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Pseudomonas aeruginosa is a Gram‐negative bacterium and an opportunistic pathogen ubiquitously present throughout nature. LecB, a fucose‐, and mannose‐binding lectin, is a prominent virulence factor of P. aeruginosa, which can be expressed on the bacterial surface but also be secreted. However, the LecB interaction with human immune cells remains to be characterized. Neutrophils comprise the first line of defense against infections and their production of reactive oxygen species (ROS) and release of extracellular traps (NETs) are critical antimicrobial mechanisms. When profiling the neutrophil glycome we found several glycoconjugates on granule and plasma membranes that could potentially act as LecB receptors. In line with this, we here show that soluble LecB can activate primed neutrophils to produce high levels of intracellular ROS (icROS), an effect that was inhibited by methyl fucoside. On the other hand, soluble LecB inhibits P. aeruginosa‐induced icROS production. In support of that, during phagocytosis of wild‐type and LecB‐deficient P. aeruginosa, bacteria with LecB induced less icROS production as compared with bacteria lacking the lectin. Hence, LecB can either induce or inhibit icROS production in neutrophils depending on the circumstances, demonstrating a novel and potential role for LecB as an immunomodulator of neutrophil functional responses. [ABSTRACT FROM AUTHOR]

Trained Immunity is defined as a biological process normally induced by exogenous or endogenous insults that triggers epigenetic and metabolic reprogramming events associated with long-term adaptation of innate immune cells. This trained phenotype confers enhanced responsiveness to subsequent triggers, resulting in an innate immune "memory" effect. Trained Immunity, in the past decade, has revealed important benefits for host defense and homeostasis, but can also induce potentially harmful outcomes associated with chronic inflammatory disorders or autoimmune diseases. Interestingly, evidence suggest that the "trainers" prompting trained immunity are frequently glycans structures. In fact, the exposure of different types of glycans at the surface of pathogens is a key driver of the training phenotype, leading to the reprogramming of innate immune cells through the recognition of those glycan-triggers by a variety of glycan-binding proteins (GBPs) expressed by the immune cells. β-glucan or mannose-enriched structures in Candida albicans are some of the examples that highlight the potential of glycans in trained immunity, both in homeostasis and in disease. In this review, we will discuss the relevance of glycans exposed by pathogens in establishing key immunological hubs with glycan-recognizing receptors expressed in immune cells, highlighting how this glycan-GBP network can impact trained immunity. Finally, we discuss the power of glycans and GBPs as potential targets in trained immunity, envisioning potential therapeutic applications. [Display omitted] • Glycans are crucial players in Trained Immunity in homeostasis and disease. • "Memory-like" phenotypes can be induced by glycans exposed by pathogens. • Glycans and Glycan-binding proteins are key regulators of Trained Immunity. • Trained Immunity induced by glycans is a potential therapeutic avenue. [ABSTRACT FROM AUTHOR]

Selective detection of disease-associated changes in the glycocalyx is an emerging field in modern targeted therapies. Detecting minor glycan changes on the cell surface is a challenge exacerbated by the lack of correspondence between cellular DNA/RNA and glycan structures. We demonstrate that multivalent displays of lectins on DNA-barcoded phages-liquid lectin array (LiLA)-detect subtle differences in density of glycans on cells. LiLA constructs displaying 73 copies of diCBM40 (CBM) lectin per virion (φ-CBM 73 ) exhibit non-linear ON/OFF-like recognition of sialoglycans on the surface of normal and cancer cells. A high-valency φ-CBM 290 display, or soluble CBM protein, cannot amplify the subtle differences detected by φ-CBM 73 . Similarly, multivalent displays of CBM and Siglec-7 detect differences in the glycocalyx between stem-like and non-stem populations in cancer. Multivalent display of lectins offer in situ detection of minor differences in glycocalyx in cells both in vitro and in vivo not feasible to currently available technologies., Competing Interests: Declaration of interests R.D. is shareholder of the start-up company 48Hour Discovery Inc. that licensed the patent application (WO2018141058A1) describing LiGA technology., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Glycans are carbohydrates that are made by all organisms and covalently conjugated to other biomolecules. Glycans cover the surface of both human cells and pathogens and are fundamental to defining the identity of a cell or an organism, thereby contributing to discriminating self from nonself. As such, glycans are a class of 'Self-Associated Molecular Patterns' that can fine-tune host inflammatory processes. In fact, glycans can be sensed and recognized by a variety of glycan-binding proteins (GBP) expressed by immune cells, such as galectins, siglecs, and C-type lectins, which recognize changes in the cellular glycosylation, instructing both pro-inflammatory and anti-inflammatory responses. In this review, we introduce glycans as cell-identification structures, discussing how glycans modulate host-pathogen interactions and how they can fine-tune inflammatory processes associated with infection, inflammation and autoimmunity. Finally, from the clinical standpoint, we discuss how glycoscience research can benefit life sciences and clinical medicine by providing a source of valuable biomarkers and therapeutic targets for immunity. [ABSTRACT FROM AUTHOR]

