RESUMEN
Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.
Asunto(s)
Dermatán Sulfato , Pez Cebra , Animales , Sulfatos de Condroitina/metabolismo , Dermatán Sulfato/genética , Dermatán Sulfato/metabolismo , Glicosiltransferasas/genética , Fenotipo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.
Asunto(s)
Borrelia burgdorferi/metabolismo , Dermatán Sulfato/metabolismo , Matriz Extracelular/metabolismo , Artropatías/metabolismo , Articulaciones/metabolismo , Enfermedad de Lyme/metabolismo , Animales , Antígenos Bacterianos , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidad , Dermatán Sulfato/genética , Matriz Extracelular/genética , Matriz Extracelular/microbiología , Matriz Extracelular/patología , Femenino , Fibronectinas/genética , Fibronectinas/metabolismo , Artropatías/genética , Artropatías/microbiología , Artropatías/patología , Articulaciones/microbiología , Articulaciones/patología , Enfermedad de Lyme/genética , Enfermedad de Lyme/microbiología , Enfermedad de Lyme/patología , Ratones , Ratones SCID , Mutación , Especificidad de ÓrganosRESUMEN
CXCL14, chemokine (C-X-C motif) ligand 14, is a novel highly conserved chemokine with unique features. Despite exhibiting the typical chemokine fold, it has a very short N-terminus of just two amino acid residues responsible for chemokine receptor activation. CXCL14 actively participates in homeostatic immune surveillance of skin and mucosae, is linked to metabolic disorders and fibrotic lung diseases and possesses strong anti-angiogenic properties in early tumor development. In this work, we investigated the interaction of CXCL14 with various glycosaminoglycans (GAGs) by nuclear magnetic resonance spectroscopy, microscale thermophoresis, analytical heparin (HE) affinity chromatography and in silico approaches to understand the molecular basis of GAG-binding. We observed different GAG-binding modes specific for the GAG type used in the study. In particular, the CXCL14 epitope for HE suggests a binding pose distinguishable from the ones of the other GAGs investigated (hyaluronic acid, chondroitin sulfate-A/C, -D, dermatan sulfate). This observation is also supported by computational methods that included molecular docking, molecular dynamics and free energy calculations. Based on our results, we suggest that distinct GAG sulfation patterns confer specificity beyond simple electrostatic interactions usually considered to represent the driving forces in protein-GAG interactions. The CXCL14-GAG system represents a promising approach to investigate the specificity of GAG-protein interactions, which represents an important topic for developing the rational approaches to novel strategies in regenerative medicine.
Asunto(s)
Quimiocinas CXC/metabolismo , Epítopos/genética , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Sitios de Unión/genética , Quimiocinas CXC/química , Quimiocinas CXC/genética , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Dermatán Sulfato/química , Dermatán Sulfato/genética , Epítopos/química , Glicosaminoglicanos/química , Glicosaminoglicanos/genética , Heparina/genética , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica/genética , Pliegue de ProteínaRESUMEN
The Mucopolysaccharidoses (MPS) are group of inherited metabolic diseases caused by the deficiency of enzymes required to degrade glycosaminoglycans (GAGs) in the lysosomes. GAGs are sulfated polysaccharides involving repeating disaccharides, uronic acid and hexosamines including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS). Hyaluronan is excluded in terms of being non-sulfated in the GAG family. Different types of mutations have been identified as the causative agent in all types of MPS. Herein, we planned to investigate the pathogenic mutations in different types of MPS including type I (IDUA gene), IIIA (SGSH) and IIIB (NAGLU) in the eight Iranian patients. Autozygosity mapping was performed to identify the potential pathogenic variants in these 8 patients indirectly with the clinical diagnosis of MPSs. so three panels of STR (Short Tandem Repeat) markres flanking IDUA, SGSH and NAGLU genes were selected for multiplex PCR amplification. Then in each family candidate gene was sequenced to identify the pathogenic mutation. Our study showed two novel mutations c.469 T > C and c.903C > G in the IDUA gene, four recurrent mutations: c.1A > C in IDUA, c.220C > T, c.1298G > A in SGSH gene and c.457G > A in the NAGLU gene. The c.1A > C in IDUA was the most common mutation in our study. In silico analysis were performed as well to predict the pathogenicity of the novel variants.
