RESUMO
Heparan sulfate (HS) and chondroitin sulfate (CS) are evolutionarily conserved glycosaminoglycans that are found in most animal species, including the genetically tractable model organism Drosophila. In contrast to extensive in vivo studies elucidating co-receptor functions of Drosophila HS proteoglycans (PGs), only a limited number of studies have been conducted for those of CSPGs. To investigate the global function of CS in development, we generated mutants for Chondroitin sulfate synthase (Chsy), which encodes the Drosophila homolog of mammalian chondroitin synthase 1, a crucial CS biosynthetic enzyme. Our characterizations of the Chsy mutants indicated that a fraction survive to adult stage, which allowed us to analyze the morphology of the adult organs. In the ovary, Chsy mutants exhibited altered stiffness of the basement membrane and muscle dysfunction, leading to a gradual degradation of the gross organ structure as mutant animals aged. Our observations show that normal CS function is required for the maintenance of the structural integrity of the ECM and gross organ architecture.
Assuntos
Sulfatos de Condroitina , Drosophila , Animais , Feminino , Drosophila/genética , Folículo Ovariano , Ovário , Glicosaminoglicanos , MamíferosRESUMO
Morphogens provide quantitative and robust signaling systems to achieve stereotypic patterning and morphogenesis. Heparan sulfate (HS) proteoglycans (HSPGs) are key components of such regulatory feedback networks. In Drosophila, HSPGs serve as co-receptors for a number of morphogens, including Hedgehog (Hh), Wingless (Wg), Decapentaplegic (Dpp) and Unpaired (Upd, or Upd1). Recently, Windpipe (Wdp), a chondroitin sulfate (CS) proteoglycan (CSPG), was found to negatively regulate Upd and Hh signaling. However, the roles of Wdp, and CSPGs in general, in morphogen signaling networks are poorly understood. We found that Wdp is a major CSPG with 4-O-sulfated CS in Drosophila. Overexpression of wdp modulates Dpp and Wg signaling, showing that it is a general regulator of HS-dependent pathways. Although wdp mutant phenotypes are mild in the presence of morphogen signaling buffering systems, this mutant in the absence of Sulf1 or Dally, molecular hubs of the feedback networks, produces high levels of synthetic lethality and various severe morphological phenotypes. Our study indicates a close functional relationship between HS and CS, and identifies the CSPG Wdp as a novel component in morphogen feedback pathways.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteoglicanas de Heparan Sulfato/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Sulfatases/genética , Sulfatases/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
A class of carbohydrate-modified proteins, heparan sulfate proteoglycans (HSPGs), play critical roles both in normal development and during disease. Genetic studies using a model organism, Drosophila, have been contributing to understanding the in vivo functions of HSPGs. Despite the many strengths of the Drosophila model for in vivo studies, biochemical analysis of Drosophila HS is somewhat limited, mainly due to the insufficient amount of the material obtained from the animal. To overcome this obstacle, we generated mutant cell lines for four HS modifying enzymes that are critical for the formation of ligand binding sites on HS, Hsepi, Hs2st, Hs6st and Sulf1, using a recently established method. Morphological and immunological analyses of the established lines suggest that they are spindle-shaped cells of mesodermal origin. The disaccharide profiles of HS from these cell lines showed characteristics of lack of each enzyme as well as compensatory modifications by other enzymes. Metabolic radiolabeling of HS allowed us to assess chain length and net charge of the total population of HS in wild-type and Hsepi mutant cell lines. We found that Drosophila HS chains are significantly shorter than those from mammalian cells. BMP signaling assay using Hs6st cells indicates that molecular phenotypes of these cell lines are consistent with previously known in vivo phenomena. The established cell lines will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.
Assuntos
Carboidratos Epimerases/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteoglicanas de Heparan Sulfato/metabolismo , Mutação , Sulfatases/genética , Sulfotransferases/genética , Animais , Carboidratos Epimerases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Fenótipo , Sulfatases/metabolismo , Sulfotransferases/metabolismoRESUMO
Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.
