RESUMO
A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.
Assuntos
Lectinas de Plantas/química , Lectinas de Plantas/genética , Fármacos Anti-HIV/química , Sequência de Carboidratos , Engenharia Genética , Mitógenos/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Musa/químicaRESUMO
Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15N and 13C HSQC NMR spectroscopy to gain structural insight into the CuSO4-mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.
Assuntos
Cisteína , Galectina 1 , Oxirredução , Galectina 1/metabolismo , Galectina 1/química , Galectina 1/genética , Humanos , Cisteína/metabolismo , Cisteína/química , Dissulfetos/metabolismo , Dissulfetos/química , Dobramento de Proteína , Desdobramento de Proteína , Modelos Moleculares , Lactose/metabolismo , Lactose/química , Mutagênese Sítio-DirigidaRESUMO
Galectin-3 (Gal-3) is a ß-galactosidase-binding protein involved in various biological processes, including neuronal growth and adhesion. The pairing of Gal-3 with ganglioside GM1's pentasaccharide chain at the outer leaflet of the plasma membrane, which triggers downstream cell-signaling cascades, seems to be involved in these processes. A crucial feature of Gal-3 is its ability to form oligomers and supramolecular assemblies that connect various carbohydrate-decorated molecules. Although we know the atomistic structure of Gal-3 bound to small carbohydrate ligands, it remains unclear how Gal-3 binds GM1 in a membrane. Furthermore, the influence of this interaction on Gal-3's structure and oligomeric assembly has to be elucidated. In this study, we used X-ray reflectivity (XR) from a model membrane to determine the structure and surface coverage of Gal-3 bound to a membrane containing GM1. We observed that the carbohydrate recognition domain interacts with GM1's pentasaccharide, while the N-terminal domain is pointed away from the membrane, likely to facilitate protein-protein interactions. In a membrane containing 20 mol % GM1, Gal-3 covered â¼50% of the membrane surface with one Gal-3 molecule bound per 2130 Å2. We used molecular dynamics simulations and Voronoi tessellation algorithms to build an atomistic model of membrane-bound Gal-3, which is supported by the XR results. Overall, this work provides structural information describing how Gal-3 can bind GM1's pentasaccharide chain, a prerequisite for triggering regulatory processes in neuronal growth and adhesion.
Assuntos
Gangliosídeo G(M1) , Galectina 3 , Gangliosídeo G(M1)/química , Galectina 3/metabolismo , Gangliosídeos , Membrana Celular/metabolismo , Simulação de Dinâmica MolecularRESUMO
The multifunctionality of galectins helps regulate a broad range of fundamental cellular processes via cis-binding and trans-bridging activities and has gained widespread attention with respect to the importance of the natural specificity/selectivity of this lectin family to its glycoconjugate receptors. Combining galectin (Gal)-1, -3, -4, and -9 variant test panels, achieved via rational protein engineering, and a synthetic α-dystroglycan (DG) O-Mannosylated core M1 glycopeptide library, a detailed comparative analysis was performed, utilizing microarray experiments to delineate the design-functionality relationships within this lectin family. Enhancement of prototype Gal-1 and chimera-type Gal-3 cis-binding toward the prepared ligands is possible by transforming these lectins into tandem-repeat type and prototypes, respectively. Furthermore, Gal-1 variants demonstrated improved trans-bridging capabilities between core M1 α-DG glycopeptides and laminins in microarray, suggesting the possible translational applications of these galectin variants in the treatment of some forms of α-dystroglycanopathy.
Assuntos
Distroglicanas , Galectinas , Galectinas/metabolismo , Glicoconjugados/metabolismo , GlicopeptídeosRESUMO
A code is defined by the nature of the symbols, which are used to generate information-storing combinations (e. g. oligo- and polymers). Like nucleic acids and proteins, oligo- and polysaccharides are ubiquitous, and they are a biochemical platform for establishing molecular messages. Of note, the letters of the sugar code system (third alphabet of life) excel in coding capacity by making an unsurpassed versatility for isomer (code word) formation possible by variability in anomery and linkage position of the glycosidic bond, ring size and branching. The enzymatic machinery for glycan biosynthesis (writers) realizes this enormous potential for building a large vocabulary. It includes possibilities for dynamic editing/erasing as known from nucleic acids and proteins. Matching the glycome diversity, a large panel of sugar receptors (lectins) has developed based on more than a dozen folds. Lectins 'read' the glycan-encoded information. Hydrogen/coordination bonding and ionic pairing together with stacking and C-H/π-interactions as well as modes of spatial glycan presentation underlie the selectivity and specificity of glycan-lectin recognition. Modular design of lectins together with glycan display and the nature of the cognate glycoconjugate account for the large number of post-binding events. They give an entry to the glycan vocabulary its functional, often context-dependent meaning(s), hereby building the dictionary of the sugar code.