Glycans decorate all cells and are critical mediators of cellular processes through recognition by glycan-binding proteins (GBPs). While targeting glycan–protein interactions has great therapeutic potential, these interactions are challenging to study as they are generally transient and exhibit low binding affinities. Glycan-based photo-crosslinkable probes have enabled covalent capture and identification of unknown GBP receptors and glycoconjugate ligands. Here, we review recent progress in photo-crosslinking approaches targeting glycan-mediated interactions. We discuss two prominent emerging strategies: 1) development of photo-crosslinkable oligosaccharide ligands to identify GBP receptors; and 2) cell-surface glyco-engineering to identify glycoconjugate ligands of GBPs. Overall, photoaffinity labeling affords valuable insights into complex glycan–protein networks and is poised to help elucidate the glycan–protein interactome, providing novel targets for therapeutic intervention. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] The synthesis of glycoprobes based on water-soluble polyacrylamide with a custom content of any low molecular weight fluorescent label is described. The synthesis is carried out in a stepwise manner: in the first step, the polymer is strictly dose-modified with a side substituent-linker containing an amino group; in the second step, the amino group is quantitatively acylated with an activated ester of the dye. The probes are designed to identify and study carbohydrate-binding proteins of plant cells. [ABSTRACT FROM AUTHOR]

Glucose is one of the most important monosaccharides. Although hyperglycemia in type 2 diabetes mellitus (T2DM) lead to a series of changes; however, little is known about the alterations of serum proteins in T2DM, especially those proteins with glucose affinity. In this study, the glucose‐binding proteins (GlcBPs) of serum were isolated from 30 health volunteer (HV) and 30 T2DM patients by glucose‐magnetic particle conjugates (GMPC) and identified by mass spectrum analysis. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated the main gene annotations and pathways of this GlcBPs, while Motif‐X webtool provided the potential glucose‐binding domains. Further docking analysis and glycan microarray were used to understand the interaction between the glucose and glucose‐binding domains. A total of 149 and 119 GlcBPs were identified from HV and T2DM cases. Four hundred and sixty‐eight GO annotations in 165 identified GlcBPs were available, while the majority involved in cellular processes and binding function. A short peptide, EGDEEITCLNGFWLE, which was derived from the Motif‐X analysis, presented a high‐binding ability to the glucose from both docking analysis and glycan analysis. GMPC provides a powerful tool for GlcBPs isolation and indicates the alteration of GlcBPs in T2DM. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polyacrylamide conjugates of glycans have long been widely used in many research areas of glycobiology, mainly for immobilizing glycans in solid-phase assays and as multivalent inhibitors. Pending biotin tag allows immobilizing Glyc-PAA quantitatively on any surface, and acts as a tracer for detection of carbohydrate-binding proteins. However, the scope of already realized capabilities of these probes is immeasurably richer than those listed above. This review is not so much about routine as about less common, but not less significant applications. Also, the data on the glycopolymers themselves, their molecular weight, size and polymer chain flexibility are presented, as well as the methods of synthesis, clusterisation and entropy factor in their interaction with proteins. [ABSTRACT FROM AUTHOR]

Glycans and glycan binding proteins (GBPs or lectins) are essential components in almost every aspect of immunology. Investigations of the interactions between glycans and GBPs have greatly advanced our understanding of the molecular basis of these fundamental immunological processes. In order to better study the glycan-GBP interactions, microscope glass slide-based glycan microarrays were conceived and proved to be an incredibly useful and successful tool. A variety of methods have been developed to better present the glycans so that they mimic natural presentations. Breakthroughs in chemical biology approaches have also made available glycans with sophisticated structures that were considered practically impossible just a few decade ago. Glycan microarrays provide a wealth of valuable information in immunological studies. They allow for discovery of detailed glycan binding preferences or novel binding epitopes of known endogenous immune receptors, which can potentially lead to the discovery of natural ligands that carry the glycans. Glycan microarrays also serve as a platform to discover new GBPs that are vital to the process of infection and invasion by microorganisms. This review summarizes the construction strategies and the immunological applications of glycan microarrays, particularly focused on those with the most comprehensive sets of glycan structures. We also review new methods and technologies that have evolved. We believe that glycan microarrays will continue to benefit the growing research community with various interests in the field of immunology.

