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.
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 is a galactoside-binding lectin expressed in multiple tissues that has pleiotropic immunomodulatory functions. We previously showed that galectin-1 activates human monocyte-derived ...dendritic cells (MDDCs) and triggers a specific genetic program that up-regulates DC migration through the extracellular matrix, an integral property of mucosal DCs. Here, we identify the galectin-1 receptors on MDDCs and immediate downstream effectors of galectin-1-induced MDDC activation and migration. Galectin-1 binding to surface CD43 and CD45 on MDDCs induced an unusual unipolar co-clustering of these receptors and activates a dose-dependent calcium flux that is abrogated by lactose. Using a kinome screen and a systems biology approach, we identified Syk and protein kinase C tyrosine kinases as mediators of the DC activation effects of galectin-1. Galectin-1, but not lipopolysaccharide, stimulated Syk phosphorylation and recruitment of phosphorylated Syk to the CD43 and CD45 co-cluster on MDDCs. Inhibitors of Syk and protein kinase C signaling abrogated galectin-1-induced DC activation as monitored by interleukin-6 production; and MMP-1, -10, and -12 gene up-regulation; and enhanced migration through the extracellular matrix. The latter two are specific features of galectin-1-activated DCs. Interestingly, we also found that galectin-1 can prime DCs to respond more quickly to low dose lipopolysaccharide stimulation. Finally, we underscore the biological relevance of galectin-1-enhanced DC migration by showing that intradermal injection of galectin-1 in MRL-fas mice, which have a defect in skin DC emigration, increased the in vivo migration of dermal DCs to draining lymph nodes.
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.
Galectin-1 (gal-1), an endogenous lectin secreted by a variety of cell types, has pleiotropic immunomodulatory functions, including regulation of lymphocyte survival and cytokine secretion in ...autoimmune, transplant disease, and parasitic infection models. However, the role of gal-1 in viral infections is unknown. Nipah virus (NiV) is an emerging pathogen that causes severe, often fatal, febrile encephalitis. The primary targets of NiV are endothelial cells. NiV infection of endothelial cells results in cell-cell fusion and syncytia formation triggered by the fusion (F) and attachment (G) envelope glycoproteins of NiV that bear glycan structures recognized by gal-1. In the present study, we report that NiV envelope-mediated cell-cell fusion is blocked by gal-1. This inhibition is specific to the Paramyxoviridae family because gal-1 did not inhibit fusion triggered by envelope glycoproteins of other viruses, including two retroviruses and a pox virus, but inhibited fusion triggered by envelope glycoproteins of the related Hendra virus and another paramyxovirus. The physiologic dimeric form of gal-1 is required for fusion inhibition because a monomeric gal-1 mutant had no inhibitory effect on cell fusion. gal-1 binds to specific N-glycans on NiV glycoproteins and aberrantly oligomerizes NiV-F and NiV-G, indicating a mechanism for fusion inhibition. gal-1 also increases dendritic cell production of proinflammatory cytokines such as IL-6, known to be protective in the setting of other viral diseases such as Ebola infections. Thus, gal-1 may have direct antiviral effects and may also augment the innate immune response against this emerging pathogen.
Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved beta-galactoside-binding lectin ...family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-alpha, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.
Increased resistance to apoptosis promotes lymphomagenesis with aberrant expression of cell survival proteins such as BCL-2 and c-MYC occurring in distinct lymphoma subtypes. Galectin-3 is an ...anti-apoptotic protein that protects T cells, macrophages, and breast carcinoma cells from death triggered by a variety of agents. We have found high levels of galectin-3 protein expression in a subset of B-cell neoplasms including diffuse large B-cell lymphoma (DLBCL), primary effusion lymphoma (PEL), and multiple myeloma (MM), in both cell lines and patient samples. However, we failed to detect galectin-3 in Burkitt lymphoma (BL), follicular lymphoma (FL), marginal zone lymphoma (MZL), MALT lymphoma or B-small lymphocytic lymphoma (B-SLL) cell lines or patient samples. To determine whether galectin-3 expression protects B cells from apoptosis, galectin-3-negative BL cells were transfected with a galectin-3 expressing plasmid, which resulted in markedly increased resistance to anti-Fas-induced cell death. In contrast, galectin-3-positive PEL cells transfected with an amino-terminal truncated galectin-3 vector showed increased sensitivity to anti-Fas induced apoptosis. During normal B-cell development, galectin-3 expression was lowest in germinal center and plasma B cells, from which DLBCL, PEL, and MM derive, and highest in long-lived naïve and memory B cells. This pattern of expression suggests that aberrantly increased galectin-3 levels in specific B-cell populations may yield a protective advantage during transformation and/or progression of certain B-cell neoplasms.
Galectins are a family of mammalian beta-galactoside-binding proteins that positively and negatively regulate T cell death. Extracellular galectin-1 directly induces death of T cells and thymocytes, ...while intracellular galectin-3 blocks T cell death. In contrast to the antiapoptotic function of intracellular galectin-3, we demonstrate that extracellular galectin-3 directly induces death of human thymocytes and T cells. However, events in galectin-3- and galectin-1-induced cell death differ in a number of ways. Thymocyte subsets demonstrate different susceptibility to the two galectins: whereas galectin-1 kills double-negative and double-positive human thymocytes with equal efficiency, galectin-3 preferentially kills double-negative thymocytes. Galectin-3 binds to a complement of T cell surface glycoprotein receptors distinct from that recognized by galectin-1. Of these glycoprotein receptors, CD45 and CD71, but not CD29 and CD43, appear to be involved in galectin-3-induced T cell death. In addition, CD7 that is required for galectin-1-induced death is not required for death triggered by galectin-3. Following galectin-3 binding, CD45 remains uniformly distributed on the cell surface, in contrast to the CD45 clustering induced by galectin-1. Thus, extracellular galectin-3 and galectin-1 induce death of T cells through distinct cell surface events. However, as galectin-3 and galectin-1 cell death are neither additive nor synergistic, the two death pathways may converge inside the cell.
Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell ...adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K1=21±6×103 M−1) than the second (K2=4±2×103 M−1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K1=20±10×103 M−1 and K2=1.67±0.07×103 M−1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure–function relationships and to protein–carbohydrate interactions in general.
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