Assuntos
Ácidos Nucleicos , Açúcares , Carboidratos/química , Lectinas/metabolismo , Polissacarídeos/químicaRESUMO
Galectin-4 (Gal-4) is a member of the galectin family, which have been identified as galactose-binding proteins. Gal-4 possesses two tandem repeat carbohydrate recognition domains and acts as a cross-linking bridge in sulfatide-dependent glycoprotein routing. We herein document its upregulation in osteoarthritis (OA) in correlation with the extent of cartilage degradation in vivo. Primary human OA chondrocytes in vitro respond to carbohydrate-inhibitable Gal-4 binding with the upregulation of pro-degradative/-inflammatory proteins such as interleukin-1ß (IL-1ß) and matrix metalloproteinase-13 (MMP-13), as documented by RT-qPCR-based mRNA profiling and transcriptome data processing. Activation of p65 by phosphorylation of Ser536 within the NF-κB pathway and the effect of three p65 inhibitors on Gal-4 activity support downstream involvement of such signaling. In 3D (pellet) cultures, Gal-4 presence causes morphological and biochemical signs of degradation. Taken together, our findings strongly support the concept of galectins acting as a network in OA pathogenesis and suggest that blocking their activity in disease progression may become clinically relevant in the future.
Assuntos
Condrócitos/química , Galectina 4/genética , Osteoartrite/genética , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Galectina 4/metabolismo , Humanos , Osteoartrite/metabolismo , Osteoartrite/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Chemokines and galectins are simultaneously upregulated and mediate leukocyte recruitment during inflammation. Until now, these effector molecules have been considered to function independently. Here, we tested the hypothesis that they form molecular hybrids. By systematically screening chemokines for their ability to bind galectin-1 and galectin-3, we identified several interacting pairs, such as CXCL12 and galectin-3. Based on NMR and MD studies of the CXCL12/galectin-3 heterodimer, we identified contact sites between CXCL12 ß-strand 1 and Gal-3 F-face residues. Mutagenesis of galectin-3 residues involved in heterodimer formation resulted in reduced binding to CXCL12, enabling testing of functional activity comparatively. Galectin-3, but not its mutants, inhibited CXCL12-induced chemotaxis of leukocytes and their recruitment into the mouse peritoneum. Moreover, galectin-3 attenuated CXCL12-stimulated signaling via its receptor CXCR4 in a ternary complex with the chemokine and receptor, consistent with our structural model. This first report of heterodimerization between chemokines and galectins reveals a new type of interaction between inflammatory mediators that can underlie a novel immunoregulatory mechanism in inflammation. Thus, further exploration of the chemokine/galectin interactome is warranted.
Assuntos
Galectinas , Inflamação , Animais , Quimiotaxia , Galectinas/genética , Galectinas/metabolismo , Inflamação/genética , Leucócitos/metabolismo , Camundongos , Transdução de SinaisRESUMO
Pairing glycans with tissue lectins controls multiple effector pathways in (patho)physiology. A clinically relevant example is the prodegradative activity of galectins-1 and -3 (Gal-1 and -3) in the progression of osteoarthritis (OA) via matrix metalloproteinases (MMPs), especially MMP-13. The design of heterobifunctional inhibitors that can block galectin binding and MMPs both directly and by preventing their galectin-dependent induction selectively offers a perspective to dissect the roles of lectins and proteolytic enzymes. We describe the synthesis of such a reagent with a bivalent galectin ligand connected to an MMP inhibitor and of two tetravalent glycoclusters with a subtle change in headgroup presentation for further elucidation of influence on ligand binding. Testing was performed on clinical material with mixtures of galectins as occurring in vivo, using sections of fixed tissue. Two-colour fluorescence microscopy monitored binding to the cellular glycome after optimization of experimental parameters. In the presence of the inhibitor, galectin binding to OA specimens was significantly reduced. These results open the perspective to examine the inhibitory capacity of custom-made ditopic compounds on binding of lectins in mixtures using sections of clinical material with known impact of galectins and MMPs on disease progression.