Asunto(s)
Análisis Mutacional de ADN/métodos , Pruebas Genéticas/métodos , Mucopolisacaridosis/genética , Mutación , Adolescente , Niño , Preescolar , Sulfatos de Condroitina/genética , Dermatán Sulfato/genética , Femenino , Heparitina Sulfato/genética , Humanos , Lactante , Sulfato de Queratano/genética , Masculino , Reacción en Cadena de la Polimerasa MultiplexRESUMEN
The main structural component of connective tissues is fibrillar, cross-linked collagen whose fibrillogenesis can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs). Not all SLRPs' effects on collagen and extracellular matrix in vivo have been elucidated; one of the less investigated SLRPs is asporin. Here we describe the successful generation of an Aspn-/- mouse model and the investigation of the Aspn-/- skin phenotype. Functionally, Aspn-/- mice had an increased skin mechanical toughness, although there were no structural changes present on histology or immunohistochemistry. Electron microscopy analyses showed 7% thinner collagen fibrils in Aspn-/- mice (not statistically significant). Several matrix genes were upregulated, including collagens (Col1a1, Col1a2, Col3a1), matrix metalloproteinases (Mmp2, Mmp3) and lysyl oxidases (Lox, Loxl2), while lysyl hydroxylase (Plod2) was downregulated. Intriguingly no differences were observed in collagen protein content or in collagen cross-linking-related lysine oxidation or hydroxylation. The glycosaminoglycan content and structure in Aspn-/- skin was profoundly altered: chondroitin/dermatan sulfate was more than doubled and had an altered composition, while heparan sulfate was halved and had a decreased sulfation. Also, decorin and biglycan were doubled in Aspn-/- skin. Overall, asporin deficiency changes skin glycosaminoglycan composition, and decorin and biglycan content, which may explain the changes in skin mechanical properties.
Asunto(s)
Biglicano/genética , Decorina/genética , Proteínas de la Matriz Extracelular/deficiencia , Efecto Fundador , Regulación de la Expresión Génica , Piel/metabolismo , Aminoácido Oxidorreductasas/genética , Aminoácido Oxidorreductasas/metabolismo , Animales , Biglicano/metabolismo , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Decorina/metabolismo , Dermatán Sulfato/análogos & derivados , Dermatán Sulfato/genética , Dermatán Sulfato/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Femenino , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Sulfato de Queratano/genética , Sulfato de Queratano/metabolismo , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Fenotipo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Piel/ultraestructuraRESUMEN
Glycosaminoglycans (GAGs), such as chondroitin sulfate (CS) and dermatan sulfate (DS) from various vertebrate and invertebrate sources are known to be involved in diverse cellular mechanisms during repair and regenerative processes. Recently, we have identified CS/DS as the major GAG in the brittlestar Amphiura filiformis, with high proportions of di- and tri-O-sulfated disaccharide units. As this echinoderm is known for its exceptional regeneration capacity, we aimed to explore the role of these GAG chains during A. filiformis arm regeneration. Analysis of CS/DS chains during the regeneration process revealed an increase in the proportion of the tri-O-sulfated disaccharides. Conversely, treatment of A. filiformis with sodium chlorate, a potent inhibitor of sulfation reactions in GAG biosynthesis, resulted in a significant reduction in arm growth rates with total inhibition at concentrations higher than 5 mM. Differentiation was less impacted by sodium chlorate exposure or even slightly increased at 1-2 mM. Based on the structural changes observed during arm regeneration we identified chondroitin synthase, chondroitin-4-O-sulfotransferase 2 and dermatan-4-O-sulfotransferase as candidate genes and sought to correlate their expression with the expression of the A. filiformis orthologue of bone morphogenetic factors, AfBMP2/4. Quantitative amplification by real-time PCR indicated increased expression of chondroitin synthase and chondroitin-4-O-sulfotransferase 2, with a corresponding increase in AfBMP2/4 during regeneration relative to nonregenerating controls. Our findings suggest that proper sulfation of GAGs is important for A. filiformis arm regeneration and that these molecules may participate in mechanisms controlling cell proliferation.
