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The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies.
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Antifúngicos , Candida albicans , Pared Celular , Antifúngicos/farmacología , Antifúngicos/química , Candida albicans/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/farmacología , Helianthus/química , Mananos/química , Mananos/farmacología , Mananos/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as ß-galactoside binding lectins. However, certain members of this family have shown selective affinity toward specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7, and -12) toward a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I versus type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity toward poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance experiments. Particularly, we identified oligosaccharide blood group A antigen tetraose 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited interleukin-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.
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The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.
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Galectins, a family of endogenous glycan-binding proteins, play crucial roles in a broad range of physiological and pathological processes. Galectin-1 (Gal-1), a proto-type member of this family, is overexpressed in several cancers and plays critical roles in tumor-immune escape, angiogenesis and metastasis. Thus, generation of high-affinity Gal-1 inhibitors emerges as an attractive therapeutic approach for a wide range of neoplastic conditions. Small-molecule carbohydrate inhibitors based on lactose (Lac) and N-acetyllactosamine (LacNAc) structures have been tested showing different results. In this study, we evaluated Lac- and LacNAc-based compounds with specific chemical modifications at key positions as Gal-1 ligands by competitive solid-phase assays (SPA) and isothermal titration calorimetry (ITC). Both assays showed excellent correlation, highlighting that lactosides bearing bulky aromatic groups at the anomeric carbon and sulfate groups at the O3' position exhibited the highest binding affinities. To dissect the atomistic determinants for preferential affinity of the different tested Gal-1 ligands, molecular docking simulations were conducted and PRODIGY-LIG structure-based method was employed to predict binding affinity in protein-ligand complexes. Notably, calculated binding free energies derived from the molecular docking were in accordance with experimental values determined by SPA and ITC, showing excellent correlation between theoretical and experimental approaches. Moreover, this analysis showed that 3'-O-sulfate groups interact with residues of the Gal-1 subsite B, mainly with Asn33, while the ester groups of the aromatic anomeric group interact with Gly69 and Thr70 at Gal-1 subsite E, extending deeper into the pocket, which could account for the enhanced binding affinity. This study contributes to the rational design of highly optimized Gal-1 inhibitors to be further studied in cancer models and other pathologic conditions.
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Galectins are a family of endogenous glycan-binding proteins that have crucial roles in a broad range of physiological and pathological processes. As a group, these proteins use both extracellular and intracellular mechanisms as well as glycan-dependent and independent pathways to reprogramme the fate and function of numerous cell types. Given their multifunctional roles in both tissue fibrosis and cancer, galectins have been identified as potential therapeutic targets for these disorders. Here, we focus on the therapeutic relevance of galectins, particularly galectin 1 (GAL1), GAL3 and GAL9 to tumour progression and fibrotic diseases. We consider an array of galectin-targeted strategies, including small-molecule carbohydrate inhibitors, natural polysaccharides and their derivatives, peptides, peptidomimetics and biological agents (notably, neutralizing monoclonal antibodies and truncated galectins) and discuss their mechanisms of action, selectivity and therapeutic potential in preclinical models of fibrosis and cancer. We also review the results of clinical trials that aim to evaluate the efficacy of galectin inhibitors in patients with idiopathic pulmonary fibrosis, nonalcoholic steatohepatitis and cancer. The rapid pace of glycobiology research, combined with the acute need for drugs to alleviate fibrotic inflammation and overcome resistance to anticancer therapies, will accelerate the translation of anti-galectin therapeutics into clinical practice.
