Melanoma tumour-derived glycans hijack dendritic cell subsets through C-type lectin receptor binding.

Dendritic cell (DC) subsets play a crucial role in shaping anti-tumour immunity. Cancer escapes from the control immune system by hijacking DC functions. Yet, bases for such subversion are only partially understood. Tumour cells display aberrant glycan motifs on surface glycoproteins and glycolipids. Such carbohydrate patterns can be sensed by DCs through C-type lectin receptors (CLRs) that are critical to shape and orientate immune responses. We recently demonstrated that melanoma tumour cells harboured an aberrant 'glyco-code,' and that circulating and tumour-infiltrating DCs from melanoma patients displayed major perturbations in their CLR profiles. To decipher whether melanoma, through aberrant glycan patterns, may exploit CLR pathways to mislead DCs and evade immune control, we explored the impact of glycan motifs aberrantly found in melanoma (neoglycoproteins [NeoGP] functionalised with Gal, Man, GalNAc, s-Tn, fucose [Fuc] and GlcNAc residues) on features of human DC subsets (cDC2s, cDC1s and pDCs). We examined the ability of glycans to bind to purified DCs, and assessed their impact on DC basal properties and functional features using flow cytometry, confocal microscopy and multiplex secreted protein analysis. DC subsets differentially bound and internalised NeoGP depending on the nature of the glycan. Strikingly, Fuc directly remodelled the expression of activation markers and immune checkpoints, as well as the cytokine/chemokine secretion profile of DC subsets. NeoGP interfered with Toll like receptor (TLR)-signalling and pre-conditioned DCs to exhibit an altered response to subsequent TLR stimulation, dampening antitumor mediators while triggering pro-tumoral factors. We further demonstrated that DC subsets can bind NeoGP through CLRs, and identified GalNAc/MGL and s-Tn/ C-type lectin-like receptor 2 (CLEC2) as potential candidates. Moreover, DC dysfunction induced by tumour-associated carbohydrate molecules may be reversed by interfering with the glycan/CLR axis. These findings revealed the glycan/CLR axis as a promising checkpoint to exploit in order to reshape potent antitumor immunity while impeding immunosuppressive pathways triggered by aberrant tumour glycosylation patterns. This may rescue DCs from tumour hijacking and improve clinical success in cancer patients.

High-Mannose Oligosaccharide Hemimimetics that Recapitulate the Conformation and Binding Mode to Concanavalin A, DC-SIGN and Langerin.

The "carbohydrate chemical mimicry" exhibited by sp2 -iminosugars has been utilized to develop practical syntheses for analogs of the branched high-mannose-type oligosaccharides (HMOs) Man3 and Man5 . In these compounds, the terminal nonreducing Man residues have been substituted with 5,6-oxomethylidenemannonojirimycin (OMJ) motifs. The resulting oligomannoside hemimimetic accurately reproduce the structure, configuration, and conformational behavior of the original mannooligosaccharides, as confirmed by NMR and computational techniques. Binding studies with mannose binding lectins, including concanavalin A, DC-SIGN, and langerin, by enzyme-linked lectin assay and surface plasmon resonance revealed significant variations in their ability to accommodate the OMJ unit in the mannose binding site. Intriguingly, OMJMan segments demonstrated "in line" heteromultivalent effects during binding to the three lectins. Similar to the mannobiose (Man2 ) branches in HMOs, the binding modes involving the external or internal monosaccharide unit at the carbohydrate binding-domain exist in equilibrium, facilitating sliding and recapture processes. This equilibrium, which influences the multivalent binding of HMOs, can be finely modulated upon incorporation of the OMJ sp2 -iminosugar caps. As a proof of concept, the affinity and selectivity towards DC-SIGN and langerin were adjustable by presenting the OMJMan epitope in platforms with diverse architectures and valencies.

Fluorinated Man9 as a High Mannose Mimetic to Unravel Its Recognition by DC-SIGN Using NMR.

