Glycan Microarray Research Articles

Molecular determinants of the interaction between HSV-1 glycoprotein D and heparan sulfate.

Literature has well-established the importance of 3-O-sulfation of neuronal cell surface glycan heparan sulfate (HS) to its interaction with herpes simplex virus type 1 glycoprotein D (gD). Previous investigations of gD to its viral receptors HVEM and nectin-1 also highlighted the conformational dynamics of gD's N- and C-termini, necessary for viral membrane fusion. However, little is known on the structural interactions of gD with HS. Here, we present our findings on this interface from both the glycan and the protein perspective. We used C-terminal and N-terminal gD variants to probe the role of their respective regions in gD/HS binding. The N-terminal truncation mutants (with Δ1-22) demonstrate equivalent or stronger binding to heparin than their intact glycoproteins, indicating that the first 22 amino acids are disposable for heparin binding. Characterization of the conformational differences between C-terminal truncated mutants by sedimentation velocity analytical ultracentrifugation distinguished between the "open" and "closed" conformations of the glycoprotein D, highlighting the region's modulation of receptor binding. From the glycan perspective, we investigated gD interacting with heparin, heparan sulfate, and other de-sulfated and chemically defined oligosaccharides using surface plasmon resonance and glycan microarray. The results show a strong preference of gD for 6-O-sulfate, with 2-O-sulfation becoming more important in the presence of 6-O-S. Additionally, 3-O-sulfation shifted the chain length preference of gD from longer chain to mid-chain length, reaffirming the sulfation site's importance to the gD/HS interface. Our results shed new light on the molecular details of one of seven known protein-glycan interactions with 3-O-sulfated heparan sulfate.

Exploring the In situ pairing of human galectins toward synthetic O-mannosylated core M1 glycopeptides of α-dystroglycan

Dystroglycan (DG), which constitutes a part of the dystrophin–glycoprotein complex, connects the extracellular matrix to the cytoskeleton. The matriglycans presented by the extracellular α-DG serve as a contact point with extracellular matrix proteins (ECM) containing laminin G-like domains, providing cellular stability. However, it remains unknown whether core M1 (GlcNAcβ1-2Man) structures can serve as ligands among the various O-Mannosylated glycans. Therefore, based on the presence of N-acetylLactosamine (LacNAc) in this glycan following the core extension, the binding interactions with adhesion/growth-regulatory galectins were explored. To elucidate this process, the interaction between galectin (Gal)-1, -3, -4 and -9 with α-DG fragment 372TRGAIIQTPTLGPIQPTRV390 core M1-based glycopeptide library were profiled, using glycan microarray and nuclear magnetic resonance studies. The binding of galectins was revealed irrespective of its modular architecture, adding galectins to the list of possible binding partners of α-DG core M1 glycoconjugates by cis-binding (via peptide- and carbohydrate-protein interactions), which can be abrogated by α2,3-sialylation of the LacNAc units. The LacNAc-terminated α-DG glycopeptide interact simultaneously with both the S- and F-faces of Gal-1, thereby inducing oligomerization. Furthermore, Gal-1 can trans-bridge α-DG core M1 structures and laminins, which proposed a possible mechanism by which Gal-1 ameliorates muscular dystrophies; however, this proposal warrants further investigation.

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.

Computationally guided conversion of the specificity of E-selectin to mimic that of Siglec-8

Sulfated glycans have been found to be associated with various diseases and therefore have significant potential in molecular pathology as biomarkers. Although lectins are useful reagents for detecting glycans, there is a paucity of sulfate-recognizing lectins, and those that exist, such as from Maackia amurensis, display mixed specificities. Recombinant lectin engineering offers an emerging tool for creating novel glycan recognition by altering and/or enhancing endogenous specificities. The present study demonstrated the use of computational approaches in the engineering of a mutated form of E-selectin that displayed highly specific recognition of 6'-sulfo-sialyl Lewis X (6'-sulfo-sLex), with negligible binding to its endogenous nonsulfated ligand, sLex. This new specificity mimics that of the unrelated protein Siglec-8, for which 6'-sulfo-sLex is its preferred ligand. Molecular dynamics simulations and energy calculations predicted that two point mutations (E92A/E107A) would be required to stabilize binding to the sulfated oligosaccharide with E-selectin. In addition to eliminating putative repulsions between the negatively charged side chains and the sulfate moiety, the mutations also abolished favorable interactions with the endogenous ligand. Glycan microarray screening of the recombinantly expressed proteins confirmed the predicted specificity change but also identified the introduction of unexpected affinity for the unfucosylated form of 6'-sulfo-sLex (6'-sulfo-sLacNAc). Three key requirements were demonstrated in this case for engineering specificity for sulfated oligosaccharide: 1) removal of unfavorable interactions with the 6'-sulfate, 2) introduction of favorable interactions for the sulfate, and 3) removal of favorable interactions with the endogenous ligand.

