Insights into Glycobiology and the Protein-Glycan Interactome Using Glycan Microarray Technologies.

Glycans linked to proteins and lipids and also occurring in free forms have many functions, and these are partly elicited through specific interactions with glycan-binding proteins (GBPs). These include lectins, adhesins, toxins, hemagglutinins, growth factors, enzymes, but antibodies can also bind glycans. While humans and other animals generate a vast repertoire of GBPs and different glycans in their glycomes, other organisms, including phage, microbes, protozoans, fungi, and plants also express glycans and GBPs, and these can also interact with their host glycans. This can be termed the protein-glycan interactome, and in nature is likely to be vast, but is so far very poorly described. Understanding the breadth of the protein-glycan interactome is also a key to unlocking our understanding of infectious diseases involving glycans, and immunology associated with antibodies binding to glycans. A key technological advance in this area has been the development of glycan microarrays. This is a display technology in which minute quantities of glycans are attached to the surfaces of slides or beads. This allows the arrayed glycans to be interrogated by GBPs and antibodies in a relatively high throughput approach, in which a protein may bind to one or more distinct glycans. Such binding can lead to novel insights and hypotheses regarding both the function of the GBP, the specificity of an antibody, and the function of the glycan within the context of the protein-glycan interactome. This article focuses on the types of glycan microarray technologies currently available to study animal glycobiology and examples of breakthroughs aided by these technologies. (254 words).

Functional implications of glycans and their curation: insights from the workshop held at the 16th Annual International Biocuration Conference in Padua, Italy.

Dynamic changes in protein glycosylation impact human health and disease progression. However, current resources that capture disease and phenotype information focus primarily on the macromolecules within the central dogma of molecular biology (DNA, RNA, proteins). To gain a better understanding of organisms, there is a need to capture the functional impact of glycans and glycosylation on biological processes. A workshop titled "Functional impact of glycans and their curation" was held in conjunction with the 16th Annual International Biocuration Conference to discuss ongoing worldwide activities related to glycan function curation. This workshop brought together subject matter experts, tool developers, and biocurators from over 20 projects and bioinformatics resources. Participants discussed four key topics for each of their resources: (i) how they curate glycan function-related data from publications and other sources, (ii) what type of data they would like to acquire, (iii) what data they currently have, and (iv) what standards they use. Their answers contributed input that provided a comprehensive overview of state-of-the-art glycan function curation and annotations. This report summarizes the outcome of discussions, including potential solutions and areas where curators, data wranglers, and text mining experts can collaborate to address current gaps in glycan and glycosylation annotations, leveraging each other's work to improve their respective resources and encourage impactful data sharing among resources. Database URL: https://wiki.glygen.org/Glycan_Function_Workshop_2023.

Recognition of Highly Branched N-Glycans of the Porcine Whipworm by the Immune System.

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array, we explored the interactions of glycans with C-type lectins, C-reactive protein, and sera from T. suis-infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

Reusable glycan microarrays using a microwave assisted wet-erase (MAWE) process.

Modern studies on binding of proteins to glycans commonly involve the use of synthetic glycans and their derivatives in which a small amount of the material is covalently printed onto a functionalized slide in a glycan microarray format. While incredibly useful to explore binding interactions with many types of samples, the common techniques involve drying the slides, which leads to irreversible association of the protein to the spots on slides to which they bound, thus limiting a microarray to a single use. We have developed a new technique which we term Microwave Assisted Wet-Erase (MAWE) glycan microarrays. In this approach we image the slides under wet conditions to acquire the data, after which the slides are cleaned of binding proteins by treatment with a denaturing SDS solution along with microwave treatment. Slides cleaned in this way can be reused multiple times, and an example here shows the reuse of a single array 15 times. We also demonstrate that this method can be used for a single-array per slide or multi-array per slide platforms. Importantly, the results obtained using this technique for a variety of lectins sequentially applied to a single array, are concordant to those obtained via the classical dry approaches on multiple slides. We also demonstrate that MAWE can be used for different types of samples, such as serum for antibody binding, and whole cells, such as yeast. This technique will greatly conserve precious glycans and prolong the use of existing and new glycan microarrays.

Recognition of highly branched N-glycans of the porcine whipworm by the immune system.

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array we explored the interactions of glycans with C-type lectins, C-reactive protein and sera from T. suis infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties, but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems, and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation.

Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1 N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

Heparan sulfate glycomimetics via iterative assembly of "clickable" disaccharides.

