Galectins and Immune Responses-Just How Do They Do Those Things They Do?

Galectins are a family of mammalian carbohydrate-binding proteins expressed by many cell types. Galectins can function intracellularly and can also be secreted to bind to cell surface glycoconjugate counterreceptors. Some galectins are made by immune cells, whereas other galectins are secreted by different cell types, such as endothelial or epithelial cells, and bind to immune cells to regulate immune responses. Galectin binding to a single glycan ligand is a low-affinity interaction, but the multivalency of galectins and the glycan ligands presented on cell surface glycoproteins results in high-avidity binding that can reversibly scaffold or cluster these glycoproteins. Galectin binding to a specific glycoprotein counterreceptor is regulated in part by the repertoire of glycosyltransferase enzymes (which make the glycan ligands) expressed by that cell, and the effect of galectin binding results from clustering or retention of specific glycoprotein counterreceptors bearing these specific ligands.

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family.

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.

The direct and indirect effects of glycans on immune function.

The biological impact of glycans is as diverse and complex as the impact of proteins on biology. Familiar roles include those as a protein folding checkpoint in the endoplasmic reticulum and as a modulator of the serum half-life of secreted glycoproteins, but it has become clear over the last several decades that glycans are key signaling moieties, participate in cell-cell interactions and modulate the function of individual proteins, to name but a few examples. In the immune system, the majority of microbial "patterns" are glycans or glycoconjugates, while virtually all cell surface receptors are glycoproteins, and antibody glycosylation critically influences antibody function. In order to provide a simple contextual framework to understand the myriad roles, glycans play in immunity, we propose that glycan effects are considered direct or indirect, depending on their direct participation or their indirect effects on other components in a given biological process or pathway. Here, we present the published evidence that supports this framework, which ultimately leads to the conclusion that we should learn to embrace the complexity inherent to the glycome and its potential as a largely uncharted but target rich area of new therapeutic investigation.

Lectin-binding characterizes the healthy human skeletal muscle glycophenotype and identifies disease-specific changes in dystrophic muscle.

Our understanding of muscle glycosylation to date has derived from studies in mouse models and a limited number of human lectin histochemistry studies. As various therapeutic approaches aimed at treating patients with muscular dystrophies are being translated from rodent models to human, it is critical to better understand human muscle glycosylation and relevant disease-specific differences between healthy and dystrophic muscle. Here, we report the first quantitative characterization of human muscle glycosylation, and identify differentiation- and disease-specific differences in human muscle glycosylation. Utilizing a panel of 13 lectins with varying glycan specificities, we surveyed lectin binding to primary and immortalized myoblasts and myotubes from healthy and dystrophic sources. Following differentiation of primary and immortalized healthy human muscle cells, we observed increased binding of Narcissus pseudonarcissus agglutinin (NPA), PNA, MAA-II and WFA to myotubes compared to myoblasts. Following differentiation of immortalized healthy and dystrophic human muscle cells, we observed disease-specific differences in binding of NPA, Jac and Tricosanthes japonica agglutinin-I (TJA-I) to differentiated myotubes. We also observed differentiation- and disease-specific differences in binding of NPA, Jac, PNA, TJA-I and WFA to glycoprotein receptors in muscle cells. Additionally, Jac, PNA and WFA precipitated functionally glycosylated α-DG, that bound laminin, while NPA and TJA-I did not. Lectin histochemistry of healthy and dystrophic human muscle sections identified disease-specific differences in binding of O-glycan and sialic acid-specific lectins between healthy and dystrophic muscle. These results indicate that specific and discrete changes in glycosylation occur following differentiation, and identify specific lectins as potential biomarkers sensitive to changes in healthy human muscle glycosylation.

Galectin-9 binds to O-glycans on protein disulfide isomerase.

Changes in the T cell surface redox environment regulate critical cell functions, such as cell migration, viral entry and cytokine production. Cell surface protein disulfide isomerase (PDI) contributes to the regulation of T cell surface redox status. Cell surface PDI can be released into the extracellular milieu or can be internalized by T cells. We have found that galectin-9, a soluble lectin expressed by T cells, endothelial cells and dendritic cells, binds to and retains PDI on the cell surface. While endogenous galectin-9 is not required for basal cell surface PDI expression, exogenous galectin-9 mediated retention of cell surface PDI shifted the disulfide/thiol equilibrium on the T cell surface. O-glycans on PDI are required for galectin-9 binding, and PDI recognition appears to be specific for galectin-9, as galectin-1 and galectin-3 do not bind PDI. Galectin-9 is widely expressed by immune and endothelial cells in inflamed tissues, suggesting that T cells would be exposed to abundant galectin-9, in cis and in trans, in infectious or autoimmune conditions.