RESUMO
Trafficking of leukocytes and their local activity profile are of pivotal importance for many (patho)physiological processes. Fittingly, microenvironments are complex by nature, with multiple mediators originating from diverse cell types and playing roles in an intimately regulated manner. To dissect aspects of this complexity, effectors are initially identified and structurally characterized, thus prompting familial classification and establishing foci of research activity. In this regard, chemokines present themselves as role models to illustrate the diversification and fine-tuning of inflammatory processes. This in turn discloses the interplay among chemokines, their cell receptors and cognate glycosaminoglycans, as well as their capacity to engage in new molecular interactions that form hetero-oligomers between themselves and other classes of effector molecules. The growing realization of versatility of adhesion/growth-regulatory galectins that bind to glycans and proteins and their presence at sites of inflammation led to testing the hypothesis that chemokines and galectins can interact with each other by protein-protein interactions. In this review, we present some background on chemokines and galectins, as well as experimental validation of this chemokine-galectin heterodimer concept exemplified with CXCL12 and galectin-3 as proof-of-principle, as well as sketch out some emerging perspectives in this arena.
Assuntos
Quimiocinas/metabolismo , Galectinas/metabolismo , Inflamação/fisiopatologia , Animais , HumanosRESUMO
Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.
Assuntos
Galectina 1/química , Galectina 3/química , Glicoconjugados/química , Polissacarídeos/química , Amino Açúcares/química , Amino Açúcares/metabolismo , Sítios de Ligação , Proteínas Sanguíneas , Adesão Celular/genética , Proliferação de Células/genética , Galectina 1/genética , Galectina 3/genética , Galectinas , Humanos , Lactose/química , Ligantes , Nanopartículas/química , Polissacarídeos/genéticaRESUMO
Glycan-lectin recognition is vital to processes that impact human health, including viral infections. Proceeding from crystallographical evidence of case studies on adeno-, corona-, and rotaviral spike proteins, the relationship of these adhesins to mammalian galectins was examined by computational similarity assessments. Intrafamily diversity among human galectins was in the range of that to these viral surface proteins. Our findings are offered to inspire the consideration of lectin-based approaches to thwart infection by present and future viral threats, also mentioning possible implications for vaccine development.
Assuntos
Galectinas , Polissacarídeos , Glicoproteína da Espícula de Coronavírus , Animais , Coronaviridae , HumanosRESUMO
Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.
Assuntos
Lectinas Tipo C/química , Mucina-1/química , Sequência de Aminoácidos , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/metabolismo , Calorimetria/métodos , Epitopos/metabolismo , Galactose , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Mucina-1/metabolismo , Ligação ProteicaRESUMO
TIM-3 has been considered as a target in cancer immunotherapy. In T cells, inhibitory as well as activating functions have been ascribed to this molecule. Its role may therefore depend on the state of T cells and on the presence of interaction partners capable to perform functional pairing. Carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) has been proposed to bind TIM-3 and to regulate its function. Using a T cell reporter platform we confirmed CEACAM1-mediated inhibition, but CEACAM1 did not functionally engage TIM-3. TIM-3 and CEACAM1 coexpression was limited to a small subset of activated T cells. Moreover, results obtained in extensive binding studies were not in support of an interaction between TIM-3 and CEACAM1. Cytoplasmic sequences derived from TIM-3 induced inhibitory signaling in our human T cell reporter system. Our results indicate that TIM-3 functions are independent of CEACAM1 and that this receptor has the capability to promote inhibitory signaling pathways in human T cells.
Assuntos
Antígenos CD/fisiologia , Moléculas de Adesão Celular/fisiologia , Receptor Celular 2 do Vírus da Hepatite A/fisiologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Receptor Celular 2 do Vírus da Hepatite A/análise , Humanos , Células Jurkat , Ativação Linfocitária , Transdução de Sinais/fisiologia , Linfócitos T/imunologiaRESUMO
Wild-type lectins have distinct types of modular design. As a step to explain the physiological importance of their special status, hypothesis-driven protein engineering is used to generate variants. Concerning adhesion/growth-regulatory galectins, non-covalently associated homodimers are commonly encountered in vertebrates. The homodimeric galectin-7 (Gal-7) is a multifunctional context-dependent modulator. Since the possibility of conversion from the homodimer to hybrids with other galectin domains, i.e. from Gal-1 and Gal-3, has recently been discovered, we designed Gal-7-based constructs, i.e. stable (covalently linked) homo- and heterodimers. They were produced and purified by affinity chromatography, and the sugar-binding activity of each lectin unit proven by calorimetry. Inspection of profiles of binding of labeled galectins to an array-like platform with various cell types, i.e. sections of murine epididymis and jejunum, and impact on neuroblastoma cell proliferation revealed no major difference between natural and artificial (stable) homodimers. When analyzing heterodimers, acquisition of altered properties was seen. Remarkably, binding properties and activity as effector can depend on the order of arrangement of lectin domains (from N- to C-termini) and on the linker length. After dissociation of the homodimer, the Gal-7 domain can build new functionally active hybrids with other partners. This study provides a clear direction for research on defining the full range of Gal-7 functionality and offers the perspective of testing applications for engineered heterodimers.
