Glycan Array Research Articles

Semi‐Synthetic Glycoconjugate Vaccine Lead Against Acinetobacter baumannii 17978

AbstractAcinetobacter baumannii is a opportunistic bacterial pathogen responsible for serious nosocomial infections that is becoming increasingly resistant against antibiotics. Capsular polysaccharides (CPS) that cover A. baumannii are a major virulence factor that play an important role in pathogenesis, are used to assign serotypes and provide the basis for vaccine development. Synthetic oligosaccharides resembling the CPS of A. baumannii 17978 were printed onto microarray slides and used to screen sera from patients infected with A. baumannii as well as a monoclonal mouse antibody (mAb C8). A synthetic oligosaccharide emerged from glycan array screening as lead for the development of a vaccine against A. baumannii 17978. Tetrasaccharide 20 is a key epitope for recognition by an antibody and is a vaccine lead.

Specificity of viscumin revised. As probed with a printed glycan array

Viscumin, a lectin used in anti-cancer therapy, was originally considered as βGal recognizing protein; later, an ability to bind 6′-sialyl N-acetyllactosamine (6′SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galβ1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral β-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a β-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.

An Automated Approach to Assess Relative Galectin-Glycan Affinity Following Glycan Microarray Analysis.

Numerous studies have highlighted the utility of glycan microarray analysis for the elucidation of protein-glycan interactions. However, most current glycan microarray studies analyze glycan binding protein (GBP)-glycan interactions at a single protein concentration. While this approach provides useful information related to a GBP’s overall binding capabilities, extrapolation of true glycan binding preferences using this method fails to account for printing variations or other factors that may confound relative binding. To overcome this limitation, we examined glycan array binding of three galectins over a range of concentrations to allow for a more complete assessment of binding preferences. This approach produced a richer data set than single concentration analysis and provided more accurate identification of true glycan binding preferences. However, while this approach can be highly informative, currently available data analysis approaches make it impractical to perform binding isotherms for each glycan present on currently available platforms following GBP evaluation. To overcome this limitation, we developed a method to directly optimize the efficiency of assessing association constants following multi-GBP concentration glycan array analysis. To this end, we developed programs that automatically analyze raw array data (kdMining) to generate output graphics (kaPlotting) following array analysis at multiple doses. These automatic programing methods reduced processing time from 32.8 h to 1.67 min. Taken together, these results demonstrate an effective approach to glycan array analysis that provides improved detail and efficiency when compared to previous methods.

Synthesis of Fungal Cell Wall Oligosaccharides and Their Ability to Trigger Plant Immune Responses.

Oligosaccharide fragments of fungal cell wall glycans are important molecular probes for studying both the biology of fungi and fungal infections of humans, animals, and plants. The fungal cell wall contains large amounts of various polysaccharides that are ligands for pattern recognition receptors (PRRs), eliciting an immune response upon recognition. Towards the establishment of a glycan array platform for the identification of new ligands of plant PRRs, tri‐, penta‐, and heptasaccharide fragments of different cell wall polysaccharides were prepared. Chito‐ and β‐(1→6)‐gluco‐oligosaccharides were synthesized by automated glycan assembly (AGA), and α‐(1→3)‐ and α‐(1→4)‐gluco‐oligosaccharides were synthesized in solution using a recently reported highly α‐selective glycosylation methodology. Incubation of plants with the synthesized oligosaccharides revealed i) length dependence for plant activation by chito‐oligosaccharides and ii) β‐1,6‐glucan oligosaccharides as a new class of glycans capable of triggering plant activation.