Vascular endothelia are covered with a dense glycocalix that is heavily sialylated. Sialylation of vascular glycoconjugates is involved in the regulation of cell–cell interactions, be it among endothelial cells at cell junctions or between endothelial and blood-borne cells. It also plays important roles in modulating the binding of soluble ligands and the signaling by vascular receptors. Here, we provide an overview over the sialylation-function relationships of glycoproteins expressed in the blood and lymphatic vasculature. We first describe cellular interactions in which sialic acid contributes in a stereospecific manner to glycan epitopes recognized by glycan-binding proteins. Our major focus is however on the rarely discussed examples of vascular glycoproteins whose biological functions are modulated by sialylation through other mechanisms. [ABSTRACT FROM AUTHOR]

Interactions of carbohydrates and proteins are essential for many biological processes and glycan microarrays have emerged as powerful tools to rapidly assess these carbohydrate-protein interactions. Diverse platforms to immobilize glycans on glass slides for subsequent probing of the specificities of glycan-binding proteins (GBPs) have evolved. It has been suggested that high local glycan density on microarrays is crucial for detecting low-affinity interactions. To determine the influence of printing efficacy on GBP binding, we compared N -hydroxyl succinimide (NHS)-ester activated glass slides from three different manufacturers and evaluated two different printing buffers. Large differences in binding efficacies of Concanavalin A, peanut agglutinin, and Ricinus communis agglutinin 120 were observed. On some slides, low affinity interactions were missed altogether. Addition of polyethylenglycol (PEG) 400 to the printing buffer significantly enhanced the sensitivity of the binding assays. After monitoring printing efficacy over prolonged printing times, substantial effects resulting from progressing hydrolysis of the NHS-esters during the printing run on one type of slides were found. Printing efficiency of glycans strongly depends on the type of NHS-ester activated slides, the printing buffer, and the printing time. We provide practical advice for selecting the right printing conditions for particular applications. Image 1 • Optimization of printing conditions afforded high density glycan microarrays. • NHS-Activated slides from different manufacturers hydrolize with different rates during printing runs. • Slides from different manufacturers provided different signal intensities after interrogation with glycan-binding proteins. • Glycan microarray printing has to be carefully fine-tuned depending on the particular application. [ABSTRACT FROM AUTHOR]

Mammalian glycan‐binding receptors, sometimes known as lectins, interact with glycans, the oligosaccharide portions of endogenous mammalian glycoproteins and glycolipids as well as sugars on the surfaces of microbes. These receptors guide glycoproteins out of and back into cells, facilitate communication between cells through both adhesion and signaling, and allow the innate immune system to respond quickly to viral, fungal, bacterial, and parasitic pathogens. For many of the roughly 100 glycan‐binding receptors that are known in humans, there are good descriptions of what types of glycans they bind and how selectivity for these ligands is achieved at the molecular level. In some cases, there is also comprehensive evidence for the roles that the receptors play at the cellular and organismal levels. In addition to highlighting these well‐understood paradigms for glycan‐binding receptors, this review will suggest where gaps remain in our understanding of the physiological functions that they can serve. [ABSTRACT FROM AUTHOR]

Abstract N-glycosylation is one of major post-translational modifications in nature, and it is essential for protein structure and function. As hydrophilic moieties of glycoproteins, N-glycans play important roles in protein stability. They protect the proteins against proteolytic degradation, aggregation, and thermal denaturation through maintaining optimal conformations. There are extensive evidences showing the involvement of N-glycans in the pharmacodynamics and pharmacokinetics of recombinant therapeutic proteins and antibodies. Highly sialylated complex-type glycans enable the longer serum half-lives of proteins against uptake through hepatic asialoglycoprotein receptor and mannose receptor for degradation in lysosomes. Moreover, the presence of nonhuman glycans results in clearance through pre-existing antibodies from serum and induces IgE-mediated anaphylaxis. N-glycans also facilitate or reduce the adverse immune responses of the proteins through interacting with multiple glycan-binding proteins, including those specific for mannose or mannose 6-phosphate. Due to the glycan impacts, a few therapeutic proteins were glycoengineered to improve the pharmacokinetics and stability. Thus, N-glycosylation should be extensively investigated and optimized for each individual protein for better efficacy and safety. [ABSTRACT FROM AUTHOR]