Asunto(s)
Galectinas , Neoplasias , Humanos , Galectinas/metabolismo , Neoplasias/tratamiento farmacológico , Anticuerpos Monoclonales , Polisacáridos/metabolismo , FibrosisRESUMEN
Blockade of vascular endothelial growth factor (VEGF) signaling with bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), or with receptor tyrosine kinase inhibitors, has improved progression-free survival and, in some indications, overall survival across several types of cancers by interrupting tumor angiogenesis. However, the clinical benefit conferred by these therapies is variable, and tumors from treated patients eventually reinitiate growth. Previously we demonstrated, in mouse tumor models, that galectin-1 (Gal1), an endogenous glycan-binding protein, preserves angiogenesis in anti-VEGF-resistant tumors by co-opting the VEGF receptor (VEGFR)2 signaling pathway in the absence of VEGF. However, the relevance of these findings in clinical settings is uncertain. Here, we explored, in a cohort of melanoma patients from AVAST-M, a multicenter, open-label, randomized controlled phase 3 trial of adjuvant bevacizumab versus standard surveillance, the role of circulating plasma Gal1 as part of a compensatory mechanism that orchestrates endothelial cell programs in bevacizumab-treated melanoma patients. We found that increasing Gal1 levels over time in patients in the bevacizumab arm, but not in the observation arm, significantly increased their risks of recurrence and death. Remarkably, plasma Gal1 was functionally active as it was able to reprogram endothelial cell biology, promoting migration, tubulogenesis, and VEGFR2 phosphorylation. These effects were prevented by blockade of Gal1 using a newly developed fully human anti-Gal1 neutralizing mAb. Thus, using samples from a large-scale clinical trial from stage II and III melanoma patients, we validated the clinical relevance of Gal1 as a potential mechanism of resistance to bevacizumab treatment.
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Melanoma , Factor A de Crecimiento Endotelial Vascular , Animales , Ratones , Humanos , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , Galectina 1 , Melanoma/tratamiento farmacológico , Melanoma/patología , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Células Endoteliales/patología , Factores de Crecimiento Endotelial Vascular , Biología , Inhibidores de la Angiogénesis/farmacologíaRESUMEN
Combined Antiretroviral therapy (cART) suppresses HIV replication but fails to eradicate the virus, which persists in a small pool of long-lived latently infected cells. Immune activation and residual inflammation during cART are considered to contribute to viral persistence. Galectins, a family of ß-galactoside-binding proteins, play central roles in host-pathogen interactions and inflammatory responses. Depending on their structure, glycan binding specificities and/or formation of distinct multivalent signaling complexes, different members of this family can complement, synergize, or oppose the function of others. Here, we identify a regulatory circuit, mediated by galectin-1 (Gal-1)-glycan interactions, that promotes reversal of HIV-1 latency in infected T cells. We found elevated levels of circulating Gal-1 in plasma from HIV-1-infected individuals, which correlated both with inflammatory markers and the transcriptional activity of the reservoir, as determined by unspliced-RNA (US-RNA) copy number. Proinflammatory extracellular vesicles (EVs) isolated from the plasma of HIV-infected individuals induced Gal-1 secretion by macrophages. Extracellularly, Gal-1 interacted with latently infected resting primary CD4+ T cells and J-LAT cells in a glycan-dependent manner and reversed HIV latency via activation of the nuclear factor κB (NF-κB). Furthermore, CD4+ T cells isolated from HIV-infected individuals showed increased HIV-1 transcriptional activity when exposed to Gal-1. Thus, by modulating reservoir dynamics, EV-driven Gal-1 secretion by macrophages links inflammation with HIV-1 persistence in cART-treated individuals. IMPORTANCE Antiretroviral therapy has led to a dramatic reduction in HIV-related morbidity and mortality. However, cART does not eradicate the virus, which persists in resting CD4+ T cells as the main viral reservoir, consequently requiring lifelong treatment. A major question is how the functional status of the immune system during antiretroviral therapy determines the activity and size of the viral reservoir. In this study, we identified a central role for galectin-1 (Gal-1), a glycan-binding protein released in response to extracellular vesicles (EVs), in modulating the activity of HIV reservoir, thus shaping chronic immune activation in HIV-infected patients. Our work unveils a central role of Gal-1 in linking chronic immune activation and reservoir dynamics, highlighting new therapeutic opportunities in HIV infection.
