RESUMO
Galectins constitute a family of soluble lectins with unique capacity to induce macroscale rearrangements upon interacting with cell membrane glycoconjugates. Galectin-8 (Gal-8) is acknowledged for its role in facilitating antigen uptake and processing upon engaging with cell surface glycoconjugates on antigen-presenting cells (APCs). Gal-8 consists of two covalently fused N- and C-terminal carbohydrate recognition domains (N- and C-CRD), each exhibiting distinct glycan specificity. In this study, we utilized single N- and C-CRD recombinant proteins to dissect the nature of Gal-8-glycan interactions during antigen internalization enhancement. Single C-CRD was able to replicate the effect of full-length Gal-8 (FLGal-8) on antigen internalization in BMDCs. Antigen uptake enhancement was diminished in the presence of lactose or when N-glycosylation-deficient macrophages served as APCs, underscoring the significance of glycan recognition. Measurement of the elastic modulus using Atomic Force Microscopy unveiled that FLGal-8- and C-CRD-stimulated macrophages exhibited heightened membrane stiffness compared to untreated cells, providing a plausible mechanism for their involvement in endocytosis. C-CRD proved to be as efficient as FLGal-8 in promoting antigen degradation, suggesting its implication in antigen-processing induction. Lastly, C-CRD was able to replicate FLGal-8-induced antigen presentation in the MHC-II context both in vitro and in vivo. Our findings support the notion that Gal-8 binds through its C-CRD to cell surface N-glycans, thereby altering membrane mechanical forces conducive to soluble antigen endocytosis, processing, and presentation to cognate CD4 T-cells. These findings contribute to a deeper comprehension of Gal-8 and its mechanisms of action, paving the way for the development of more efficacious immunotherapies.
RESUMO
Galectin-8 (Gal-8) is a tandem-repeat type galectin with affinity for ß-galactosides, bearing two carbohydrate recognition domains (CRD) connected by a linker peptide. The N- and C-terminal domains (Gal-8N and Gal-8C) share 35% homology, and their glycan ligand specificity is notably dissimilar: while Gal-8N shows strong affinity for α(2-3)-sialylated oligosaccharides, Gal-8C has higher affinity for non-sialylated oligosaccharides, including poly-N-acetyllactosamine and/ or A and B blood group structures. Particularly relevant for understanding the biological role of this lectin, full-length Gal-8 can bind cell surface glycoconjugates with broader affinity than the isolated Gal-8N and Gal-8C domains, a trait also described for other tandem-repeat galectins. Herein, we aim to discuss the potential use of separate CRDs in modelling tandem-repeat galectin-8 and its biological functions. For this purpose, we will cover several aspects of the structure-function relationship of this protein including crystallographic structures, glycan specificity, cell function and biological roles, with the ultimate goal of understanding the potential role of each CRD in predicting full-length Gal-8 involvement in relevant biological processes.