DC-SIGN is a cell-surface receptor for several pathogenic threats, such as HIV, Ebola virus, or Mycobacterium tuberculosis. Multiple attempts to develop inhibitors of the underlying carbohydrate-protein interactions have been undertaken in the past fifteen years. Still, drug-like DC-SIGN ligands are sparse, which is most likely due to its hydrophilic, solvent-exposed carbohydrate-binding site. Herein, we report on a parallel fragment screening against DC-SIGN applying SPR and a reporter displacement assay, which complements previous screenings using 19F NMR spectroscopy and chemical fragment microarrays. Hit validation by SPR and 1H-15N HSQC NMR spectroscopy revealed that although no fragment bound in the primary carbohydrate site, five secondary sites are available to harbor drug-like molecules. Building on key interactions of the reported fragment hits, these pockets will be targeted in future approaches to accelerate the development of DC-SIGN inhibitors. [ABSTRACT FROM AUTHOR]

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan-GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan-GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction., (© 2023 The Author(s).)

Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the "nested" covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 µM and 900 µM, assigned to domains B and B' and domains A and A' respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner. [ABSTRACT FROM AUTHOR]

Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBP) expressed by dendritic cells (DCs) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by DCs for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglec-5 and Siglec-9 showed weak binding to a few glycans. By contrast, most hGBP bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2--fucosyl-lactose (2'-FL) and 3-fucosyl-lactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2--FL or 3-FL, as well as the ability of the free glycans to inhibit DCSIGN binding. 2--FL had an IC50 of ∼1 mMfor DC-SIGN, which is within the physiological concentration of 2--FL in human milk. These results demonstrate that DC-SIGN among the many hGBP expressed by DCs binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. [ABSTRACT FROM AUTHOR]

Virtually all cells and extracellular material are heavily decorated by various glycans, yet our understanding of the structure and function of these moieties lags behind the understanding of nucleic acids, lipids, and proteins. Recent years have seen a tremendous acceleration of knowledge in the field of glycobiology, revealing many intricacies and functional contributions that were previously poorly appreciated or even unrecognized. This review highlights several topics relevant to glycoimmunology in which mammalian and pathogen-derived glycans displayed on glycoproteins and other scaffolds are recognized by specific glycan-binding proteins (GBPs), leading to a variety of proinflammatory and anti-inflammatory cellular responses. The focus for this review is mainly on 2 families of GBPs, sialic acid–binding immunoglobulin-like lectins (siglecs) and selectins, that are involved in multiple steps of the immune response, including distinguishing pathogens from self, cell trafficking to sites of inflammation, fine-tuning of immune responses leading to activation or tolerance, and regulation of cell survival. Importantly for the clinician, accelerated rates of discovery in the field of glycoimmunology are being translated into innovative medical approaches that harness the interaction of glycans and GBPs to the benefit of the host and might soon lead to novel diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Cyanovirin-N (CV-N) is an antiviral lectin with potent activity against enveloped viruses, including HIV. The mechanism of action involves high affinity binding to mannose-rich glycans that decorate the surface of enveloped viruses. In the case of HIV, antiviral activity of CV-N is postulated to require multivalent interactions with envelope protein gp120, achieved through a pseudo-repeat of sequence that adopts two near-identical glycan-binding sites, and possibly involves a 3D-domain-swapped dimeric form of CV-N. Here, we present a covalent dimer of CV-N that increases the number of active glycan-binding sites, and we characterize its ability to recognize four glycans in solution. A CV-N variant was designed in which two native repeats were separated by the “nested” covalent insertion of two additional repeats of CV-N, resulting in four possible glycan-binding sites. The resulting Nested CV-N folds into a wild-type-like structure as assessed by circular dichroism and NMR spectroscopy, and displays high thermal stability with a Tm of 59 °C, identical to WT. All four glycan-binding domains encompassed by the sequence are functional as demonstrated by isothermal titration calorimetry, which revealed two sets of binding events to dimannose with dissociation constants Kd of 25 μM and 900 μM, assigned to domains B and B’ and domains A and A’ respectively. Nested CV-N displays a slight increase in activity when compared to WT CV-N in both an anti-HIV cellular assay and a fusion assay. This construct conserves the original binding specifityies of domain A and B, thus indicating correct fold of the two CV-N repeats. Thus, rational design can be used to increase multivalency in antiviral lectins in a controlled manner.