Biologically Derived Neoproteoglycans for Profiling Protein–Glycosaminoglycan Interactions

Glycosaminoglycans (GAGs) are a class of highly negatively charged membrane-associated and extracellular matrix polysaccharides involved in the regulation of myriad biological functions, including cell adhesion, migration, signaling, and differentiation, among others. GAGs are typically attached to core proteins, termed proteoglycans (PGs), and can engage >500 binding proteins, making them prominent relays for sensing external stimuli and transducing cellular responses. However, their unique substructural protein-recognition domains that confer their binding specificity remain elusive. While the emergence of glycan arrays has rapidly enabled the profiling of ligand specificities of a range of glycan-binding proteins, their adaptation for the analysis of GAG-binding proteins has been considerably more challenging. Current GAG microarrays primarily employ synthetically defined oligosaccharides, which capture only a fraction of the structural diversity of native GAG polysaccharides. Augmenting existing array platforms to include GAG structures purified from tissues or produced in cells with engineered glycan biosynthetic pathways may significantly advance the understanding of structure-activity relationships in GAG-protein interactions. Here, we demonstrate an efficient and tunable strategy to mimic cellular proteoglycan architectures by conjugating biologically derived GAG chains to a protein scaffold, defined as neoproteoglycans (neoPGs). The use of a reactive fluorogenic linker enabled real-time monitoring of the conjugation reaction efficiency and tuning of the neoPG valency. Immobilization of the reagents on a 96-well array platform allowed for efficient probing of ligand binding and enzyme-substrate specificity, including growth factors and the human sulfatase 1. The neoPGs can also be used directly as soluble probes to evaluate GAG-dependent growth factor signaling in cells.

Novel fold of rotavirus glycan-binding domain predicted by AlphaFold2 and determined by X-ray crystallography.

The VP8* domain of spike protein VP4 in group A and C rotaviruses, which cause epidemic gastroenteritis in children, exhibits a conserved galectin-like fold for recognizing glycans during cell entry. In group B rotavirus, which causes significant diarrheal outbreaks in adults, the VP8* domain (VP8*B) surprisingly lacks sequence similarity with VP8* of group A or group C rotavirus. Here, by using the recently developed AlphaFold2 for ab initio structure prediction and validating the predicted model by determining a 1.3-Å crystal structure, we show that VP8*B exhibits a novel fold distinct from the galectin fold. This fold with a β-sheet clasping an α-helix represents a new fold for glycan recognition based on glycan array screening, which shows that VP8*B recognizes glycans containing N-acetyllactosamine moiety. Although uncommon, our study illustrates how evolution can incorporate structurally distinct folds with similar functionality in a homologous protein within the same virus genus.

Genetically encoded multivalent liquid glycan array displayed on M13 bacteriophage

Abnormal cell surface glycosylation plays a major role in disease processes such as immune evasion. However, the underlying role of glycans is yet to be fully understood. Binding information obtained from glycan arrays can provide critical starting points for downstream applications such as the development of carbohydrate‐based inhibitors, vaccines, and other therapeutics. However, it is challenging to use powerful techniques like DNA deep sequencing to analyze glycan recognition due to the lack of 1:1 correspondence between DNA and glycan structures. Therefore, we have developed Liquid Glycan Array (LiGA), a technology that allows for genetic encoding of glycans. LiGA provides a 1:1 correspondence between the glycan displayed in multiple copies on a bacteriophage carrier and the phage genetic material. LiGA is generated by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido‐modified glycans. The display of glycans on each phage virion can be controlled from 30‐1500 copies to probe the critical variables in glycan recognition: valency and density. A simple pulldown of the LiGA along with lectins followed by deep sequencing of the DNA in the bound phage decodes the recognized glycans. LiGA is target agnostic and measures binding profile of lectins expressed on intact cells, such as hCD22 (Siglec‐2) and DC‐SIGN (Dendritic Cell‐Specific Intercellular adhesion molecule‐3‐Grabbing Non‐integrin), and in live mice (Nat. Chem. Bio. 17, 806–816, 2021). From a mixture of 50‐100 multivalent glycan probes, LiGA identifies the glycan‐phage conjugates with optimal valency and density for binding to antibodies and lectins on cells in vitro and in vivo.Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) expressed on the surface of immune cells are exploited by cancer to evade immune response. We applied LiGA to study the binding specificity of Siglec‐7, a cell surface receptor that cancer cells use to evade immune response from natural killer (NK) cells. Additionally, we explored the roles of valency and density in ganglioside interaction with Siglec‐1 using a cell‐based assay. Building on these successes, we plan to use LiGA to identify the valency and affinity required by trans‐ glycan to overcome the cis‐ masking on the surface of immune cells.

