isolation from organisms and readily available glycoproteins has become an increasingly convenient source of N-glycans for multiple applications including glycan microarrays, as reference standards in glycan analysis or as reagents that improve bioavailability of protein and peptide therapeutics through conjugation. A problematic step in the isolation process on a preparative scale can be the attachment of a linker for the improved purification, separation, immobilization and quantification of the glycan structures. Addressing this issue, we firstly aimed for the development of an UV active linker for a fast and reliable attachment to anomeric glycosylamines via urea bond formation. Secondly, we validated the new linker on glycan arrays in a comparative study with a collection of N-glycans which were screened against various lectins. In total, we coupled four structurally varied N-glycans to four different linkers, immobilized all constructs on a microarray and compared their binding affinities to four plant and fungal lectins of widely described specificity. Our study shows that the urea type linker showed an overall superior performance for lectin binding and once more, highlights the often neglected influence of the choice of linker on lectin recognition.

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.

A galactoside-specific Dalbergieae legume lectin from seeds of Vataireopsis araroba (Aguiar) Ducke.

The Dalbergieae lectin group encompasses several lectins with significant differences in their carbohydrate specificities and biological properties. The current work reports on the purification and characterization of a GalNAc/Gal-specific lectin from Vataireopsis araroba (Aguiar) Ducke, designated as VaL. The lectin was purified from the seeds in a single step using guar gum affinity chromatography. The lectin migrated as a single band of about 35 kDa on SDS-PAGE and, in native conditions, occurs as a homodimer. The purified lectin is stable at temperatures up to 60 °C and in a pH range from 7 to 8 and requires divalent cations for its activity. Sugar-inhibition assays demonstrate the lectin specificity towards N-acetyl-D-galactosamine, D-galactose and related sugars. Furthermore, glycan array analyses show that VaL interacts preferentially with glycans containing terminal GalNAc/Galß1-4GlcNAc. Biological activity assays were performed using three insect cell lines: CF1 midgut cells from the spruce budworm Choristoneura fumiferana, S2 embryo cells from the fruit fly Drosophila melanogaster, and GutAW midgut cells from the corn earworm Helicoverpa zea. In vitro assays indicated a biostatic effect for VaL on CF1 cells, but not on S2 and GutAW cells. The lectin presented a biostatic effect by reducing the cell growth and inducing cell agglutination, suggesting an interaction with glycans on the cell surface. VaL has been characterized as a galactoside-specific lectin of the Dalbergieae tribe, with sequence similarity to lectins from Vatairea and Arachis.

Immobilization of Biantennary N-Glycans Leads to Branch Specific Epitope Recognition by LSECtin.

The molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcß1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment.

N-Glycans in Immortalized Mesenchymal Stromal Cell-Derived Extracellular Vesicles Are Critical for EV-Cell Interaction and Functional Activation of Endothelial Cells.

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are widely considered as a cell-free therapeutic alternative to MSC cell administration, due to their immunomodulatory and regenerative properties. However, the interaction mechanisms between EV and target cells are not fully understood. The surface glycans could be key players in EV-cell communication, being specific molecular recognition patterns that are still little explored. In this study, we focused on the role of N-glycosylation of MSC-EV as mediators of MSC-EV and endothelial cells' interaction for subsequent EV uptake and the induction of cell migration and angiogenesis. For that, EV from immortalized Wharton's Jelly MSC (iWJ-MSC-EV) were isolated by size exclusion chromatography (SEC) and treated with the glycosidase PNGase-F in order to remove wild-type N-glycans. Then, CFSE-labelled iWJ-MSC-EV were tested in the context of in vitro capture, agarose-spot migration and matrigel-based tube formation assays, using HUVEC. As a result, we found that the N-glycosylation in iWJ-MSC-EV is critical for interaction with HUVEC cells. iWJ-MSC-EV were captured by HUVEC, stimulating their tube-like formation ability and promoting their recruitment. Conversely, the removal of N-glycans through PNGase-F treatment reduced all of these functional activities induced by native iWJ-MSC-EV. Finally, comparative lectin arrays of iWJ-MSC-EV and PNGase-F-treated iWJ-MSC-EV found marked differences in the surface glycosylation pattern, particularly in N-acetylglucosamine, mannose, and fucose-binding lectins. Taken together, our results highlight the importance of N-glycans in MSC-EV to permit EV-cell interactions and associated functions.

COVID-BIOCHIP: A Web Tool to Analyse COVID-19 Antigen Microarrays.

During the COVID-19 pandemic, there was a growing need to characterise the disease. A very important aspect is the ability to measure the immunisation extent, which can be achieved using antigen microarrays that quantitively measure the presence of COVID-related antibodies. A significant limitation for these tests was the complexity of manually analysing the results, and the limited availability of software for its analysis. In this paper, we describe the development of COVID-BIOCHIP, an ad-hoc web-based solution for the automatic analysis and visualisation of COVID-19 antigen microarray data results.

Cross-reactivity of glycan-reactive HIV-1 broadly neutralizing antibodies with parasite glycans.