Previously, we demonstrated that porcine spermatozoa bind selectively to Lewisx (LeX) and bi-antennary α2-6 sialylated N-acetyllactosamine (bi-SiaLN) glycan motifs found in the oviduct. Recognition of these motifs mediates sperm binding to oviduct epithelial cells to form a reservoir. Here, we characterize proteins that bind to LeX and bi-SiaLN in ejaculated and cauda epididymal spermatozoa. Fluoresceinated 3′- O-sulphated-LeX (suLeX) and bi-SiaLN were used to locate the binding proteins on spermatozoa. Binding to N-acetyllactosamine (LN), a glycan that spermatozoa bind less, was assessed as a control. Over 60% of ejaculated and epididymal spermatozoa bound to suLeX and bi-SiaLN, but only 25% bound to LN. Specific binding to suLeX and bi-SiaLN required divalent cations. Fluoresceinated suLeX bound mostly to the plasma membrane at the apex of the head, whereas bi-SiaLN bound to a broader area of the head. Further characterization of receptors was accomplished by probing, with specific glycans, sperm proteins that had been separated by SDS-PAGE and transferred to blots. Glycan blot assays identified several suLeX-binding bands that migrated as 12-250 kDa. Migration of some of the bands that bound bi-SiaLN was similar to those that bound suLeX. A 12-kDa band protein was detected in seminal fluid and ejaculated spermatozoa, but absent in epididymal spermatozoa. In summary, we determined that: (i) multiple glycan-binding proteins recognize suLeX and bi-SiaLN motifs, (ii) specific binding required divalent cations and (iii) glycan-binding proteins were identified before and after sperm contact with accessory gland fluids. While the identity of these molecules is still unknown, this information predicts characteristics of the authentic oviduct suLeX and bi-SiaLN receptors on spermatozoa. [ABSTRACT FROM AUTHOR]

Angiogenesis, the growth OF new capillaries from preexisting blood vessels, is a complex process involving endothelial cell (EC) activation, disruption of vascular basement membranes, and migration and proliferation of ECs. Glycan-mediated recognition has been proposed to play an instrumental role in mediating cell-cell and cell-matrix interactions. Galectins (Gal), a family of glycan-binding proteins with affinity for (ß-galactosides and a conserved sequence motif, can decipher glycan- containing information and mediate cell-cell communication. Galectin-8 (Gal-8), a member of this family, is a bivalent "tandem-repeat"-type galectin, which possesses 2 CRDs connected by a linker peptide. Here, we show that Gal-8 is endowed with proangiogeneic properties. Functional assays revealed a critical role for this lectin in the regulation of capillary-tube formation and EC migration. Moreover, Matrigel, either supplemented with Gal-8 or vascular endothelial growth factor (VEGF), injected in mice resulted in induction of in vivo angiogenesis. Remarkably, Gal-8 was expressed both in the cytoplasm and nucleus in ECs of normal and tumor vessels. Furthermore, CD 166 [activated leukocyte cell adhesion molecule (ALCAM)] was identified as a specific Gal-8-bind-ing partner in normal vascular ECs. Collectively, these data provide the first evidence demonstrating an essential role for Gal-8 in the regulation of angiogenesis with critical implications in tumor biology. [ABSTRACT FROM AUTHOR]