Lectins are capable of reading out the structural information contained in carbohydrates through specific recognition processes. Determining the binding epitope of the sugar is fundamental to understanding this recognition event. Nuclear magnetic resonance (NMR) is a powerful tool to obtain this structural information in solution; however, when the sugar involved is a complex oligosaccharide, such as high mannose, the signal overlap found in the NMR spectra precludes an accurate analysis of the interaction. The introduction of tags into these complex oligosaccharides could overcome these problems and facilitate NMR studies. Here, we show the preparation of the Man9 of high mannose with some fluorine tags and the study of the interaction with its receptor, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This fluorinated ligand has allowed us to apply heteronuclear two-dimensional (2D) 1H,19F STD-TOCSYreF NMR experiments, using the initial slope approach, which has facilitated the analysis of the Man9/DC-SIGN interaction, unequivocally providing the binding epitope.

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist.

The efficient spread of SARS-CoV-2 resulted in a unique pandemic in modern history. Despite early identification of ACE2 as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. We evaluated the contribution of C-type lectin receptors (CLRS) of antigen-presenting cells, widely present in respiratory mucosa and lung tissue. DC-SIGN, L-SIGN, Langerin and MGL bind to diverse glycans of the spike using multiple interaction areas. Using pseudovirus and cells derived from monocytes or T-lymphocytes, we demonstrate that while virus capture by the CLRs examined does not allow direct cell infection, DC/L-SIGN, among these receptors, promote virus transfer to permissive ACE2+ Vero E6 cells. A glycomimetic compound designed against DC-SIGN, enable inhibition of this process. These data have been then confirmed using authentic SARS-CoV-2 virus and human respiratory cell lines. Thus, we described a mechanism potentiating viral spreading of infection.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin.

Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.

Microbe-focused glycan array screening platform.

Interactions between glycans and glycan binding proteins are essential for numerous processes in all kingdoms of life. Glycan microarrays are an excellent tool to examine protein-glycan interactions. Here, we present a microbe-focused glycan microarray platform based on oligosaccharides obtained by chemical synthesis. Glycans were generated by combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis, and chemoenzymatic methods. The current library of more than 300 glycans is as diverse as the mammalian glycan array from the Consortium for Functional Glycomics and, due to its microbial focus, highly complementary. This glycan platform is essential for the characterization of various classes of glycan binding proteins. Applications of this glycan array platform are highlighted by the characterization of innate immune receptors and bacterial virulence factors as well as the analysis of human humoral immunity to pathogenic glycans.

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.

Synthesis, self-assembly and Langerin recognition studies of a resorcinarene-based glycocluster exposing a hyaluronic acid thiodisaccharide mimetic.

Herein, we report the synthesis of an octavalent glycocluster exposing a thiodisaccharide mimetic of the repetitive unit of hyaluronic acid, ßSGlcA(1 → 3)ßSGlcNAc, constructed on a calix[4]resorcinarene scaffold by CuAAC reaction of suitable precursors. This glycocluster showed a strong tendency toward self-aggregation. DOSY-NMR and DLS experiments demonstrated the formation of spherical micelles of d ≅ 6.2 nm, in good agreement. TEM micrographs showed the presence of particles of different sizes, depending on the pH of the starting solution, thus evidencing that the negative charge on the micelle surface due to ionization of the GlcA residues plays an important role in the aggregation process. STD-NMR and DLS experiments provided evidence of the interaction between the synthetic glycocluster and Langerin, a relevant C-type lectin. This interaction was not observed in the STD-NMR experiments performed with the basic disaccharide, providing evidence of a multivalent effect.

New lipophilic glycomimetic DC-SIGN ligands: Stereoselective synthesis and SPR-based binding inhibition assays.