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Vesículas Extracelulares , Infecciones por VIH , VIH-1 , Linfocitos T CD4-Positivos , Galectina 1/uso terapéutico , VIH-1/fisiología , Humanos , Inflamación , ARN , Latencia del Virus , Replicación ViralRESUMEN
Development of an aberrant vascular network is a hallmark of the multistep pathological process of tumor growth and metastasis. In response to hypoxia, several pro-angiogenic factors are synthesized to support vascularization programs required for cancer progression. Emerging data indicate the involvement of glycans and glycan-binding proteins as critical regulators of vascular circuits in health and disease. Galectins may be regulated by hypoxic conditions and control angiogenesis in different physiopathological settings. These ß-galactoside-binding proteins may promote sprouting angiogenesis by interacting with different glycosylated receptors and triggering distinct signaling pathways. Understanding the role of galectins in tumor neovascularization will contribute to the design of novel anti-angiogenic therapies aimed at complementing current anti-cancer modalities and overcoming resistance to these treatments. Here we describe selected strategies and methods used to study the role of hypoxia-regulated galectins in the regulation of blood vessel formation.
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Galectinas , Hipoxia , Neoplasias , Neovascularización Patológica , Galectinas/metabolismo , Humanos , Hipoxia/fisiopatología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/fisiopatología , Transducción de SeñalRESUMEN
The rapid spread of the SARS-CoV-2, the causative agent of the emergent pandemic disease COVID-19, requires the urgent commitment of the immunology community to understand the adaptive immune response developed by COVID-19 convalescent patients and individuals vaccinated with different strategies and schemes, with the ultimate goal of implementing and optimizing health care and prevention policies. Currently, assessment of SARS-CoV-2-specific immunity is mainly focused on the measurement of the antibody titers and analysis of their neutralizing capacity. However, a considerable proportion of individuals lack humoral responses or show a progressive decline of SARS-CoV-2-specific neutralizing antibodies. In order to study the cellular response of convalescent patients and vaccinated individuals, we have developed the "COVID-T Platform", an optimized strategy to study SARS-CoV-2-specific T cell responses. This platform allows assessment of the nature, magnitude and persistence of antigen-specific T-cell immunity in COVID-19-convalescent patients and vaccinated individuals. Moreover, it gives the opportunity to study cellular responses against emerging coronavirus variants and to identify individuals with cross-reactive immunity against seasonal coronaviruses.
La rápida propagación del coronavirus SARS-CoV-2, agente causal de la enfermedad pandémica emergente COVID-19 y sus nuevas variantes, requiere del compromiso de la comunidad inmunológica para comprender la magnitud y naturaleza de la respuesta inmunológica adaptativa desarrollada por pacientes recuperados de COVID-19 e individuos vacunados con diferentes estrategias y protocolos, a los fines de implementar nuevas políticas sanitarias. En la actualidad, la determinación de la inmunidad contra SARS-CoV-2 se basa principalmente en la detección de anticuerpos específicos y la determinación de su actividad neutralizante. Sin embargo, a pesar de la alta sensibilidad de estos ensayos, un número considerable de pacientes e individuos vacunados carecen de respuesta humoral detectable, o evidencian una disminución rápida de la misma en el tiempo. Con el objetivo de estudiar la respuesta inmune celular desencadenada frente a SARS-CoV-2, en nuestro laboratorio desarrollamos la "Plataforma COVID-T" estrategia integral optimizada dirigida a caracterizar y monitorear la respuesta de linfocitos T específicos de SARS-CoV-2 a partir de muestras de sangre de individuos vacunados y/o recuperados de COVID-19. Esta plataforma permite evaluar la naturaleza, magnitud y persistencia de la inmunidad celular T generada tanto por la infección con SARS-CoV-2, como por distintos esquemas y protocolos de vacunación en diferentes poblaciones de individuos. Asimismo, permite evaluar la respuesta inmunológica T generada frente a nuevas variantes del virus e identificar individuos sanos resistentes a SARS-CoV-2 con inmunidad pre-existente hacia coronavirus estacionales.