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

The molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcβ1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment.

Microarray-guided evaluation of the frequency, B-cell origins, and selectivity of human glycan-binding antibodies reveals new insights and novel antibodies

The immune system produces a diverse collection of antiglycan antibodies that are critical for host defense. At present, however, we know very little about the binding properties, origins, and sequences of these antibodies because of a lack of access to a variety of defined individual antibodies. To address this challenge, we used a glycan microarray with over 800 different components to screen a panel of 516 human monoclonal antibodies that had been randomly cloned from different B-cell subsets originating from healthy human subjects. We obtained 26 antiglycan antibodies, most of which bound microbial carbohydrates. The majority of the antiglycan antibodies identified in the screen displayed selective binding for specific glycan motifs on our array and lacked polyreactivity. We found that antiglycan antibodies were about twice as likely than expected to originate from IgG+ memory B cells, whereas none were isolated from naïve, early emigrant, or immature B cells. Therefore, our results indicate that certain B-cell subsets in our panel are enriched in antiglycan antibodies, and IgG+ memory B cells may be a promising source of such antibodies. Furthermore, some of the newly identified antibodies bound glycans for which there are no reported monoclonal antibodies available, and these may be useful as research tools, diagnostics, or therapeutic agents. Overall, the results provide insight into the types and properties of antiglycan antibodies produced by the human immune system and a framework for the identification of novel antiglycan antibodies in the future.

Antibody Recognition of Different Staphylococcus aureus Wall Teichoic Acid Glycoforms.

Wall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibody-mediated treatments against Staphylococcus aureus, including methicillin-resistant (MRSA) strains. Through a combination of glycan microarray, synthetic chemistry, crystallography, NMR, and computational studies, we unraveled the molecular and structural details of fully defined synthetic WTA fragments recognized by previously described monoclonal antibodies (mAbs 4461 and 4497). Our results unveiled the structural requirements for the discriminatory recognition of α- and β-GlcNAc-modified WTA glycoforms by the complementarity-determining regions (CDRs) of the heavy and light chains of the mAbs. Both mAbs interacted not only with the sugar moiety but also with the phosphate groups as well as residues in the ribitol phosphate (RboP) units of the WTA backbone, highlighting their significant role in ligand specificity. Using elongated WTA fragments, containing two sugar modifications, we also demonstrated that the internal carbohydrate moiety of α-GlcNAc-modified WTA is preferentially accommodated in the binding pocket of mAb 4461 with respect to the terminal moiety. Our results also explained the recently documented cross-reactivity of mAb 4497 for β-1,3/β-1,4-GlcNAc-modified WTA, revealing that the flexibility of the RboP backbone is crucial to allow positioning of both glycans in the antibody binding pocket.

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.

Chemical Synthesis of the Highly Sterically Hindered Core Undecasaccharide of Helicobacter pylori Lipopolysaccharide for Antigenicity Evaluation with Human Serum.

