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1.
Development ; 150(17)2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-37694610

RESUMEN

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.


Asunto(s)
Sulfatos de Condroitina , Drosophila , Animales , Femenino , Drosophila/genética , Folículo Ovárico , Ovario , Glicosaminoglicanos , Mamíferos
2.
J Cell Sci ; 136(7)2023 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-36897575

RESUMEN

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.


Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteoglicanos de Heparán Sulfato/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Sulfatasas/genética , Sulfatasas/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo
3.
J Biol Chem ; 291(46): 24105-24120, 2016 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-27758869

RESUMEN

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling.


Asunto(s)
Hexosaminas/biosíntesis , Ácido Hialurónico/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Transducción de Señal , Animales , Femenino , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Hexosaminas/genética , Ácido Hialurónico/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , Transferasas de Grupos Nitrogenados/genética , Transferasas de Grupos Nitrogenados/metabolismo
4.
J Biol Chem ; 291(44): 23294-23304, 2016 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-27645998

RESUMEN

Chondroitin sulfate (CS)/chondroitin (Chn) chains are indispensable for embryonic cell division and cytokinesis in the early developmental stages in Caenorhabditis elegans and mice, whereas heparan sulfate (HS) is essential for axon guidance during nervous system development. These data indicate that the fundamental functions of CS and HS are conserved from worms to mammals and that the function of CS/Chn differs from that of HS. Although previous studies have shown that C. elegans produces HS and non-sulfated Chn, whether the organism produces CS remains unclear. Here, we demonstrate that C. elegans produces a small amount of 4-O-sulfated Chn and report the identification of C41C4.1, an orthologue of the human chondroitin 4-O-sulfotransferase gene. Loss of C41C4.1 in C. elegans resulted in a decline in 4-O-sulfation of CS and an increase in the number of sulfated units in HS. C41C4.1 deletion mutants exhibited reduced survival rates after synchronization with sodium hypochlorite. Collectively, these results show for the first time that CS glycans are present in C. elegans and that the Chn 4-O-sulfotransferase responsible for the sulfation plays an important role in protecting nematodes from oxidative stress.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/crecimiento & desarrollo , Sulfatos de Condroitina/metabolismo , Condroitín/metabolismo , Estrés Oxidativo , Sulfotransferasas/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , División Celular , Femenino , Heparitina Sulfato/metabolismo , Masculino , Eliminación de Secuencia , Sulfatos/metabolismo , Sulfotransferasas/genética
5.
J Biol Chem ; 290(12): 7576-85, 2015 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-25653284

RESUMEN

Glypican-3 (GPC3) is one of the six members of the mammalian glypican family. We have previously reported that GPC3 inhibits Hedgehog (Hh) signaling by competing with Patched (Ptc) for Hh binding. We also showed that GPC3 binds with high affinity to Hh through its core protein, but that it does not interact with Ptc. Several members of the glypican family, including GPC3, are subjected to an endoproteolytic cleavage by the furin-like convertase family of endoproteases. Surprisingly, however, we have found that a mutant GPC3 that cannot be processed by convertases is as potent as wild-type GPC3 in stimulating Wnt activity in hepatocellular carcinoma cell lines and 293T cells and in promoting hepatocellular carcinoma growth. In this study, we show that processing by convertases is essential for GPC3-induced inhibition of Hh signaling. Moreover, we show that a convertase-resistant GPC3 stimulates Hh signaling by increasing the binding of this growth factor to Ptc. Consistent with this, we show that the convertase-resistant mutant binds to both Hh and Ptc through its heparan sulfate (HS) chains. Unexpectedly, we found that the mutant core protein does not bind to Hh. We also report that the convertase-resistant mutant GPC3 carries HS chains with a significantly higher degree of sulfation than those of wild-type GPC3. We propose that the structural changes generated by the lack of cleavage determine a change in the sulfation of the HS chains and that these hypersulfated chains mediate the interaction of the mutant GPC3 with Ptc.