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COVID-19 , SARS-CoV-2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Humanos , Linfocitos TRESUMEN
Resumen La rápida propagación del coronavirus SARS-CoV-2, agente causal de la enfermedad pandémica emergente COVID-19 y sus nuevas variantes, requiere del compromiso de la comunidad inmunológica para comprender la magnitud y naturaleza de la respuesta inmunológica adaptativa desarrollada por pacientes recuperados de COVID-19 e individuos vacunados con diferentes estrategias y protocolos, a los fines de imple mentar nuevas políticas sanitarias. En la actualidad, la determinación de la inmunidad contra SARS-CoV-2 se basa principalmente en la detección de anticuerpos específicos y la determinación de su actividad neutralizante. Sin embargo, a pesar de la alta sensibilidad de estos ensayos, un número considerable de pacientes e indivi duos vacunados carecen de respuesta humoral detectable, o evidencian una disminución rápida de la misma en el tiempo. Con el objetivo de estudiar la respuesta inmune celular desencadenada frente a SARS-CoV-2, en nuestro laboratorio desarrollamos la "Plataforma COVID-T" estrategia integral optimizada dirigida a caracte rizar y monitorear la respuesta de linfocitos T específicos de SARS-CoV-2 a partir de muestras de sangre de individuos vacunados y/o recuperados de COVID-19. Esta plataforma permite evaluar la naturaleza, magnitud y persistencia de la inmunidad celular T generada tanto por la infección con SARS-CoV-2, como por distintos esquemas y protocolos de vacunación en diferentes poblaciones de individuos. Asimismo, permite evaluar la respuesta inmunológica T generada frente a nuevas variantes del virus e identificar individuos sanos resistentes a SARS-CoV-2 con inmunidad pre-existente hacia coronavirus estacionales.
Abstract The rapid spread of the SARS-CoV-2, the caus ative agent of the emergent pandemic disease COVID-19, requires the urgent commitment of the immunology community to understand the adaptive immune response developed by COVID-19 convalescent patients and individuals vaccinated with different strategies and schemes, with the ultimate goal of implementing and optimizing health care and prevention policies. Currently, assessment of SARS-CoV-2-specific immunity is mainly focused on the measurement of the antibody titers and analysis of their neutralizing capacity. However, a considerable proportion of individuals lack humoral responses or show a progressive decline of SARS-CoV-2-specific neutral izing antibodies. In order to study the cellular response of convalescent patients and vaccinated individuals, we have developed the 'COVID-T Platform', an optimized strategy to study SARS-CoV-2-specific T cell responses. This platform allows assessment of the nature, magnitude and persistence of antigen-specific T-cell immunity in COVID-19-convalescent patients and vaccinated individuals. Moreover, it gives the opportunity to study cellular responses against emerging coronavirus variants and to identify individuals with cross-reactive immunity against seasonal coronaviruses.
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Humanos , SARS-CoV-2 , COVID-19 , Linfocitos T , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Galectins, soluble lectins widely expressed intra- and extracellularly in different cell types, play major roles in deciphering the cellular glycocode. Galectin-1 (Gal-1), a prototype member of this family, presents a carbohydrate recognition domain (CRD) with specific affinity for ß-galactosides such as N-acetyllactosamine (ß-d-Galp-(1 â 4)-d-GlcpNAc), and mediate numerous physiological and pathological processes. In this work, Gal-1 binding affinity for ß-(1 â 6) galactosides, including ß-d-Galp-(1 â 6)-ß-d-GlcpNAc-(1 â 4)-d-GlcpNAc was evaluated, and their performance was compared to that of ß-(1 â 4) and ß-(1 â 3) galactosides. To this end, the trisaccharide ß-d-Galp-(1 â 6)-ß-d-GlcpNAc-(1 â 4)-d-GlcpNAc was enzymatically synthesized, purified and structurally characterized. To evaluate the affinity of Gal-1 for the galactosides, competitive solid phase assays (SPA) and isothermal titration calorimetry (ITC) studies were carried out. The experimental dissociation constants and binding energies obtained were compared to those calculated by molecular docking. These analyses evidenced the critical role of the glycosidic linkage between the terminal galactopyranoside residue and the adjacent monosaccharide, as galactosides bearing ß-(1 â 6) glycosidic linkages showed dissociation constants six- and seven-fold higher than those involving ß-(1 â 4) and ß-(1 â 3) linkages, respectively. Moreover, docking experiments revealed the presence of hydrogen bond interactions between the N-acetyl group of the glucosaminopyranose moiety of the evaluated galactosides and specific amino acid residues of Gal-1, relevant for galectin-glycan affinity. Noticeably, the binding free energies (ΔGbindcalc) derived from the molecular docking were in good agreement with experimental values determined by ITC measurements (ΔGbindexp), evidencing a good correlation between theoretical and experimental approaches, which validates the in silico simulations and constitutes an important tool for the rational design of future optimized ligands.
