RESUMEN
The rapidly evolvable influenza A virus has caused pandemics linked to millions of deaths in the past century. Influenza A viruses are categorized by H (hemagglutinin; HA) and N (neuraminidase; NA) proteins expressed on the viral envelope surface. Analyses of past pandemics suggest that the HA gene segment comes from a nonhuman virus, which is then introduced into an immunologically naïve human population with potentially devastating consequences. As a prerequisite for infection, the nonhuman HA molecules of H1-H16 viruses must be able to bind to specific sialyl receptors on the host cell surface along the human respiratory tract. Thus, additional insight into the structures of host cell glycans and how different HAs interact with different glycans might provide new insight into the mechanisms underlying sustained infection and transmission in humans. In this work, we identified the sialyl N-glycans found in normal human alveoli and characterized the influenza viruses that preferentially bound to these different structures. We also determined the amino acid changes in HA that were linked to a switch of receptor-binding preference from nonhuman to pandemic, as well as pandemic to seasonal. Our data provide insight into why seasonal viruses are associated with reduced alveolar infection and damage and suggest new considerations for designing anti-HA vaccines and drugs. The results provide a better understanding of viral tropism and pathogenesis in humans that will be important for prediction and surveillance of zoonotic, pandemic, and epidemic influenza outbreaks. DATABASE: The novel hemagglutinin nucleotide sequences reported here were deposited in GISAID under the accession numbers of EPI685738 for A/Yamaguchi/20/2006(H1N1) and EPI685740 for A/Kitakyushu/10/2006(H1N1).
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Virus de la Influenza A/fisiología , Gripe Aviar/virología , Gripe Humana/virología , Infecciones por Orthomyxoviridae/virología , Polisacáridos/fisiología , Enfermedades de las Aves de Corral/virología , Alveolos Pulmonares/patología , Receptores Virales/química , Tropismo Viral/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Unión Competitiva , Secuencia de Carbohidratos , Brotes de Enfermedades , Perros , Patos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/genética , Gripe Aviar/epidemiología , Gripe Aviar/patología , Gripe Humana/epidemiología , Gripe Humana/patología , Células de Riñón Canino Madin Darby , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/patología , Pandemias , Polisacáridos/química , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/patología , Unión Proteica , Alveolos Pulmonares/química , Alveolos Pulmonares/virología , ARN Viral/genética , Estaciones del Año , Ácidos Siálicos/química , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/patología , Enfermedades de los Porcinos/virología , Replicación Viral , ZoonosisRESUMEN
Both mucin 1 (MUC1) and galectin-3 are known to be overexpressed in various malignant tumors and associated with a poor prognosis. It has been extensively reported that MUC1 is involved in potentiation of growth factor-dependent signal transduction. Because some carbohydrate moieties carried on MUC1 change to preferable ones for binding of galectin-3 in cancer cells, we speculated that MUC1-mediated signaling may occur through direct binding of galectin-3. Immunochemical studies showed that the distribution of galectin-3 coincided with that of MUC1 in various human tumor tissues but not in human nonmalignant tissues, and the level of galectin-3 retained on the surface of various cancer cells paralleled that of MUC1. Treatment of MUC1-expressing cells with galectin-3 induced phosphorylation of ERK1/2 and Akt following enhanced phosphorylation of MUC1 C-terminal domain, consistently promoting tumor cell malignancy. It is also noted that this enhanced phosphorylation occurred independently of EGF receptor-mediated signaling in both EGF receptor- and MUC1-expressing cells, and multivalency of galectin-3 was important for initiation of MUC1-mediated signaling. Expectedly, both silencing of endogenous galectin-3 and treatment with galectin-3 antagonists down-regulated cell proliferation of MUC1-expressing cells. These results suggest that the binding of galectin-3 to MUC1 plays a key role in MUC1-mediated signaling. Thus, constitutive activation of MUC1-mediated signaling in an autocrine/paracrine manner caused by ligation of galectin-3 promotes uncontrolled tumor cell malignancy. This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated signaling pathway.
Asunto(s)
Neoplasias del Colon/patología , Galectina 3/metabolismo , Sistema de Señalización de MAP Quinasas , Mucina-1/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proliferación Celular , Neoplasias del Colon/enzimología , Neoplasias del Colon/metabolismo , Células HCT116 , Humanos , Fosforilación , Unión Proteica , Transducción de SeñalRESUMEN
UNLABELLED: A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses. IMPORTANCE: Circulation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism.