The design and synthesis of efficient ligands for DC-SIGN is a topic of high interest, because this C-type lectin has been implicated in the early stages of many infection processes. DC-SIGN membrane-protein presents four carbohydrate-binding domains (CRD) that specifically recognize mannose and fucose. Therefore, antagonists of minimal disaccharide epitope Manα(1,2)Man, represent potentially interesting antibacterial and antiviral agents. In the recent past, we were able to develop efficient antagonists, mimics of the natural moiety, characterized by the presence of a real d-carbamannose unit which confers greater stability to enzymatic breakdown than the corresponding natural disaccharide ligand. Herein, we present the challenging stereoselective synthesis of four new amino or azide glycomimetic DC-SIGN antagonists with attractive orthogonal lipophilic substituents in C(3), C(4) or C(6) positions of the real carba unit, which were expected to establish crucial interactions with lipophilic areas of DC-SIGN CRD. The activity of the new ligands was evaluated by SPR binding inhibition assays. The interesting results obtained, allow to acquire important information about the influence of the lipophilic substituents present in specific positions of the carba scaffold. Furthermore, C(6) benzyl C(4) tosylamide pseudodisaccharide displayed a good affinity for DC-SIGN with a more favorable IC50 value than those of the previously described real carba-analogues. This study provides valuable knowledge for the implementation of further structural modifications towards improved inhibitors.

Interdomain Flexibility within NADPH Oxidase Suggested by SANS Using LMNG Stealth Carrier.

Small angle neutron scattering (SANS) provides a method to obtain important low-resolution information for integral membrane proteins (IMPs), challenging targets for structural determination. Specific deuteration furnishes a "stealth" carrier for the solubilized IMP. We used SANS to determine a structural envelope of SpNOX, the Streptococcus pneumoniae NADPH oxidase (NOX), a prokaryotic model system for exploring structure and function of eukaryotic NOXes. SpNOX was solubilized in the detergent lauryl maltose neopentyl glycol, which provides optimal SpNOX stability and activity. Using deuterated solvent and protein, the lauryl maltose neopentyl glycol was experimentally undetected in SANS. This affords a cost-effective SANS approach for obtaining novel structural information on IMPs. Combining SANS data with molecular modeling provided a first, to our knowledge, structural characterization of an entire NOX enzyme. It revealed a distinctly less compact structure than that predicted from the docking of homologous crystal structures of the separate transmembrane and dehydrogenase domains, consistent with a flexible linker connecting the two domains.

Rapid On-Chip Synthesis of Complex Glycomimetics from N-Glycan Scaffolds for Improved Lectin Targeting.

C-type lectin receptor (CLR) carbohydrate binding proteins found on immune cells with important functions in pathogen recognition as well as self and non-self-differentiation are increasingly moving into the focus of drug developers as targets for the immune therapy of cancer autoimmune diseases and inflammation and to improve the efficacy of vaccines. The development of molecules with increased affinity and selectivity over the natural glycan binders has largely focused on the synthesis of mono and disaccharide mimetics but glycan array binding experiments have shown increased binding selectivity and affinity for selected larger oligosaccharides that are able to engage in additional favorable interactions beyond the primary binding site. Here, a platform for the rapid preparation and screening of N-glycan mimetics on microarrays is presented that turns a panel of complex glycan core structures into structurally diverse glycomimetics by a combination of enzymatic glycosylation with a nonnatural donor and subsequent cycloaddition with a collection of alkynes. All surface-based reactions were monitored by MALDI-TOF MS to assess conversion and purity of spot compositions. Screening the collection of 374 N-glycomimetics against the plant lectin WFA and the 2 human immune lectins MGL ECD and Langerin ECD produced a number of high affinity binders as lead structures for more selective lectin targeting probes.

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.

Second-Generation Dendrimers with Chondroitin Sulfate Type-E Disaccharides as Multivalent Ligands for Langerin.

Chondroitin sulfate type-E (CS-E) is a sulfated polysaccharide that shows several interesting biological activities, such as modulation of the neuronal growth factor signaling and its interaction with langerin, a C-type lectin with a crucial role in the immunological system. However, applications of CS-E are hampered by the typical heterogeneous structure of the natural polysaccharide. Well-defined, homogeneous CS-E analogues are highly demanded. Here, we report the synthesis of monodispersed, structurally well-defined second-generation glycodendrimers displaying up to 18 CS-E disaccharide units. These complex multivalent systems have a molecular weight and a number of disaccharide repeating units comparable with those of the natural polysaccharides. In addition, surface plasmon resonance experiments revealed a calcium-independent interaction between these glycodendrimers and langerin, in the micromolar range, highlighting the utility of these compounds as CS-E mimetics.

Influence of the reducing-end anomeric configuration of the Man9 epitope on DC-SIGN recognition.