Chemically synthesized plant oligosaccharides have recently evolved as powerful molecular tools for plant cell wall biology. Synthetic plant glycan microarrays equipped with these oligosaccharides enable high-throughput analyses of glycan-binding proteins and carbohydrate-active enzymes. To produce these glycan microarrays, small amounts of glycan solution are printed on suitable surfaces for covalent or non-covalent immobilization. Synthetic plant glycan microarrays have been used for example to map the epitopes of plant cell wall-directed antibodies, to characterize glycosyl hydrolases and glycosyl transferases, and to analyze lectin binding. In this chapter, detailed experimental procedures for the production of synthetic glycan microarrays and their use for the characterization of cell wall glycan-directed antibodies are described., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The pleiotropic functions of macrophages in immune defense, tissue repair, and maintenance of tissue homeostasis are supported by the heterogeneity in macrophage sub-populations that differ both in ontogeny and polarization. Although glycans and glycan-binding proteins (GBPs) are integral to macrophage function and may contribute to macrophage diversity, little is known about the factors governing their expression. Here, we provide a resource for characterizing the N-/O-glycomes of various murine peritoneal macrophage sub-populations, demonstrating that glycosylation primarily reflects developmental origin and, to a lesser degree, cellular polarization. Furthermore, comparative analysis of GBP-coding genes in resident and elicited macrophages indicated that GBP expression is consistent with specialized macrophage functions and correlates with specific types of displayed glycans. An integrated, semi-quantitative approach was used to confirm distinct expression patterns of glycans and their binding proteins across different macrophages. The data suggest that regulation of glycan-protein complexes may be central to macrophage residence and recruitment. [Display omitted] • Glycan alterations distinguish macrophage populations primarily by lineage • Resident macrophages exhibit a less heterogeneous glycan profile than elicited cells • C-Type lectins are downregulated in elicited macrophages • Siglec expression is correlated with prevalent sialyltransferase expression Park et al. combines native glycan staining, metabolic tagging, structural glycomics, and transcript analysis to generate a map of glycan and glycan-binding protein expression in primary murine peritoneal macrophage sub-populations. The study serves as a resource to identify distinct molecular differences in macrophages according to cellular ontogeny and polarization. [ABSTRACT FROM AUTHOR]

Glycans and glycan binding proteins (GBPs or lectins) are essential components in almost every aspect of immunology. Investigations of the interactions between glycans and GBPs have greatly advanced our understanding of the molecular basis of these fundamental immunological processes. In order to better study the glycan-GBP interactions, microscope glass slide-based glycan microarrays were conceived and proved to be an incredibly useful and successful tool. A variety of methods have been developed to better present the glycans so that they mimic natural presentations. Breakthroughs in chemical biology approaches have also made available glycans with sophisticated structures that were considered practically impossible just a few decade ago. Glycan microarrays provide a wealth of valuable information in immunological studies. They allow for discovery of detailed glycan binding preferences or novel binding epitopes of known endogenous immune receptors, which can potentially lead to the discovery of natural ligands that carry the glycans. Glycan microarrays also serve as a platform to discover new GBPs that are vital to the process of infection and invasion by microorganisms. This review summarizes the construction strategies and the immunological applications of glycan microarrays, particularly focused on those with the most comprehensive sets of glycan structures. We also review new methods and technologies that have evolved. We believe that glycan microarrays will continue to benefit the growing research community with various interests in the field of immunology., (Copyright © 2019 Gao, Wei, McKitrick, McQuillan, Heimburg-Molinaro and Cummings.)

The glycan ligands recognized by Siglecs, influenza viruses, and galectins, as well as many plant lectins, are not well defined. To explore their binding to asparagine (Asn)-linked N -glycans, we synthesized a library of isomeric multiantennary N -glycans that vary in terminal non-reducing sialic acid, galactose, and N -acetylglucosamine residues, as well as core fucose. We identified specific recognition of N -glycans by several plant lectins, human galectins, influenza viruses, and Siglecs, and explored the influence of sialic acid linkages and branching of the N -glycans. These results show the unique recognition of complex-type N -glycans by a wide variety of glycan-binding proteins and their abilities to distinguish isomeric structures, which provides new insights into the biological roles of these proteins and the uses of lectins in biological applications to identify glycans. • A library of isomeric complex Asn-linked glycans was chemoenzymatically synthesized • Free reducing N -glycans can be generated by treatment with sodium hypochlorite • Unique N -glycan interactions identified with a suite of human proteins and viruses • Differential N -glycan recognition provides insights into biomedical applications Gao et al. chemoenzymatically synthesized a library of naturally occurring asparagine-linked glycan isomers and identified unique glycan-binding specificities of plant lectins, human galectins, influenza viruses, and Siglecs. These results provide important guidance on the biological and clinical applications of these proteins in distinguishing isomeric glycans with certain structural features. [ABSTRACT FROM AUTHOR]