Asunto(s)
Sulfatos de Condroitina/biosíntesis , Dermatán Sulfato/biosíntesis , Glicosaminoglicanos/biosíntesis , Regeneración/genética , Animales , Proliferación Celular/genética , Cloratos/farmacología , Sulfatos de Condroitina/genética , Dermatán Sulfato/genética , Disacáridos/genética , Disacáridos/metabolismo , Equinodermos/genética , Equinodermos/crecimiento & desarrollo , Glicosaminoglicanos/genética , Sulfotransferasas/genéticaRESUMEN
Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (ß4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of ß4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in ß4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.
Asunto(s)
Sulfatos de Condroitina/biosíntesis , Dermatán Sulfato/análogos & derivados , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Heparitina Sulfato/biosíntesis , Ácido Hialurónico/biosíntesis , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Sulfatos de Condroitina/antagonistas & inhibidores , Sulfatos de Condroitina/genética , Dermatán Sulfato/antagonistas & inhibidores , Dermatán Sulfato/biosíntesis , Dermatán Sulfato/genética , Células Epiteliales/patología , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Heparitina Sulfato/antagonistas & inhibidores , Heparitina Sulfato/genética , Humanos , Hialuronano Sintasas/antagonistas & inhibidores , Hialuronano Sintasas/genética , Hialuronano Sintasas/metabolismo , Ácido Hialurónico/antagonistas & inhibidores , Ácido Hialurónico/genética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , N-Acetil-Lactosamina Sintasa/antagonistas & inhibidores , N-Acetil-Lactosamina Sintasa/genética , N-Acetil-Lactosamina Sintasa/metabolismo , Proteínas de Transporte de Nucleótidos/antagonistas & inhibidores , Proteínas de Transporte de Nucleótidos/genética , Proteínas de Transporte de Nucleótidos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulphate/dermatan sulphate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. This study was carried out to determine whether diet-induced hypercholesterolemia had any effect on erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulphated and 4-O-sulphated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding.
Asunto(s)
Sulfatos de Condroitina/sangre , Dermatán Sulfato/sangre , Eritrocitos/metabolismo , Hipercolesterolemia/sangre , Animales , Adhesión Celular/genética , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/genética , Dermatán Sulfato/biosíntesis , Dermatán Sulfato/genética , Diabetes Mellitus/sangre , Diabetes Mellitus/patología , Matriz Extracelular/metabolismo , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/patología , Ratas , Relación Estructura-ActividadRESUMEN
Widespread skeletal muscle degeneration and impaired regeneration lead to progressive muscle weakness and premature death in patients with Duchenne muscular dystrophy (DMD). Dystrophic muscles are progressively replaced by nonfunctional tissue because of exhaustion of muscle precursor cells and excessive accumulation of extracellular matrix (ECM). Sulfated glycosaminoglycans (GAGs) are components of the ECM and are increasingly implicated in the regulation of biologic processes, but their possible role in the progression of DMD pathology is not understood. In the present study, we performed immunohistochemical and biochemical analyses of endogenous GAGs in skeletal muscle biopsies of 10 DMD patients and 11 healthy individuals (controls). Immunostaining targeted to specific GAG species showed greater deposition of chondroitin sulfate (CS)/dermatan (DS) sulfate in DMD patient biopsies versus control biopsies. The selective accumulation of CS/DS in DMD biopsies was confirmed by biochemical quantification assay. In addition, high-performance liquid chromatography analysis demonstrated a modification of the sulfation pattern of CS/DS disaccharide units in DMD muscles. In conclusion, our data open up a new path of investigation and suggest that GAGs could represent a new and original therapeutic target for improving the success of gene or cell therapy for the treatment of muscular dystrophies.