Assuntos
Metabolismo dos Carboidratos , Galectinas/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Galectinas/química , Humanos , Ligantes , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Galectin-8 (Gal-8), a 'tandem-repeat'-type galectin, has been described as a modulator of cellular functions including adhesion, spreading, growth arrest, apoptosis, pathogen recognition, autophagy, and immunomodulation. We have previously shown that activated leukocyte cell adhesion molecule (ALCAM), also known as CD166, serves as a receptor for endogenous Gal-8. ALCAM is a member of the immunoglobulin superfamily involved in cell-cell adhesion through homophilic (ALCAM-ALCAM) and heterophilic (i.e. ALCAM-CD6) interactions in different tissues. Here we investigated the physiologic relevance of ALCAM-Gal-8 association and glycosylation-dependent mechanisms governing these interactions. We found that silencing of ALCAM in MDA-MB-231 triple negative breast cancer cells decreases cell adhesion and migration onto Gal-8-coated surfaces in a glycan-dependent fashion. Remarkably, either Gal-8 or ALCAM silencing also disrupted cell-cell adhesion, and led to reduced tumor growth in a murine model of triple negative breast cancer. Moreover, structural characterization of endogenous ALCAM N-glycosylation showed abundant permissive structures for Gal-8 binding. Importantly, we also found that cell sialylation controls Gal-8-mediated cell adhesion. Altogether, these findings demonstrate a central role of either ALCAM or Gal-8 (or both) in controlling triple negative breast cancer.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/metabolismo , Galectinas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antígenos CD/genética , Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular Tumoral , Feminino , Proteínas Fetais/genética , Galectinas/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas de Neoplasias/genética , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Galectin-8 (Gal-8) is a mammalian ß-galactoside-binding lectin, endowed with proinflammatory properties. Given its capacity to enhance antigen-specific immune responses in vivo, we investigated whether Gal-8 was also able to promote APC activation to sustain T cell activation after priming. Both endogenous [dendritic cells (DCs)] and bone marrow-derived DCs (BMDCs) treated with exogenous Gal-8 exhibited a mature phenotype characterized by increased MHC class II (MHCII), CD80, and CD86 surface expression. Moreover, Gal-8-treated BMDCs (Gal-8-BMDCs) stimulated antigen-specific T cells more efficiently than immature BMDCs (iBMDCs). Proinflammatory cytokines IL-3, IL-2, IL-6, TNF, MCP-1, and MCP-5, as well as growth factor G-CSF, were augmented in Gal-8-BMDC conditioned media, with IL-6 as the most prominent. Remarkably, BMDCs from Gal-8-deficient mice (Lgals8-/- BMDC) displayed reduced CD86 and IL-6 expression and an impaired ability to promote antigen-specific CD4 T cell activation. To test if Gal-8-induced activation correlates with the elicitation of an effective immune response, soluble Gal-8 was coadministrated with antigen during immunization of BALB/cJ mice in the experimental foot-and-mouth disease virus (FMDV) model. When a single dose of Gal-8 was added to the antigen formulation, an increased specific and neutralizing humoral response was developed, sufficient to enhance animal protection upon viral challenge. IL-6 and IFN-γ, as well as lymphoproliferative responses, were also incremented in Gal-8/antigen-immunized animals only at 48 h after immunization, suggesting that Gal-8 induces the elicitation of an inflammatory response at an early stage. Taking together, these findings argue in favor of the use of Gal-8 as an immune-stimulator molecule to enhance the adaptive immune response.
Assuntos
Apresentação de Antígeno , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Febre Aftosa/imunologia , Galectinas/imunologia , Imunidade Adaptativa , Animais , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Linfócitos T CD4-Positivos/virologia , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Células Dendríticas/virologia , Febre Aftosa/genética , Febre Aftosa/prevenção & controle , Febre Aftosa/virologia , Vírus da Febre Aftosa/crescimento & desenvolvimento , Vírus da Febre Aftosa/imunologia , Galectinas/genética , Galectinas/farmacologia , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/imunologia , Imunização , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-3/genética , Interleucina-3/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quimioatraentes de Monócitos/genética , Proteínas Quimioatraentes de Monócitos/imunologia , Transdução de Sinais , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model. METHODS: We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS. RESULTS: Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin-glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30-40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels. CONCLUSIONS: Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.
