RESUMEN
Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.
Asunto(s)
Sulfatos de Condroitina , Proteínas de la Matriz Extracelular , Heparitina Sulfato , Animales , Humanos , Ratones , Biomarcadores de Tumor/química , Proteínas de Unión al Calcio/química , Sulfatos de Condroitina/análisis , Heparitina Sulfato/análisis , Inmunohistoquímica/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Triptasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genéticaRESUMEN
T antigen (Galß1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 ß1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAß1-3Galß1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila ß1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization, basement membrane formation, and NMJ arborization on larval muscles.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Membrana Basal/metabolismo , Proteínas de Drosophila/metabolismo , Músculos/metabolismo , Unión Neuromuscular/metabolismo , Animales , Western Blotting , Drosophila/genética , Glucuronosiltransferasa/metabolismo , Immunoblotting , Larva/metabolismo , Espectrometría de Masas , Fenotipo , Polisacáridos/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Glycan structures are synthesized by a series of reactions conducted by glycosylation-related (GR) proteins such as glycosyltransferases, glycan-modifying enzymes, and nucleotide-sugar transporters. For example, the common core region of glycosaminoglycans (GAGs) is sequentially synthesized by peptide-O-xylosyltransferase, ß1,4-galactosyltransferase I, ß1,3-galactosyltransferase II, and ß1,3-glucuronyltransferase. This raises the possibility that functional impairment of GR proteins involved in synthesis of the same glycan might result in the same phenotypic abnormality. To examine this possibility, comprehensive silencing of genes encoding GR and proteoglycan core proteins was conducted in Drosophila. Drosophila GR candidate genes (125) were classified into five functional groups for synthesis of GAGs, N-linked, O-linked, Notch-related, and unknown glycans. Spatiotemporally regulated silencing caused a range of malformed phenotypes that fell into three types: extra veins, thick veins, and depigmentation. The clustered phenotypes reflected the biosynthetic pathways of GAGs, Fringe-dependent glycan on Notch, and glycans placed at or near nonreducing ends (herein termed terminal domains of glycans). Based on the phenotypic clustering, CG33145 was predicted to be involved in formation of terminal domains. Our further analysis showed that CG33145 exhibited galactosyltransferase activity in synthesis of terminal N-linked glycans. Phenotypic clustering, therefore, has potential for the functional prediction of novel GR genes.
Asunto(s)
Silenciador del Gen , Familia de Multigenes , Fenotipo , Interferencia de ARN , Animales , Drosophila , Glicosilación , Glicosiltransferasas/metabolismo , Datos de Secuencia Molecular , Polisacáridos/genéticaRESUMEN
MOTIVATION: It is well known influenza viruses recognize and bind terminal sialic acid (SA) on glycans that are found on the cell surface. In this work, we used a data mining technique to analyze the glycan array data of influenza viruses to find novel glycan structures other than SA that may be involved in viral infection. RESULTS: In addition to SA structures noted previously, we noted the sulfated structures in the mining results. For verification, we overexpressed the sulfotransferase that is involved in synthesizing these structures, and we performed a viral infection experiment to assess changes in infection in these cells. In our results, we found that there is a 70-fold increase in these cells compared with the control. Thus, we have found a novel pattern in glycan structures that may be involved in viral infection. AVAILABILITY AND IMPLEMENTATION: The Glycan Miner Tool is available from the RINGS resource at http://www.rings.t.soka.ac.jp.