Heparan sulfate (HS) glycosaminoglycans are widely expressed on the mammalian cell surfaces and extracellular matrices and play important roles in a variety of cell functions. Studies on the structure-activity relationships of HS have long been hampered by the challenges in obtaining chemically defined HS structures with unique sulfation patterns. Here, we report a new approach to HS glycomimetics based on iterative assembly of clickable disaccharide building blocks that mimic the disaccharide repeating units of native HS. Variably sulfated clickable disaccharides were facilely assembled into a library of mass spec-sequenceable HS-mimetic oligomers with defined sulfation patterns by solution-phase iterative syntheses. Microarray and surface plasmon resonance (SPR) binding assays corroborated molecular dynamics (MD) simulations and confirmed that these HS-mimetic oligomers bind protein fibroblast growth factor 2 (FGF2) in a sulfation-dependent manner consistent with that of the native HS. This work established a general approach to HS glycomimetics that can potentially serve as alternatives to native HS in both fundamental research and disease models.

Increased Galectin-9 Levels Correlate with Disease Activity in Patients with DMARD-Naïve Rheumatoid Arthritis and Modulate the Secretion of MCP-1 and IL-6 from Synovial Fibroblasts.

Background: Fibroblast-like synoviocytes (FLSs) are essential mediators in the expansive growth and invasiveness of rheumatoid synovitis, and patients with a fibroblastic-rich pauci-immune pathotype respond poorly to currently approved antirheumatic drugs. Galectin-9 (Gal-9) has been reported to directly modulate rheumatoid arthritis (RA) FLSs and to hold both pro- and anti-inflammatory properties. The objective of this study was to evaluate clinical and pathogenic aspects of Gal-9 in RA, combining national patient cohorts and cellular models. Methods: Soluble Gal-9 was measured in plasma from patients with newly diagnosed, treatment-naïve RA (n = 98). The disease activity score 28-joint count C-reactive protein (DAS28CRP) and total Sharp score were used to evaluate the disease course serially over a two-year period. Plasma and synovial fluid samples were examined for soluble Gal-9 in patients with established RA (n = 18). A protein array was established to identify Gal-9 binding partners in the extracellular matrix (ECM). Synovial fluid mononuclear cells (SFMCs), harvested from RA patients, were used to obtain synovial-fluid derived FLSs (SF-FLSs) (n = 7). FLSs from patients suffering from knee Osteoarthritis (OA) were collected from patients when undergoing joint replacement surgery (n = 5). Monocultures of SF-FLSs (n = 6) and autologous co-cultures of SF-FLSs and peripheral blood mononuclear cells (PBMCs) were cultured with and without a neutralizing anti-Gal-9 antibody (n = 7). The mono- and co-cultures were subsequently analyzed by flow cytometry, MTT assay, and ELISA. Results: Patients with early and established RA had persistently increased plasma levels of Gal-9 compared with healthy controls (HC). The plasma levels of Gal-9 were associated with disease activity and remained unaffected when adding a TNF-inhibitor to their standard treatment. Gal-9 levels were elevated in the synovial fluid of established RA patients with advanced disease, compared with corresponding plasma samples. Gal-9 adhered to fibronectin, laminin and thrombospondin, while not to interstitial collagens in the ECM protein array. In vitro, a neutralizing Gal-9 antibody decreased MCP-1 and IL-6 production from both RA FLSs and OA FLSs. In co-cultures of autologous RA FLSs and PBMCs, the neutralization of Gal-9 also decreased MCP-1 and IL-6 production, without affecting the proportion of inflammatory FLSs. Conclusions: In RA, pretreatment plasma Gal-9 levels in early RA were increased and correlated with clinical disease activity. Gal-9 levels remained increased despite a significant reduction in the disease activity score in patients with early RA. The in vitro neutralization of Gal-9 decreased both MCP-1 and IL-6 production in an inflammatory subset of RA FLSs. Collectively these findings indicate that the persistent overexpression of Gal-9 in RA may modulate synovial FLS activities and could be involved in the maintenance of subclinical disease activity in RA.

N-glycosylation of mannose receptor (CD206) regulates glycan binding by C-type lectin domains.

The macrophage mannose receptor (MR, CD206) is a transmembrane endocytic lectin receptor, expressed in selected immune and endothelial cells, and is involved in immunity and maintaining homeostasis. Eight of the ten extracellular domains of the MR are C-type lectin domains (CTLDs) which mediate the binding of mannose, fucose, and GlcNAc in a calcium-dependent manner. Previous studies indicated that self-glycosylation of MR regulates its glycan binding. To further explore this structure-function relationship, we studied herein a recombinant version of mouse MR CTLD4-7 fused to human Fc-portion of IgG (MR-Fc). The construct was expressed in different glycosylation-mutant cell lines to study the influence of differential glycosylation on receptor glycan-binding properties. We conducted site-specific N- and O-glycosylation analysis and glycosylation site characterization using mass spectrometry by which several novel O-glycosylation sites were identified in mouse MR and confirmed in human full-length MR. This information guided experiments evaluating the receptor functionality by glycan microarray analysis in combination with glycan-modifying enzymes. Treatment of active MR-Fc with combinations of exoglycosidases, including neuraminidase and galactosidases, resulted in the loss of trans-binding (binding of MR CTLDs to non-MR glycans), due to unmasking of terminal, nonreducing GlcNAc in N-glycans of the MR CTLDs. Regalactosylation of N-glycans rescues mannose binding by MR-Fc. Our results indicate that glycans within the MR CTLDs act as a regulatory switch by masking and unmasking self-ligands, including terminal, nonreducing GlcNAc in N-glycans, which could control MR activity in a tissue- and cell-specific manner or which potentially affect bacterial pathogenesis in an immunomodulatory fashion.

Galectin-3 Decreases 4-1BBL Bioactivity by Crosslinking Soluble and Membrane Expressed 4-1BB.

4-1BB is a T cell costimulatory receptor and a member of the tumor necrosis factor receptor superfamily. Here, we show that Galectin-3 (Gal-3) decreases the cellular response to its ligand (4-1BBL). Gal-3 binds to both soluble 4-1BB (s4-1BB) and membrane-bound 4-1BB (mem4-1BB), without blocking co-binding of 4-1BBL. In plasma, we detected complexes composed of 4-1BB and Gal-3 larger than 100 nm in size; these complexes were reduced in synovial fluid from rheumatoid arthritis. Both activated 4-1BB+ T cells and 4-1BB-transfected HEK293 cells depleted these complexes from plasma, followed by increased expression of 4-1BB and Gal-3 on the cell surface. The increase was accompanied by a 4-fold decrease in TNFα production by the 4-1BBhighGal-3+ T cells, after exposure to 4-1BB/Gal-3 complexes. In RA patients, complexes containing 4-1BB/Gal-3 were dramatically reduced in both plasma and SF compared with healthy plasma. These results support that Gal-3 binds to 4-1BB without blocking the co-binding of 4-1BBL. Instead, Gal-3 leads to formation of large soluble 4-1BB/Gal-3 complexes that attach to mem4-1BB on the cell surfaces, resulting in suppression of 4-1BBL's bioactivity.

Differential recognition of oligomannose isomers by glycan-binding proteins involved in innate and adaptive immunity.

The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.

Tumor cells express pauci- and oligomannosidic N-glycans in glycoproteins recognized by the mannose receptor (CD206).

The macrophage mannose receptor (CD206, MR) is an endocytic lectin receptor which plays an important role in homeostasis and innate immunity, however, the endogenous glycan and glycoprotein ligands recognized by its C-type lectin domains (CTLD) have not been well studied. Here we used the murine MR CTLD4-7 coupled to the Fc-portion of human IgG (MR-Fc) to investigate the MR glycan and glycoprotein recognition. We probed 16 different cancer and control tissues using the MR-Fc, and observed cell- and tissue-specific binding with varying intensity. All cancer tissues and several control tissues exhibited MR-Fc ligands, intracellular and/or surface-located. We further confirmed the presence of ligands on the surface of cancer cells by flow cytometry. To characterize the fine specificity of the MR for glycans, we screened a panel of glycan microarrays. Remarkably, the results indicate that the CTLD4-7 of the MR is highly selective for specific types of pauci- and oligomannose N-glycans among hundreds of glycans tested. As lung cancer tissue and the lung cancer cell line A549 showed intense MR-Fc binding, we further investigated the MR glycoprotein ligands in those cells by immunoprecipitation and glycoproteomic analysis. All enriched glycoproteins, of which 42 were identified, contained pauci- or oligomannose N-glycans, confirming the microarray results. Our study demonstrates that the MR CTLD4-7 is highly selective for pauci- and oligomannosidic N-glycans, structures that are often elevated in tumor cells, and suggest a potential role for the MR in tumor biology.

Unique repertoire of anti-carbohydrate antibodies in individual human serum.

Humoral immunity to pathogens and other environmental challenges is paramount to maintain normal health, and individuals lacking or unable to make antibodies are at risk. Recent studies indicate that many human protective antibodies are against carbohydrate antigens; however, little is known about repertoires and individual variation of anti-carbohydrate antibodies in healthy individuals. Here we analyzed anti-carbohydrate antibody repertoires (ACARs) of 105 healthy individual adult donors, aged 20-60+ from different ethnic backgrounds to explore variations in antibodies, as defined by binding to glycan microarrays and by affinity purification. Using microarrays that contained > 1,000 glycans, including antigens from animal cells and microbes, we profiled the IgG and IgM ACARs from all donors. Each donor expressed many ACAs, but had a relatively unique ACAR, which included unanticipated antibodies to carbohydrate antigens not well studied, such as chitin oligosaccharides, Forssman-related antigens, globo-type antigens, and bacterial glycans. We also saw some expected antibodies to ABO(H) blood group and α-Gal-type antigens, although these also varied among individuals. Analysis suggests differences in ACARs are associated with ethnicity and age. Thus, each individual ACAR is relatively unique, suggesting that individualized information could be useful in precision medicine for predicting and monitoring immune health and resistance to disease.