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Galactósidos/química , Galectina 1/química , Azúcares/química , Acetilación , Conformación de Carbohidratos , Humanos , Simulación del Acoplamiento MolecularRESUMEN
Tn is a tumor-associated carbohydrate antigen that constitutes both a diagnostic tool and an immunotherapeutic target. It originates from interruption of the mucin O-glycosylation pathway through defects involving, at least in part, alterations in core-1 synthase activity, which is highly dependent on Cosmc, a folding chaperone. Tn antigen is recognized by the Macrophage Galactose-type Lectin (MGL), a C-type lectin receptor present on dendritic cells and macrophages. Specific interactions between Tn and MGL shape anti-tumoral immune responses by regulating several innate and adaptive immune cell programs. In this work, we generated and characterized a variant of the lung cancer murine cell line LL/2 that expresses Tn by mutation of the Cosmc chaperone gene (Tn+ LL/2). We confirmed Tn expression by lectin glycophenotyping and specific anti-Tn antibodies, verified abrogation of T-synthase activity in these cells, and confirmed its recognition by the murine MGL2 receptor. Interestingly, Tn+ LL/2 cells were more aggressive in vivo, resulting in larger and highly vascularized tumors than those generated from wild type Tn- LL/2 cells. In addition, Tn+ tumors exhibited an increase in CD11c+ F4/80+ cells with high expression of MGL2, together with an augmented expression of IL-10 in infiltrating CD4+ and CD8+ T cells. Importantly, this immunosuppressive microenvironment was dependent on the presence of MGL2+ cells, since depletion of these cells abrogated tumor growth, vascularization and recruitment of IL-10+ T cells. Altogether, our results suggest that expression of Tn in tumor cells and its interaction with MGL2-expressing CD11c+F4/80+ cells promote immunosuppression and angiogenesis, thus favoring tumor progression.
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Antígenos de Carbohidratos Asociados a Tumores/inmunología , Galactosa/inmunología , Lectinas Tipo C/inmunología , Neoplasias Pulmonares/inmunología , Macrófagos/inmunología , Neovascularización Patológica/inmunología , Animales , Antígeno CD11c/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Femenino , Terapia de Inmunosupresión/métodos , Interleucina-10/inmunología , Ratones , Ratones Endogámicos C57BL , Microambiente Tumoral/inmunologíaRESUMEN
Diverse immunoregulatory circuits operate to preserve intestinal homeostasis and prevent inflammation. Galectin-1 (Gal1), a ß-galactoside-binding protein, promotes homeostasis by reprogramming innate and adaptive immunity. Here, we identify a glycosylation-dependent "on-off" circuit driven by Gal1 and its glycosylated ligands that controls intestinal immunopathology by targeting activated CD8+ T cells and shaping the cytokine profile. In patients with inflammatory bowel disease (IBD), augmented Gal1 was associated with dysregulated expression of core 2 ß6-N-acetylglucosaminyltransferase 1 (C2GNT1) and α(2,6)-sialyltransferase 1 (ST6GAL1), glycosyltransferases responsible for creating or masking Gal1 ligands. Mice lacking Gal1 exhibited exacerbated colitis and augmented mucosal CD8+ T cell activation in response to 2,4,6-trinitrobenzenesulfonic acid; this phenotype was partially ameliorated by treatment with recombinant Gal1. While C2gnt1-/- mice exhibited aggravated colitis, St6gal1-/- mice showed attenuated inflammation. These effects were associated with intrinsic T cell glycosylation. Thus, Gal1 and its glycosylated ligands act to preserve intestinal homeostasis by recalibrating T cell immunity.