Asunto(s)
Glipicanos/fisiología , Proteínas Hedgehog/metabolismo , Proproteína Convertasas/metabolismo , Transducción de Señal/fisiología , Células 3T3 , Animales , Células HEK293 , Humanos , Ratones
6.
J Biol Chem ; 290(9): 5438-48, 2015 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-25568321

RESUMEN

A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAß1-3Galß1-3Galß1-4Xyl(2-O-phosphate), in ChGn-1(-/-) growth plate cartilage but not in ChGn-2(-/-) or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAß1-3Galß1-3Galß1-4Xyl was detected in wild-type, ChGn-1(-/-), and ChGn-2(-/-) growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.


Asunto(s)
Acetilgalactosamina/metabolismo , Sulfatos de Condroitina/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Oligosacáridos/metabolismo , Fosfatos/metabolismo , Animales , Animales Recién Nacidos , Vías Biosintéticas/genética , Western Blotting , Células COS , Secuencia de Carbohidratos , Cartílago/citología , Cartílago/embriología , Cartílago/metabolismo , Células Cultivadas , Chlorocebus aethiops , Condrocitos/metabolismo , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Placa de Crecimiento/embriología , Placa de Crecimiento/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , N-Acetilgalactosaminiltransferasas/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Especificidad por Sustrato , Xilosa/metabolismo
7.
J Biol Chem ; 289(10): 6695-6708, 2014 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-24425863

RESUMEN

Recently, we demonstrated that FAM20B is a kinase that phosphorylates the xylose (Xyl) residue in the glycosaminoglycan-protein linkage region of proteoglycans. The phosphorylation of Xyl residues by FAM20B enhances the formation of the linkage region. Rapid dephosphorylation is probably induced just after synthesis of the linker and just before polymerization initiates. Indeed, in vitro chondroitin or heparan sulfate polymerization does not occur when the Xyl residue of the tetrasaccharide linkage region is phosphorylated. However, the enzyme responsible for the dephosphorylation of Xyl remains unknown. Here, we identified a novel protein that dephosphorylates the Xyl residue and designated it 2-phosphoxylose phosphatase. The phosphatase efficiently removed the phosphate from the phosphorylated trisaccharide, Galß1-3Galß1-4Xyl(2-O-phosphate), but not from phosphorylated tetrasaccharide, GlcUAß1-3Galß1-3Galß1-4Xyl(2-O-phosphate). Additionally, RNA interference-mediated inhibition of 2-phosphoxylose phosphatase resulted in increased amounts of GlcNAcα1-4GlcUAß1-3Galß1-3Galß1-4Xyl(2-O-phosphate), Galß1-3Galß1-4Xyl(2-O-phosphate), and Galß1-4Xyl(2-O-phosphate) in the cells. Gel filtration analysis of the glycosaminoglycan chains synthesized in the knockdown cells revealed that these cells produced decreased amounts of glycosaminoglycan chains and that the chains had similar lengths to those in the mock-transfected cells. Transcripts encoding this phosphatase were ubiquitously, but differentially, expressed in human tissues. Moreover, the phosphatase localized to the Golgi and interacted with the glucuronyltransferase-I involved in the completion of the glycosaminoglycan-protein linkage region. Based on these findings, we conclude that transient phosphorylation of the Xyl residue in the glycosaminoglycan-protein linkage region controls the formation of glycosaminoglycan chains of proteoglycans.


Asunto(s)
Glicosaminoglicanos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteoglicanos/metabolismo , Xilosa/metabolismo , Secuencia de Aminoácidos , Condroitín/metabolismo , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Polimerizacion
8.
Biochem Biophys Res Commun ; 460(2): 233-7, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25772620

RESUMEN

Thrombomodulin (TM) is a cell-surface glycoprotein and a critical mediator of endothelial anticoagulant function. TM exists as both a chondroitin sulfate (CS) proteoglycan (PG) form and a non-PG form lacking a CS chain (α-TM); therefore, TM can be described as a part-time PG. Previously, we reported that α-TM bears an immature, truncated linkage tetrasaccharide structure (GlcAß1-3Galß1-3Galß1-4Xyl). However, the biosynthetic mechanism to generate part-time PGs remains unclear. In this study, we used several mutants to demonstrate that the amino acid sequence surrounding the CS attachment site influences the efficiency of chondroitin polymerization. In particular, the presence of acidic residues surrounding the CS attachment site was indispensable for the elongation of CS. In addition, mutants defective in CS elongation did not exhibit anti-coagulant activity, as in the case with α-TM. Together, these data support a model for CS chain assembly in which specific core protein determinants are recognized by a key biosynthetic enzyme involved in chondroitin polymerization.