Asunto(s)
Variación Genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H5N1 del Virus de la Influenza A/clasificación , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Mutación Missense , Adaptación Biológica , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Epiteliales/virología , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/virología , Análisis de SupervivenciaRESUMEN
A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5 kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528 bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manß1-4GlcNAcß1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.
Asunto(s)
Agaricales/química , Lectinas de Unión a Manosa/aislamiento & purificación , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Lectinas de Unión a Manosa/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We designed and synthesized three tetravalent sialo-glycoclusters that had different separations between the terminal sialic acids and the linking carboxy groups of the ethylene glycol bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetate scaffold to serve as ligands for the sialic acid-binding lectin Sambucus sieboldiana agglutinin (SSA). The interaction between each glycocluster and SSA was characterized by hemagglutination inhibition, quantitative precipitation, and double-diffusion assays. For the precipitation assays, the precipitin curves indicated that the ligands and SSA bound in either a 1:1 or a 1:2 ratio, i.e., stoichiometrically. The strong interactions of these sialo-glycoclusters with SSA could be ascribed to a combination of multivalency and spacer effects. We also assessed the nature of the ligand-SSA complexes by isothermal titration calorimetry and dynamic light scattering. The results of those experiments indicated that formation of intermolecular complexes occurred at less than stoichiometric ratios of ligand to SSA concentrations and that, as the concentrations of the ligands increased, larger cross-linked aggregates formed. Large aggregates that were present concurrently with visible precipitation and with a particle size centered at ~600 to 800 nm were identified by dynamic light scattering.
Asunto(s)
Acetatos/química , Acetatos/síntesis química , Lectinas de Plantas/análisis , Lectinas de Plantas/química , Sambucus/química , Ácidos Siálicos/química , Acetatos/análisis , Sitios de Unión , Estructura MolecularRESUMEN
A series of dansyl-labeled glycosides with di-, tetra-, and hexasaccharides carrying the terminal N-acetyllactosamine (LacNAc) sequence were synthesized as acceptor substrates for α2,6- and α2,3-sialyltransferases. As an alternative design, dansyl-labeled LacNAc glycoside carrying a long-spacer linked glycan was engineered by replacement of the LacNAc or lactose units with an alkyl chain. In addition, we designed a dansyl-labeled bi-antennary LacNAc glycoside as an N-linked oligosaccharide mimetic, such as asialo-α(1)-acid glycoprotein. The kinetic parameters for the transfer reaction of synthesized dansyl-labeled glycosides by sialyltransferases were determined by the fluorescent HPLC method. The catalytic efficiencies (V(max)/K(m)) of acceptor substrates carrying the terminal LacNAc sequence with various length glycans in the array for α2,6- and α2,3-sialyltransferases decreased in a glycan length-dependent manner. Furthermore, of the acceptor substrates tested, dansyl-labeled bi-antennary LacNAc glycoside displayed the most favorable K(m) value for α2,6- and α2,3-sialyltransferases.
Asunto(s)
Colorantes Fluorescentes/química , Glicósidos/química , Sialiltransferasas/metabolismo , Amino Azúcares , Compuestos de Dansilo , Glicósidos/metabolismo , Cinética , Oligosacáridos , Unión ProteicaRESUMEN
Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state. Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.
Asunto(s)
Antígenos CD/metabolismo , Carcinoma/inmunología , Células Dendríticas/inmunología , Lectinas/metabolismo , Monocitos/inmunología , Mucinas/metabolismo , Neoplasias/inmunología , Escape del Tumor , Línea Celular Tumoral , Humanos , Inmunomodulación , Mucinas/sangre , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS-PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galbeta1-->4GlcNAcbeta1-->4Manbeta1-->4GlcNAcbeta1-->4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats.
Asunto(s)
Agaricales/química , Lectinas/aislamiento & purificación , Lectinas de Plantas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Asialoglicoproteínas , Carbohidratos/química , Diarrea/inducido químicamente , Electroforesis en Gel de Poliacrilamida , Fetuínas , Glicoproteínas/química , Lectinas/química , Lectinas/toxicidad , Ratones , Micotoxinas/química , Micotoxinas/aislamiento & purificación , Micotoxinas/toxicidad , Lectinas de Plantas/química , Lectinas de Plantas/toxicidad , Ratas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , alfa-FetoproteínasRESUMEN
Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N'-diacetylchitobiose [(GlcNAc)(2)] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.