Galectin-1 regulates tissue exit of specific dendritic cell populations.

During inflammation, dendritic cells emigrate from inflamed tissue across the lymphatic endothelium into the lymphatic vasculature and travel to regional lymph nodes to initiate immune responses. However, the processes that regulate dendritic cell tissue egress and migration across the lymphatic endothelium are not well defined. The mammalian lectin galectin-1 is highly expressed by vascular endothelial cells in inflamed tissue and has been shown to regulate immune cell tissue entry into inflamed tissue. Here, we show that galectin-1 is also highly expressed by human lymphatic endothelial cells, and deposition of galectin-1 in extracellular matrix selectively regulates migration of specific human dendritic cell subsets. The presence of galectin-1 inhibits migration of immunogenic dendritic cells through the extracellular matrix and across lymphatic endothelial cells, but it has no effect on migration of tolerogenic dendritic cells. The major galectin-1 counter-receptor on both dendritic cell populations is the cell surface mucin CD43; differential core 2 O-glycosylation of CD43 between immunogenic dendritic cells and tolerogenic dendritic cells appears to contribute to the differential effect of galectin-1 on migration. Binding of galectin-1 to immunogenic dendritic cells reduces phosphorylation and activity of the protein-tyrosine kinase Pyk2, an effect that may also contribute to reduced migration of this subset. In a murine lymphedema model, galectin-1(-/-) animals had increased numbers of migratory dendritic cells in draining lymph nodes, specifically dendritic cells with an immunogenic phenotype. These findings define a novel role for galectin-1 in inhibiting tissue emigration of immunogenic, but not tolerogenic, dendritic cells, providing an additional mechanism by which galectin-1 can dampen immune responses.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

Timing of galectin-1 exposure differentially modulates Nipah virus entry and syncytium formation in endothelial cells.

UNLABELLED: Nipah virus (NiV) is a deadly emerging enveloped paramyxovirus that primarily targets human endothelial cells. Endothelial cells express the innate immune effector galectin-1 that we have previously shown can bind to specific N-glycans on the NiV envelope fusion glycoprotein (F). NiV-F mediates fusion of infected endothelial cells into syncytia, resulting in endothelial disruption and hemorrhage. Galectin-1 is an endogenous carbohydrate-binding protein that binds to specific glycans on NiV-F to reduce endothelial cell fusion, an effect that may reduce pathophysiologic sequelae of NiV infection. However, galectins play multiple roles in regulating host-pathogen interactions; for example, galectins can promote attachment of HIV to T cells and macrophages and attachment of HSV-1 to keratinocytes but can also inhibit influenza entry into airway epithelial cells. Using live Nipah virus, in the present study, we demonstrate that galectin-1 can enhance NiV attachment to and infection of primary human endothelial cells by bridging glycans on the viral envelope to host cell glycoproteins. In order to exhibit an enhancing effect, galectin-1 must be present during the initial phase of virus attachment; in contrast, addition of galectin-1 postinfection results in reduced production of progeny virus and syncytium formation. Thus, galectin-1 can have dual and opposing effects on NiV infection of human endothelial cells. While various roles for galectin family members in microbial-host interactions have been described, we report opposing effects of the same galectin family member on a specific virus, with the timing of exposure during the viral life cycle determining the outcome. IMPORTANCE: Nipah virus is an emerging pathogen that targets endothelial cells lining blood vessels; the high mortality rate (up to 70%) in Nipah virus infections results from destruction of these cells and resulting catastrophic hemorrhage. Host factors that promote or prevent Nipah virus infection are not well understood. Endogenous human lectins, such as galectin-1, can function as pattern recognition receptors to reduce infection and initiate immune responses; however, lectins can also be exploited by microbes to enhance infection of host cells. We found that galectin-1, which is made by inflamed endothelial cells, can both promote Nipah virus infection of endothelial cells by "bridging" the virus to the cell, as well as reduce production of progeny virus and reduce endothelial cell fusion and damage, depending on timing of galectin-1 exposure. This is the first report of spatiotemporal opposing effects of a host lectin for a virus in one type of host cell.