Asunto(s)
Sulfatos de Condroitina/metabolismo , Condroitín/química , Trombomodulina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Cartilla de ADN , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Polimerizacion , Trombomodulina/química , Trombomodulina/genética
9.
Biochim Biophys Acta ; 1830(10): 4806-12, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23811343

RESUMEN

BACKGROUND: Previously, we identified two missense mutations in the chondroitin N-acetylgalactosaminyltransferase-1 gene in patients with neuropathy. These mutations are associated with a profound decrease in chondroitin N-acetylgalactosaminyltransferase-1 enzyme activity. Here, we describe a patient with neuropathy who is heterozygous for a chondroitin synthase-1 mutation. Chondroitin synthase-1 has two glycosyltransferase activities: it acts as a GlcUA and a GalNAc transferase and is responsible for adding repeated disaccharide units to growing chondroitin sulfate chains. METHODS: Recombinant wild-type chondroitin synthase-1 enzyme and the F362S mutant were expressed. These enzymes and cells expressing them were then characterized. RESULTS: The mutant chondroitin synthase-1 protein retained approximately 50% of each glycosyltransferase activity relative to the wild-type chondroitin synthase-1 protein. Furthermore, unlike chondroitin polymerase comprised of wild-type chondroitin synthase-1 protein, the non-reducing terminal 4-O-sulfation of GalNAc residues synthesized by chondroitin N-acetylgalactosaminyltransferase-1 did not facilitate the elongation of chondroitin sulfate chains when chondroitin polymerase that consists of the mutant chondroitin synthase-1 protein was used as the enzyme source. CONCLUSIONS: The chondroitin synthase-1 F362S mutation in a patient with neuropathy resulted in a decrease in chondroitin polymerization activity and the mutant protein was defective in regulating the number of chondroitin sulfate chains via chondroitin N-acetylgalactosaminyltransferase-1. Thus, the progression of peripheral neuropathies may result from defects in these regulatory systems. GENERAL SIGNIFICANCE: The elongation of chondroitin sulfate chains may be tightly regulated by the cooperative expression of chondroitin synthase-1 and chondroitin N-acetylgalactosaminyltransferase-1 in peripheral neurons and peripheral neuropathies may result from synthesis of abnormally truncated chondroitin sulfate chains.


Asunto(s)
Sulfatos de Condroitina/metabolismo , Mutación Missense , N-Acetilgalactosaminiltransferasas/metabolismo , Enfermedades del Sistema Nervioso/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/genética , Enfermedades del Sistema Nervioso/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Homología de Secuencia de Aminoácido
10.
Biochem J ; 441(2): 697-705, 2012 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-21942880

RESUMEN

Recently, it has been shown that a deficiency in ChGn-1 (chondroitin N-acetylgalactosaminyltransferase-1) reduced the numbers of CS (chondroitin sulfate) chains, leading to skeletal dysplasias in mice. Although these results indicate that ChGn-1 regulates the number of CS chains, the mechanism mediating this regulation is not clear. ChGn-1 is thought to initiate CS biosynthesis by transferring the first GalNAc (N-acetylgalactosamine) to the tetrasaccharide in the protein linkage region of CS. However, in vitro chondroitin polymerization does not occur on the non-reducing terminal GalNAc-linkage pentasaccharide structure. In the present study we show that several different heteromeric enzyme complexes composed of different combinations of four chondroitin synthase family members synthesized more CS chains when a GalNAc-linkage pentasaccharide structure with a non-reducing terminal 4-O-sulfation was the CS acceptor. In addition, C4ST-2 (chondroitin 4-O-sulfotransferase-2) efficiently transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of non-reducing terminal GalNAc-linkage residues, and the number of CS chains was regulated by the expression levels of C4ST-2 and of ChGn-1. Taken together, the results of the present study indicate that C4ST-2 plays a key role in regulating levels of CS synthesized via ChGn-1.