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Colorectal cancer (CRC) represents the third most common malignancy and the second leading cause of cancer-related deaths worldwide. Although immunotherapy has taken center stage in mainstream oncology, it has shown limited clinical efficacy in CRC, generating an urgent need for discovery of new biomarkers and potential therapeutic targets. Galectin-1 (Gal-1), an endogenous glycan-binding protein, induces tolerogenic programs and contributes to tumor cell evasion of immune responses. Here, we investigated the relevance of Gal-1 in CRC and explored its modulatory activity within the CD8+ regulatory T cell (Treg) compartment. Mice lacking Gal-1 (Lgals1-/- ) developed a lower number of tumors and showed a decreased frequency of a particular population of CD8+CD122+PD-1+ Tregs in the azoxymethane-dextran sodium sulfate model of colitis-associated CRC. Moreover, silencing of tumor-derived Gal-1 in the syngeneic CT26 CRC model resulted in reduced number and attenuated immunosuppressive capacity of CD8+CD122+PD-1+ Tregs, leading to slower tumor growth. Moreover, stromal Gal-1 also influenced the fitness of CD8+ Tregs, highlighting the contribution of both tumor and stromal-derived Gal-1 to this immunoregulatory effect. Finally, bioinformatic analysis of a colorectal adenocarcinoma from The Cancer Genome Atlas dataset revealed a particular signature characterized by high CD8+ Treg score and elevated Gal-1 expression, which delineates poor prognosis in human CRC. Our findings identify CD8+CD122+PD-1+ Tregs as a target of the immunoregulatory activity of Gal-1, suggesting a potential immunotherapeutic strategy for the treatment of CRC.
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Adenocarcinoma/genética , Linfocitos T CD8-positivos/inmunología , Colitis/genética , Neoplasias Colorrectales/genética , Galectina 1/genética , Linfocitos T Reguladores/inmunología , Adenocarcinoma/inmunología , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Animales , Atlas como Asunto , Azoximetano/administración & dosificación , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Colitis/inducido químicamente , Colitis/inmunología , Colitis/mortalidad , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Biología Computacional , Sulfato de Dextran/administración & dosificación , Modelos Animales de Enfermedad , Galectina 1/deficiencia , Galectina 1/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/inmunología , Ratones , Ratones Noqueados , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal , Análisis de Supervivencia , Linfocitos T Reguladores/patología , Carga TumoralRESUMEN
Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for ß-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of ß-galactoside and ß-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable ß-N- and ß-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of ß-N- and ß-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.