Asunto(s)
Minería de Datos/métodos , Orthomyxoviridae/fisiología , Polisacáridos/química , Programas Informáticos , Animales , Perros , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Orthomyxoviridae/metabolismo , Polisacáridos/metabolismo , Sulfotransferasas/metabolismoRESUMEN
Honeybees (Apis mellifera) produce unique complex-type N-glycans bearing a Galß1-3GalNAc (T-antigen) unit, and honeybee-specific N-glycans are linked to royal jelly glycoproteins. In this study, we identified two novel honeybee ß1,3-galactosyltransferase (ß1,3-GalT) genes responsible for biosynthesis of the T-antigen in insect N-glycans. The products of the two putative ß1,3-GalT genes (ß1,3-GalT1 and ß1,3-GalT2), which were expressed in Sf21 insect cells, transferred galactose (Gal) residues to GalNAc2GlcNAc2Man3GlcNAc2-PA to form the Galß1-3GalNAc unit, indicating that the identified genes were involved in biosynthesis of the ß1-3 Gal-containing N-glycan. Therefore, using biochemistry and molecular biology techniques, we revealed a unique N-glycan biosynthesis mechanism in the cephalic region of honeybees, which has not previously been found in other animal or plant cells.
Asunto(s)
Antígenos Virales de Tumores/metabolismo , Abejas/enzimología , Galactosiltransferasas/metabolismo , Proteínas de Insectos/metabolismo , Polisacáridos/biosíntesis , Secuencia de Aminoácidos , Animales , Abejas/genética , Conformación de Carbohidratos , Secuencia de Carbohidratos , Clonación Molecular , Proteínas de Drosophila/química , Galactosa/metabolismo , Galactosiltransferasas/genética , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Polisacáridos/química , Homología de Secuencia de AminoácidoRESUMEN
Late-onset non-syndromic autosomal dominant hearing loss 9 (DFNA9) is a hearing impairment caused by mutations in the coagulation factor C homology gene (COCH). COCH encodes for cochlin, a major component of the cochlear extracellular matrix. Though biochemical and genetic studies have characterized the properties of wild-type and mutated cochlins derived from DFNA9, little is known about the underlying pathogenic mechanism. In this study, we established a cochlin reporter cell, which allowed us to monitor the interaction of cochlin with its ligand(s) by means of a ß-galactosidase assay. We found a class of highly sulfated glycosaminoglycans (GAGs), heparin, that were selectively bound to cochlin. The interaction was distinctly abrogated by N-desulfation, but not by 2-O- or 6-O-desulfation. The binding of cochlin to GAG was diminished by all of the point mutations found in DFNA9 patients. Through GAG composition analysis and immunostaining using mouse cochlin/immunoglobulin-Fc fusion protein, we identified moderately sulfated GAGs in mouse cochlea tissue; this implies that cochlin binds to such sulfated GAGs in the cochlea. Since GAGs play an important role in cell growth and survival as co-receptors of signal transduction mechanisms, the interaction of cochlin with GAGs in the extracellular matrix could aid the pathological research of autosomal dominant late-onset hearing loss in DFNA9.
Asunto(s)
Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Animales , Ratones , Sordera/genética , Proteínas de la Matriz Extracelular/metabolismo , Pérdida Auditiva/genética , Pérdida Auditiva Sensorineural/genética , Heparina , Heparitina Sulfato , SulfatosRESUMEN
Synthesis of extracellular sulfated molecules requires active 3'-phosphoadenosine 5'-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Regulación del Desarrollo de la Expresión Génica , Aparato de Golgi/metabolismo , Heparitina Sulfato/metabolismo , Proteínas de Transporte de Nucleótidos/fisiología , Alelos , Animales , Caenorhabditis elegans , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Reporteros , Glicosaminoglicanos/química , Proteínas Fluorescentes Verdes/química , Mutación , Fracciones Subcelulares , Especificidad por Sustrato , TransgenesRESUMEN
Sulfation represents an essential modification for various molecules and regulates many biological processes. The sulfation of glycans requires a specific transporter for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) on the Golgi apparatus. This study investigated the expression of PAPS transporter genes in colorectal carcinomas and the significance of Golgi-specific sulfation in the proliferation of colorectal carcinoma cells. The relative amount of PAPST1 transcripts was found to be higher than those of PAPST2 in colorectal cancerous tissues. Immunohistochemically, the enhanced expression of PAPST1 was observed in fibroblasts in the vicinity of invasive cancer cells, whereas the expression of PAPST2 was decreased in the epithelial cells. RNA interference of either of the two PAPS transporter genes reduced the extent of sulfation of cellular proteins and cellular proliferation of DLD-1 human colorectal carcinoma cells. Silencing the PAPS transporter genes reduced fibroblast growth factor signaling in DLD-1 cells. These findings indicate that PAPS transporters play a role in the proliferation of colorectal carcinoma cells themselves and take part in a desmoplastic reaction to support cancer growth by controlling their sulfation status.
