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.

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.

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.

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.

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.

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.

Fluorinated carbohydrates as chemical probes for molecular recognition studies. Current status and perspectives.

This review provides an extensive summary of the effects of carbohydrate fluorination with regard to changes in physical, chemical and biological properties with respect to regular saccharides. The specific structural, conformational, stability, reactivity and interaction features of fluorinated sugars are described, as well as their applications as probes and in chemical biology.

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.

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Glycans in drug discovery.

Glycans are key players in many biological processes. They are essential for protein folding and stability and act as recognition elements in cell-cell and cell-matrix interactions. Thus, being at the heart of medically relevant biological processes, glycans have come onto the scene and are considered hot spots for biomedical intervention. The progress in biophysical techniques allowing access to an increasing molecular and structural understanding of these processes has led to the development of effective therapeutics. Indeed, strategies aimed at designing glycomimetics able to block specific lectin-carbohydrate interactions, carbohydrate-based vaccines mimicking self- and non-self-antigens as well as the exploitation of the therapeutic potential of glycosylated antibodies are being pursued. In this mini-review the most prominent contributions concerning recurrent diseases are highlighted, including bacterial and viral infections, cancer or immune-related pathologies, which certainly show the great promise of carbohydrates in drug discovery.

Metabolomics Applied to the Study of Extracellular Vesicles.

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities-particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies.

Assessing the role of surface glycans of extracellular vesicles on cellular uptake.

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. However, there is ambiguity around the fundamental mechanisms by which these effects are transduced, particularly in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Lectin microarray technology was employed to define the surface glycosylation patterns of EVs. Glycosidases PNGase F and neuraminidase which cleave N-glycans and terminal sialic acids, respectively, were used to analyze the relevance of these modifications to EV surface glycans on the uptake of fluorescently labelled EVs by a panel of cells representing a variety of tissues. Flow cytometry revealed an increase in affinity for EVs modified by both glycosidase treatments. High-content screening exhibited a broader range of responses with different cell types preferring different vesicle glycosylation states. We also found differences in vesicle charge after treatment with glycosidases. We conclude that glycans are key players in the tuning of EV uptake, through charge-based effects, direct glycan recognition or both, supporting glycoengineering as a toolkit for therapy development.

Molecular Insights into DC-SIGN Binding to Self-Antigens: The Interaction with the Blood Group A/B Antigens.

The dendritic cell-specific intracellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is an important receptor of the immune system. Besides its role as pathogen recognition receptor (PRR), it also interacts with endogenous glycoproteins through the specific recognition of self-glycan epitopes, like LeX. However, this lectin represents a paradigmatic case of glycan binding promiscuity, and it also has been shown to recognize antigens with α1-α2 linked fucose, such as the histo blood group antigens, with similar affinities to LeX. Herein, we have studied the interaction in solution between DC-SIGN and the blood group A and B antigens, to get insights into the atomic details of such interaction. With a combination of different NMR experiments, we demonstrate that the Fuc coordinates the primary Ca2+ ion with a single binding mode through 3-OH and 4-OH. The terminal αGal/αGalNAc affords marginal direct polar contacts with the protein, but provides a hydrophobic hook in which V351 of the lectin perfectly fits. Moreover, we have found that αGal, but not αGalNAc, is a weak binder itself for DC-SIGN, which could endow an additional binding mode for the blood group B antigen, but not for blood group A.

Microarray assessment of N-glycan-specific IgE and IgG profiles associated with Schistosoma mansoni infection in rural and urban Uganda.

Core ß-1,2-xylose and α-1,3-fucose are antigenic motifs on schistosome N-glycans, as well as prominent IgE targets on some plant and insect glycoproteins. To map the association of schistosome infection with responses to these motifs, we assessed plasma IgE and IgG reactivity using microarray technology among Ugandans from rural Schistosoma mansoni (Sm)-endemic islands (n = 209), and from proximate urban communities with lower Sm exposure (n = 62). IgE and IgG responses to core ß-1,2-xylose and α-1,3-fucose modified N-glycans were higher in rural versus urban participants. Among rural participants, IgE and IgG to core ß-1,2-xylose were positively associated with Sm infection and concentration peaks coincided with the infection intensity peak in early adolescence. Responses to core α-1,3-fucose were elevated regardless of Sm infection status and peaked before the infection peak. Among urban participants, Sm infection intensity was predominantly light and positively associated with responses to both motifs. Principal component and hierarchical cluster analysis reduced the data to a set of variables that captured core ß-1,2-xylose- and α-1,3-fucose-specific responses, and confirmed associations with Sm and the rural environment. Responses to core ß-1,2-xylose and α-1,3-fucose have distinctive relationships with Sm infection and intensity that should further be explored for associations with protective immunity, and cross-reactivity with other exposures.

