RESUMEN
Heparan sulfate (HS), a sulfated linear carbohydrate that decorates the cell surface and extracellular matrix, is ubiquitously distributed throughout the animal kingdom and represents a key regulator of biological processes and a largely untapped reservoir of potential therapeutic targets. The temporal and spatial variations in the HS structure underpin the concept of "heparanome" and a complex network of HS binding proteins. However, despite its widespread biological roles, the determination of direct structure-to-function correlations is impaired by HS chemical heterogeneity. Attempts to correlate substitution patterns (mostly at the level of sulfation) with a given biological activity have been made. Nonetheless, these do not generally consider higher-level conformational effects at the carbohydrate level. Here, the use of NMR chemical shift analysis, NOEs, and spin-spin coupling constants sheds new light on how different sulfation patterns affect the polysaccharide backbone geometry. Furthermore, the substitution of native O-glycosidic linkages to hydrolytically more stable S-glycosidic forms leads to observable conformational changes in model saccharides, suggesting that alternative chemical spaces can be accessed and explored using such mimetics. Employing a series of systematically modified heparin oligosaccharides (as a proxy for HS) and chemically synthesized O- and S-glycoside analogues, the chemical space occupied by such compounds is explored and described.
RESUMEN
The linear anionic class of polysaccharides, glycosaminoglycans (GAGs), are critical throughout the animal kingdom for developmental processes and the maintenance of healthy tissues. They are also of interest as a means of influencing biochemical processes. One member of the GAG family, heparin, is exploited globally as a major anticoagulant pharmaceutical and there is a growing interest in the potential of other GAGs for diverse applications ranging from skin care to the treatment of neurodegenerative conditions, and from the treatment and prevention of microbial infection to biotechnology. To realize the potential of GAGs, however, it is necessary to develop effective tools that are able to exploit the chemical manipulations to which GAGs are susceptible. Here, the current knowledge concerning the chemical modification of GAGs, one of the principal approaches for the study of the structure-function relationships in these molecules, is reviewed. Some additional methods that were applied successfully to the analysis and/or processing of other carbohydrates, but which could be suitable in GAG chemistry, are also discussed.
Asunto(s)
Glicosaminoglicanos/química , Polisacáridos/química , Animales , Anticoagulantes/química , Heparina/química , Humanos , Relación Estructura-ActividadRESUMEN
The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/enzimología , Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Heparitina Sulfato/biosíntesis , Transporte Biológico/efectos de los fármacos , Brefeldino A/farmacología , Vesículas Cubiertas por Proteínas de Revestimiento/genética , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Retículo Endoplásmico/química , Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica , Aparato de Golgi/química , Aparato de Golgi/efectos de los fármacos , Heparina/farmacología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , Transfección , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The cytotoxic mode of action of four antimicrobial peptides (AMPs) (gomesin, tachyplesin, protegrin, and polyphemusin) against a HeLa cell tumor model is discussed. A study of cell death by AMP stimulation revealed some similarities, including annexin-V externalization, reduction of mitochondrial potential, insensitivity against inhibitors of cell death, and membrane permeabilization. Evaluation of signaling proteins and gene expression that control cell death revealed wide variation in the responses to AMPs. However, the ability to cross cell membranes emerged as an important characteristic of AMP-dependent cell death, where endocytosis mediated by dynamin is a common mechanism. Furthermore, the affinity between AMPs and glycosaminoglycans (GAGs) and GAG participation in the cytotoxicity of AMPs were verified. The results show that, despite their primary and secondary structure homology, these peptides present different modes of action, but endocytosis and GAG participation are an important and common mechanism of cytotoxicity for ß-hairpin peptides.
Asunto(s)
Péptidos Antimicrobianos , Glicosaminoglicanos , Humanos , Muerte Celular , Endocitosis , Células HeLaRESUMEN
3-O-Sulfotransferase enzyme (sHS) from Litopenaeus vannamei was cloned and its substrate specificity was investigated against a number of GAG structures, including modified heparin polysaccharides and model oligosaccharides. For the heparin polysaccharides, derived from porcine intestinal mucosa heparin, sulfate groups were incorporated into glucosamine residues containing both N-sulfated and N-acetylated substitution within the regions of the predominant repeating disaccharide, either I-ANS or I-ANAc. However, the resulting polysaccharides did not stabilize antithrombin, which is correlated with anticoagulant activity. It was also shown that the enzyme was able to sulfate disaccharides, I2S-ANS and G-ANAc. The results further illustrate that 3-O-sulfation can be induced outside of the classical heparin-binding pentasaccharide sequence, show that 3-O-sulfation of glucosamine is not a sufficient condition for antithrombin stabilization and suggest that the use of this enzyme during HS biosynthesis may not occur as the final enzymatic step.