Assuntos
Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Sialoglicoproteínas/metabolismo , Humanos , Sialoglicoproteínas/química , Sialoglicoproteínas/genéticaRESUMO
Podocalyxin (PC) was first identified as a heavily sialylated transmembrane protein of glomerular podocytes. Recent studies suggest that PC is a remarkable glycoconjugate that acts as a universal glyco-carrier. The glycoforms of PC are responsible for multiple functions in normal tissue, human cancer cells, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). PC is employed as a major pluripotent marker of hESCs and hiPSCs. Among the general antibodies for human PC, TRA-1-60 and TRA-1-81 recognize the keratan sulfate (KS)-related structures. Therefore, It is worthwhile to summarize the outstanding chemical characteristic of PC, including the KS-related structures. Here, we review the glycoforms of PC and discuss the potential of PC as a novel KS proteoglycan in undifferentiated hESCs and hiPSCs.
Assuntos
Antígenos de Diferenciação/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Sulfato de Queratano/metabolismo , Sialoglicoproteínas/metabolismo , Anticorpos/química , HumanosRESUMO
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.
Assuntos
Inativação Gênica , Família Multigênica , Fenótipo , Interferência de RNA , Animais , Drosophila , Glicosilação , Glicosiltransferases/metabolismo , Dados de Sequência Molecular , Polissacarídeos/genéticaRESUMO
The biosynthesis of heparan sulfate proteoglycans is tightly regulated by multiple feedback mechanisms, which support robust developmental systems. One of the regulatory network systems controlling heparan sulfate (HS) biosynthesis is sulfation compensation. A previous study using Drosophila HS 2-O- and 6-O-sulfotransferase (Hs2st and Hs6st) mutants showed that loss of sulfation at one position is compensated by increased sulfation at other positions, supporting normal FGF signaling. Here, we show that HS sulfation compensation rescues both Decapentaplegic and Wingless signaling, suggesting a universal role of this regulatory system in multiple pathways in Drosophila. Furthermore, we identified Sulf1, extracellular HS 6-O-endosulfatase, as a novel component of HS sulfation compensation. Simultaneous loss of Hs2st and Sulf1 led to 6-O-oversulfation, leading to patterning defects, overgrowth, and lethality. These phenotypes are caused at least partly by abnormal up-regulation of Hedgehog signaling. Thus, sulfation compensation depends on the coordinated activities of Hs2st, Hs6st, and Sulf1.
Assuntos
Proteínas de Drosophila/metabolismo , Sulfatases/metabolismo , Sulfotransferases/metabolismo , Animais , Padronização Corporal/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Sulfatases/genética , Sulfotransferases/genéticaRESUMO
During the biosynthesis of heparan sulfate (HS), glucuronyl C5-epimerase (Hsepi) catalyzes C5-epimerization of glucuronic acid (GlcA), converting it to iduronic acid (IdoA). Because HS 2-O-sulfotransferase (Hs2st) shows a strong substrate preference for IdoA over GlcA, C5-epimerization is required for normal HS sulfation. However, the physiological significance of C5-epimerization remains elusive. To understand the role of Hsepi in development, we isolated Drosophila Hsepi mutants. Homozygous mutants are viable and fertile with only minor morphological defects, including the formation of an ectopic crossvein in the wing, but they have a short lifespan. We propose that two mechanisms contribute to the mild phenotypes of Hsepi mutants: HS sulfation compensation and possible developmental roles of 2-O-sulfated GlcA (GlcA2S). HS disaccharide analysis showed that loss of Hsepi resulted in a significant impairment of 2-O-sulfation and induced compensatory increases in N- and 6-O-sulfation. Simultaneous block of Hsepi and HS 6-O-sulfotransferase (Hs6st) activity disrupted tracheoblast formation, a well established FGF-dependent process. This result suggests that the increase in 6-O-sulfation in Hsepi mutants is critical for the rescue of FGF signaling. We also found that the ectopic crossvein phenotype can be induced by expression of a mutant form of Hs2st with a strong substrate preference for GlcA-containing units, suggesting that this phenotype is associated with abnormal GlcA 2-O-sulfation. Finally, we show that Hsepi formed a complex with Hs2st and Hs6st in S2 cells, raising the possibility that this complex formation contributes to the close functional relationships between these enzymes.