Fluorescent Neoglycoprotein Gold Nanoclusters: Synthesis and Applications in Plant Lectin Sensing and Cell Imaging.

Carbohydrate-protein interactions mediate fundamental biological processes, such as fertilization, cell signaling, or host-pathogen communication. However, because of the enormous complexity of glycan recognition events, new tools enabling their analysis or applications emerge in recent years. Here, we describe the first preparation of neoglycoprotein functionalized fluorescent gold nanoclusters, containing a biantennary N-glycan G0 as targeting molecule, ovalbumin as carrier/model antigen, and a fluorescent gold core as imaging probe (G0-OVA-AuNCs). Subsequently, we demonstrate the utility of generated G0-OVA-AuNCs for specific sensing of plant lectins and in vitro imaging of dendritic cells.

Glycan Microarray-Assisted Identification of IgG Subclass Targets in Schistosomiasis.

Infection with schistosomes is accompanied by the induction of antibodies against the parasite. Despite having IgG against both protein and glycan antigens, infected individuals remain chronically infected until treated, and re-infection is common in endemic areas as immunity does not develop effectively. Parasite specific IgG subclasses may differ in functionality and effectivity with respect to effector functions that contribute to parasite killing and immunity. In this study, we investigated if specific IgG subclasses target specific antigenic schistosome glycan motifs during human infection. Sera from 41 S. mansoni infected individuals from an endemic area in Uganda were incubated on two glycan microarrays, one consisting of a large repertoire of schistosome glycoprotein- and glycolipid- derived glycans and the other consisting of chemically synthesized core xylosylated and fucosylated N-glycans also expressed by schistosomes. Our results show that highly antigenic glycan motifs, such as multi-fucosylated terminal GalNAc(ß1-4)GlcNAc (LDN) can be recognized by all IgG subclasses of infection sera, however with highly variable intensities. Detailed examination of core-modified N-glycan targets revealed individual antibody responses specific for core-xylosylated and core α3-fucosylated glycan motifs that are life stage specifically expressed by schistosomes. IgG1 and IgG3 were detected against a range of N-glycan core structures, but IgG2 and IgG4, when present, were specific for the core α3-fucose and xylose motifs that were previously found to be IgE targets in schistosomiasis, and in allergies. This study is the first to address IgG subclass responses to defined helminth glycans.

Measuring Bacterial Glycosyl Hydrolase Activity with a Soluble Capture Probe by Mass Spectrometry.

A solution-phase enzymatic assay has been developed to track bacterial glycosyl hydrolase activity by surface-assisted MALDI-TOF mass spectrometry. Lactose was equipped with an azide-functionalized linker and was supplemented to bacterial cultures as an artificial substrate for bacterial ß-galactosidase enzyme. The azide linked glycoside probe was then covalently captured on an alkyne-functionalized indium tin oxide sample plate via a bio-orthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). The noncovalent immobilization of the alkyne capture tag via hydrophobic interactions on the ITO-sample plate allowed the analysis of the probe conjugate by surface-based mass spectrometry. The ratio of digested to nondigested lactose probe was then employed as a measure for bacterial hydrolase activity, which correlated well with bacterial growth measured by optical density. In addition, we established in a proof of concept experiment that the setup was well suited to identify antibiotic susceptibility of bacterial strains with a performance comparable to current state-of-the-art methods. While the proof of concept version is limited to the identification of a single enzyme activity, we envisage that the use of multiple substrate probes in a multiplexed version will allow the quantification of various glycosyl hydrolase activities with clinical relevance in a single experiment.