Asunto(s)
Sulfatos de Condroitina/metabolismo , Dermatán Sulfato/análogos & derivados , Glicosaminoglicanos/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/patología , Adolescente , Adulto , Niño , Sulfatos de Condroitina/genética , Cromatografía Líquida de Alta Presión , Dermatán Sulfato/genética , Dermatán Sulfato/metabolismo , Progresión de la Enfermedad , Femenino , Glicosaminoglicanos/genética , Humanos , Masculino , ARN Mensajero/genética , Adulto JovenRESUMEN
The hyaluronan (HA) receptor for endocytosis (HARE; Stab2) clears 14 systemic ligands, including HA and heparin. Here, we used NF-κB promoter-driven luciferase reporter assays to test HARE-mediated intracellular signaling during the uptake of eight ligands, whose binding sites in the HARE ectodomain were mapped by competition studies (Harris, E. N., and Weigel, P. H. (2008) Glycobiology 18, 638-648). Unique intermediate size Select-HA(TM), heparin, dermatan sulfate, and acetylated LDL stimulated dose-dependent HARE-mediated NF-κB activation of luciferase expression, with half-maximal values of 10-25 nM. In contrast, chondroitin sulfate types A, C, D, and E did not stimulate NF-κB activation. Moreover, degradation of endogenous IkB-α (an NF-κB inhibitor) was stimulated only by the signaling ligands. The stimulatory activities of pairwise combinations of the four signaling ligands were additive. The four nonstimulatory chondroitin sulfate types, which compete for HA binding, also effectively blocked HA-stimulated signaling. Clathrin siRNA decreased clathrin expression by â¼50% and completely eliminated NF-κB-mediated signaling by all four ligands, indicating that activation of signaling complexes occurs after endocytosis. These results indicate that HARE not only binds and clears extracellular matrix degradation products (e.g. released normally or during infection, injury, tumorigenesis, or other stress situations) but that a subset of ligands also serves as signaling indicator ligands. HARE may be part of a systemic tissue-stress sensor feedback system that responds to abnormal tissue turnover or damage as a danger signal; the signaling indicator ligands would reflect the homeostatic status, whether normal or pathological, of tissue cells and biomatrix components.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Sulfatos de Condroitina/metabolismo , Dermatán Sulfato/metabolismo , Endocitosis/fisiología , Regulación de la Expresión Génica/fisiología , Ácido Hialurónico/metabolismo , Lipoproteínas LDL/metabolismo , FN-kappa B/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Sulfatos de Condroitina/genética , Dermatán Sulfato/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Ácido Hialurónico/genética , Lipoproteínas LDL/genética , FN-kappa B/genética , Transducción de Señal/fisiologíaRESUMEN
A number of genetic disorders are caused by mutations in the genes encoding glycosyltransferases and sulfotransferases, enzymes responsible for the synthesis of sulfated glycosaminoglycan (GAG) side chains of proteoglycans, including chondroitin sulfate, dermatan sulfate, and heparan sulfate. The phenotypes of these genetic disorders reflect disturbances in crucial biological functions of GAGs in human. Recent studies have revealed that mutations in genes encoding chondroitin sulfate and dermatan sulfate biosynthetic enzymes cause various disorders of connective tissues. This minireview focuses on growing glycobiological studies of recently described genetic diseases caused by disturbances in biosynthetic enzymes for sulfated GAGs.
