Disassembly and phagocytic removal of dying cells is critical to maintain immune homeostasis. The factors regulating 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. CD45 is not required for membrane changes or DNA degradation during galectin-1 death. However, here we show that fodrin degradation occurs during galectin-1 T cell death, and CD45 is essential for fodrin degradation to occur. In the absence of CD45 and fodrin degradation, cell death is not accompanied by membrane blebbing, indicating that fodrin degradation occurs via a distinct pathway compared to the pathway that initiates apoptotic membrane changes and DNA degradation. Moreover, there is slower phagocytic uptake of cells in which fodrin degradation is blocked relative to 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.
By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(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 15N–1H 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 β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.
agglutinin; glycolipid; glycoprotein; lectin; sugar code
Galectin-1, a β-galactoside binding protein, is produced by thymic epithelial cells and binds to human thymocytes. We have previously reported that galectin-1 induces the apoptosis of activated T lymphocytes. Because the majority of thymocytes die via apoptosis while still within the thymus, we tested whether galectin-1 could induce the apoptosis of these cells. We now report that in vitro exposure to galectin-1 induced apoptosis of two subsets of CD4lo CD8lo thymocytes. The phenotypes of susceptible thymocytes were consistent with that of both negatively selected and nonselected cells. Galectin-1–induced apoptosis was enhanced by preexposure of thymocytes to antibody to CD3, suggesting that galectin-1 may be a participant in T-cell– receptor mediated apoptosis. In contrast, pretreatment of thymocytes with dexamethasone had no effect on galectin-1 susceptibility. We noted that 71% of the cells undergoing apoptosis after galectin-1 treatment had a DNA content greater than 2N, indicating that proliferating thymocytes were most sensitive to galectin-1. We propose that galectin-1 plays a role in the apoptosis of both negatively selected and nonselected thymocytes, and that the susceptibility of thymocytes to galectin-1 is regulated, in part, by entry or exit from the cell cycle.
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.
apoptosis; galectin; glycan microarray; lattice; tandem repeat
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.
Galectin; leukocyte; migration; vascular endothelium; lymphatic endothelium
Type 1 diabetes (T1D) is a T-cell-mediated autoimmune disease that targets the β-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 β-cells. We demonstrated that in NOD mice, gal-1-therapy reduces significantly the amount of Th1 cells and augments the number of T-cells secreting IL-4 or IL-10 specific for islet cell-Ag, and causes peripheral deletion of β-cell-reactive T-cells. Administration of gal-1 prevented onset of hyperglycemia in NOD mice at early and sub-clinical 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 β-cell damage, while sparing naïve T-cells, Th2 lymphocytes and regulatory T-cells in NOD mice. Importantly, gal-1 reversed β-cell autoimmunity in NOD mice with ongoing T1D, with reversal of hyperglycemia. Since gal-1-therapy did not cause major side effects or β-cell toxicity in NOD mice, the use of gal-1 to control β-cell autoimmunity represents a novel alternative for treatment of sub-clinical or ongoing T1D.
Diabetes; Autoimmunity; Th1/Th2 cells
The formation of multivalent complexes of soluble galectins with glycoprotein receptors on the plasma membrane helps to organize glycoprotein assemblies on the surface of the cell. In some cell types, this formation of galectin–glycan lattices or scaffolds is critical for organizing plasma membrane domains, such as lipid rafts, or for targeted delivery of glycoproteins to the apical or basolateral surface. Galectin–glycan lattice formation is also involved in regulating the signalling threshold of some cell-surface glycoproteins, including T-cell receptors and growth factor receptors. Finally, galectin–glycan lattices can determine receptor residency time by inhibiting endocytosis of glycoprotein receptors from the cell surface, thus modulating the magnitude or duration of signalling from the cell surface. This paper reviews recent evidence in vitro and in vivo for critical physiological and cellular functions that are regulated by galectin–glycoprotein interactions.
apoptosis; endocytosis; galectin; glycoprotein; lattice; lipid raft
Supramolecular chemistry has been employed to develop flexible and adaptable multivalent neoglycoconjugates for binding galectin-1 (Gal-1). Gal-1, a dimeric lectin with two galactoside-binding sites, regulates cancer progression and immune responses. Self-assembled pseudopolyrotaxanes comprised of lactoside-displaying cyclodextrin (LCD) “beads” threaded onto polyviologen “strings,” display mobile ligands as a result of cyclodextrin rotation about, and limited translation along, the polymer chain. The pseudopolyrotaxanes rapidly and efficiently precipitate Gal-1 and provide valency-corrected enhancements of up to 30-fold over native lactose and 20-fold over free LCD in a T-cell agglutination assay. A supramolecular stastical effect was observed, wherein the efficacy of Gal-1 inhibition correlates with the number of ligands connected to each other solely through mechanical and noncovalent interactions. Such flexible and adaptable self-assembled pseudopolyrotaxanes show promise for the study of multivalent interactions and targeting of therapeutically relevant lectins.