Cell Surface Glycan Engineering to Examine Receptor Specificities of Influenza and Other Viruses

The transmission of viruses from animal reservoirs to humans poses great threats to public health. Preparedness for future zoonotic outbreaks requires a fundamental understanding how viruses of animal origin have adapted to replication in humans. In particular, it necessitates insight how viruses have evolved to binding to a cell surface component, or receptor, of the new host. We have developed glycan arrays and glycan cell surface engineering strategies to examine receptor specificities of viruses. The approaches were employed to examine receptor specificities of evolutionary distinct A/H3N2 influenza viruses. Recent A/H3N2 influenza viruses exhibit altered receptor specificities, and as a result they have lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. Glycan array technology showed that these viruses have compensated for decreased binding of the prototypic human receptor by recognizing α2,6‐sialosides on extended LacNAc moieties. Erythrocyte glycomics showed an absence of extended glycans, providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installed functional receptors on erythrocytes, allowing antigenic characterization of recent A/H3N2 viruses and confirming the cocirculation of antigenically different viruses in humans. Computational studies of HAs in complex with sialosides having extended LacNAc moieties reveal that mutations distal to the RBD reoriented the Y159 side chain, resulting in an extended receptor binding site. The approach was extended to examining the receptor specificity of Lass Virus.

Serial crystallography and kinetics reveal how the FimH bacterial lectin tweaks between mono‐ and multivalent binding of high‐mannose N‐glycans

BackgroundThe lectin domain of the fimbrial adhesin FimH from Escherichia coli recognizes with the highest affinity and in seemingly monovalent fashion oligomannosides‐3 and ‐5 N‐glycans. This happens with a high affinity (Kd=20 nM) because the alpha1,3‐linked arm remains free from any further alpha1,2‐mannose substitution. The latter causes an at least 10‐fold affinity loss in creating the oligomannose‐6 N‐glycan (1).MethodsThe binding of FimH to pauci‐ and oligomannoside‐containing N‐glycans and similarly glycosylated proteins, as well as its dimannoside endings Manalpha1,2, Manalpha1,3 and Manalpha1,6, have been profiled using the FimH Lectprofile kit (GlycoDiag, Applied Glycomics, France) and a newly designed paucimannose‐containing glycan array. The kinetics of binding, ka and kd, were analysed using surface plasmon resonance and compared with earlier incomplete data (2).ResultsMono‐ and multivalent binding were found in co‐crystal structures of the FimH lectin domain with alpha1,6‐fucosylated oligomannose‐3 and with oligoannose‐6, respectively. The kinetics of binding indicate that once alpha1,2‐linked mannose comes into play, the complexes are not so stable, as previously also measured using calorimetry (3), and accompanied with a high dissociation constant. Molecular dynamics simulations further support that it is not the total interaction energy that determines the final retained complex (metastable complexes), but the kinetics of interaction with the three possible dimannoside endings.ConclusionsKinetics and stability of the complexes drive the most favoured arrangement, via either a unique or multivalent interaction, between the N‐glycan and the FimH protein in the crystal.References :(1) Bouckaert, J., Mackenzie, J., de Paz, J. L., Chipwaza, B., Choudhury, D., Zavialov, A., Mannerstedt, K., Anderson, J., Pierard, D., Wyns, L., Seeberger, P. H., Oscarson, S., De Greve, H., Knight, S. D. (2006) The affinity of the FimH fimbrial adhesin is receptor‐driven and quasi‐independent of Escherichia colipathotypes. Molecular Microbiology 61, 1556‐1568(2) Sauer, M. M., Jakob, R. P., Luber, T., Canonica, F., Navarra, G., Ernst, B., Maier, T., Glockshuber, R. (2019) Binding of the bacterial adhesin FimH to its natural, multivalent high‐mannose type glycan targets. J Am Chem Soc 141, 936‐944(3) Dumych, T., Bridot, C., Gouin, S. G., Paryzhak, S., Szunerits, S., Bilyy, R., Bouckaert, J., (2018) A novel integrated way for deciphering the glycan code for the FimH lectin. Molecules 23, 2794

Antigenic and virological properties of an H3N2 variant that continues to dominate the 2021-22 Northern Hemisphere influenza season.