Asunto(s)
Sulfatos de Condroitina/genética , Enfermedades del Tejido Conjuntivo , Dermatán Sulfato/genética , Glicosaminoglicanos/genética , Heparitina Sulfato/genética , Errores Innatos del Metabolismo , Sulfatos de Condroitina/biosíntesis , Enfermedades del Tejido Conjuntivo/enzimología , Enfermedades del Tejido Conjuntivo/genética , Dermatán Sulfato/biosíntesis , Glicosaminoglicanos/biosíntesis , Heparitina Sulfato/biosíntesis , Humanos , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genéticaRESUMEN
The outermost positions of mammalian cell-surface glycans are predominantly occupied by the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). To date, hydroxylation of CMP-Neu5Ac resulting in the conversion into CMP-Neu5Gc is the only known enzymatic reaction in mammals to synthesize a monosaccharide carrying an N-glycolyl group. In our accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, jbc.M112.363549), we report a metabolic pathway for degradation of Neu5Gc, demonstrating that N-acetylhexosamine pathways are tolerant toward the N-glycolyl substituent of Neu5Gc breakdown products. In this study, we show that exogenously added N-glycolylgalactosamine (GalNGc) serves as a precursor for Neu5Gc de novo biosynthesis, potentially involving seven distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with the endogenous UDP-GlcNAc for the sialic acid biosynthetic pathway. Using UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase-deficient cells, we confirm that conversion of GalNGc into Neu5Gc depends on this key enzyme of sialic acid biosynthesis. Furthermore, we demonstrate by mass spectrometry that the metabolic intermediates UDP-GalNGc and UDP-GlcNGc serve as substrates for assembly of most major classes of cellular glycans. We show for the first time incorporation of GalNGc and GlcNGc into chondroitin/dermatan sulfates and heparan sulfates, respectively. As demonstrated by structural analysis, N-glycolylated hexosamines were found in cellular gangliosides and incorporated into Chinese hamster ovary cell O-glycans. Remarkably, GalNAc derivatives altered the overall O-glycosylation pattern as indicated by the occurrence of novel O-glycan structures. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are surprisingly tolerant toward the N-glycolyl substituent.
Asunto(s)
Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Células CHO , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Cricetinae , Cricetulus , Dermatán Sulfato/genética , Dermatán Sulfato/metabolismo , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Ratones , Ratones Noqueados , Ácido N-Acetilneuramínico/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Azúcares de Uridina Difosfato/genética , Azúcares de Uridina Difosfato/metabolismoRESUMEN
Chondroitin sulfate/dermatan sulfate (CS/DS) polysaccharides have been reported to play a crucial role in the proliferation and maintenance of neural stem cells (NSCs). However, little is known about the structural changes and functional role of CS/DS chains in the differentiation of NSCs. Western blots of NSCs, neurons and astrocytes in culture, with three CS-polysaccharide antibodies of different specificities, revealed marked differences in CS structure among the three cell types. To confirm this finding, we measured gene expression levels of CS sulfotransferases and C5-epimerase in these cell types, as these are responsible for producing the high structural diversity of CS/DS. Expressions of chondroitin 4-O-sulfotransferase, chondroitin 6-O-sulfotransferase, and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase mRNAs were low in cultures of differentiated neural cells, such as neurons and astrocytes, in comparison to NSCs. In contrast, expressions of uronyl 2-O-sulfotransferase and C5-epimerase mRNAs were higher in the differentiated neural cells than NSCs. Thus, we first provide evidence to support the hypothesis that CS/DS undergoes structural changes during NSC differentiation. The structural changes in CS/DS may be implicated in the regulation of NSC differentiation through interactions with growth/neurotrophic factors and cytokines.