Asunto(s)
Sulfatos de Condroitina/biosíntesis , N-Acetilgalactosaminiltransferasas/metabolismo , Sulfotransferasas/fisiología , Acetilgalactosamina/metabolismo , Animales , Células L , Ratones , Sulfotransferasas/metabolismo
11.
Biochem Biophys Res Commun ; 420(3): 523-9, 2012 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-22440395

RESUMEN

Bone formation in the vertebrate skeleton occurs via the processes of endochondral and membranous ossification. Bone matrices contain chondroitin sulfate (CS) chains that regulate endochondral ossification. However, the function of CS in membranous ossification is unclear. Here, using preosteoblastic MC3T3-E1 cells we demonstrate that chondroitin sulfate-E (CS-E) promotes osteoblast differentiation by binding to both N-cadherin and cadherin-11. Differentiated MC3T3-E1 cells exhibited an increase in the total amount of CS and of E-disaccharide units of CS over time. In addition, CS-E polysaccharide, but not CS-A polysaccharide, bound to N-cadherin and cadherin-11 and enhanced osteoblast differentiation. In contrast, osteoblast differentiation was inhibited in chondroitinase ABC-digested MC3T3-E1 cells. Notably, CS-E polysaccharide and hexasaccharide activated intracellular signaling during osteoblast differentiation in non-contacting MC3T3-E1 cells, decreased ERK1/2 phosphorylation, and activated Smad3 and Smad1/5/8; these reactions were blocked by neutralizing antibodies against N-cadherin or cadherin-11, even though cell-cell adhesion is reported to be required for initiation of MC3T3-E1 cell differentiation. Furthermore, CS-E-unit overexpression in MC3T3-E1 cells increased adhesion of the cells to N-cadherin and cadherin-11, and promoted osteoblast differentiation. Collectively, these results suggest that CS-E is a selective ligand for the potential CS receptors, N-cadherin and cadherin-11, leading to osteoblast differentiation of MC3T3-E1 cells.


Asunto(s)
Diferenciación Celular , Sulfatos de Condroitina/metabolismo , Osteoblastos/citología , Osteogénesis , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Sulfatos de Condroitina/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteoblastos/metabolismo , Osteogénesis/efectos de los fármacos , Proteína Smad1/metabolismo , Proteína smad3/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo
12.
Biochem J ; 434(2): 321-31, 2011 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21138417

RESUMEN

Previously, we demonstrated that sog9 cells, a murine L cell mutant, are deficient in the expression of C4ST (chondroitin 4-O-sulfotransferase)-1 and that they synthesize fewer and shorter CS (chondroitin sulfate) chains. These results suggested that C4ST-1 regulates not only 4-O-sulfation of CS, but also the length and amount of CS chains; however, the mechanism remains unclear. In the present study, we have demonstrated that C4ST-1 regulates the chain length and amount of CS in co-operation with ChGn-2 (chondroitin N-acetylgalactosaminyltransferase 2). Overexpression of ChGn-2 increased the length and amount of CS chains in L cells, but not in sog9 mutant cells. Knockdown of ChGn-2 resulted in a decrease in the amount of CS in L cells in a manner proportional to ChGn-2 expression levels, whereas the introduction of mutated C4ST-1 or ChGn-2 lacking enzyme activity failed to increase the amount of CS. Furthermore, the non-reducing terminal 4-O-sulfation of N-acetylgalactosamine residues facilitated the elongation of CS chains by chondroitin polymerase consisting of chondroitin synthase-1 and chondroitin-polymerizing factor. Overall, these results suggest that the chain length of CS is regulated by C4ST-1 and ChGn-2 and that the enzymatic activities of these proteins play a critical role in CS elongation.