Asunto(s)
Heparitina Sulfato/biosíntesis , Sulfotransferasas/metabolismo , Animales , Estabilidad de Enzimas , Heparitina Sulfato/química , Modelos Moleculares , Penaeidae/enzimología , TemperaturaRESUMEN
Heparin, the widely used pharmaceutical anticoagulant, has been in clinical use for well over half a century. Its introduction reduced clotting risks substantially and subsequent developments, including the introduction of low-molecular-weight heparin, made possible many major surgical interventions that today make heparin an indispensable drug. There has been a recent burgeoning of interest in heparin and related glycosaminoglycan (GAG) polysaccharides, such as chondroitin sulfates, heparan sulfate, and hyaluronate, as potential agents in various applications. This ability arises mainly from the ability of GAGs to interact with, and alter the activity of, a wide range of proteins. Here, we review new developments (since 2010) in the application of heparin and related GAGs across diverse fields ranging from thrombosis and neurodegenerative disorders to microbiology and biotechnology.
Asunto(s)
Anticoagulantes/química , Anticoagulantes/farmacología , Glicosaminoglicanos/química , Glicosaminoglicanos/farmacología , Anticoagulantes/uso terapéutico , Biotecnología/métodos , Glicosaminoglicanos/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Enfermedades Neurodegenerativas/tratamiento farmacológico , Trombosis/tratamiento farmacológicoRESUMEN
Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of 19F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing 19F NMR spectroscopy is followed. Furthermore, the ability of 19F labelled GAGs to be imaged using CARS microscopy is demonstrated. 19F labelled GAGs enable both 19F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals.
Asunto(s)
Imagen por Resonancia Magnética con Fluor-19/métodos , Flúor/química , Glicosaminoglicanos/química , Sondas Moleculares/química , Coloración y Etiquetado/métodos , Acetilación , Antitrombinas/química , Glicosaminoglicanos/análisis , Halogenación , Espectroscopía de Resonancia Magnética/métodos , Sondas Moleculares/análisis , Unión Proteica , Soluciones , Espectrometría Raman/métodosRESUMEN
Heparan sulfate (HS) polysaccharides are ubiquitous components of the cell surface and extracellular matrix of all multicellular animals, whereas heparin is present within mast cells and can be viewed as a more sulfated, tissue-specific, HS variant. HS and heparin regulate biological processes through interactions with a large repertoire of proteins. Owing to these interactions and diverse effects observed during in vitro, ex vivo and in vivo experiments, manifold biological/pharmacological activities have been attributed to them. The properties that have been thought to bestow protein binding and biological activity upon HS and heparin vary from high levels of sequence specificity to a dependence on charge. In contrast to these opposing opinions, we will argue that the evidence supports both a level of redundancy and a degree of selectivity in the structure-activity relationship. The relationship between this apparent redundancy, the multi-dentate nature of heparin and HS polysaccharide chains, their involvement in protein networks and the multiple binding sites on proteins, each possessing different properties, will also be considered. Finally, the role of cations in modulating HS/heparin activity will be reviewed and some of the implications for structure-activity relationships and regulation will be discussed.
Asunto(s)
Heparina , Heparitina Sulfato , Mastocitos , Proteínas , Animales , Sitios de Unión , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Mastocitos/química , Mastocitos/metabolismo , Proteínas/química , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
UNLABELLED: Heparan sulfate endosulfatase-1 and -2 (SULF1 and SULF2) are two important extracellular 6-O-endosulfatases that remove 6-O sulfate groups of N-glucosamine along heparan sulfate (HS) proteoglycan chains often found in the extracellular matrix. The HS sulfation pattern influences signaling events at the cell surface, which are critical for interactions with growth factors and their receptors. SULFs are overexpressed in several types of human tumors, but their role in cancer is still unclear because their molecular mechanism has not been fully explored and understood. To further investigate the functions of these sulfatases in tumorigenesis, stable overexpression models of these genes were generated in the colorectal cancer cells, Caco-2 and HCT-116. Importantly, mimicking overexpression of these sulfatases resulted in increased viability and proliferation, and augmented cell migration. These effects were reverted by shRNA-mediated knockdown of SULF1 or SULF2 and by the addition of unfractionated heparin. Detailed structural analysis of HS from cells overexpressing SULFs showed reduction in the trisulfated disaccharide UA(2S)-GlcNS(6S) and corresponding increase in UA(2S)-GlcNS disaccharide, as well as an unexpected rise in less common disaccharides containing GlcNAc(6S) residues. Moreover, cancer cells transfected with SULFs demonstrated increased Wnt signaling. In summary, SULF1 or SULF2 overexpression contributes to colorectal cancer cell proliferation, migration, and invasion. IMPLICATIONS: This study reveals that sulfatases have oncogenic effects in colon cancer cells, suggesting an important role for these enzymes in cancer progression.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Sulfotransferasas/metabolismo , Células CACO-2 , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Neoplasias Colorrectales/genética , Células HCT116 , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Sulfatasas , Sulfotransferasas/genética , Vía de Señalización WntRESUMEN
The structural characterization and the anticoagulant potential of a novel heparin/heparan sulfate-like compound from the heads of Litopenaeus vannamei shrimp are described. While it is distinct from either heparin or heparan sulfate, enzymatic depolymerization and nuclear magnetic resonance spectroscopy analyses revealed that this molecule does share some structural features with heparin, such as the high degree of N- and 6-O-sulfation and minor N-acetylation, and with heparan sulfate, in the glucuronic acid content. Its ability to stabilize human antithrombin explains its significant anticoagulant activity in aPTT and Factor-Xa inhibition assays. Interestingly, in contrast to mammalian heparin, the shrimp compound displayed negligible hemorrhagic effect. Together, these findings have particular interest since they reveal a novel molecule with significant anti-Xa activity coupled with low bleeding effects which make the shrimp heparin/HS-like compound a potential alternative for mammalian heparin.