Assuntos
Neoplasias Encefálicas/patologia , Galectinas/fisiologia , Glioblastoma/patologia , Animais , Apoptose/fisiologia , Neoplasias Encefálicas/genética , Bovinos , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Citometria de Fluxo/métodos , Galectina 1/análise , Galectina 1/fisiologia , Galectina 3/análise , Galectina 3/fisiologia , Galectinas/análise , Galectinas/farmacologia , Glioblastoma/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Células Tumorais CultivadasRESUMO
BACKGROUND: We previously demonstrated that the activated leukocyte cell adhesion molecule (ALCAM/CD166) can interact with galectin-8 (Gal-8) in endothelial cells. ALCAM is a member of the immunoglobulin superfamily that promotes homophilic and heterophilic cell-cell interactions. Gal-8 is a "tandem-repeat"-type galectin, known as a matricellular protein involved in cell adhesion. Here, we analyzed the physical interaction between both molecules in breast cancer cells and the functional relevance of this phenomenon. METHODS: We performed binding assays by surface plasmon resonance to study the interaction between Gal-8 and the recombinant glycosylated ALCAM ectodomain or endogenous ALCAM from MDA-MB-231 breast cancer cells. We also analyzed the binding of ALCAM-silenced or control breast cancer cells to immobilized Gal-8 by SPR. In internalization assays, we evaluated the influence of Gal-8 on ALCAM surface localization. RESULTS: We showed that recombinant glycosylated ALCAM and endogenous ALCAM from breast carcinoma cells physically interacted with Gal-8 in a glycosylation-dependent fashion displaying a differential behavior compared to non-glycosylated ALCAM. Moreover, ALCAM-silenced breast cancer cells exhibited reduced binding to Gal-8 relative to control cells. Importantly, exogenously added Gal-8 provoked ALCAM segregation, probably trapping this adhesion molecule at the surface of breast cancer cells. CONCLUSIONS: Our data indicate that Gal-8 interacts with ALCAM at the surface of breast cancer cells through glycosylation-dependent mechanisms. GENERAL SIGNIFICANCE: A novel heterophilic interaction between ALCAM and Gal-8 is demonstrated here, suggesting its physiologic relevance in the biology of breast cancer cells.
Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Fetais/metabolismo , Galectinas/metabolismo , Mapas de Interação de Proteínas/genética , Antígenos CD/genética , Neoplasias da Mama/patologia , Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Comunicação Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Células Endoteliais/metabolismo , Feminino , Proteínas Fetais/genética , Galectinas/genética , Glicosilação , Humanos , Ligação Proteica , Propriedades de SuperfícieRESUMO
BACKGROUND: Glioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell mode.l METHODS: We used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS. RESULTS: Gal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin-glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30-40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels. CONCLUSIONS: Gal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.
Assuntos
Humanos , Animais , Bovinos , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Galectinas/fisiologia , Fatores de Tempo , Neoplasias Encefálicas/genética , Células Tumorais Cultivadas , Movimento Celular/fisiologia , Apoptose/fisiologia , Glioblastoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Galectinas/análise , Galectinas/farmacologia , Galectina 1/análise , Galectina 1/fisiologia , Galectina 3/análise , Galectina 3/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Citometria de Fluxo/métodosRESUMO
Galectin-8 belongs to a family of mammalian lectins that recognize glycoconjugates present on different cell surface components and modulate a variety of cellular processes. A role of Gal-8 in the immune system has been proposed based on its effects in immune cells, including T and B lymphocytes, as well as the presence of anti-Gal-8 autoantibodies in the prototypic autoimmune disease systemic lupus erythematosus (SLE). We have previously described that Gal-8 induces apoptosis in activated T cells interacting with certain β1 integrins and this effect is counteracted by the anti-Gal-8 autoantibodies. Given that Gal-8 can potentially interact with several glycoproteins, here we analyzed the β2 integrin Lymphocyte Function-Associated Antigen-1 (LFA-1), which is involved in leukocyte cell adhesion and immunological synapses. We show by GST-pull down assays that Gal-8 interacts with LFA-1 and this interaction is inhibited by anti-Gal-8 autoantibodies isolated from SLE patients. In cell adhesion assays, Gal-8 precluded the interaction of LFA-1 with its ligand Intracellular Adhesion Molecule-1 (ICAM-1). These results suggest that Gal-8 can exert immunosuppressive action not only by inducing apoptosis in activated T cells but also by negatively modulating the crucial function of LFA-1 in the immune system, while function-blocking autoantibodies counteract these effects.