Asunto(s)
Astrocitos/enzimología , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Dermatán Sulfato/análogos & derivados , Regulación Enzimológica de la Expresión Génica/genética , Células-Madre Neurales/enzimología , Neuronas/enzimología , Animales , Astrocitos/citología , Conformación de Carbohidratos , Diferenciación Celular/genética , Células Cultivadas , Sulfatos de Condroitina/biosíntesis , Dermatán Sulfato/biosíntesis , Dermatán Sulfato/química , Dermatán Sulfato/genética , Femenino , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/genética , Células-Madre Neurales/citología , Neuronas/citología , Embarazo , Ratas , Relación Estructura-ActividadRESUMEN
Decorin is known to influence the size of collagen fibrils in ligaments and tendons and it has been hypothesized to provide a structural link between collagen fibrils in connective tissues, including cartilage. Coincidently, mechanical properties of skin, ligament, and tendons are altered in decorin knockout mice, suggesting it may influence the structural properties of tissue or tissue matrix organization. To further examine the role of decorin in the extracellular matrix development and subsequent material properties of cartilage, tissue (neocartilage) was grown in a 3D culture model using a pure population of genetically modified chondrocytes stably overexpressing decorin (DCN) or decorin lacking dermatan sulfate (MDCN). An empty vector (CON) served as a virus control. Following generation of the cartilage-like tissues, mechanical properties in tension and compression, collagen fibril diameter, matrix organization, and biochemistry of the tissue were determined. There were no differences between CON and DCN tissues in any parameter measured. In contrast, tissue generated in MDCN cultures was thinner, had higher collagen density, and higher elastic moduli as compared to both CON and DCN tissues. Considering there was no difference in stiffness between CON and DCN tissues, the notion that decorin contributes to the mechanical properties via load transfer was refuted in this model. However, contrasts in the mechanical properties of the MDCN tissue suggest that the dermatan sulfate chains on decorin influences the organization/maturation and resultant mechanical properties of the matrix by as an yet-unidentified regulatory mechanism.
Asunto(s)
Cartílago Articular/fisiología , Condrocitos/fisiología , Dermatán Sulfato/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteoglicanos/metabolismo , Animales , Fenómenos Biomecánicos , Cartílago Articular/citología , Células Cultivadas , Condrocitos/citología , Colágeno/metabolismo , Decorina , Dermatán Sulfato/deficiencia , Dermatán Sulfato/genética , Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Vectores Genéticos , Masculino , Proteoglicanos/genética , Conejos , Estrés Mecánico , Ingeniería de Tejidos , Transducción GenéticaRESUMEN
Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.
Asunto(s)
Carbohidrato Epimerasas/metabolismo , Colágeno/metabolismo , Dermatán Sulfato/metabolismo , Ácido Idurónico/metabolismo , Piel/metabolismo , Animales , Carbohidrato Epimerasas/genética , Colágeno/ultraestructura , Decorina , Dermatán Sulfato/genética , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Proteoglicanos/metabolismo , Piel/enzimología , Piel/ultraestructuraRESUMEN
Dermatan sulfate is a highly sulfated polysaccharide and has a variety of biological functions in development and disease. Iduronic acid domains in dermatan sulfate, which are formed by the action of two DS-epimerases, have a key role in mediating these functions. We have identified the catalytic site and three putative catalytic residues in DS-epimerase 1, His-205, Tyr-261, and His-450, by tertiary structure modeling and amino acid conservation to heparinase II. These residues were systematically mutated to alanine or more conserved residues, which resulted in complete loss of epimerase activity. Based on these data and the close relationship between lyase and epimerase reactions, we propose a model where His-450 functions as a general base abstracting the C5 proton from glucuronic acid. Subsequent cleavage of the glycosidic linkage by Tyr-261 generates a 4,5-unsaturated hexuronic intermediate, which is protonated at the C5 carbon by His-205 from the side of the sugar plane opposite to the side of previous proton abstraction. Concomitant recreation of the glycosidic linkage ends the reaction, generating iduronic acid. In addition, we show that proper N-glycosylation of DS-epimerase 1 is required for enzyme activity. This study represents the first description of the structural basis for epimerization by a glycosaminoglycan epimerase.
Asunto(s)
Antígenos de Neoplasias/química , Dominio Catalítico/fisiología , Proteínas de Unión al ADN/química , Modelos Moleculares , Proteínas de Neoplasias/química , Sustitución de Aminoácidos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dermatán Sulfato/biosíntesis , Dermatán Sulfato/química , Dermatán Sulfato/genética , Glicosilación , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Mapeo Peptídico/métodos , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Estructura Terciaria de Proteína/fisiologíaRESUMEN
The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.