Assuntos
Carboidratos Epimerases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crescimento & desenvolvimento , Glucuronatos/metabolismo , Heparitina Sulfato/biossíntese , Sulfotransferases/metabolismo , Animais , Carboidratos Epimerases/genética , Drosophila/enzimologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ácido Glucurônico/metabolismo , Ácido Idurônico/metabolismo , Longevidade/genética , Mutagênese Sítio-Dirigida , Mutação , Transdução de Sinais , Sulfotransferases/genéticaRESUMO
Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.
Assuntos
Osso e Ossos/embriologia , Cartilagem/embriologia , Sulfatos de Condroitina/biossíntese , Proteínas de Transporte de Monossacarídeos/fisiologia , Proteínas de Transporte de Nucleotídeos/fisiologia , Animais , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Cartilagem/metabolismo , Cartilagem/patologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Epífises/embriologia , Epífises/metabolismo , Epífises/patologia , Ossos Faciais/anormalidades , Ossos Faciais/embriologia , Ossos Faciais/metabolismo , Humanos , Deformidades Congênitas dos Membros/embriologia , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Transporte de Monossacarídeos/deficiência , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Nucleotídeos/genéticaRESUMO
Heparan sulfate proteoglycans (HSPGs) serve as co-receptors for growth factor signaling during development. It is well known that the level and patterns of sulfate groups of heparan sulfate (HS) chains, or HS fine structures, have a major impact on HSPG function. On the other hand, the physiological significance of other structural features of HS, including NS/NA domain organization, remains to be elucidated. A blueprint of the HS domain structures is mainly controlled by HS N-deacetylase/N-sulfotransferases (NDSTs). To analyze in vivo activities of differentially modified HS, we established two knock-in (KI) Drosophila strains with the insertion of mouse Ndst1 (mNdst1) or Ndst2 (mNdst2) in the locus of sulfateless (sfl), the only Drosophila NDST. In these KI lines, mNDSTs are expressed from the sfl locus, in the level and patterns identical to the endogenous sfl gene. Thus, phenotypes of Ndst1 KI and Ndst2KI animals reflect the ability of HS structures made by these enzymes to rescue sfl mutation. Remarkably, we found that mNdst1 completely rescued the loss of sfl. mNdst2 showed a limited rescue ability, despite a higher level of HS sulfation compared to HS in mNdst1 KI. Our study suggests that independent of sulfation levels, additional HS structural features controlled by NDSTs play key roles during tissue patterning.
RESUMO
ZG16p is a soluble 16 kDa pancreatic protein having structural similarities with plant ß-prism fold lectins such as the banana lectin BanLec and the jackfruit lectin jacalin. ZG16p is postulated to be involved in the formation of zymogen granules by interacting with proteoglycans (PGs) localized in pancreatic exocrine granule membranes, but direct evidence was lacking. We characterized the structural properties of rat pancreatic zymogen granule PGs and examined their interaction with ZG16p. Structural analysis of the glycosaminoglycans (GAGs) showed that rat pancreatic zymogen granule PGs have heparan sulfate chains with a unique property, a high degree of sulfation (ΔUA-GlcNAc:ΔUA-GlcNS:ΔUA-GlcNAc6S:ΔUA-GlcNS6S:ΔUA2S-GlcNS:ΔUA2S-GlcNS6S, 27.9:16.6:5.7:22.5:6.2:21.1). After heparin lyase II digestion, the core proteins derived from the PGs were detected at molecular weights of 66,000 and 35,000-40,000. An overlay binding assay revealed that ZG16p binds specifically to heparan sulfate PGs by recognizing their GAG chains. Affinity chromatography demonstrated that ZG16p binds most strongly to heparin among the zymogen granule proteins. Site-directed mutational analysis revealed that the basic amino acid residues located in two putative carbohydrate-binding sites (CBSs) of ZG16p, which were found in association with the crystal structure of BanLec, are responsible for the recognition of heparin. These observations suggest that ZG16p is the primary binding partner of the granule heparan sulfate PGs. ZG16p may cross-link the granule heparan sulfate chains via two CBSs and facilitate the formation of a submembranous matrix, a sorting platform for enzyme proteins on the luminal side of the zymogen granule membrane.