Asunto(s)
Proteínas de Transporte de Anión , Expresión Génica , Proteínas de Transporte de Membrana , Polisacáridos , Sulfamonometoxina , Trimetoprim , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Transporte Biológico , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Combinación de Medicamentos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunohistoquímica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Invasividad Neoplásica , Fosfoadenosina Fosfosulfato/metabolismo , Polisacáridos/genética , Polisacáridos/metabolismo , Interferencia de ARN , ARN Mensajero/biosíntesis , Transducción de Señal/genética , Sulfamonometoxina/metabolismo , Transportadores de Sulfato , Trimetoprim/metabolismo , Células Tumorales CultivadasRESUMEN
When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galß1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galß1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.
Asunto(s)
Embrión de Pollo/virología , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Polisacáridos/metabolismo , Sulfatos/metabolismo , Alantoides/metabolismo , Amnios/metabolismo , Animales , Embrión de Pollo/metabolismo , Perros , Galactósidos/química , Galactósidos/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Células de Riñón Canino Madin Darby , Mutación , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/química , Receptores Virales/metabolismo , Sulfatos/química , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Carbohidrato SulfotransferasasRESUMEN
T antigen (Galbeta1-3GalNAcalpha1-Ser/Thr), the well-known tumor-associated antigen, is a core 1 mucin-type O-glycan structure that is synthesized by core 1 beta1,3-galactosyltransferase (C1beta3GalT), which transfers Gal from UDP-Gal to Tn antigen (GalNAcalpha1-Ser/Thr). Three putative C1beta3GalTs have been identified in Drosophila. However, although all three are expressed in embryos, their roles during embryogenesis have not yet been clarified. In this study, we used P-element inserted mutants to show that CG9520, one of the three putative C1beta3GalTs, synthesizes T antigen expressed on the central nervous system (CNS) during embryogenesis. We also found that T antigen was expressed on a subset of the embryonic hemocytes. CG9520 mutant embryos showed the loss of T antigens on the CNS and on a subset of hemocytes. Then, the loss of T antigens was rescued by precise excision of the P-element inserted into the CG9520 gene. Our data demonstrate that T antigens expressed on the CNS and on a subset of hemocytes are synthesized by CG9520 in the Drosophila embryo. In addition, we found that the number of circulating hemocytes was reduced in third instar larvae of CG9520 mutant. We, therefore, named the CG9520 gene Drosophila core 1 beta1,3-galactosyltransferase 1 because it is responsible for the synthesis and function of T antigen in vivo.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/biosíntesis , Proteínas de Drosophila/genética , Drosophila/embriología , Drosophila/enzimología , Galactosiltransferasas/genética , Hemocitos/metabolismo , Animales , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Sistema Nervioso Central/metabolismo , Drosophila/genética , Embrión no Mamífero/enzimología , Histocitoquímica , Mutación , FilogeniaRESUMEN
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP), a member of the 2H phosphoesterase superfamily, is firmly bound to brain white matter and found mainly in the central nervous system of vertebrates, and it catalyzes the hydrolysis of 2',3'-cyclic nucleotide to produce 2'-nucleotide. Recent studies on CNP-knockout mice have revealed that the absence of CNP causes axonal swelling and neuronal degeneration. Here, the crystal structure of the catalytic fragment (CF) of human CNP (hCNP-CF) is solved at 1.8A resolution. It is an alpha+beta type structure consisting of three alpha-helices and nine beta-strands. The structural core of the molecule is comprised of two topologically equivalent three-stranded antiparallel beta-sheets that are related by a pseudo 2-fold symmetry. Each