Asunto(s)
Sulfatos de Condroitina/metabolismo , N-Acetilgalactosaminiltransferasas/metabolismo , Sulfotransferasas/metabolismo , Acetilgalactosamina/metabolismo , Animales , Línea Celular Tumoral , Condroitín/metabolismo , Humanos , Células L , Ratones , Especificidad por Sustrato , Sulfotransferasas/genética , Transfección
13.
J Biol Chem ; 285(16): 12190-6, 2010 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-20164174

RESUMEN

We have revealed that in Caenorhabditis elegans, non-sulfated chondroitin is required for normal cell division and cytokinesis at an early developmental stage, whereas heparan sulfate is essential for embryonic morphogenesis in the later stages of development. To clarify the roles of chondroitin sulfate and heparan sulfate in early embryogenesis in mammals, we generated glucuronyltransferase-I (GlcAT-I) knock-out mice by gene targeting. GlcAT-I is an enzyme required for the synthesis of both chondroitin sulfate and heparan sulfate. Here we report that mice with a deletion of GlcAT-I showed remarkable reduction of the synthesis of chondroitin sulfate and heparan sulfate and embryonic lethality before the 8-cell stage because of failed cytokinesis. In addition, treatment of wild-type 2-cell embryos with chondroitinase ABC had marked effects on cell division, although many heparitinase-treated embryos normally developed to blastocysts. Taken together, these results suggest that chondroitin sulfate in mammals, as with non-sulfated chondroitin in C. elegans, is indispensable for embryonic cell division.


Asunto(s)
División Celular/fisiología , Fase de Segmentación del Huevo/citología , Fase de Segmentación del Huevo/metabolismo , Glucuronosiltransferasa/deficiencia , Glicosaminoglicanos/biosíntesis , Animales , Secuencia de Bases , Caenorhabditis elegans/citología , Caenorhabditis elegans/embriología , Caenorhabditis elegans/metabolismo , Sulfatos de Condroitina/biosíntesis , Cruzamientos Genéticos , Citocinesis/fisiología , Cartilla de ADN/genética , Técnicas de Cultivo de Embriones , Desarrollo Embrionario/fisiología , Femenino , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Heparitina Sulfato/biosíntesis , Heterocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de la Especie
14.
J Biol Chem ; 285(32): 24717-28, 2010 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-20529843

RESUMEN

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 , Transgenes
15.
Biochem Biophys Res Commun ; 415(1): 109-13, 2011 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-22020094

RESUMEN

HNK-1 (human natural killer-1) carbohydrate epitope (HSO(3)-3GlcAß1-3Galß1-4GlcNAc-) recognized by a HNK-1 monoclonal antibody is highly expressed in the nervous system and biosynthesized by a glucuronyltransferase (GlcAT-P or GlcAT-S), and sulfotransferase (HNK-1ST). A similar oligosaccharide (HSO(3)-3GlcAß1-3Galß1-3Galß1-4Xyl) also recognized by the HNK-1 antibody had been found in a glycosaminoglycan (GAG)-protein linkage region of α-thrombomodulin (TM) from human urine. However, which sulfotransferase is involved in sulfation of the terminal GlcA in the GAG-protein linkage region remains unclear. In this study, using CHO-K1 cells in which neither GlcAT-P nor GlcAT-S is endogenously expressed, we found that HNK-1ST has the ability to produce HNK-1 immunoreactivity on α-TM. We also demonstrated that HNK-1ST caused the suppression of chondroitin sulfate (CS) synthesis on TM and a reduction of its anti-coagulant activity. Moreover, using an in vitro enzyme assay system, the HNK-1-positive TM was found not to be utilized as a substrate for CS-polymerizing enzymes (chondroitin synthase (ChSy) and chondroitin polymerizing factor (ChPF)). These results suggest that HNK-1ST is involved in 3-O-sulfation of the terminal GlcA of the linkage tetrasaccharide which acts as an inhibitory signal for the initiation of CS biosynthesis on TM.


Asunto(s)
Sulfatos de Condroitina/biosíntesis , Ácido Glucurónico/metabolismo , Oligosacáridos/metabolismo , Sulfotransferasas/metabolismo , Trombomodulina/metabolismo , Animales , Secuencia de Carbohidratos , Línea Celular , Sulfatos de Condroitina/química , Cricetinae , Cricetulus , Humanos , Datos de Secuencia Molecular , Oligosacáridos/química , Sulfatos , Trombomodulina/química
16.
Biochem Biophys Res Commun ; 406(1): 36-41, 2011 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-21284936