Influenza viruses circulated at very low levels during the beginning of the COVID-19 pandemic, and population immunity against these viruses is low. An H3N2 strain (3C.2a1b.2a2) with a hemagglutinin (HA) that has several substitutions relative to the 2021–22 H3N2 vaccine strain is dominating the 2021–22 Northern Hemisphere influenza season. Here, we show that one of these substitutions eliminates a key glycosylation site on HA and alters sialic acid binding. Using glycan array profiling, we show that the 3C.2a1b.2a2 H3 maintains binding to an extended biantennary sialoside and replicates to high titers in human airway cells. We find that antibodies elicited by the 2021–22 Northern Hemisphere influenza vaccine poorly neutralize the 3C.2a1b.2a2 H3N2 strain. Together, these data indicate that 3C.2a1b.2a2 H3N2 viruses efficiently replicate in human cells and escape vaccine-elicited antibodies.

Modeling the mucinous glycocalyx to unravel receptor pattern recognition by influenza A viruses

Influenza A viruses (IAVs) exploit host glycans in airway epithelial mucosa for entry and infection. Rapid detection of changes in IAV glycan‐binding phenotype can provide early indication of virus transmissibility and infection potential. IAVs use hemagglutinins (HAs) to bind sialic acids, and a switch in HA specificity is considered a prerequisite for transmission from birds to humans. Likewise, understanding the molecular mechanisms used by the viruses to overcome the mucin barrier may guide the development of more effective antiviral therapies. This presentation will describe the development of a glycan array platform, which uses synthetic mimetics of mucin glycoproteins to model how receptor presentation in the mucinous glycocalyx (i.e., glycan type, valency of the glycoconjugates, and their surface density) may impact IAV recognition. This platform utilizes machine learning to identify preferred receptor displays in the mucinous glycocalyx for adhesion of H1N1 and H3N2 viruses. Intriguingly, the arrays revealed a change in receptor pattern recognition by H1N1 upon transition from avian to mammalian cell culture. Without altering its HA affinity, the mammalian cell‐produced virus showed reduced sensitivity to surface crowding of the mucin mimetics. Using our mucin mimetics, we have also evaluated the physical protective functions of the glycocalyx on viral adhesion to cells. We found that bulky mucin mimetics lacking sialoglycan receptors inhibited the rate of H1N1 adhesion in a size‐ and density‐dependent manner, consistent with current view of the mucins as providing a protective shield against pathogens. Counterintuitively, increasing density of the mucin mimetics enhanced the retention of bound viruses. Careful characterization of SNA behavior at the RBC surface using a range of biophysical and imaging techniques revealed lectin‐induced crowding and reorganization of the glycocalyx with concomitant enhancement in lectin clustering, presumably through the formation of a more extensive glycan receptor patch at the cell membrane. Our findings indicate that glycan‐targeting pathogens may exploit the biophysical and biomechanical properties of mucins to overcome the mucosal glycocalyx barrier.

CarboGrove: a resource of glycan-binding specificities through analyzed glycan-array datasets from all platforms.

Glycan arrays continue to be the primary resource for determining the glycan-binding specificity of proteins. The volume and diversity of glycan-array data are increasing, but no common method and resource exist to analyze, integrate, and use the available data. To meet this need, we developed a resource of analyzed glycan-array data called CarboGrove. Using the ability to process and interpret data from any type of glycan array, we populated the database with the results from 35 types of glycan arrays, 13 glycan families, 5 experimental methods, and 19 laboratories or companies. In meta-analyses of glycan-binding proteins, we observed glycan-binding specificities that were not uncovered from single sources. In addition, we confirmed the ability to efficiently optimize selections of glycan-binding proteins to be used in experiments for discriminating between closely related motifs. Through descriptive reports and a programmatically accessible Application Programming Interface, CarboGrove yields unprecedented access to the wealth of glycan-array data being produced and powerful capabilities for both experimentalists and bioinformaticians.