Helicobacter pylori, listed as a human carcinogen by the Department of Health and Human Services, colonizes the gastric mucosa of more than half of the world's population. The individuals infected with H. pylori have a high risk to develop chronic gastritis, peptic ulcers, and even gastric cancer. The conserved core structure of H. pylori lipopolysaccharide (LPS) has been regarded as a promising candidate structure for development of a glycoconjugate vaccine targeting multiple serotypes. Here, we report a total synthesis of the core undecasaccharide of H. pylori LPS and its subunit antigens. The match and mismatch between the glycosyl donor and acceptor caused by the inert hydroxyl groups were addressed by a judicious choice of orthogonal protection strategies and glycosylation conditions. A combination of acyl remote participation and solvent effects has been applied for selective formation of the five 1,2-cis-glucosidic bonds. The high steric hindrance induced by the high carbon sugars and trinacriform architecture required that the core undecasaccharide was synthesized through a finely tuned linear assembly [2 + (1 + (3 + (1 + (1 + 3))))] rather than convergent strategies. An antigenicity evaluation using glycan microarrays showed that an α-(1 → 6)-glucan trisaccharide is recognized by IgG antibodies in sera of H. pylori-infected patients. The phosphate group of the inner core trisaccharide key epitope is very important for IgG recognition. These findings are an important step toward designing carbohydrate-based vaccines against H. pylori.

α2,3-Linked Sialic Acids Are the Potential Attachment Receptor for Shaan Virus Infection in MARC-145 Cells.

ABSTRACTShaan virus (ShaV), a novel species of the genus Jeilongvirus, family Paramyxoviridae, was isolated from an insectivore bat (Miniopterus schreibersii) in Korea in 2016. ShaV particles contain a hemagglutinin-neuraminidase (HN) glycoprotein in their envelope that allows the virus to target cells. Typically, diverse paramyxoviruses with HN glycoprotein are reported to interact predominantly with sialic acids, but there are no studies of receptors for ShaV. In this study, the identification of potential receptors for ShaV was demonstrated using sialidase treatments, glycan microarray, magnetic bead-based virus binding assay, and neuraminidase inhibitor treatments. Pretreatment of MARC-145 cells with sialidase, which cleaves α2,3-linked sialic acids, showed higher inhibition of viral infection than α2,6-linked-specific sialidase. These data were supported by the binding of ShaV to predominantly α2,3-linked sialylated glycans in the screening of sialyl linkage patterns through glycan microarray. To further confirm the direct interaction between ShaV and α2,3-linked sialic acids, ShaV was incubated with α2,3- or α2,6-linked sialylated glycans conjugated to magnetic beads, and binding signals were detected only for α2,3-linked sialylated glycans. In addition, the potential of sialic acids as a receptor was demonstrated by the viral replication inhibitory effect of the neuraminidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminicacid (DANA) in the mature virion release steps. Collectively, these results support that α2,3-linked sialic acids are the potential receptor for ShaV infection in MARC-145 cells.IMPORTANCE Bats host major mammalian paramyxoviruses, and novel paramyxoviruses are increasingly being reported around the world. Shaan virus (ShaV), from the genus Jeilongvirus, family Paramyxoviridae, has a potential attachment glycoprotein, HN. Here, we identify that ShaV binds to sialic acid and demonstrate that α2,3-linked sialic acids are the potential receptor for ShaV infection. The presented data regarding ShaV receptor specificity will enable studies into the viral tropism for the host and contribute to the development of new antiviral strategies targeting viral receptors.

Chemical Synthesis and Antigenic Evaluation of Inner Core Oligosaccharides from Acinetobacter baumannii Lipopolysaccharide.

Acinetobacter baumannii is currently posing a serious threat to global health. Lipopolysaccharide (LPS) is a potent virulence factor of pathogenic Gram-negative bacteria. To explore the antigenic properties of A. baumannii LPS, four Kdo-containing inner core glycans from A. baumannii strain ATCC 17904 were synthesized. A flexible and divergent method based on the use of the orthogonally substituted α-Kdo-(2→5)-Kdo disaccharides was developed. Selective removal of different protecting groups in these key precursors and elongation of sugar chain via α-stereocontrolled coupling with 5,7-O-di-tert-butylsilylene or 5-O-benzoyl protected Kdo thioglycosides and 2-azido-2-deoxyglucosyl thioglycoside allowed efficient assembly of the target molecules. Glycan microarray analysis of sera from infected patients revealed that the 4,5-branched Kdo trimer was a potential antigenic epitope, which is attractive for further immunological research to develop carbohydrate vaccines against A. baumannii.