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Lectinas/metabolismo , Pâncreas/metabolismo , Proteoglicanas/metabolismo , Vesículas Secretórias/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Proteoglicanas de Heparan Sulfato/química , Heparitina Sulfato/química , Lectinas/química , Lectinas/isolamento & purificação , Dados de Sequência Molecular , Pâncreas/química , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Proteoglicanas/química , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vesículas Secretórias/químicaRESUMO
Autophagy is a catabolic process that provides cells with energy and molecular building blocks during nutritional stress. Autophagy also removes misfolded proteins and damaged organelles, a critical mechanism for cellular repair. Earlier work demonstrated that heparan sulfate proteoglycans, an abundant class of carbohydrate-modified proteins found on cell surfaces and in the extracellular matrix, suppress basal levels of autophagy in several cell types during development in Drosophila melanogaster In studies reported here, we examined the capacity of heparan sulfate synthesis to influence events affected by autophagy, including lifespan, resistance to reactive oxygen species (ROS) stress, and accumulation of ubiquitin-modified proteins in the brain. Compromising heparan sulfate synthesis increased autophagy-dependent processes, evident by extended lifespan, increased resistance to ROS, and reduced accumulation of ubiquitin-modified proteins in the brains of ROS exposed adults. The capacity of altering heparan sulfate biosynthesis to protect cells from injury was also evaluated in two different models of neurodegeneration, overexpression of Presenilin and parkin mutants. Presenilin overexpression in the retina produces cell loss, and compromising heparan sulfate biosynthesis rescued retinal patterning and size abnormalities in these animals. parkin is the fly homolog of human PARK2, one of the genes responsible for juvenile onset Parkinson's Disease. Parkin is involved in mitochondrial surveillance and compromising parkin function results in degeneration of both flight muscle and dopaminergic neurons in Drosophila Altering heparan sulfate biosynthesis suppressed flight muscle degeneration and mitochondrial dysmorphology, indicating that activation of autophagy-mediated removal of mitochondria (mitophagy) is potentiated in these animals. These findings provide in vivo evidence that altering the levels of heparan sulfate synthesis activates autophagy and can provide protection from a variety of cellular stressors.
Assuntos
Autofagia , Proteínas de Drosophila/genética , Heparitina Sulfato/biossíntese , Longevidade , Estresse Oxidativo , Ubiquitina-Proteína Ligases/genética , Animais , Encéfalo/metabolismo , Olho Composto de Artrópodes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Heparitina Sulfato/genética , Músculos/metabolismo , Mutação , Presenilinas/genética , Presenilinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A chemical method for the determination of hyaluronan (hyaluronic acid, HA) has been developed and applied to the human blood plasma. Human blood plasma HA was converted to the ΔDi-HA by digestion with hyaluronidase SD and determined by a sensitive and selective high-performance liquid chromatography (HPLC). The HPLC includes the separation and detection of ΔDi-HA using a graphitized carbon column and fluorometric reaction with 2-cyanoacetamide in an alkaline eluent. The calibration graph for ΔDi-HA was linear over the range 0.2 ng-1 µg. It was revealed that the concentration of HA in normal human blood plasma is very low levels (about 24 ng/ml) in comparison to low-sulfated chondroitin 4-sulfate (about 13 µg/ml).