Assuntos
Galectinas/metabolismo , Engenharia de Proteínas , Linhagem Celular Tumoral , Galectinas/análise , Galectinas/isolamento & purificação , Humanos , Espectrometria de MassasRESUMO
Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance. Their study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as S- and Se-glycosides. The latter enable extensive analyses by specific 77 Se NMR spectroscopy, but still remain underexplored. By using the example of selenodigalactoside (SeDG) and the human galectin-1 and -3, we have evaluated diverse 77 Se NMR detection methods and propose selective 1 H,77 Se heteronuclear Hartmann-Hahn transfer for efficient use in competitive NMR screening against a selenoglycoside spy ligand. By fluorescence anisotropy, circular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDG binding by galectins are similar to thiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact. ITC data in D2 O versus H2 O are similar for TDG and LacNAc binding by both galectins, but a solvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAc dimers with extended chain length.
Assuntos
Galectinas , Ressonância Magnética Nuclear Biomolecular , Polissacarídeos , Sítios de Ligação , Óxido de Deutério , Galectinas/metabolismo , Humanos , Isótopos , Ligantes , Polissacarídeos/metabolismo , Ligação Proteica , Selênio , SolventesRESUMO
Glycans have been shown to function as versatile molecular signals in cells. This prompted us to look at their roles in endocytosis, endolysosomal system and autophagy. We start by introducing the cell biological aspects of these pathways, the concept of the sugar code, and provide an overview on the role of glycans in the targeting of lysosomal proteins and in lysosomal functions. Moreover, we review evidence on the regulation of endocytosis and autophagy by glycans. Finally, we discuss the emerging concept that cytosolic exposure of luminal glycans, and their detection by endogenous lectins, provides a mechanism for the surveillance of the integrity of the endolysosomal compartments, and serves their eventual repair or disposal.
Assuntos
Autofagia/fisiologia , Endocitose/fisiologia , Lisossomos/fisiologia , Polissacarídeos/metabolismo , Regulação da Expressão Gênica , Proteínas/genética , Proteínas/metabolismoRESUMO
Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease of the central nervous system with unknown etiology. Currently approved disease-modifying treatment modalities are immunomodulatory or immunosuppressive. While the applied drugs reduce the frequency and severity of the attacks, their efficacy to regenerate myelin membranes and to halt disease progression is limited. To achieve such therapeutic aims, understanding biological mechanisms of remyelination and identifying factors that interfere with remyelination in MS can give respective directions. Such a perspective is given by the emerging functional profile of galectins. They form a family of tissue lectins, which are potent effectors in processes as diverse as adhesion, apoptosis, immune mediator release or migration. This review focuses on endogenous and exogenous roles of galectins in glial cells such as oligodendrocytes, astrocytes and microglia in the context of de- and (re)myelination and its dysregulation in MS. Evidence is arising for a cooperation among family members so that timed expression and/or secretion of galectins-1, -3 and -4 result in modifying developmental myelination, (neuro)inflammatory processes, de- and remyelination. Dissecting the mechanisms that underlie the distinct activities of galectins and identifying galectins as target or tool to modulate remyelination have the potential to contribute to the development of novel therapeutic strategies for MS.
Assuntos
Galectinas/metabolismo , Esclerose Múltipla/metabolismo , Esclerose Múltipla/terapia , Remielinização , Animais , Autoanticorpos/sangue , Humanos , Modelos Biológicos , Esclerose Múltipla/sangue , Esclerose Múltipla/patologia , Bainha de Mielina/metabolismoRESUMO
Human galectin-7 (Gal-7; also termed p53-induced gene 1 product) is a multifunctional effector by productive pairing with distinct glycoconjugates and protein counter-receptors in the cytoplasm and nucleus, as well as on the cell surface. Its structural analysis by NMR spectroscopy detected doubling of a set of particular resonances, an indicator of Gal-7 existing in two conformational states in slow exchange on the chemical shift time scale. Structural positioning of this set of amino acids around the P4 residue and loss of this phenomenon in the bioactive P4L mutant indicated cis-trans isomerization at this site. Respective resonance assignments confirmed our proposal of two Gal-7 conformers. Mapping hydrogen bonds and considering van der Waals interactions in molecular dynamics simulations revealed a structural difference for the N-terminal peptide, with the trans-state being more exposed to solvent and more mobile than the cis-state. Affinity for lactose or glycan-inhibitable neuroblastoma cell surface contact formation was not affected, because both conformers associated with an overall increase in order parameters (S2). At low µM concentrations, homodimer dissociation is more favored for the cis-state of the protein than its trans-state. These findings give direction to mapping binding sites for protein counter-receptors of Gal-7, such as Bcl-2, JNK1, p53 or Smad3, and to run functional assays at low concentration to test the hypothesis that this isomerization process provides a (patho)physiologically important molecular switch for Gal-7.