Asunto(s)
Sulfatos de Condroitina/biosíntesis , Condroitinsulfatasas/metabolismo , Regulación Enzimológica de la Expresión Génica , N-Acetilgalactosamina-4-Sulfatasa/metabolismo , Anticuerpos/química , Línea Celular Tumoral , Condroitina ABC Liasa/química , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Condroitinsulfatasas/genética , Decorina , Dermatán Sulfato/biosíntesis , Dermatán Sulfato/química , Dermatán Sulfato/genética , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Silenciador del Gen , Heparina/biosíntesis , Heparina/química , Heparina/genética , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/química , Heparitina Sulfato/genética , Humanos , Mucopolisacaridosis VI/enzimología , Mucopolisacaridosis VI/genética , N-Acetilgalactosamina-4-Sulfatasa/genética , Proteoglicanos/biosíntesis , Proteoglicanos/genética , ARN Interferente Pequeño , Sindecano-1/biosíntesis , Sindecano-1/genéticaRESUMEN
The principal extracellular matrix (ECM) chondroitin/dermatan sulfate proteoglycans include members of two gene families--the large aggregating chondroitin sulfate proteoglycans (lecticans) and the small leucine-rich proteoglycans (SLRPs). These families of proteoglycans are widely distributed within the interstitial matrix, where they are known to bind a variety of both soluble and insoluble ligands. Extensive structural studies and data concerning the synthesis of these proteoglycans have been published over the last few years. This review focuses on the regulation of the expression of the lectican, versican, and the SLRPs--decorin and biglycan, as well--studied and widely distributed examples of these families of ECM proteoglycans. In addition, the effects of these proteoglycans on the formation of the ECM and the response of cells to growth factors and cytokines are examined as mechanisms by which versican, decorin and biglycan, both directly and indirectly influence cellular proliferation, migration, and phenotype.
Asunto(s)
Proliferación Celular , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanos/metabolismo , Animales , Biglicano , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Proteoglicanos Tipo Condroitín Sulfato/genética , Decorina , Dermatán Sulfato/genética , Dermatán Sulfato/metabolismo , Proteínas de la Matriz Extracelular , Sustancias de Crecimiento/metabolismo , Humanos , Lectinas Tipo C , Proteoglicanos/genética , VersicanosRESUMEN
The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (A(L) and B(L)) or short (A(S) and B(S)) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AA(L) and PDGF-BB(S) isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.
Asunto(s)
Glicosaminoglicanos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Especificidad de Anticuerpos , Becaplermina , Células CHO , Sulfatos de Condroitina/genética , Sulfatos de Condroitina/metabolismo , Cricetinae , Dermatán Sulfato/genética , Dermatán Sulfato/metabolismo , Glicosaminoglicanos/genética , Glicosaminoglicanos/farmacología , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Isomerismo , Lipoproteína Lipasa/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis , Factor de Crecimiento Derivado de Plaquetas/química , Factor de Crecimiento Derivado de Plaquetas/inmunología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas c-sisRESUMEN
Decorin, a small proteoglycan containing a dermatan sulfate (DS) chain, is expressed abnormally in human colon cancer stroma. The aim of this study was to determine neoplastic changes in DS chains from human colon cancer and normal colonic mucosa. Proteoglycans were extracted from human colon cancer and normal colonic mucosa and successively digested with enzymes. The glycosaminoglycan obtained was fluoro-labeled with 2-aminopyridine at reducing terminals and fractionated by HPLC. Fluoro-labeled DS chains were collected and digested with bovine testicular hyaluronidase, followed by HPLC. The repeating disaccharide connected to the linkage region [glucuronosyl-galactosyl-galactosyl-xylosyl(2-aminopyridine)] of pyridylaminated DS chains from both types of tissue was glucuronosyl-N-acetylgalactosamine. The other glucuronic acid of the pyridylaminated DS chain was located 12 saccharides from the reducing terminal in colon cancer, and 18 saccharides from the reducing terminal in normal colon. The saccharide sequence of DS chains from human colon cancer is altered from that in normal colon.