Asunto(s)
Acetilglucosamina/química , Amino Azúcares/química , Reactivos de Enlaces Cruzados/química , Disacáridos/química , Lectinas de Plantas/química , Aglutininas del Germen de Trigo/química , Animales , Reactivos de Enlaces Cruzados/farmacología , Diseño de Fármacos , Eritrocitos/efectos de los fármacos , Glicósidos/química , Pruebas de Inhibición de Hemaglutinación , Estructura Molecular , Conejos , Resonancia por Plasmón de SuperficieRESUMEN
A series of spacer-N-linked glycopolymers carrying long/short α2,3/6 sialylated glycan were designed as polymeric inhibitors of influenza virus. Lactose (Lac) and N-acetyllactosamine (LN: Galß1,4GlcNAc) were first converted to spacer-N-linked disaccharide glycosides, followed by consecutive enzymatic addition of GlcNAc and Gal residues to the glycosides. The resulting spacer-N-linked glycosides with di-, tetra-, and hexasaccharides carrying a Lac, LN, lacto-N-neotetraose (LNnT: Galß1,4GlcNAcß1,3Galß1,4Glc), and LNß1,3LNnT were coupled to the carboxy group of γ-polyglutamic acid (γ-PGA) and enzymatically converted to glycopolypeptides carrying α2,3/6 sialylated glycans. The interactions of a series of sialoglycopolypeptides with avian and human influenza virus strains were investigated using a hemagglutination inhibition assay. The avian virus A/Duck/HongKong/313/4/78 (H5N3) bound specifically, regardless of the structure of the asialo portion. In contrast, human virus A/Aichi/2/68 (H3N2) bound preferentially to long α2,6sialylated glycans with penta- or heptasaccharides in a glycan length-dependent manner. Furthermore, the Sambucus sieboldiana (SNA) lectin was also useful as a model of human virus hemagglutinin (HA) for understanding the carbohydrate binding properties, because the recognition motifs of the inner sugar in the receptor were very similar.
Asunto(s)
Alphainfluenzavirus/efectos de los fármacos , Gripe Aviar/tratamiento farmacológico , Gripe Humana/tratamiento farmacológico , Gripe Humana/prevención & control , Sialoglicoproteínas/farmacología , Animales , Diseño de Fármacos , Patos , Pruebas de Inhibición de Hemaglutinación , Humanos , Subtipo H3N2 del Virus de la Influenza A , Gripe Aviar/prevención & control , Sialoglicoproteínas/síntesis química , Sialoglicoproteínas/uso terapéuticoRESUMEN
A crude enzyme preparation from Penicillium multicolor efficiently produced mainly gentiotriose to gentiopentaose (d.p. 3-5) by transglycosylation using a high concentration of gentiobiose as the substrate. The resulting gentiotriose was examined in a gustatory sensation test using human volunteers, and was determined to have one-fifth of the bitterness of gentiobiose. The crude enzyme preparation was analyzed by chromatography to determine the enzyme responsible for formation of the gentiooligosaccharides. The transglycosylation was shown to take place in two stages by a combination of beta-glucosidase and beta-(1-->6)-glucanase. In the initial stage, which was the rate-limiting step in the overall process, beta-glucosidase produced mainly gentiotriose from gentiobiose. In the second step, beta-(1-->6)-glucanase acted on the resulting gentiotriose, which served as both donor and acceptor, to produce a series of gentiooligosaccharides (d.p. 4-9) by transglycosylation.
Asunto(s)
Oligosacáridos/química , Oligosacáridos/síntesis química , Penicillium/enzimología , beta-Glucosidasa/metabolismo , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Glicosilación , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
A lectin, PPL, was purified from the mushroom Pleurocybella porrigens. The results of SDS-PAGE, gel filtration, and MALDI-TOF-mass of PPL indicated that its molecular mass was 56 kDa, and it was composed of four 14 kDa subunits with no disulfide bonds. In hemagglutination inhibition assay, PPL exhibited the strongest binding specificity toward GalNAc among the mono- and oligo-saccharides tested. Among the glycoproteins, asialo-bovine submaxillary mucin (asialo-BSM) showed the strongest inhibitory effect. In surface plasmon resonance analysis, asialo-BSM, porcine stomach mucin (PSM), and BSM exhibited potent binding affinity. The complete amino acid sequence was determined by amino acid sequencing of intact and of enzyme-digested PPL. The cDNA of PPL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA of the protein consisted of 411 bp, encoding 137 amino acids. This is the first report of isolation of a lectin of the genus Pleurocybella.