beta-sheet contains an H-X-T-X motif, which is strictly conserved among members of the 2H phosphoesterase superfamily. The phosphate ion is bound to the side-chains of His and Thr from each of the two motifs. Structural comparison of hCNP-CF with plant 1'',2''-cyclic nucleotide phosphodiesterase (CPDase) and bacterial 2'-5' RNA ligase reveals that the H-X-T-X motifs are structurally conserved among these enzymes, but the surface properties of the active site are quite different among the enzymes, reflecting the differences in their substrates. On the basis of the present crystal structure of the hCNP-CF/phosphate complex, the available structure of the CPDase/cyclic-nucleotide analogue complex, and the recent functional studies of rat CNP-CF, we propose a possible substrate-binding mode and catalytic mechanism of CNP, which employs the nucleophilic water molecule activated by His310. The proposed mechanism is basically equivalent to the second step of the well-accepted reaction mechanism of RNase A. Since the overall structure of hCNP-CF differs considerably from that of RNase A, it is likely that the similar active sites with two catalytic histidine residues in these enzymes arose through convergent evolution.
Asunto(s)
Encéfalo/enzimología , Hidrolasas Diéster Fosfóricas/química , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Electricidad Estática , Especificidad por SustratoRESUMEN
This study focuses on clarifying the contribution of sulfation to radiation-induced apoptosis in human Burkitt's lymphoma cell lines, using 3'-phosphoadenosine 5'-phosphosulfate transporters (PAPSTs). Overexpression of PAPST1 or PAPST2 reduced radiation-induced apoptosis in Namalwa cells, whereas the repression of PAPST1 expression enhanced apoptosis. Inhibition of PAPST slightly decreased keratan sulfate (KS) expression, so that depletion of KS significantly increased radiation-induced apoptosis. In addition, the repression of all three N-acetylglucosamine-6-O-sulfotransferases (CHST2, CHST6, and CHST7) increased apoptosis. In contrast, PAPST1 expression promoted the phosphorylation of p38 MAPK and Akt in irradiated Namalwa cells. These findings suggest that 6-O-sulfation of GlcNAc residues in KS reduces radiation-induced apoptosis of human Burkitt's lymphoma cells.
Asunto(s)
Linfoma de Burkitt/metabolismo , Sulfato de Queratano/metabolismo , Proteoglicanos/metabolismo , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Apoptosis/fisiología , Apoptosis/efectos de la radiación , Secuencia de Bases , Linfoma de Burkitt/patología , Linfoma de Burkitt/radioterapia , Línea Celular Tumoral , Humanos , Sulfato de Queratano/química , Sistema de Señalización de MAP Quinasas , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteoglicanos/química , ARN Interferente Pequeño/genética , Tolerancia a Radiación/fisiología , Transportadores de Sulfato , Ésteres del Ácido Sulfúrico/metabolismoRESUMEN
Maintenance of self-renewal and pluripotency in mouse embryonic stem cells (mESCs) is regulated by the balance between several extrinsic signaling pathways. Recently, we demonstrated that heparan sulfate (HS) chains play important roles in the maintenance and differentiation of mESCs by regulating extrinsic signaling. Sulfated HS structures are modified by various sulfotransferases during development. However, the significance of specific HS structures during development remains unclear. Here, we show that 3-O-sulfated HS structures synthesized by HS 3-O-sulfotransferases (3OSTs) and recognized by the antibody HS4C3 increase during differentiation of mESCs. Furthermore, expression of Fas on the cell surface of the differentiated cells also increased. Overexpression of the HS4C3-binding epitope in mESCs induced apoptosis and spontaneous differentiation even in the presence of LIF and serum. These data showed that the HS4C3-binding epitope was required for differentiation of mESCs. Up-regulation of the HS4C3-binding epitope resulted in the recruitment of Fas from the cytoplasm to lipid rafts on the cell surface followed by activation of