Galectin-3 binds to CD45 on diffuse large B-cell lymphoma cells to regulate susceptibility to cell death.

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma and an aggressive malignancy. Galectin-3 (gal-3), the only antiapoptotic member of the galectin family, is overexpressed in DLBCL. While gal-3 can localize to intracellular sites, gal-3 is secreted by DLBCL cells and binds back to the cell surface in a carbohydrate-dependent manner. The major counterreceptor for gal-3 on DLBCL cells was identified as the transmembrane tyrosine phosphatase CD45. Removal of cell-surface gal-3 from CD45 with the polyvalent glycan inhibitor GCS-100 rendered DLBCL cells susceptible to chemotherapeutic agents. Binding of gal-3 to CD45 modulated tyrosine phosphatase activity; removal of endogenous cell-surface gal-3 from CD45 with GCS-100 increased phosphatase activity, while addition of exogenous gal-3 reduced phosphatase activity. Moreover, the increased susceptibility of DLBCL cells to chemotherapeutic agents after removal of gal-3 by GCS-100 required CD45 phosphatase activity. Gal-3 binding to a subset of highly glycosylated CD45 glycoforms was regulated by the C2GnT-1 glycosyltransferase, indicating that specific glycosylation of CD45 is important for regulation of gal-3-mediated signaling. These data identify a novel role for cell-surface gal-3 and CD45 in DLBCL survival and suggest novel therapeutic targets to sensitize DLBCL cells to death.

Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry.

Interaction of cell surface glycoproteins with endogenous lectins on the cell surface regulates formation and maintenance of plasma membrane domains, clusters signaling complexes, and controls the residency time of glycoproteins on the plasma membrane. Galectin-9 is a soluble, secreted lectin that binds to glycoprotein receptors to form galectin-glycoprotein lattices on the cell surface. Whereas galectin-9 binding to specific glycoprotein receptors induces death of CD4 Th1 cells, CD4 Th2 cells are resistant to galectin-9 death due to alternative glycosylation. On Th2 cells, galectin-9 binds cell surface protein disulfide isomerase (PDI), increasing retention of PDI on the cell surface and altering the redox status at the plasma membrane. Cell surface PDI regulates integrin function on platelets and also enhances susceptibility of T cells to infection with HIV. We find that galectin-9 binding to PDI on Th2 cells results in increased cell migration through extracellular matrix via ß3 integrins, identifying a unique mechanism to regulate T-cell migration. In addition, galectin-9 binding to PDI on T cells potentiates infection with HIV. We identify a mechanism for regulating cell surface redox status via a galectin-glycoprotein lattice, to regulate distinct T-cell functions.

High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding.

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein ß-dystroglycan (ß-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. ß-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.

Endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation.

Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell.

N- and O-glycans modulate galectin-1 binding, CD45 signaling, and T cell death.

Galectin-1, a beta-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N- and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N- and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by alpha2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an alpha2,6- or alpha2,3-linkage. Thus, N- and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.

Galectin multimerization and lattice formation are regulated by linker region structure.

Galectins regulate cellular functions by binding to glycan ligands on cell surface glycoprotein receptors. Prototype galectins, such as galectin-1, are one carbohydrate recognition domain (CRD) monomers that noncovalently dimerize, whereas tandem-repeat galectins, such as galectin-9, have two non-identical CRDs connected by a linker domain. Dimerization of prototype galectins, or both CRDs in tandem-repeat galectins, is typically required for the crosslinking of glycoprotein receptors and subsequent cellular signaling. Several studies have found that tandem-repeat galectins are more potent than prototype galectins in triggering many cell responses, including cell death. These differences could be due to CRD specificity, the presence or absence of a linker domain between CRDs, or both. To interrogate the basis for the increased potency of tandem-repeat galectins compared with prototype galectins in triggering cell death, we created three tandem-repeat galectin constructs with different linker regions joining identical galectin-1 CRDs, so that any differences we observed would be due to the contribution of the linker region rather than due to CRD specificity. We found that random-coil or rigid α-helical linkers that permit separation of the two galectin-1 CRDs facilitated the formation of higher-order galectin multimers and that these galectins were more potent in binding to glycan ligands and cell surface glycoprotein receptors, as well as triggering T cell death, compared with native galectin-1 or a construct with a short rigid linker. Thus, the increased potency of tandem-repeat galectins compared with prototype galectins is likely due to the ability of the linker domain to permit intermolecular CRD interactions, resulting in the formation of higher-order multimers with increased valency, rather than differences in CRD specificity.