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Galactósidos , Modelos Moleculares , Aglutinina de Mani , Galactósidos/química , Ligandos , Aglutinina de Mani/química , Unión ProteicaRESUMEN
Herein, we report the synthesis of calix[4]resorcinarene-based multivalent ligands bearing ß-S-GlcNAc and ß-S-AllNAc recognition elements. A clickable ß-S-AllNAc derivative was successfully prepared from a ß-thioalkynyl GlcNAc precursor, making use of a 2,3-oxazoline intermediate, easily formed by intramolecular displacement of a triflate group located at the 3-position by the 2-N-acetate group. By reaction of these alkynyl-functionalized derivatives with an octaazido-calix[4]resorcinarene macrocycle having undecyl chains, two octavalent glycoclusters exposing the epimeric N-acetylhexosamines were obtained. In addition, a related calix[4]resorcinarene-based glycocluster having methyl groups instead of undecyl chains and ß-S-GlcNAc residues was also synthesized. After an initial evaluation of the interaction of the undecyl-functionalized ß-S-GlcNAc octavalent derivative with Wheat Germ Agglutinin (WGA) by a turbidimetry experiment, the interaction of the three synthesized glycoclusters towards WGA was studied by Isothermal Titration Calorimetry. The results showed a favorable effect due to the presence of the undecyl chains in terms of affinity. Surprisingly, the ß-S-AllNAc octavalent compound showed the highest affinity among the evaluated glycoclusters, showing for the first time that WGA interacts with ß-AllNAc-bearing ligands. Molecular docking studies of ß-AllNAc with WGA in comparison with ß-GlcNAc contributed to the understanding of the atomic interactions responsible for this unexpected affinity.
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Aglutininas del Germen de TrigoRESUMEN
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.
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Metabolismo de los Hidratos de Carbono , Galectinas/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Galectinas/química , Humanos , Ligandos , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12-/-) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.
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Adipocitos/metabolismo , Células Endoteliales/metabolismo , Galectinas/metabolismo , Neovascularización Patológica/metabolismo , Tejido Adiposo/metabolismo , Animales , Fenómenos Fisiológicos Celulares/fisiología , Resistencia a la Insulina/fisiología , Gotas Lipídicas/metabolismo , Lipólisis/fisiología , Ratones Noqueados , Polisacáridos/metabolismoRESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas Fetales/metabolismo , Galectinas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Antígenos CD/genética , Adhesión Celular/genética , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular Tumoral , Femenino , Proteínas Fetales/genética , Galectinas/genética , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Proteínas de Neoplasias/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Galectins are a family of carbohydrate-recognizing proteins that by interacting with specific glycoepitopes can mediate important biological processes, including immune cell homeostasis and activation of tolerogenic circuits. Among the different members of this family, Galectin 1 and 3 have shown pro-tumorigenic effects, being overexpressed in numerous neoplasic diseases, proving to be relevant in tumor immune escape, tumor progression and resistance to drug-induced apoptosis. Thus, generation of specific glycosides that could inhibit their pro-tumorigenic ability by blocking their carbohydrate recognition domain is one of the current major challenges in the field. Considering that galectin-ligand binding strength is closely related to the ligand structure, analysis of this relationship provides valuable information for rational design of high-affinity ligands that could work as effective galectin inhibitors. Taking profit of the ability of glycosidases to catalyze transglycosylation reactions we achieved the enzymatic synthesis of ß-d-Galp-(1â¯ââ¯6)-ß-d-Galp-(1â¯ââ¯4)-d-Glcp(2), a mixture of ß-d-Galp-(1â¯ââ¯6)-ß-d-Glcp-(1â¯ââ¯4)-d-Glcp(5) and ß-d-Galp-(1â¯ââ¯3)-ß-d-Glcp-(1â¯ââ¯4)-d-Glcp(6), and finally benzyl ß-d-galactopyranoside (9), with reaction yields between 16 and 27%. All the galactosides were purified, and characterized using 1H and 13C nuclear magnetic resonance spectroscopy. Docking results performed between the synthesized compounds and human Galectin 1 (hGal-1) and human Galectin 3 (hGal-3) showed that the replacement of a glucose moiety linked to the terminal galactose with a galactose moiety, decreases the affinity for these galectins. Moreover, regarding the interglycosidic bond the most favorable ß-Gal linkage seems to be ß(1â¯ââ¯4) followed by ß(1â¯ââ¯3) and ß(1â¯ââ¯6) for hGal-1, and ß(1â¯ââ¯4) followed by ß(1â¯ââ¯6) and ß(1â¯ââ¯3) for hGal-3. These results were in accordance with the IC50 values obtained with in vitro solid phase inhibition assays. Therefore, docking results obtained in this work proved to be a very good approximation for predicting binding affinity of novel galactosides.