RESUMEN

Subendothelial retention of lipoproteins by proteoglycans (PGs) is the initiating event in atherosclerosis. The elongation of chondroitin sulfate (CS) chains is associated with increased low-density lipoprotein (LDL) binding and progression of atherosclerosis. Recently, it has been shown that 2 Golgi enzymes, chondroitin 4-O-sulfotransferase-1 (C4ST-1) and chondroitin N-acetylgalactosaminyltransferase-2 (ChGn-2), play a critical role in CS chain elongation. However, the roles of C4ST-1 and ChGn-2 during the progression of atherosclerosis are not known. The aim of this study was to analyze the expression of C4ST-1 and ChGn-2 in atherosclerotic lesions in vivo and determine whether their expression correlated with CS chain elongation. Low-density lipoprotein receptor knockout (LDLr KO) mice were fed a western diet for 2, 4, and 8weeks to stimulate development of atherosclerosis. The binding of LDL and CS PG in this mouse model was confirmed by chondroitinase ABC (ChABC) digestion and apolipoprotein B (apo B) staining. Gel filtration analysis revealed that the CS chains began to elongate as early as 2weeks after beginning a western diet and continued as the atherosclerosis progressed. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the mRNA levels of C4ST-1 and ChGn-2 increased after 8weeks of this diet. In contrast, the mRNA levels of their homologs, C4ST-2 and ChGn-1, were unchanged. In addition, immunohistochemical analysis demonstrated that the expression of C4ST-1 and ChGn-2 appeared to have similar site-specific patterns and coincided with biglycan expression at the aortic root. Our results suggested that C4ST-1 and ChGn-2 may be involved in the elongation of CS chains in the arterial wall during the progression of atherosclerosis. Therefore, modulating their expression and activity might be a novel therapeutic strategy for atherosclerosis.


Asunto(s)
Aterosclerosis/metabolismo , Sulfatos de Condroitina/metabolismo , N-Acetilgalactosaminiltransferasas/biosíntesis , Sulfotransferasas/biosíntesis , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/patología , Biglicano/metabolismo , Lipoproteínas LDL/metabolismo , Ratones , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/genética , Receptores de LDL/genética , Sulfotransferasas/genética
17.
J Hum Genet ; 56(2): 143-6, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21160489

RESUMEN

Chondroitin sulfate proteoglycans (CSPGs) in the peripheral nervous system likely participate as regulatory molecules in the process of axonal degeneration and regeneration. We investigated the chondroitin beta1,4-N-acetylgalactosaminyltransferase-1 (ChGn-1) gene in 114 patients affected with neuropathies including Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, hereditary motor and sensory neuropathy (HMSN) and unknown etiology. The controls were 196 patients with other neurological diseases. We found novel missense mutations in two patients with neuropathy (Bell's palsy, unknown HMSN) in exons 5 (H234R) and 10 (M509R), respectively. None of the patients with other neurological diseases had either of these mutations. We then synthesized the two soluble forms of ChGn-1, containing each of the above mutations. Each of the soluble mutants was expressed in COS-1 cells and the mutant proteins were purified. The purified mutant proteins were used for western blotting analysis using an anti-ChGn-1 antibody and evaluated for glycosyltransferase activities. Although the expression of the ChGn-1 mutant proteins was confirmed by western blotting, they exhibited no N-acetylgalactosamineT-II activities. It is possible that these mutations are associated with the pathogenetic mechanisms of the peripheral neuropathies.


Asunto(s)
Enfermedades Desmielinizantes/genética , Mutación Missense , N-Acetilgalactosaminiltransferasas/genética , Enfermedades del Sistema Nervioso Periférico/genética , Animales , Células COS , Estudios de Casos y Controles , Chlorocebus aethiops , Síndrome de Guillain-Barré/genética , Neuropatía Hereditaria Motora y Sensorial/genética , Humanos , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/genética
18.
Biochem J ; 432(1): 47-55, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20812917

RESUMEN

CS (chondroitin sulfate) is a glycosaminoglycan species that is widely distributed in the extracellular matrix. To understand the physiological roles of enzymes involved in CS synthesis, we produced CSGalNAcT1 (CS N-acetylgalactosaminyltransferase 1)-null mice. CS production was reduced by approximately half in CSGalNAcT1-null mice, and the amount of short-chain CS was also reduced. Moreover, the cartilage of the null mice was significantly smaller than that of wild-type mice. Additionally, type-II collagen fibres in developing cartilage were abnormally aggregated and disarranged in the homozygous mutant mice. These results suggest that CSGalNAcT1 is required for normal CS production in developing cartilage.