Assuntos
Galectinas/química , Multimerização Proteica , Sítios de Ligação , Linhagem Celular Tumoral , Galectinas/genética , Humanos , Isomerismo , Espectroscopia de Ressonância MagnéticaRESUMO
Pancreatic ductal adenocarcinoma (PDA) remains one of the most lethal tumor types, with extremely low survival rates due to late diagnosis and resistance to standard therapies. A more comprehensive understanding of the complexity of PDA pathobiology, and especially of the role of the tumor microenvironment in disease progression, should pave the way for therapies to improve patient response rates. In this study, we identify galectin-1 (Gal1), a glycan-binding protein that is highly overexpressed in PDA stroma, as a major driver of pancreatic cancer progression. Genetic deletion of Gal1 in a Kras-driven mouse model of PDA (Ela-KrasG12Vp53-/- ) results in a significant increase in survival through mechanisms involving decreased stroma activation, attenuated vascularization, and enhanced T cell infiltration leading to diminished metastasis rates. In a human setting, human pancreatic stellate cells (HPSCs) promote cancer proliferation, migration, and invasion via Gal1-driven pathways. Moreover, in vivo orthotopic coinjection of pancreatic tumor cells with Gal1-depleted HPSCs leads to impaired tumor formation and metastasis in mice. Gene-expression analyses of pancreatic tumor cells exposed to Gal1 reveal modulation of multiple regulatory pathways involved in tumor progression. Thus, Gal1 hierarchically regulates different events implicated in PDA biology including tumor cell proliferation, invasion, angiogenesis, inflammation, and metastasis, highlighting the broad therapeutic potential of Gal1-specific inhibitors, either alone or in combination with other therapeutic modalities.
Assuntos
Carcinoma Ductal Pancreático/terapia , Galectina 1/fisiologia , Galectinas/fisiologia , Terapia de Alvo Molecular , Neoplasias Pancreáticas/terapia , Animais , Carcinoma Ductal Pancreático/irrigação sanguínea , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Divisão Celular/genética , Movimento Celular/genética , Meios de Cultivo Condicionados , Galectinas/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Xenoenxertos , Humanos , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Metástase Neoplásica , Neovascularização Patológica , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/transplante , Comunicação Parácrina , RNA Interferente Pequeno/genética , Células Estromais/metabolismo , Microambiente TumoralRESUMO
Precise translation of glycan-encoded information into cellular activity depends critically on highly specific functional pairing between glycans and their human lectin counter receptors. Sulfoglycolipids, such as sulfatides, are important glycolipid components of the biological membranes found in the nervous and immune systems. The optimal molecular and spatial design aspects of sulfated and nonsulfated glycans with high specificity for lectin-mediated bridging are unknown. To elucidate how different molecular and spatial aspects combine to ensure the high specificity of lectin-mediated bridging, a bottom-up toolbox is devised. To this end, negatively surface-charged glycodendrimersomes (GDSs), of different nanoscale dimensions, containing sulfo-lactose groups are self-assembled in buffer from a synthetic sulfatide mimic: Janus glycodendrimer (JGD) containing a 3'-O-sulfo-lactose headgroup. Also prepared for comparative analysis are GDSs with nonsulfated lactose, a common epitope of human membranes. These self-assembled GDSs are employed in aggregation assays with 15 galectins, comprising disease-related human galectins, and other natural and engineered variants from four families, having homodimeric, heterodimeric, and chimera architectures. There are pronounced differences in aggregation capacity between human homodimeric and heterodimeric galectins, and also with respect to their responsiveness to the charge of carbohydrate-derived ligand. Assays reveal strong differential impact of ligand surface charge and density, as well as lectin concentration and structure, on the extent of surface cross-linking. These findings demonstrate how synthetic JGD-headgroup tailoring teamed with protein engineering and network assays can help explain how molecular matchmaking operates in the cellular context of glycan and lectin complexity.