Fas signaling. Indeed, the HS4C3-binding epitope interacted with a region that included the heparin-binding domain (KLRRRVH) of Fas. Reduced self-renewal capability in cells overexpressing 3OST resulted from the degradation of Nanog by activated caspase-3, which is downstream of Fas signaling, and was rescued by the inhibition of Fas signaling. We also found that knockdown of 3OST and inhibition of Fas signaling reduced the potential for differentiation into the three germ layers during embryoid body formation. This is the first demonstration that activation of Fas signaling is mediated by an increase in the HS4C3-binding epitope and indicates a novel signaling pathway for differentiation in mESCs.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Heparitina Sulfato/química , Heparitina Sulfato/farmacología , Receptor fas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Immunoblotting , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacosRESUMEN
There is increasing evidence that soluble glycosaminoglycans such as heparin can interfere with the infectivity of various viruses, including ecotropic murine leukemia viruses (MLVs). The ecotropic MLV, Friend MLV (F-MLV) and the neuropathogenic variants A8 MLV and PVC-211 MLV, were susceptible to heparin-mediated inhibition of infection of NIH 3T3 cells. To investigate the interaction between the envelope glycoprotein (Env) of MLV and heparin, we prepared vesicular stomatitis virus-based pseudotyped viruses carrying the Env of F-, A8, or PVC-211 MLVs. Surface plasmon resonance analyses indicated that the Env of A8 and PVC-211 MLVs had a higher binding activity to heparin than that of F-MLV. We examined the influence of N- or O-sulfation of heparin on binding activity to Env and on the inhibition of the infectivity of MLV and pseudotyped viruses carrying Env. This analysis indicated that the O-sulfate groups of heparin play a major role in determining Env-dependent inhibitory effects.
Asunto(s)
Regulación hacia Abajo , Heparina/química , Heparina/metabolismo , Virus de la Leucemia Murina/fisiología , Infecciones por Retroviridae/veterinaria , Enfermedades de los Roedores/metabolismo , Animales , Virus de la Leucemia Murina/genética , Ratones , Estructura Molecular , Células 3T3 NIH , Infecciones por Retroviridae/metabolismo , Infecciones por Retroviridae/virología , Enfermedades de los Roedores/virología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Walker-Warburg syndrome, a progressive muscular dystrophy, is a severe disease with various kinds of symptoms such as muscle weakness and occasional seizures. The genes of protein O-mannosyltransferases 1 and 2 (POMT1 and POMT2), fukutin, and fukutin-related protein are responsible for this syndrome. In our previous study, we cloned Drosophila orthologs of human POMT1 and POMT2 and identified their activity. However, the mechanism of onset of this syndrome is not well understood. Furthermore, little is known about the behavioral properties of the Drosophila POMT1 and POMT2 mutants, which are called rotated abdomen (rt) and twisted (tw), respectively. First, we performed various kinds of behavioral tests and described in detail the muscle structures by using these mutants. The mutant flies exhibited abnormalities in heavy exercises such as climbing or flight but not in light movements such as locomotion. Defective motor function in mutants appeared immediately after eclosion and was exaggerated with aging. Along with motor function, muscle ultrastructure in the tw mutant was altered, as seen in human patients. We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively. Flies expressing RNAi had reduced lifespans. These findings clearly demonstrate that Drosophila POMT mutants are models for human muscular dystrophy. We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity. In this paper, we propose a novel mechanism for the development of muscular dystrophy: POMT mutation causes high myoblast density and position derangement, which result in apoptosis, muscle disorganization, and muscle cell defects.