Glycans in polysaccharides and glycoconjugates of the hydrophilic exterior of all animal cells participate in signal transduction, cellular adhesion, intercellular signaling, and sites for binding of pathogens largely through protein-glycan interactions. Microarrays of defined glycans have been used to study the binding specificities of biologically relevant glycan-binding proteins (GBP), but such arrays are limited by their lack of diversity or relevance to the GBP being investigated. Shotgun glycan microarrays are made up of structurally undefined glycans that were released from natural sources, labeled with bifunctional reagents so that they can be monitored during their purification using multidimensional chromatographic procedures, stored as a tagged glycan library (TGL) and subsequently printed onto microarrays at equal molar concentrations. The shotgun glycan microarray is then interrogated with a biologically relevant GBP and the corresponding glycan ligands can be retrieved from the TGL for detailed structural analysis and further functional analysis. Shotgun glycomics extended the defined glycan microarray to a discovery platform that supports functional glycomic analyses and may provide a useful process for ultimately defining the human glycome., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Glycan-binding proteins are widely distributed in human and play an essential role in biological processes. Their involvements in inflammatory and immune responses make it increasingly likely that the glycan-binding proteins may represent valuable therapeutic targets., Objective: The current review aims to provide information on recent advancements in clinical developments of antibodies against glycan-binding proteins as potential targets., Results and Conclusion: There are several therapeutic antibodies being developed targeting glycanbinding proteins, including CD22, CD33, DEC-205, and CD62P, for different diseases. The clinical investigations demonstrated benefits of treatments with one antibody-drug conjugate against CD22 being approved by the regulatory agencies. The recent progresses in clinical developments of these antibodies have provided great promises in therapeutic targeting of more glycan-binding proteins for treating multiple diseases, including inflammation, autoimmune diseases, and hematological malignancies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The C-type lectin receptor Langerin is a glycan-binding protein that serves as an uptake receptor on Langerhans cells and is essential for the formation of Birbeck granules. Whereas most Langerin ligands are recognized by a canonical Ca 2+ -dependent binding site, heparins have been proposed to make additional contacts to a secondary, Ca 2+ -independent site. Glycan array screening and biomolecular NMR spectroscopy were employed to investigate the molecular mechanism of these interactions. We observed that binding of heparin hexasaccharides to a secondary site did not require the presence of Ca 2+ and activated a previously identified intradomain allosteric network of Langerin (thus far only associated with Ca 2+ affinity and release). We propose a communication hub between these two binding sites, which sheds new light on modulatory functions of Langerin-heparin interactions., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Glycan-binding proteins (GBPs) play an important role in cell adhesion, bacterial/viral infection, and cellular signaling pathways. However, little is known about the precision alteration of GBPs referred to pathological changes in hepatic stellate cells (HSCs) during liver fibrosis. Here, the carbohydrate microarrays were used to probe the alteration of GBPs in the activated HSCs and quiescent HSCs. As a result, 12 carbohydrates (e.g. Gal, GalNAc, and Man-9Glycan) showed increased signal, while seven carbohydrates (e.g. NeuAc, Lac, and GlcNAc-O-Ser) showed decreased signal in activated HSCs. Three carbohydrates (Gal, GalNAc, and NeuAc) were selected and subsequently used to validate the results of the carbohydrate microarrays as well as assess the distribution and localization of their binding proteins in HSCs and liver tissues by cy/histochemistry; the results showed that GBPs mainly distributed in the cytoplasma membrane and perinuclear region of cytoplasm. The immunocytochemistry was further used to verify some GBPs really exist in Golgi apparatus of the cells. The precision alteration and localization of GBPs referred to pathological changes in HSCs may provide pivotal information to help understand the biological functions of glycans how to exert through their recognition by a wide variety of GBPs. This study could lead to the development of new anti-fibrotic strategies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent research has uncovered unexpected ways that glycans contribute to biology, as well as new strategies for combatting disease using approaches involving glycans. To make full use of glycans for clinical applications, we need more detailed information on the location, nature, and dynamics of glycan expression in vivo. Such studies require the use of specimens acquired directly from patients. Effective studies of clinical specimens require low-volume assays, high precision measurements, and the ability to process many samples. Assays using affinity reagents-lectins and glycan-binding antibodies-can meet these requirements, but further developments are needed to make the methods routine and effective. Recent advances in the use of glycan-binding proteins involve improved determination of specificity using glycan arrays; the availability of databases for mining and analyzing glycan array data; lectin engineering methods; and the ability to quantitatively interpret lectin measurements. Here, we describe many of the challenges and opportunities involved in the application of these new approaches to the study of biological samples. The new tools hold promise for developing methods to improve the outcomes of patients afflicted with diseases characterized by aberrant glycan expression., (© 2015 Elsevier Inc. All rights reserved.)