Chemical Synthesis and Antigenic Evaluation of Inner Core Oligosaccharides from Acinetobacter baumannii Lipopolysaccharide

AbstractAcinetobacter baumannii is currently posing a serious threat to global health. Lipopolysaccharide (LPS) is a potent virulence factor of pathogenic Gram‐negative bacteria. To explore the antigenic properties of A. baumannii LPS, four Kdo‐containing inner core glycans from A. baumannii strain ATCC 17904 were synthesized. A flexible and divergent method based on the use of the orthogonally substituted α‐Kdo‐(2→5)‐Kdo disaccharides was developed. Selective removal of different protecting groups in these key precursors and elongation of sugar chain via α‐stereocontrolled coupling with 5,7‐O‐di‐tert‐butylsilylene or 5‐O‐benzoyl protected Kdo thioglycosides and 2‐azido‐2‐deoxyglucosyl thioglycoside allowed efficient assembly of the target molecules. Glycan microarray analysis of sera from infected patients revealed that the 4,5‐branched Kdo trimer was a potential antigenic epitope, which is attractive for further immunological research to develop carbohydrate vaccines against A. baumannii.

CarbArrayART: a new software tool for carbohydrate microarray data storage, processing, presentation, and reporting.

Glycan microarrays are essential tools in glycobiology and are being widely used for assignment of glycan ligands in diverse glycan recognition systems. We have developed a new software, called Carbohydrate microArray Analysis and Reporting Tool (CarbArrayART), to address the need for a distributable application for glycan microarray data management. The main features of CarbArrayART include: (i) Storage of quantified array data from different array layouts with scan data and array-specific metadata, such as lists of arrayed glycans, array geometry, information on glycan-binding samples, and experimental protocols. (ii) Presentation of microarray data as charts, tables, and heatmaps derived from the average fluorescence intensity values that are calculated based on the imaging scan data and array geometry, as well as filtering and sorting functions according to monosaccharide content and glycan sequences. (iii) Data export for reporting in Word, PDF, and Excel formats, together with metadata that are compliant with the guidelines of MIRAGE (Minimum Information Required for A Glycomics Experiment). CarbArrayART is designed for routine use in recording, storage, and management of any slide-based glycan microarray experiment. In conjunction with the MIRAGE guidelines, CarbArrayART addresses issues that are critical for glycobiology, namely, clarity of data for evaluation of reproducibility and validity.

The minimum information required for a glycomics experiment (MIRAGE): reporting guidelines for capillary electrophoresis.

The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectrometry methods, liquid chromatography analysis, exoglycosidase digestions, glycan microarray methods, and nuclear magnetic resonance methods. Here, we present the first version of reporting guidelines for glyco(proteo)mics analysis by capillary electrophoresis (CE) for standardized and high-quality reporting of experimental conditions in the scientific literature. The guidelines cover all aspects of a glyco(proteo)mics CE experiment including sample preparation, CE operation mode (CZE, CGE, CEC, MEKC, cIEF, cITP), instrument configuration, capillary separation conditions, detection, data analysis, and experimental descriptors. These guidelines are linked to other MIRAGE guidelines and are freely available through the project website https://www.beilstein-institut.de/en/projects/mirage/guidelines/#ce_analysis (doi:10.3762/mirage.7).

Selective Recognition of Carbohydrate Antigens by Germline Antibodies Isolated from AID Knockout Mice.

Germline antibodies, the initial set of antibodies produced by the immune system, are critical for host defense, and information about their binding properties can be useful for designing vaccines, understanding the origins of autoantibodies, and developing monoclonal antibodies. Numerous studies have found that germline antibodies are polyreactive with malleable, flexible binding pockets. While insightful, it remains unclear how broadly this model applies, as there are many families of antibodies that have not yet been studied. In addition, the methods used to obtain germline antibodies typically rely on assumptions and do not work well for many antibodies. Herein, we present a distinct approach for isolating germline antibodies that involves immunizing activation-induced cytidine deaminase (AID) knockout mice. This strategy amplifies antigen-specific B cells, but somatic hypermutation does not occur because AID is absent. Using synthetic haptens, glycoproteins, and whole cells, we obtained germline antibodies to an assortment of clinically important tumor-associated carbohydrate antigens, including Lewis Y, the Tn antigen, sialyl Lewis C, and Lewis X (CD15/SSEA-1). Through glycan microarray profiling and cell binding, we demonstrate that all but one of these germline antibodies had high selectivity for their glycan targets. Using molecular dynamics simulations, we provide insights into the structural basis of glycan recognition. The results have important implications for designing carbohydrate-based vaccines, developing anti-glycan monoclonal antibodies, and understanding antibody evolution within the immune system.