Asunto(s)
Apoptosis/fisiología , Distrofias Musculares/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Animales , Apoptosis/genética , Western Blotting , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Inmunohistoquímica , Locomoción/genética , Locomoción/fisiología , Longevidad/genética , Longevidad/fisiología , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Microscopía Electrónica de Transmisión , Distrofias Musculares/genética , Reacción en Cadena de la Polimerasa , Interferencia de ARNRESUMEN
Recently, we have identified two 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent K(m) value of 1.54 microM or 1.49 microM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fosfoadenosina Fosfosulfato/metabolismo , Animales , Proteínas de Transporte de Anión/genética , Proliferación Celular , Sulfatos de Condroitina/metabolismo , Regulación hacia Abajo , Embrión de Mamíferos/citología , Técnicas de Silenciamiento del Gen , Estratos Germinativos/citología , Heparitina Sulfato/metabolismo , Cinética , Ratones , Modelos Biológicos , Neurogénesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Especificidad por Sustrato , Sulfatos/metabolismoRESUMEN
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is found mainly in the central nervous system of vertebrates and catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides in vitro. Recently, Several 2H phosphodiesterase super family protein structures have been determined by X-ray crystallography and NMR spectroscopy. Here we report the structure-function relationship studies of two hydrophobic residues in CNP family proteins.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Histidina/química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/clasificación , Hidrolasas Diéster Fosfóricas/metabolismo , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The catalytic fragment of human 2',3'-cyclic nucleotide 3'-phosphodiesterase (hCNP-CF) has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 300 as the precipitating agent. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 44.39, b = 55.35, c = 78.76 A. There is one molecule per asymmetric unit. The crystals diffract to at least 1.8 A resolution using synchrotron radiation and are suitable for X-ray structure analysis at high resolution.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , 2',3'-Nucleótido Cíclico 3'-Fosfodiesterasa , Cristalización , Cristalografía por Rayos X , Humanos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/aislamiento & purificaciónRESUMEN
2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is found mainly in the central nervous system of vertebrates and catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides in vitro. Recently, CNP has been identified as a member of the 2H phosphoesterase superfamily. Here we have determined the crystal structure of the catalytic fragment of human CNP (hCNP-CF) at 1.3 A resolution.
Asunto(s)
2',3'-Nucleótido Cíclico Fosfodiesterasas/química , Encéfalo/enzimología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/aislamiento & purificación , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Cristalización , Cristalografía por Rayos X , HumanosRESUMEN
Walker-Warburg syndrome, caused by mutations in protein O-mannosyltransferase-1 (POMT1), is an autosomal recessive disorder characterized by severe brain malformation, muscular dystrophy, and structural eye abnormalities. As humans have a second POMT, POMT2, we cloned each Drosophila ortholog of the human POMT genes and carried out RNA interference (RNAi) knock-down to investigate the function of these proteins in vivo. Drosophila POMT2 (dPOMT2) RNAi mutant flies showed a "twisted abdomen phenotype," in which the abdomen is twisted 30-60 degrees , similar to the dPOMT1 mutant. Moreover, dPOMT2 interacted genetically with dPOMT1, suggesting that the dPOMTs function in collaboration with each other in vivo. We expressed dPOMTs in Sf21 cells and measured POMT activity. dPOMT2 transferred a mannose to the dystroglycan protein only when it was coexpressed with dPOMT1. Likewise, dPOMT1 showed POMT activity only when coexpressed with dPOMT2, and neither dPOMT showed any activity by itself. Each dPOMT RNAi fly totally reduced POMT activity, despite the specific reduction in the level of each dPOMT mRNA. The expression pattern of dPOMT2 mRNA was found to be similar to that of dPOMT1 mRNA using whole mount in situ hybridization. These results demonstrate that the two dPOMTs function as a protein O-mannosyltransferase in association with each other, in vitro and in vivo, to generate and maintain normal muscle development.