The HIV-1 Envelope glycoprotein (Env) is the sole target for broadly neutralizing antibodies (bnAbs). Env is heavily glycosylated with host-derived N-glycans, and many bnAbs bind to, or are dependent upon, Env glycans for neutralization. Although glycan-binding bnAbs are frequently detected in HIV-infected individuals, attempts to elicit them have been unsuccessful because of the poor immunogenicity of Env N-glycans. Here, we report cross-reactivity of glycan-binding bnAbs with self- and non-self N-glycans and glycoprotein antigens from different life-stages of Schistosoma mansoni. Using the IAVI Protocol C HIV infection cohort, we examine the relationship between S. mansoni seropositivity and development of bnAbs targeting glycan-dependent epitopes. We show that the unmutated common ancestor of the N332/V3-specific bnAb lineage PCDN76, isolated from an HIV-infected donor with S. mansoni seropositivity, binds to S. mansoni cercariae while lacking reactivity to gp120. Overall, these results present a strategy for elicitation of glycan-reactive bnAbs which could be exploited in HIV-1 vaccine development.

Purification and characterization of a highly thermostable GlcNAc-binding lectin from Collaea speciosa seeds.

Lectins from plants of the Diocleinae subtribe often exhibit specificity towards mannose/glucose and derived sugars, with some plants also displaying a second lectin specific to lactose/GalNAc. Here, we present a novel lectin from Collaea speciosa, named CsL, that displays specificity for GlcNAc/glucose. The lectin was extracted from Collaea speciosa seeds and purified by a single chromatographic step on a Sephadex G-50 matrix. In solution, the lectin appears as a dimeric protein composed of 25 kDa monomers. The protein is stable at pH 7-8 and dependent on divalent cations. CsL maintained its agglutination activity after heating to 90 °C for 1 h. Glycan array studies revealed that CsL binds to N-glycans with terminal GlcNAc residues, chitobiose and chitotriose moieties. The partial amino acid sequence of the lectin is similar to that of some lactose-specific lectins from the same subtribe. In contrast to other ConA-like lectins, CsL is not toxic to Artemia. Because of its remarkably different properties and specificity, this lectin could be the first member of a new group inside the Diocleinae lectins.

Glycosylation reduces the glycan-independent immunomodulatory effect of recombinant Orysata lectin in Drosophila S2 cells.

Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins.

Controlled density glycodendron microarrays for studying carbohydrate-lectin interactions.

Glycodendron microarrays with defined valency have been constructed by on-chip synthesis on hydrophobic indium tin oxide (ITO) coated glass slides and employed in lectin-carbohydrate binding studies with several plant and human lectins. Glycodendrons presenting sugar epitopes at different valencies were prepared by spotwise strain-promoted azide-alkyne cycloaddition (SPAAC) between immobilised cyclooctyne dendrons and azide functionalised glycans. The non-covalent immobilisation of dendrons on the ITO surface by hydrophobic interaction allowed us to study dendron surface density and SPAAC conversion rate by in situ MALDI-TOF MS analysis. By diluting the dendron surface density we could study how the carbohydrate-lectin interactions became exclusively dependant on the valency of the immobilised glycodendron.

Longitudinal Development of Antibody Responses in COVID-19 Patients of Different Severity with ELISA, Peptide, and Glycan Arrays: An Immunological Case Series.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). A better understanding of its immunogenicity can be important for the development of improved diagnostics, therapeutics, and vaccines. Here, we report the longitudinal analysis of three COVID-19 patients with moderate (#1) and mild disease (#2 and #3). Antibody serum responses were analyzed using spike glycoprotein enzyme linked immunosorbent assay (ELISA), full-proteome peptide, and glycan microarrays. ELISA immunoglobulin A, G, and M (IgA, IgG, and IgM) signals increased over time for individuals #1 and #2, whereas #3 only showed no clear positive IgG and IgM result. In contrast, peptide microarrays showed increasing IgA/G signal intensity and epitope spread only in the moderate patient #1 over time, whereas early but transient IgA and stable IgG responses were observed in the two mild cases #2 and #3. Glycan arrays showed an interaction of antibodies to fragments of high-mannose and core N-glycans, present on the viral shield. In contrast to protein ELISA, microarrays allow for a deeper understanding of IgA, IgG, and IgM antibody responses to specific epitopes of the whole proteome and glycans of SARS-CoV-2 in parallel. In the future, this may help to better understand and to monitor vaccination programs and monoclonal antibodies as therapeutics.

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.

FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment.

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-ß-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Cross-reactive carbohydrate determinant-specific IgE obscures true atopy and exhibits ⍺-1,3-fucose epitope-specific inverse associations with asthma.