The effect of enzyme treatment on polyphenol and cell wall polysaccharide extraction from the grape berry and subsequent sensory attributes in Cabernet Sauvignon wines

Pectolytic enzyme maceration is common for producing red wines, but the effects on bitterness and astringency are not well understood. Glycan microarrays assessed polysaccharide diversity and with polyphenol analysis was correlated with sensory data on descriptors of astringency and their perceived levels in enzyme-crafted Cabernet Sauvignon wines. Enzyme use is shown to have no effect on bitterness, but enzyme-macerated wines are more astringent. The data suggests that pectolytic enzymes are much more pronounced in their effect on the cell wall matrix than the ripeness of the berries at harvest and subsequent sensory perception. Enzyme-macerated red wines showed higher levels of polyphenol which were more polymerized and galloylated. The polyphenol-rich wines were described as hard, chalky, grippy, grainy and dry. The non-enzyme wines had elevated levels of arabinogalactan protein and pectin epitopes (notably biomarker mAbs JIM8 and JIM13) with the wines being characterized as soft, fine and velvety.

Galectin-9 recognizes and exhibits antimicrobial activity toward microbes expressing blood group–like antigens

While adaptive immunity recognizes a nearly infinite range of antigenic determinants, immune tolerance renders adaptive immunity vulnerable to microbes decorated in self-like antigens. Recent studies suggest that sugar-binding proteins galectin-4 and galectin-8 bind microbes expressing blood group antigens. However, the binding profile and potential antimicrobial activity of other galectins, particularly galectin-9 (Gal-9), has remained incompletely defined. Here, we demonstrate that while Gal-9 possesses strong binding preference for ABO(H) blood group antigens, each domain exhibits distinct binding patterns, with the C-terminal domain (Gal-9C) exhibiting higher binding to blood group B than the N-terminal domain (Gal-9N). Despite this binding preference, Gal-9 readily killed blood group B–positive Escherichia coli, whereas Gal-9N displayed higher killing activity against this microbe than Gal-9C. Utilization of microarrays populated with blood group O antigens from a diverse array of microbes revealed that Gal-9 can bind various microbial glycans, whereas Gal-9N and Gal-9C displayed distinct and overlapping binding preferences. Flow cytometric examination of intact microbes corroborated the microbial glycan microarray findings, demonstrating that Gal-9, Gal-9N, and Gal-9C also possess the capacity to recognize distinct strains of Providencia alcalifaciens and Klebsiella pneumoniae that express mammalian blood group–like antigens while failing to bind related strains that do not express mammalian-like glycans. In each case of microbial binding, Gal-9, Gal-9N, and Gal-9C induced microbial death. In contrast, while Gal-9, Gal-9N, and Gal-9C engaged red blood cells, each failed to induce hemolysis. These data suggest that Gal-9 recognition of distinct microbial strains may provide antimicrobial activity against molecular mimicry.

A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities.

Glycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving; however, the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well-defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use a combination of machine learning algorithms and expert annotation to define lectin specificity for this important probe set. Our analysis uses comprehensive glycan microarray analysis of commercially available lectins we obtained using version 5.0 of the Consortium for Functional Glycomics glycan microarray (CFGv5). This data set was made public in 2011. We report the creation of this data set and its use in large-scale evaluation of lectin-glycan binding behaviors. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides and linkages. Combining machine learning with manual annotation, we create a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.

Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.

Engineering the Ligand Specificity of the Human Galectin-1 by Incorporation of Tryptophan Analogues.

Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3'-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.