Asunto(s)
Agaricales/genética , ADN Complementario/genética , Lectinas/genética , Lectinas/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Glicoproteínas/metabolismo , Glicoproteínas/farmacología , Hemaglutinación/efectos de los fármacos , Concentración de Iones de Hidrógeno , Proteínas Inmovilizadas/metabolismo , Cinética , Lectinas/química , Lectinas/metabolismo , Datos de Secuencia Molecular , Monosacáridos/farmacología , Oligosacáridos/farmacología , Estabilidad Proteica , TemperaturaRESUMEN
We designed a series of gamma-polyglutamic acid (gamma-PGA)-based glycopolypeptides carrying long/short alpha2,3/6 sialylated glycans to act inhibitors of the influenza virus. As an alternative design, sialoglycopolypeptides carrying long-spacer linked glycans were engineered by replacement of the N-acetyllactosamine (LN) unit by an alkyl chain. The structure-activity relationship of the resulting sialoglycopolypeptides with different glycans in the array has been investigated by in vitro and in vivo infection experiments. The avian viruses specifically bound to glycopolypeptides carrying a short sialoglycan with higher affinity than to a long glycan. In contrast, human viruses, preferentially bound not only to long alpha2,3/6 sialylated glycan with LN repeats in the receptors, but also to more spacer-linked glycan in which the inner sugar has been replaced by a nonsugar structural unit such as a pentylamido group. Taken together, our results indicate that a spaced tandem/triplet pentylamido repeat is a good mimetic of a tandem/triplet LN repeat. Our strategy provides a facile way to design strong polymeric inhibitors of infection by avian and human influenza viruses.
Asunto(s)
Antivirales/uso terapéutico , Gripe Aviar/prevención & control , Gripe Humana/prevención & control , Orthomyxoviridae/efectos de los fármacos , Ácido Poliglutámico/uso terapéutico , Polisacáridos/uso terapéutico , Amino Azúcares/química , Animales , Antivirales/síntesis química , Antivirales/química , Antivirales/farmacología , Aves/virología , Línea Celular , Femenino , Hemaglutininas/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ácido Poliglutámico/síntesis química , Ácido Poliglutámico/química , Ácido Poliglutámico/farmacología , Polisacáridos/síntesis química , Polisacáridos/química , Polisacáridos/farmacologíaRESUMEN
Formation of membrane microdomain is critical for cell migration (epiboly) during gastrulation of medaka fish [Adachi et al. (Biochem. Biophys. Res. Commun. 358:848-853, 2007)]. In this study, we characterized membrane microdomain from gastrula embryos to understand its roles in epiboly. A cell adhesion molecule (E-cadherin), its associated protein (beta-catenin), transducer proteins (PLCgamma, cSrc), and a cytoskeleton protein (beta-actin) were enriched in the membrane microdomain. Le(X)-containing glycolipids and glycoproteins (Le(X)-gp) were exclusively enriched in the membrane microdomain. Interestingly, the isolated membrane microdomain had the ability to bind to each other in the presence of Ca(2+). This membrane microdomain binding was achieved through the E-cadherin homophilic and the Le(X)-glycan-mediated interactions. E-cadherin and Le(X)-gp were co-localized on the same membrane microdomain, suggesting that these two interactions are operative at the same time. Thus, the membrane microdomain functions as a platform of the E-cadherin- and Le(X)-glycan-mediated cell adhesion and signal transduction.