BACKGROUND: In high-income, temperate countries, IgE to allergen extracts is a risk factor for, and mediator of, allergy-related diseases (ARDs). In the tropics, positive IgE tests are also prevalent, but rarely associated with ARD. Instead, IgE responses to ubiquitous cross-reactive carbohydrate determinants (CCDs) on plant, insect and parasite glycoproteins, rather than to established major allergens, are dominant. Because anti-CCD IgE has limited clinical relevance, it may impact ARD phenotyping and assessment of contribution of atopy to ARD. METHODS: Using an allergen extract-based test, a glycan and an allergen (glyco)protein microarray, we mapped IgE fine specificity among Ugandan rural Schistosoma mansoni (Sm)-endemic communities, proximate urban communities, and importantly in asthmatic and nonasthmatic schoolchildren. RESULTS: Overall, IgE sensitization to extracts was highly prevalent (43%-73%) but allergen arrays indicated that this was not attributable to established major allergenic components of the extracts (0%-36%); instead, over 40% of all participants recognized CCD-bearing components. Using glycan arrays, we dissected IgE responses to specific glycan moieties and found that reactivity to classical CCD epitopes (core ß-1,2-xylose, α-1,3-fucose) was positively associated with sensitization to extracts, rural environment and Sm infection, but not with skin reactivity to extracts or sensitization to their major allergenic components. Interestingly, we discovered that reactivity to only a subset of core α-1,3-fucose-carrying N-glycans was inversely associated with asthma. CONCLUSIONS: CCD reactivity is not just an epiphenomenon of parasite exposure hampering specificity of allergy diagnostics; mechanistic studies should investigate whether specific CCD moieties identified here are implicated in the protective effect of certain environmental exposures against asthma.

Chemo-Enzymatic Synthesis of S. mansoni O-Glycans and Their Evaluation as Ligands for C-Type Lectin Receptors MGL, DC-SIGN, and DC-SIGNR.

Due to their interactions with C-type lectin receptors (CLRs), glycans from the helminth Schistosoma mansoni represent promising leads for treatment of autoimmune diseases, allergies or cancer. We chemo-enzymatically synthesized nine O-glycans based on the two predominant O-glycan cores observed in the infectious stages of schistosomiasis, the mucin core 2 and the S. mansoni core. The O-glycans were fucosylated next to a selection of N-glycans directly on a microarray slide using a recombinant fucosyltransferase and GDP-fucose or GDP-6-azidofucose as donor. Binding assays with fluorescently labelled human CLRs DC-SIGN, DC-SIGNR and MGL revealed the novel O-glycan O8 as the best ligand for MGL from our panel. Significant binding to DC-SIGN was also found for azido-fucosylated glycans. Contrasting binding specificities were observed between the monovalent carbohydrate recognition domain (CRD) and the tetravalent extracellular domain (ECD) of DC-SIGNR.

The Interaction of Fluorinated Glycomimetics with DC-SIGN: Multiple Binding Modes Disentangled by the Combination of NMR Methods and MD Simulations.

Fluorinated glycomimetics are frequently employed to study and eventually modulate protein-glycan interactions. However, complex glycans and their glycomimetics may display multiple binding epitopes that enormously complicate the access to a complete picture of the protein-ligand complexes. We herein present a new methodology based on the synergic combination of experimental 19F-based saturation transfer difference (STD) NMR data with computational protocols, applied to analyze the interaction between DC-SIGN, a key lectin involved in inflammation and infection events with the trifluorinated glycomimetic of the trimannoside core, ubiquitous in human glycoproteins. A novel 2D-STD-TOCSYreF NMR experiment was employed to obtain the experimental STD NMR intensities, while the Complete Relaxation Matrix Analysis (CORCEMA-ST) was used to predict that expected for an ensemble of geometries extracted from extensive MD simulations. Then, an in-house built computer program was devised to find the ensemble of structures that provide the best fit between the theoretical and the observed STD data. Remarkably, the experimental STD profiles obtained for the ligand/DC-SIGN complex could not be satisfactorily explained by a single binding mode, but rather with a combination of different modes coexisting in solution. Therefore, the method provides a precise view of those ligand-receptor complexes present in solution.

TETRALEC, Artificial Tetrameric Lectins: A Tool to Screen Ligand and Pathogen Interactions.

C-type lectin receptor (CLR)/carbohydrate recognition occurs through low affinity interactions. Nature compensates that weakness by multivalent display of the lectin carbohydrate recognition domain (CRD) at the cell surface. Mimicking these low affinity interactions in vitro is essential to better understand CLR/glycan interactions. Here, we present a strategy to create a generic construct with a tetrameric presentation of the CRD for any CLR, termed TETRALEC. We applied our strategy to a naturally occurring tetrameric CRD, DC-SIGNR, and compared the TETRALEC ligand binding capacity by synthetic N- and O-glycans microarray using three different DC-SIGNR constructs i) its natural tetrameric counterpart, ii) the monomeric CRD and iii) a dimeric Fc-CRD fusion. DC-SIGNR TETRALEC construct showed a similar binding profile to that of its natural tetrameric counterpart. However, differences observed in recognition of low affinity ligands underlined the importance of the CRD spatial arrangement. Moreover, we further extended the applications of DC-SIGNR TETRALEC to evaluate CLR/pathogens interactions. This construct was able to recognize heat-killed Candida albicans by flow cytometry and confocal microscopy, a so far unreported specificity of DC-SIGNR. In summary, the newly developed DC-SIGNR TETRALEC tool proved to be useful to unravel novel CLR/glycan interactions, an approach which could be applied to other CLRs.