High-mannose (Man9GlcNAc2) is the main carbohydrate unit present in viral envelope glycoproteins such as gp120 of HIV and the GP1 of Ebola virus. This oligosaccharide comprises the Man9 epitope conjugated to two terminal N-acetylglucosamines by otherwise rarely-encountered ß-mannose glycosidic bond. Formation of this challenging linkage is the bottleneck of the few synthetic approaches described to prepare high mannose. Herein, we report the synthesis of the Man9 epitope with both alpha and beta configurations at the reducing end, and subsequent evaluation of the impact of this configuration on binding to natural receptor of high-mannose, DC-SIGN. Using fluorescence polarization assays, we demonstrate that both anomers bind to DC-SIGN with comparable affinity. These relevant results therefore indicate that the more synthetically-accesible Man9 alpha epitope may be deployed as ligand for DC-SIGN in both in vitro and in vivo biological assays.

Development of C-type lectin-oriented surfaces for high avidity glycoconjugates: towards mimicking multivalent interactions on the cell surface.

Multivalent interactions between complex carbohydrates and oligomeric C-type lectins govern a wide range of immune responses. Up to date, standard SPR (surface plasmon resonance) competitive assays have largely been to evaluate binding properties from monosaccharide units (low affinity, mM) to multivalent elemental antagonists (moderate affinity, µM). Herein, we report typical case-studies of SPR competitive assays showing that they underestimate the potency of glycoclusters to inhibit the interaction between DC-SIGN and immobilized glycoconjugates. This paper describes the design and implementation of a SPR direct interaction over DC-SIGN oriented surfaces, extendable to other C-type lectin surfaces as such Langerin. This setup provides an overview of intrinsic avidity generation emanating simultaneously from multivalent glycoclusters and from DC-SIGN tetramers organized in nanoclusters at the cell membrane. To do so, covalent biospecific capture of DC-SIGN via StreptagII/StrepTactin interaction preserves tetrameric DC-SIGN, accessibility and topology of its active sites, that would have been dissociated using standard EDC-NHS procedure under acidic conditions. From the tested glycoclusters libraries, we demonstrated that the scaffold architecture, the valency and the glycomimetic-based ligand are crucial to reach nanomolar affinities for DC-SIGN. The glycocluster 3·D illustrates the tightest binding partner in this set for a DC-SIGN surface (KD = 18 nM). Moreover, the selectivity at monovalent scale of glycomimetic D can be easily analyzed at multivalent scale comparing its binding over different C-type lectin immobilized surfaces. This approach may give rise to novel insights into the multivalent binding mechanisms responsible for avidity and make a major contribution to the full characterization of the binding potency of promising specific and multivalent immodulators.

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.

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide.

Carbohydrate-lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOSR1 mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.

Enhancing Potency and Selectivity of a DC-SIGN Glycomimetic Ligand by Fragment-Based Design: Structural Basis.

Chemical modification of pseudo-dimannoside ligands guided by fragment-based design allowed for the exploitation of an ammonium-binding region in the vicinity of the mannose-binding site of DC-SIGN, leading to the synthesis of a glycomimetic antagonist (compound 16) of unprecedented affinity and selectivity against the related lectin langerin. Here, the computational design of pseudo-dimannoside derivatives as DC-SIGN ligands, their synthesis, their evaluation as DC-SIGN selective antagonists, the biophysical characterization of the DC-SIGN/16 complex, and the structural basis for the ligand activity are presented. On the way to the characterization of this ligand, an unusual bridging interaction within the crystals shed light on the plasticity and potential secondary binding sites within the DC-SIGN carbohydrate recognition domain.

On-Chip Screening of a Glycomimetic Library with C-Type Lectins Reveals Structural Features Responsible for Preferential Binding of Dectin-2 over DC-SIGN/R and Langerin.

A library of mannose- and fucose-based glycomimetics was synthesized and screened in a microarray format against a set of C-type lectin receptors (CLRs) that included DC-SIGN, DC-SIGNR, langerin, and dectin-2. Glycomimetic ligands able to interact with dectin-2 were identified for the first time. Comparative analysis of binding profiles allowed their selectivity against other CLRs to be probed.