Asunto(s)
Condrogénesis , Sulfatos de Condroitina/biosíntesis , Placa de Crecimiento/enzimología , N-Acetilgalactosaminiltransferasas/metabolismo , Animales , Western Blotting , Línea Celular , Proliferación Celular , Condrocitos/citología , Condrocitos/enzimología , Condrocitos/metabolismo , Colágeno Tipo II/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Placa de Crecimiento/embriología , Placa de Crecimiento/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , N-Acetilgalactosaminiltransferasas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
19.
Biochem J ; 421(2): 157-62, 2009 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-19473117

RESUMEN

2-O-phosphorylation of xylose has been detected in the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans. Recent mutant analyses in zebrafish suggest that xylosyltransferase I and FAM20B, a protein of unknown function that shows weak similarity to a Golgi kinase encoded by four-jointed, operate in a linear pathway for proteoglycan production. In the present study, we identified FAM20B as a kinase that phosphorylates the xylose residue in the linkage region. Overexpression of FAM20B increased the amount of both chondroitin sulfate and heparan sulfate in HeLa cells, whereas the RNA interference of FAM20B resulted in a reduction of their amount in the cells. Gel-filtration analysis of the glycosaminoglycan chains synthesized in the overexpressing cells revealed that the glycosaminoglycan chains had a similar length to those in mock-transfected cells. These results suggest that FAM20B regulates the number of glycosaminoglycan chains by phosphorylating the xylose residue in the glycosaminoglycan-protein linkage region of proteoglycans.


Asunto(s)
Glicosaminoglicanos/metabolismo , Fosfotransferasas/metabolismo , Xilosa/metabolismo , Animales , Células COS , Chlorocebus aethiops , Cromatografía en Gel , ADN Complementario/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación , Fosfotransferasas/genética , Interferencia de ARN , Transfección
20.
Cell Death Dis ; 10(11): 803, 2019 10 23.
Artículo en Inglés | MEDLINE | ID: mdl-31645543

RESUMEN

The hexosamine biosynthetic pathway (HBP) metabolically regulates dynamic cellular events by linking nutrient availability to numerous signaling networks. Significant alterations in the HBP are often associated with cancer pathogenesis. In this study, we investigated the molecular events underlying cancer pathogenesis associated with enhanced HBP flux. Multidimensional analysis of microarray datasets demonstrated up-regulation of genes encoding HBP enzymes in clinical breast cancers and revealed that co-expression of hyaluronan synthase 2 (HAS2) and glutamine:fructose-6-phosphate amidotransferase (GFAT), a rate-limiting enzyme of the HBP, was strongly correlated with a poor prognosis in advanced cancer patients. Consistently with the clinical data, comparative analyses of distinct breast cancer mouse models demonstrated enhancement of the HBP gene expression in primary carcinoma cells, with elevation of Has2 expression and hyaluronan production in aggressive breast cancer cells. The silencing of GFAT reduced CD44high/CD24low cancer stem cell (CSC)-like subpopulations, aldehyde dehydrogenase-positive cell populations, and mammosphere size, which were further diminished by gene targeting of Has2. Has2 gene disruption reduced the in vivo growth of aggressive cancer cells and attenuated pro-tumorigenic Akt/GSK3ß/ß-catenin signaling and cisplatin resistance. Overall protein O-GlcNAcylation was also elevated in association with HBP enhancement in aggressive cancer cells, and the modification exhibited overlapping but distinct roles from the hyaluronan signal in the regulation of CSC-like features. The current data therefore demonstrate that enhanced hexosamine metabolism drives pro-tumorigenic signaling pathways involving hyaluronan and O-GlcNAcylation in aggressive breast cancer.


Asunto(s)
Neoplasias de la Mama/metabolismo , Hexosaminas/metabolismo , Ácido Hialurónico/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones Transgénicos , Transducción de Señal
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