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fluorometria/métodos , Grafite/química , Ácido Hialurônico/sangue , Adulto , Sulfatos de Condroitina/química , Sulfatos de Condroitina/metabolismo , Condroitinases e Condroitina Liases/metabolismo , Cromatografia Líquida de Alta Pressão/instrumentação , Feminino , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Masculino , Nitrilas/química , Nitrilas/metabolismo , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Proliferação de Células , Sulfatos de Condroitina/metabolismo , Regulação para Baixo , Embrião de Mamíferos/citologia , Técnicas de Silenciamento de Genes , Camadas Germinativas/citologia , Heparitina Sulfato/metabolismo , Cinética , Camundongos , Modelos Biológicos , Neurogênese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Especificidade por Substrato , Sulfatos/metabolismoRESUMO
Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Heparitina Sulfato/farmacologia , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Endoderma/metabolismo , Humanos , Camundongos , Modelos Biológicos , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteína Wnt3RESUMO
The GalNAcbeta1,4GlcNAc (LacdiNAc or LDN) structure is a more common structural feature in invertebrate glycoconjugates when compared with the Galbeta1,4GlcNAc structure. Recently, beta1,4-N-acetylgalactosaminyltransferase (beta4GalNAcT) was identified in some invertebrates including Drosophila. However, the LDN structure has not been reported in Drosophila, and the biological function of LDN remains to be determined. In this study, we examined acceptor substrate specificity of Drosophila beta4GalNAcTA by using some N- and O-glycans on glycoproteins and neutral glycosphingolipids (GSLs). GalNAc was efficiently transferred toward N-glycans, O-glycans, and the arthro-series GSLs. Moreover, we showed that dbeta4GalNAcTA contributed to the synthesis of the LDN structure in vivo. The dbeta4GalNAcTA mRNA was highly expressed in the developmental and adult neuronal tissues. Thus, these results suggest that dbeta4GalNAcTA acts on the terminal GlcNAc residue of some glycans for the synthesis of LDN, and the LDN structure may play a role in the physiological or neuronal development of Drosophila.
Assuntos
Dissacarídeos/biossíntese , Proteínas de Drosophila/biossíntese , Glicoproteínas/biossíntese , Glicoesfingolipídeos/biossíntese , Lactose/análogos & derivados , N-Acetilgalactosaminiltransferases/fisiologia , Animais , Sequência de Carboidratos , Dissacarídeos/química , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Lactose/biossíntese , Lactose/química , Dados de Sequência Molecular , N-Acetilgalactosaminiltransferases/químicaRESUMO
Heparan sulfate has been isolated for the first time from the mosquito Anopheles stephensi, a known vector for Plasmodium parasites, the causative agents of malaria. Chondroitin sulfate, but not dermatan sulfate or hyaluronan, was also present in the mosquito. The glycosaminoglycans were isolated, from salivary glands and midguts of the mosquito in quantities sufficient for disaccharide microanalysis. Both of these organs are invaded at different stages of the Plasmodium life cycle. Mosquito heparan sulfate was found to contain the critical trisulfated disaccharide sequence, -->4)beta-D-GlcNS6S(1-->4)-alpha-L-IdoA2S(1-->, that is commonly found in human liver heparan sulfate, which serves as the receptor for apolipoprotein E and is also believed to be responsible for binding to the circumsporozoite protein found on the surface of the Plasmodium sporozoite. The heparan sulfate isolated from the whole mosquito binds to circumsporozoite protein, suggesting a role within the mosquito for infection and transmission of the Plasmodium parasite.