Asunto(s)
Cadherinas/metabolismo , Metabolismo de los Hidratos de Carbono , Comunicación Celular , Gástrula/citología , Gástrula/metabolismo , Microdominios de Membrana/metabolismo , Oryzias/embriología , Animales , Western Blotting , Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Moléculas de Adhesión Celular/metabolismo , Comunicación Celular/efectos de los fármacos , Colesterol/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Gástrula/efectos de los fármacos , Gastrulación/efectos de los fármacos , Glucolípidos/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Antígeno Lewis X/metabolismo , Microdominios de Membrana/efectos de los fármacos , Modelos Biológicos , Oryzias/metabolismo , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacosRESUMEN
Galectins are a family of beta-galactoside-specific lectins bearing a conserved carbohydrate recognition domain. Interactions between galectins and poly-N-acetyllactosamine sequences are critical in a variety of biological processes. Galectin-9, a member of the galectin family, has two carbohydrate recognition domains at both the N- and C-terminal regions. Here we report the crystal structure of the human galectin-9 N-terminal carbohydrate recognition domain in complex with N-acetyllactosamine dimers and trimers. These complex structures revealed that the galectin-9 N-terminal carbohydrate recognition domain can recognize internal N-acetyllactosamine units within poly-N-acetyllactosamine chains. Based on these complex structures, we propose two putative recognition modes for poly-N-acetyllactosamine binding by galectins.
Asunto(s)
Galectinas/química , Galectinas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Many tumors arising from epithelial tissues produce mucins, which readily come into contact with infiltrating cells in cancer tissues. MUC2 mucins were purified from the conditioned medium of a colorectal cancer cell line, LS180 cells. It is known that in cancer patients, the number of dendritic cells (DCs) is reduced and their function is impaired. Mature DCs were generated from human peripheral blood monocytes through successive treatments with GM-CSF and IL-4, and then with proinflammatory mediators. When monocytes were cultured in the presence of MUC2 mucins in addition to GM-CSF and IL-4 at an early stage of development, mature DCs expressing CD83 decreased and apoptotic cells increased in a dose-dependent manner. During the development of DCs, sialic acid-binding Ig-like lectin (Siglec)-3 was constantly expressed. We prepared recombinant soluble Siglec-3 corresponding to the ectodomain of Siglec-3 and confirmed the binding of soluble Siglec-3 to the MUC2 mucins, probably through alpha2,6-sialic acid-containing O-glycans including a sialyl Tn antigen, which is known to bind to Siglec-3. Apoptosis was partially inhibited by anti-Siglec-3 mAb or recombinant soluble Siglec-3. These results suggest that apoptosis was partially induced through the ligation of the MUC2 mucins with Siglec-3.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Monocitos/efectos de los fármacos , Mucina 2/farmacología , Anticuerpos Monoclonales/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Células Dendríticas/citología , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-4/farmacología , Lectinas/genética , Lectinas/inmunología , Lectinas/metabolismo , Monocitos/citología , Mucina 2/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Células U937RESUMEN
To control interspecies transmission of influenza viruses, it is essential to elucidate the molecular mechanisms of the interaction of influenza viruses with sialo-glycoconjugate receptors expressed on different host cells. Competitive inhibitors containing mimetic receptor carbohydrates that prevent virus entry may be useful tools to address such issues. We chemoenzymatically synthesized and characterized the glycopolymers that were carrying terminal 2,6-sialic acid on lactosamine repeats as influenza virus inhibitors. In vitro and in vivo infection experiments using these glycopolymers demonstrated marked differences in inhibitory activity against different species of viruses. Human viruses, including clinically isolated strains, were consistently inhibited by glycopolymers carrying lactosamine repeats with higher activity than those containing a single lactosamine. A swine virus also showed the same recognition properties as those from human hosts. In contrast, avian and equine viruses were not inhibited by any of the glycopolymers examined carrying single, tandem, or triplet lactosamine repeats. Hemagglutination inhibition and solid-phase binding analyses indicated that binding affinity of glycopolymers with influenza viruses contributes dominantly to the inhibitory activity against viral infection. Sequence analysis and molecular modeling of human viruses indicated that specific amino acid substitutions on hemagglutinin may affect binding affinity of glycopolymers carrying lactosamine repeats with viruses. In conclusion, glycopolymers carrying lactosamine repeats of different lengths are useful to define molecular mechanisms of virus recognition. The core carbohydrate portion as well as sialyl linkages on the receptor glycoconjugate may affect host cell recognition of human and swine viruses.