Host glycocalyx captures HIV proximal to the cell surface via oligomannose-GlcNAc glycan-glycan interactions to support viral entry.

Here, we present ultrastructural analyses showing that incoming HIV are captured near the lymphocyte surface in a virion-glycan-dependent manner. Biophysical analyses show that removal of either virion- or cell-associated N-glycans impairs virus-cell binding, and a similar glycan-dependent relationship is observed between purified HIV envelope (Env) and primary Tcells. Trimming of N-glycans from either HIV or Env does not inhibit protein-protein interactions. Glycan arrays reveal HIV preferentially binds to N-acetylglucosamine and mannose. Interfering with these glycan-based interactions reduces HIV infectivity. These glycan interactions are distinct from previously reported glycan-lectin and non-specific electrostatic charge-based interactions. Specific glycan-glycan-mediated attachment occurs prior to virus entry and enhances efficiency of infection. Binding and fluorescent imaging data support glycan-glycan interactions as being responsible, at least in part, for initiating contact between HIV and the host cell, prior to viral Env-cellular CD4 engagement.

Molecular Insights into the Assembly and Functional Diversification of Typhoid Toxin.

ABSTRACTTyphoid toxin is an A2B5 protein toxin and an important virulence factor for the human-adapted bacterial pathogen Salmonella enterica serovar Typhi, the causative agent of typhoid fever. Typhoid toxin contains two enzymatic subunits, PltA and CdtB, which dock onto a pentameric delivery platform composed of the protein PltB. It was recently reported that the same enzymatic subunits can assemble with a different delivery platform composed of the protein PltC, forming a distinct version of typhoid toxin. However, the differences in structure and receptor specificity between the PltC and PltB typhoid toxins remain unknown. Here, we determined atomic-level structures of the pentameric PltC subunit, the fully assembled PltC typhoid toxin, and the PltC pentamers in complex with glycan receptors. Biochemical and structural analyses indicate that PltB and PltC are unable to form heteromeric delivery complexes due to electrostatic repulsion at the subunit interface and thus form separate toxins only. We further observed that, despite low sequence similarity between PltB and PltC, they interact with PltA in a similar manner but that PltC exhibits stronger electrostatic interactions with PltA, enabling it to outcompete PltB in toxin assembly. The ligand-bound atomic structures of PltC show an additional glycan binding site not found in PltB and glycan array analysis indicates that PltB and PltC exhibit significant differences in glycan binding specificity. Collectively, this study offers atomic-level insights into how S. Typhi produces two distinct versions of typhoid toxin, thereby generating functional diversity in this key virulence factor.

Cloning, Expression, and Purification of Galectins for In Vitro Studies.

Galectins are best known for their ability to bind glycoconjugates containing β-galactose, but classification of these small proteins within the galectin family is also defined by amino acid homology within structural domains and exon/intron junctions within genes. As galectins are expressed by organisms as diverse as some fungi, C. elegans, fish, birds and mammals, and biological activities attributed to galectins are equally diverse, it becomes essential to identify, clone, and characterize galectins from many sources. Glutathione S-transferase (GST) fused to the amino-terminus of galectin cDNAs has proven to be especially useful for the preparation of recombinant galectins in bacteria for use on glycan arrays, in experiments with cultured or isolated cells, and in pull-down assays with immunopurified glycoproteins. Many galectins are stabilized by reducing reagents, such that binding and elution of GST-galectins from glutathione-conjugated Sepharose with excess glutathione is both efficient and innocuous. The ability to bind and elute GST-galectins from lactose-conjugated Sepharose with excess lactose provides a relatively easy means to insure that galectins are competent for glycoconjugate binding prior to experimentation. This chapter focuses primarily on the varied approaches to use GST-galectin binding to glutathione- and lactose-conjugated Sepharose to purify recombinant galectins and then develop effective experimental protocols to characterize the specificity, interactions and function of galectins cloned from any source. We provide one example where a pull-down assay with all the GST-tagged canine galectins reveals that the C-terminal carbohydrate recognition domain of galectin-9 (Gal-9C) specifically recognizes the glycan-dependent apical targeting signal from the glycoprotein MUC1.

Preparation of Glycan Arrays Using Glycopeptides Derived From Biomaterials.

In general, viruses recognize host cell surface glycans, but the measurement of virus-host cell glycan interaction is not widely operated. This is not only because commercially available, structure-defined glycans are limited, but also because such interactions, if any, between viruses and isolated glycans are relatively weak, and thus, difficult to detect by conventional methods, e.g., enzyme-linked immune-sorbent assay. We describe a practical method to detect virus binding to glycans; for this, preparation of glycan arrays using glycopeptides derived from biomaterials is necessary. In this context, neoglycoprotein is produced using bovine serum albumin (BSA) and commercially available glycopeptides, with which influenza viruses are detected using an evanescent-field-activated fluorescence scanner. It is clearly shown that H1N1 strains of influenza virus recognize BSA, to which DiNeuα2-6bianntena-peptide (SGP) is covalently linked, while on the other hand H5N1 strains recognize BSA linked to DiNeuα2-3bianntena-peptide (α2,3SGP).

GlyNet: a multi-task neural network for predicting protein-glycan interactions.

Advances in diagnostics, therapeutics, vaccines, transfusion, and organ transplantation build on a fundamental understanding of glycan–protein interactions. To aid this, we developed GlyNet, a model that accurately predicts interactions (relative binding strengths) between mammalian glycans and 352 glycan-binding proteins, many at multiple concentrations. For each glycan input, our model produces 1257 outputs, each representing the relative interaction strength between the input glycan and a particular protein sample. GlyNet learns these continuous values using relative fluorescence units (RFUs) measured on 599 glycans in the Consortium for Functional Glycomics glycan arrays and extrapolates these to RFUs from additional, untested glycans. GlyNet's output of continuous values provides more detailed results than the standard binary classification models. After incorporating a simple threshold to transform such continuous outputs the resulting GlyNet classifier outperforms those standard classifiers. GlyNet is the first multi-output regression model for predicting protein–glycan interactions and serves as an important benchmark, facilitating development of quantitative computational glycobiology.

Multiplex Glycan Bead Array (MGBA ) for High Throughput and High Content Analyses of Glycan-Binding Proteins Including Natural Anti-Glycan Antibodies.

We present here detailed protocols for the newly developed multiplex glycan bead array (MGBA) for the high throughput and high content analyses of various glycan-binding proteins including anti-glycan antibodies. This platform takes advantage of the commercially available Luminex beads to construct glycan arrays that are easily customizable at will and anytime by researchers. The platform allows the simultaneous analyses of up to 500 glycans and 384 samples at a time. By using multiple arrays, a researcher can analyze thousands of glycans and tens of thousands of samples within a short period. The assay is highly sensitive, specific, reproducible, economic, and fast. Furthermore, the bead array platform is approved for use in clinical settings, speeding up the translation of laboratory discoveries into patient care.

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid–binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1–3GalNAcα1–O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

Glycan Activation of Clec4b Induces Reactive Oxygen Species Protecting against Neutrophilia and Arthritis.

Animal models for complex diseases are needed to position and analyze the function of interacting genes. Previous positional cloning identified Ncf1 and Clec4b to be major regulators of arthritis models in rats. Here, we investigate epistasis between Ncf1 and Clec4b, two major regulators of arthritis in rats. We find that Clec4b and Ncf1 exert an additive effect on arthritis given by their joint ability to regulate neutrophils. Both genes are highly expressed in neutrophils, together regulating neutrophil availability and their capacity to generate reactive oxygen species. Using a glycan array, we identify key ligands of Clec4b and demonstrate that Clec4b-specific stimulation triggers neutrophils into oxidative burst. Our observations highlight Clec4b as an important regulator of neutrophils and demonstrate how epistatic interactions affect the susceptibility to, and severity of, autoimmune arthritis.