Asunto(s)
Anticoagulantes/química , Hemorragia/prevención & control , Heparina/química , Heparitina Sulfato/química , Penaeidae/química , Acetilación , Animales , Anticoagulantes/aislamiento & purificación , Anticoagulantes/farmacología , Antitrombinas/antagonistas & inhibidores , Antitrombinas/química , Antitrombinas/aislamiento & purificación , Bovinos , Cromatografía por Intercambio Iónico , Factor Xa/química , Inhibidores del Factor Xa , Ácido Glucurónico/química , Cabeza , Heparina/aislamiento & purificación , Heparina/farmacología , Heparitina Sulfato/aislamiento & purificación , Heparitina Sulfato/farmacología , Humanos , Intestinos/química , Páncreas/química , Tiempo de Tromboplastina Parcial , Ratas , Porcinos , Cola (estructura animal)/irrigación sanguínea , Cola (estructura animal)/efectos de los fármacosRESUMEN
Recently, oversulfated chondroitin sulfate (OSCS) was identified in contaminated heparin preparations, which were linked to several adverse clinical events and deaths. Orthogonal analytical techniques, namely nuclear magnetic resonance (NMR) and capillary electrophoresis (CE), have since been applied by several authors for the evaluation of heparin purity and safety. NMR identification and quantification of residual solvents and non-volatile low molecular contaminants with USP acceptance levels of toxicity was achieved 40-fold faster than the traditional GC-headspace technique, which takes ~120 min against ~3 min to obtain a (1)H NMR spectrum with a signal/noise ratio of at least 1000/1. The procedure allowed detection of Class 1 residual solvents at 2 ppm and quantification was possible above 10 ppm. 2D NMR techniques (edited-HSQC (1)H/(13)C) permitted visualization of otherwise masked EDTA signals at 3.68/59.7 ppm and 3.34/53.5 ppm, which may be overlapping mononuclear heparin signals, or those of ethanol and methanol. Detailed NMR and ESI-MS/MS studies revealed a hitherto unknown contaminant, tris(2-n-butoxyethyl) phosphate (TBEP), which has potential health risks.
Asunto(s)
Heparina/química , Espectroscopía de Resonancia Magnética/métodos , Organofosfatos/análisis , Electroforesis Capilar/métodos , Solventes/químicaRESUMEN
The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities.
Asunto(s)
Heparina/normas , Espectroscopía de Resonancia Magnética , Análisis de Componente Principal , Espectrofotometría Ultravioleta , Contaminación de Medicamentos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Espectroscopía de Resonancia Magnética/métodos , Métodos , Estándares de Referencia , Espectrofotometría Ultravioleta/métodosRESUMEN
The oxidation-reduction (redox) of the environment characterizes the Bacteroides fragilis pathogenic potential. Previously, using 3D confocal laser scanning microscopy, the bacteria prepared from cultures grown under oxidizing conditions (Eh(7)ca. + 100 mV) were able to penetrate into Hela cell monolayers. In contrast, when grown under reducing conditions (Eh(7)ca. - 60 mV), there were no bacteria evident within Hela cells. The influence of the anaerobe/aerobe environmental flux during the process of the anaerobe infection could be significant. In B. fragilis peritonitis, this may depend on the occurrence of aerobiosis as opposed to anaerobiosis. To this end, three clinical B. fragilis strains, two infectious and one non-infectious, were grown under oxidizing and reducing conditions; then, the outer membrane protein expressions derived from these strains were assessed, following sarcosyl extraction and SDS-PAGE. The differences between the protein profiles from these strains when cultured under oxidizing and reducing conditions were found to be statistically significant for the two infectious strains, but not for the non-infectious strain. OMP profiles under aerobic conditions compared to anaerobic conditions exhibited products with a range of apparent molecular weights suggestive of unique participation in the interaction with the host cell.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/biosíntesis , Bacteroides fragilis/metabolismo , Medios de Cultivo/química , Regulación de la Expresión Génica , Aerobiosis , Anaerobiosis , Humanos , Oxidación-ReducciónRESUMEN
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100 mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.
Asunto(s)
Adhesión Bacteriana , Bacteroides fragilis/crecimiento & desarrollo , Proteínas Portadoras/metabolismo , Laminina/metabolismo , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/metabolismo , Bacteroides fragilis/ultraestructura , Matriz Extracelular/metabolismo , Immunoblotting , Microscopía Electrónica de Transmisión , Oxidación-Reducción , Polisacáridos Bacterianos/metabolismo , Factores de TiempoRESUMEN
The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.