Structural aspects of binding of α-linked digalactosides to human galectin-1.

By definition, adhesion/growth-regulatory galectins are known for their ability to bind ß-galactosides such as Galß(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH-π stacking. These issues are resolved by a combination of (15)N-(1)H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH-π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH-π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and ß-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.

The road less traveled: regulation of leukocyte migration across vascular and lymphatic endothelium by galectins.

Leukocyte entry from the blood into inflamed tissues, exit into the lymphatics, and migration to regional lymph nodes are all crucial processes for mounting an effective adaptive immune response. Leukocytes must cross two endothelial cell layers, the vascular and the lymphatic endothelial cell layers, during the journey from the blood to the lymph node. The proteins and cellular interactions which regulate leukocyte migration across the vascular endothelium are well studied; however, little is known about the factors that regulate leukocyte migration across the lymphatic endothelium. Here, we will summarize evidence for a role for galectins, a family of carbohydrate-binding proteins, in regulating leukocyte migration across the vascular endothelium and propose that galectins are also involved in leukocyte migration across the lymphatic endothelium.

CD45-mediated fodrin cleavage during galectin-1 T cell death promotes phagocytic clearance of dying cells.

Disassembly and phagocytic removal of dying cells is critical to maintain immune homeostasis. The factors that regulate fragmentation and uptake of dying lymphocytes are not well understood. Degradation of fodrin, a cytoskeletal linker molecule that attaches CD45 to the actin cytoskeleton, has been described in apoptotic cells, although no specific initiator of fodrin degradation has been identified. CD45 is a glycoprotein receptor for galectin-1, an endogenous lectin that can trigger lymphocyte apoptosis, although CD45 is not required for phosphatidylserine externalization or DNA degradation during galectin-1 death. In this study, we show that fodrin degradation occurs during galectin-1 T cell death and that CD45 is essential for fodrin degradation to occur. In the absence of CD45, or if fodrin degradation is prevented, galectin-1-induced cell death is not accompanied by membrane blebbing, although phosphatidylserine externalization and DNA degradation proceed, indicating that fodrin degradation occurs via a distinct pathway compared with the pathway that leads to these other hallmarks of cell death. Moreover, there is slower phagocytic uptake by macrophages of T cells in which fodrin degradation is prevented, relative to T cells in which CD45-mediated fodrin degradation occurs. These studies identify a novel role for CD45 in regulating cellular disassembly and promoting phagocytic clearance during galectin-1-induced T cell death.

Suppression of autoimmune diabetes by soluble galectin-1.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that targets the beta-cells of the pancreas. We investigated the ability of soluble galectin-1 (gal-1), an endogenous lectin that promotes T cell apoptosis, to down-regulate the T cell response that destroys the pancreatic beta-cells. We demonstrated that in nonobese diabetic (NOD) mice, gal-1 therapy reduces significantly the amount of Th1 cells, augments the number of T cells secreting IL-4 or IL-10 specific for islet cell Ag, and causes peripheral deletion of beta-cell-reactive T cells. Administration of gal-1 prevented the onset of hyperglycemia in NOD mice at early and subclinical stages of T1D. Preventive gal-1 therapy shifted the composition of the insulitis into an infiltrate that did not invade the islets and that contained a significantly reduced number of Th1 cells and a higher percentage of CD4(+) T cells with content of IL-4, IL-5, or IL-10. The beneficial effects of gal-1 correlated with the ability of the lectin to trigger apoptosis of the T cell subsets that cause beta-cell damage while sparing naive T cells, Th2 lymphocytes, and regulatory T cells in NOD mice. Importantly, gal-1 reversed beta-cell autoimmunity and hyperglycemia in NOD mice with ongoing T1D. Because gal-1 therapy did not cause major side effects or beta-cell toxicity in NOD mice, the use of gal-1 to control beta-cell autoimmunity represents a novel alternative for treatment of subclinical or ongoing T1D.