RESUMEN
Heparan sulfate glycosaminoglycans (HS GAGs) attached to proteoglycans harbor high affinity binding sites for various growth factors (GFs) and direct their organization and activity across the cell-matrix interface. Here, we describe a mild and efficient method for generating HS-protein conjugates. The two-step process utilizes a "copper-free click" coupling between differentially sulfated heparinoids primed at their reducing end with an azide handle and a bovine serum albumin protein modified with complementary cyclooctyne functionality. When adsorbed on tissue culture substrates, the glycoconjugates served as extracellular matrix proteoglycan models with the ability to sequester FGF2 and influence mesenchymal stem cell proliferation based on the structure of their HS GAG component.
Asunto(s)
Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/química , Heparinoides/química , Células Madre/metabolismo , Animales , Glicosaminoglicanos/químicaRESUMEN
In the mucosal epithelium, the cellular glycocalyx can project tens to hundreds of nanometers into the extracellular space, erecting a physical barrier that provides protective functions, mediates the exchange of nutrients and regulates cellular interactions. Little is understood about how the physical properties of the mucosal glycocalyx influence molecular recognition at the cellular boundary. Here, we report the synthesis of PEG-based glycopolymers with tunable glycan composition, which approximate the extended architecture of mucin glycoproteins, and tether them to the plasma membranes of red blood cells (RBC) to construct an artificial mucin brush-like glycocalyx. We evaluated the association of two lectins, ConA and SNA, with their endogenous glycan ligands on the surface of the remodelled cells. The extended glycocalyx provided protection against agglutination of RBCs by both lectins; however, the rate and magnitude of ConA binding were attenuated to a greater degree in the presence of the glycopolymer spectators compared to those measured for SNA. The different sensitivity of ConA and SNA to glycocalyx crowding likely arises from the distinct presentation of their mannoside and sialoside receptors, respectively, within the native RBC glycocalyx.
Asunto(s)
Materiales Biomiméticos/metabolismo , Eritrocitos/metabolismo , Glicocálix/metabolismo , Hemaglutinación , Polietilenglicoles/metabolismo , Materiales Biomiméticos/química , Concanavalina A/metabolismo , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Eritrocitos/citología , Glicocálix/química , Glicoconjugados/química , Glicoconjugados/metabolismo , Humanos , Mucinas/química , Mucinas/metabolismo , Lectinas de Plantas/metabolismo , Polietilenglicoles/química , Polímeros/química , Polímeros/metabolismo , Proteínas Inactivadoras de Ribosomas/metabolismo , Sambucus nigra/metabolismoRESUMEN
Despite the development of promising direct oral anticoagulants, which are all orthosteric inhibitors, a sizable number of patients suffer from bleeding complications. We have hypothesized that allosterism based on the heparin-binding exosites presents a major opportunity to induce sub-maximal inhibition of coagulation proteases, thereby avoiding/reducing bleeding risk. We present the design of a group of sulfated benzofuran dimers that display heparin-binding site-dependent partial allosteric inhibition of thrombin against fibrinogen (ΔYâ¯=â¯55-75%), the first time that a small molecule (MWâ¯â¯<â¯800) has been found to thwart macromolecular cleavage by a monomeric protease in a controlled manner. The work leads to the promising concept that it should be possible to develop allosteric inhibitors that reduce clotting, but do not completely eliminate it, thereby avoiding major bleeding complications that beset anticoagulants today.
Asunto(s)
Benzofuranos/farmacología , Inhibidores de Serina Proteinasa/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfatos/farmacología , Trombina/antagonistas & inhibidores , Benzofuranos/síntesis química , Benzofuranos/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Sulfatos/química , Trombina/metabolismoRESUMEN
This article describes a protocol for remodeling cells with synthetic glycoprotein and glycolipid mimetics that are functionalized with lipid anchors, allowing for cell surface display of specific glycan structures in predefined nanoscale arrangements. The complex chemical heterogeneity of glycans found on the cell surface or the glycocalyx renders analysis of the individual contributions of glycans difficult. This technique allows for the precise study of individual glycans at different regions of the glycocalyx, and may be useful for interrogating glycan interactions in infection or immunity or in stem cell differentiation. CHO-Lec2 cells are prepared as adherent monolayers and, after reaching confluence, are incubated with the glycomaterials. Synthetic glycopolymers bearing α-2,3-sialyllactose glycans are used to decorate cellular surfaces in the form of 3D multivalent ligands projecting away from the cell surface, while α-2,6-sialyllactose glycolipid conjugates are used to anchor glycans in dynamic 2D arrays proximal to the cell membrane. Following washing, mimetic incorporation and glycan display can be analyzed using lectins with specificity for α-2,3- or α-2,6-linked sialic acids. Flow cytometry data reveals that cell surface remodeling with either glycoconjugate mimetic occurs efficiently in a dose-dependent manner. Combinations of glycoconjugates can also be employed simultaneously to generate a mixed glycocalyx with tunable composition and organization. © 2018 by John Wiley & Sons, Inc.
Asunto(s)
Membrana Celular/metabolismo , Glicocálix/metabolismo , Polisacáridos/metabolismo , Animales , Células CHO , Membrana Celular/química , Cricetulus , Citometría de Flujo , Glicocálix/química , Estructura Molecular , Polisacáridos/química , Propiedades de SuperficieRESUMEN
We report a method for programming complexity into the glycocalyx of live cells. Via a combination of glycomaterial synthesis and membrane remodeling, we have engineered cells to display native-like, mixed sialoglycan populations, while confining the activity of each glycan into a specific nanoscale presentation.
Asunto(s)
Glicocálix/metabolismo , Nanoestructuras/química , Polisacáridos/química , Polisacáridos/metabolismo , Animales , Células CHO , Técnicas de Química Sintética , Cricetinae , CricetulusRESUMEN
Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80-100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding.
Asunto(s)
Cumarinas/farmacología , Inhibidores de Proteasas/farmacología , Sulfatos/farmacología , Trombina/antagonistas & inhibidores , Regulación Alostérica/efectos de los fármacos , Factores de Coagulación Sanguínea/metabolismo , Dominio Catalítico , Cumarinas/química , Humanos , Inhibidores de Proteasas/química , Conformación Proteica , Sulfatos/química , Trombina/química , Trombina/metabolismoRESUMEN
In nearly all cases of biological activity of sulfated GAGs, the sulfate group(s) are critical for interacting with target proteins. A growing paradigm is that appropriate small, sulfated, nonsaccharide GAG mimetics can be designed to either mimic or interfere with the biological functions of natural GAG sequences resulting in the discovery of either antagonist or agonist agents. A number of times these sulfated NSGMs can be computationally designed based on the parent GAG-protein interaction. The small sulfated NSGMs may possess considerable aromatic character so as to engineer hydrophobic, hydrogen-bonding, Coulombic or cation-pi forces in their interactions with target protein(s) resulting in higher specificity of action relative to parent GAGs. The sulfated NSGMs can be easily synthesized in one step from appropriate natural polyphenols through chemical sulfation under microwave-based conditions. We describe step-by-step procedures to perform microwave-based sulfation of several small polyphenol scaffolds so as to prepare homogenous NSGMs containing one to more than 10 sulfate groups per molecule in high yields.