Galectin-1, a mammalian lectin expressed in many tissues, induces death of diverse cell types, including lymphocytes and tumor cells. The galectin-1 T cell death pathway is novel and distinct from other death pathways, including those initiated by Fas and corticosteroids. We have found that galectin-1 binding to human T cell lines triggered rapid translocation of endonuclease G from mitochondria to nuclei. However, endonuclease G nuclear translocation occurred without cytochrome c release from mitochondria, without nuclear translocation of apoptosis inducing factor, and prior to loss of mitochondrial membrane potential. Galectin-1 treatment did not result in caspase activation, nor was death blocked by caspase inhibitors. However, galectin-1 cell death was inhibited by intracellular expression of galectin-3, and galectin-3 expression inhibited the eventual loss of mitochondrial membrane potential. Galectin-1 induced cell death proceeds via a caspase-independent pathway that involves a unique pattern of mitochondrial events, and different galectin family members can coordinately regulate susceptibility to cell death.
galectin; apoptosis; T lymphocyte; Endonuclease G; human; phosphatidylserine (PS); z-Val-Ala-Asp(OMe)-CH2F (zVAD-fmk); z-Asp-Glu-Val-Asp(OMe)-CH2F (zDEVD-fmk); poly(ADP-ribose)polymerase (PARP); 7-amino-actinomycin D (7AAD); z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (zDEVD-AFC); mitochondrial membrane potential (Δψm); endonuclease G (EndoG); 10-N-nonyl acridine orange (NAO); Apoptosis inducing factor (AIF); truncated Bid (tBid); propidium iodide (PI); fluorescein isothiocyanate (FITC)
Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein “displacement” model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this “shielding” role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.
Currently there are few reliable cell surface markers that can clearly discriminate effector from memory T cells. To determine if there are changes in O-glycosylation between these two cell types, we analyzed virus-specific CD8 T cells at various time points after lymphocytic choriomeningitis virus infection of mice. Antigen-specific CD8 T cells were identified using major histocompatibility complex class I tetramers, and glycosylation changes were monitored with a monoclonal antibody (1B11) that recognizes O-glycans on mucin-type glycoproteins. We observed a striking upregulation of a specific cell surface O-glycan epitope on virus-specific CD8 T cells during the effector phase of the primary cytotoxic T lymphocyte (CTL) response. This upregulation showed a strong correlation with the acquisition of effector function and was downregulated on memory CD8 T cells. Upon reinfection, there was again increased expression of this specific O-glycan epitope on secondary CTL effectors, followed once more by decreased expression on memory cells. Thus, this study identifies a new cell surface marker to distinguish between effector and memory CD8 T cells. This marker can be used to isolate pure populations of effector CTLs and also to determine the proportion of memory CD8 T cells that are recruited into the secondary response upon reencounter with antigen. This latter information will be of value in optimizing immunization strategies for boosting CD8 T cell responses.
memory T cells; effector T cells; cytotoxic T lymphocytes; viral immunity; O-glycosylation
Genetic and biologic observations suggest that pigs may serve as “mixing vessels” for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.
Background: Genetic alteration of muscle cell glycosylation in muscular dystrophy models has ameliorated disease.
Results: A high throughput screen identified a small molecule, lobeline, which altered muscle cell glycosylation and improved laminin binding.
Conclusion: Lobeline increased abundance of sarcolemmal glycoproteins and increased laminin binding in an N-glycan-dependent manner.
Significance: A novel approach revealed an unexpected role for N-glycans in muscle cell function.
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 β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-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.
Glycobiology; Glycosylation Inhibitors; Laminin; Lectin; Muscular Dystrophy; Duchenne Muscular Dystrophy; N-Glycan; Dystroglycan; Lobeline; Muscle
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.
Nipah virus (NiV) is classified as a “priority pathogen” by the NIH. NiV infection of humans results in multi-organ hemorrhage due to endothelial syncytia formation, and also causes fatal encephalitis in up to 70% of patients. As there are no effective vaccines or therapeutics for NiV, understanding the mechanism of endothelial damage by NiV is a critical goal. Our present work defines the interaction between galectin-1, an innate immune lectin that is secreted by human endothelial cells, with the fusion glycoprotein of NiV. We demonstrate that galectin-1 can block the function of the NiV-F protein via three distinct mechanisms, and thus reduce the ability of NiV-F to cause endothelial cell-cell fusion. Importantly, in this study, we use human endothelial cells, the primary target of Nipah virus in vivo, and demonstrate that endogenous galectin-1 made by endothelial cells contributes to limiting cell-cell fusion caused by NiV-F. As endothelial syncytia formation is one of the primary pathophysiologic events in Nipah virus infection, contributing to the hemorrhagic diathesis seen in infected patients, understanding the mechanism of endothelial cell fusion and the ability of galectin-1 to ameliorate cell fusion are critical for development of new approaches to mitigate these events.