Tools for generating and analyzing glycan microarray data.

Glycans are one of the major biological polymers found in the mammalian body. They play a vital role in a number of physiologic and pathologic conditions. Glycan microarrays allow a plethora of information to be obtained on protein-glycan binding interactions. In this review, we describe the intricacies of the generation of glycan microarray data and the experimental methods for studying binding. We highlight the importance of this knowledge before moving on to the data analysis. We then highlight a number of tools for the analysis of glycan microarray data such as data repositories, data visualization and manual analysis tools, automated analysis tools and structural informatics tools.

Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.

GlycoGlyph: a glycan visualizing, drawing and naming application.

MOTIVATION: Glycan structures are commonly represented using symbols or linear nomenclature such as that from the Consortium for Functional Glycomics (also known as modified IUPAC-condensed nomenclature). No current tool allows for writing the name in such format using a graphical user interface (GUI); thus, names are prone to errors or non-standardized representations. RESULTS: Here we present GlycoGlyph, a web application built using JavaScript, which is capable of drawing glycan structures using a GUI and providing the linear nomenclature as an output or using it as an input in a dynamic manner. GlycoGlyph also allows users to save the structures as an SVG vector graphic, and allows users to export the structure as condensed GlycoCT. AVAILABILITY AND IMPLEMENTATION: The application can be used at: https://glycotoolkit.com/Tools/GlycoGlyph/. The application is tested to work in modern web browsers such as Firefox or Chrome. CONTACT: aymehta@bidmc.harvard.edu or rcummin1@bidmc.harvard.edu.

GlyMDB: Glycan Microarray Database and analysis toolset.

MOTIVATION: Glycan microarrays are capable of illuminating the interactions of glycan-binding proteins (GBPs) against hundreds of defined glycan structures, and have revolutionized the investigations of protein-carbohydrate interactions underlying numerous critical biological activities. However, it is difficult to interpret microarray data and identify structural determinants promoting glycan binding to glycan-binding proteins due to the ambiguity in microarray fluorescence intensity and complexity in branched glycan structures. To facilitate analysis of glycan microarray data alongside protein structure, we have built the Glycan Microarray Database (GlyMDB), a web-based resource including a searchable database of glycan microarray samples and a toolset for data/structure analysis. RESULTS: The current GlyMDB provides data visualization and glycan-binding motif discovery for 5203 glycan microarray samples collected from the Consortium for Functional Glycomics. The unique feature of GlyMDB is to link microarray data to PDB structures. The GlyMDB provides different options for database query, and allows users to upload their microarray data for analysis. After search or upload is complete, users can choose the criterion for binder versus non-binder classification. They can view the signal intensity graph including the binder/non-binder threshold followed by a list of glycan-binding motifs. One can also compare the fluorescence intensity data from two different microarray samples. A protein sequence-based search is performed using BLAST to match microarray data with all available PDB structures containing glycans. The glycan ligand information is displayed, and links are provided for structural visualization and redirection to other modules in GlycanStructure.ORG for further investigation of glycan-binding sites and glycan structures. AVAILABILITY AND IMPLEMENTATION: http://www.glycanstructure.org/glymdb. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Novel Reversible Fluorescent Glycan Linker for Functional Glycomics.

To aid in generating complex and diverse natural glycan libraries for functional glycomics, more efficient and reliable methods are needed to derivatize glycans. Here we present our development of a reversible, cleavable bifunctional linker 3-(methoxyamino)propylamine (MAPA). As the fluorenylmethyloxycarbonate (Fmoc) version (F-MAPA), it is highly fluorescent and efficiently derivatizes free reducing glycans to generate closed-ring derivatives that preserve the structural integrity of glycans. A library of glycans were derivatized and used to generate a covalent glycan microarray using N-hydroxysuccinimide derivatization. The array was successfully interrogated by a variety of lectins and antibodies, demonstrating the importance of closed-ring chemistry. The glycan derivatization was also performed at large scale using milligram quantities of glycans and excess F-MAPA, and the reaction system was successfully recycled up to five times, without an apparent decrease in conjugation efficiency. The MAPA-glycan is also easy to link to protein to generate neoglycoproteins with equivalent glycan densities. Importantly, the MAPA linker can be reversibly cleaved to regenerate free reducing glycans for detailed structural analysis (catch-and-release), often critical for functional studies of undefined glycans from natural sources. The high conjugation efficiency, bright fluorescence, and reversible cleavage of the linker enable access to natural glycans for functional glycomics.