Asunto(s)
Amino Azúcares/síntesis química , Amino Azúcares/metabolismo , Antivirales/síntesis química , Antivirales/metabolismo , Gripe Humana/tratamiento farmacológico , Internalización del Virus/efectos de los fármacos , Amino Azúcares/química , Animales , Antivirales/química , Antivirales/farmacología , Biopolímeros/química , Biopolímeros/metabolismo , Biopolímeros/farmacología , Metabolismo de los Hidratos de Carbono , Carbohidratos/química , Línea Celular , Perros , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Ratones , Modelos Moleculares , Estructura MolecularRESUMEN
Divalent glycosides carrying N-acetyl-d-glucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. First, hexan-1,6-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Hx-GlcNAc) and 3,6-dioxaoct-1,8-diyl bis-(2-acetamido-2-deoxy-beta-d-glucopyranoside) (GlcNAc-Doo-GlcNAc) were enzymatically synthesized by transglycosylation of an N,N'N'',N'''-tetraacetylchitotetraose [(GlcNAc)(4)] donor with a primary diol acceptor, utilizing a chitinolytic enzyme from Amycolatopsis orientalis. The resulting divalent glycosides were further converted to the respective hexan-1,6-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Hx-LacNAc) and 6-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-hexyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Hx-GlcNAc), and respective 3,6-dioxaoct-1,8-diyl bis-[beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside] (LacNAc-Doo-LacNAc) and 8-(2-acetamido-2-deoxy-beta-d-glucopyranosyl)-3,6-dioxaoctyl beta-d-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-d-glucopyranoside (LacNAc-Doo-GlcNAc) by galactosyltransferase. The interaction of wheat germ agglutinin (WGA) with a series of divalent glycosides and related compounds were studied using a biosensor based on surface plasmon resonance (SPR) and by precipitation analysis. Our results demonstrated that divalent glycosides carrying GlcNAc on both sides and GlcNAc and LacNAc on each side are capable of precipitating WGA as divalent ligands, but that the corresponding monovalent controls and divalent glycosides carrying LacNAc on both sides are unable to precipitate the lectin and bind as univalent ligands.
Asunto(s)
Acetilglucosamina/química , Amino Azúcares/química , Reactivos de Enlaces Cruzados/química , Glicósidos/biosíntesis , Glicósidos/química , Aglutininas del Germen de Trigo/metabolismo , Actinomycetales/enzimología , Precipitación Química , Reactivos de Enlaces Cruzados/metabolismo , Galactosiltransferasas/metabolismo , Glicósidos/metabolismo , Pruebas de Inhibición de Hemaglutinación , Ligandos , Resonancia por Plasmón de SuperficieRESUMEN
We describe here an efficient synthetic route to spacer-N-linked double-headed glycosides via a simple two-step procedure. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose [(GlcNAc)(2)] were treated with ammonia and the resulting N-beta-glycosylamines were coupled to a series of dicarboxylic acids. Condensation with each dicarboxylic acid proceeded stereoselectively to give the corresponding beta-N-linked double-headed glycoside without the need for any protection/deprotection steps. Interaction of the resulting N-linked double-headed glycosides with wheat germ agglutinin (WGA) were then investigated using a precipitation assay and an optical biosensor based on surface plasmon resonance (SPR). Spacer-N-linked double-headed glycosides bearing GlcNAc and (GlcNAc)(2) were found to be capable of binding and precipitating WGA as divalent ligands. However, the length of the spacer groups between the two terminal sugar residues was found to greatly influence the cross-linking activities with the lectin.
Asunto(s)
Reactivos de Enlaces Cruzados/síntesis química , Glicósidos/síntesis química , Aglutininas del Germen de Trigo/metabolismo , Acetilglucosamina , Precipitación Química , Disacáridos , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Shiga toxin (Stx) exerts toxic activity by binding to glycosphingolipids, mainly globotriaosyl (Gb(3)) ceramide, on the surface of target cells. The inhibition of toxin-receptor binding is a promising therapeutic approach to prevent Stx-mediated diseases. In this study, we synthesized monovalent Stx-ligands of phosphatidylethanolamine dipalmitoyl-Gb(3) (Gb(3)-PEDP) and galabiosyl (Gb(2))-PEDP and we examined their neutralizing activity against Stx-1 and Stx-2 in vitro. Both Gb(3)-PEDP and Gb(2)-PEDP strongly neutralized the cytotoxicity of Stx-1 and Stx-2. It is likely that the mechanism of neutralization involved formation of liposomes and consequently clustering of sugar units. We propose monovalent Gb(3)-/Gb(2)-derivatives conjugated with phosphatidyl residue as a novel class of Stx-neutralizing agent.