Assuntos
Anopheles/metabolismo , Heparitina Sulfato/metabolismo , Fígado/metabolismo , Malária Falciparum/transmissão , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Anopheles/química , Anopheles/parasitologia , Sequência de Carboidratos , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Dissacarídeos/química , Dissacarídeos/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/genética , Humanos , Fígado/química , Fígado/parasitologia , Malária Falciparum/metabolismo , Plasmodium falciparum/química , Ligação Proteica , Proteínas de Protozoários/química , Glândulas Salivares/química , Glândulas Salivares/metabolismo , Glândulas Salivares/parasitologiaRESUMO
Glycosaminoglycans (GAGs) play a critical role in binding and activation of growth factors involved in cell signaling critical for developmental biology. The biosynthetic pathways for GAGs have been elucidated over the past decade and now analytical methodology makes it possible to determine GAG composition in as few as 10 million cells. A glycomics approach was used to examine GAG content, composition, and the level of transcripts encoding for GAG biosynthetic enzymes as murine embryonic stem cells (mESCs) differentiate to embryoid bodies (EBs) and to extraembryonic endodermal cells (ExE) to better understand the role of GAGs in stem cell differentiation. Hyaluronan synthesis was enhanced by 13- and 24-fold, most likely due to increased expression of hyaluronan synthase-2. Chondroitin sulfate (CS)/dermatan sulfate (DS) synthesis was enhanced by 4- and 6-fold, and heparan sulfate (HS) synthesis was enhanced by 5- and 8-fold following the transition from mESC to EB and ExE. Transcripts associated with the synthesis of the early precursors were largely unaltered, suggesting other factors account for enhanced GAG synthesis. The composition of both CS/DS and HS also changed upon differentiation. Interestingly, CS type E and highly sulfated HS both increase as mESCs differentiate to EBs and ExE. Differentiation was also accompanied by enhanced 2-sulfation in both CS/DS and HS families. Transcript levels for core proteins generally showed increases or remained constant upon mESC differentiation. Finally, transcripts encoding selected enzymes and isoforms, including GlcNAc-4,6-O-sulfotransferase, C5-epimerases, and 3-O-sulfotransferases involved in late GAG biosynthesis, were also enriched. These biosynthetic enzymes are particularly important in introducing GAG fine structure, essential for intercellular communication, cell adhesion, and outside-in signaling. Knowing the changes in GAG fine structure should improve our understanding the biological properties of differentiated stem cells.
Assuntos
Células-Tronco Embrionárias/citologia , Glicômica/métodos , Proteoglicanas/química , Animais , Diferenciação Celular , Proliferação de Células , Dissacarídeos/química , Perfilação da Expressão Gênica , Glicosaminoglicanos/química , Camundongos , Modelos Biológicos , Modelos Químicos , Polissacarídeo-Liases/química , RNA Mensageiro/metabolismo , Tretinoína/químicaRESUMO
Sulfation of macromolecules requires the translocation of a high energy form of nucleotide sulfate, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), from the cytosol into the Golgi apparatus. In this study, we identified a novel Drosophila PAPS transporter gene dPAPST2 by conducting data base searches and screening the PAPS transport activity among the putative nucleotide sugar transporter genes in Drosophila. The amino acid sequence of dPAPST2 showed 50.5 and 21.5% homology to the human PAPST2 and SLALOM, respectively. The heterologous expression of dPAPST2 in yeast revealed that the dPAPST2 protein is a PAPS transporter with an apparent K(m) value of 2.3 microm. The RNA interference of dPAPST2 in cell line and flies showed that the dPAPST2 gene is essential for the sulfation of cellular proteins and the viability of the fly. In RNA interference flies, an analysis of the genetic interaction between dPAPST2 and genes that contribute to glycosaminoglycan synthesis suggested that dPAPST2 is involved in the glycosaminoglycan synthesis and the subsequent signaling. The dPAPST2 and sll genes showed a similar ubiquitous distribution. These results indicate that dPAPST2 may be involved in Hedgehog and Decapentaplegic signaling by controlling the sulfation of heparan sulfate.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Fosfoadenosina Fosfossulfato/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Ânions/química , Transporte Biológico , Proteínas de Drosophila/química , Glicosaminoglicanos/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Asas de Animais/metabolismoRESUMO
A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. Here we show that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disk cells are functionally diverse. The UDP-sugar transporter FRINGE-CONNECTION (FRC) is localized to a subset of the Golgi units distinct from those harboring SULFATELESS (SFL), which modifies glucosaminoglycans (GAGs), and from those harboring the protease RHOMBOID (RHO), which processes the glycoprotein SPITZ (SPI). Whereas the glycosylation and function of NOTCH are affected in imaginal disks of frc mutants, those of SPI and of GAG core proteins are not, even though FRC transports a broad range of glycosylation substrates, suggesting that Golgi units containing FRC and those containing SFL or RHO are functionally separable. Distinct Golgi units containing FRC and RHO in embryos could also be separated biochemically by immunoisolation techniques. We also show that Tn-antigen glycan is localized only in a subset of the Golgi units distributed basally in a polarized cell. We propose that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification.