RESUMEN
Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.
Asunto(s)
Ciclofilinas/metabolismo , Regulación Enzimológica de la Expresión Génica , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/metabolismo , Sulfotransferasas/genética , Animales , Bovinos , Línea Celular Tumoral , Regulación hacia Abajo , Heparina/metabolismo , Heparitina Sulfato/química , Humanos , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Nitrógeno/metabolismo , Especificidad de Órganos , Unión Proteica , Interferencia de ARN , Especificidad por Sustrato , Sulfatos/metabolismo , Sulfotransferasas/deficiencia , Sulfotransferasas/metabolismo , Linfocitos T/metabolismoRESUMEN
Fucan is a term used to denominate a family of sulfated L-fucose-rich polysaccharides. The brown alga Spatoglossum schröederi (Dictyotaceae) has three heterofucans namely fucan A, B and C. The 21 kDa fucan A is composed of a core of a beta (1-3) glucuronic acid-containing oligosaccharide of 4.5 kDa with branches at C4 of the fucose chains alpha (1-3) linked. The fucose is mostly substituted at C4 with a sulfate group and at C2 with chains of beta (1-4) xylose. This fucan has neither anticoagulant (from from 0.1 to 100 microg) nor hemorrhagic activities (from 50 to 800 microg/mL). The antithrombotic test in vivo showed that fucan A has no activity in any of the concentrations (from 0.2 to 20 microg/g/day) tested 1 h after polysaccharide administration. However, when fucan A was injected endovenously 24 h before the ligature of the venae cavae, we observed a dose-dependent effect, reaching saturation at around 20 microg/g of rat weight. In addition, this effect is also time-dependent, reaching saturation around 16 h after fucan administration. In addition, regardless of the administration route, fucan A displayed antithrombotic activity. The exception was the oral pathway. Of particular importance was the finding that fucan A stimulates the synthesis of an antithrombotic heparan sulfate from endothelial cells like heparin. The hypothesis has been raised that the in vivo antithrombotic activity of fucan A is related to the increased production of this heparan. Taken together with the fact that the compound is practically devoid of anticoagulant and hemorrhagic activity, the data suggest that it may be an ideal antithrombotic agent in vivo.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Fibrinolíticos/aislamiento & purificación , Heparitina Sulfato/biosíntesis , Phaeophyceae/química , Polisacáridos/aislamiento & purificación , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Células Endoteliales/metabolismo , Fibrinolíticos/administración & dosificación , Humanos , Masculino , Polisacáridos/efectos adversos , Conejos , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The brown seaweed Spatoglossum schröederi (Dictyotaceae) contains three main fucans (fucans A, B and C) with different mobility in electrophoresis. The fucan with highest mobility (fucan C) was precipitated with 2.0 volumes of acetone, purified using a combination of ion exchange chromatography and electrophoresis. It showed an MW of 24 kDa determined by HPLC and Sephadex G-75 chromatography and migrates as a single band in three distinct electrophoretic systems. This fucan contains fucose, xylose, galactose and sulfate in a molar ratio 1 : 0.6 : 2:2.3. The fucan has neither anticoagulant (from 10 to 100 microg) nor hemorrhagic activities (100 microg/mL). In addition, fucan C is neither cytotoxic nor cytostatic. However, fucan C (100 microg/mL) stimulated the synthesis of an antithrombotic heparan sulfate from endothelial cells of rabbit aorta. The results suggest that fucan C might be used as an antithrombotic therapeutic compound.
Asunto(s)
Anticoagulantes/farmacología , Heparitina Sulfato/biosíntesis , Fitoterapia , Extractos Vegetales/farmacología , Algas Marinas , Trombina/antagonistas & inhibidores , Anticoagulantes/administración & dosificación , Anticoagulantes/uso terapéutico , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéuticoRESUMEN
Heparan sulfate structure differs significantly between various cell types and during different developmental stages. The diversity is created during biosynthesis by sulfotransferases, which add sulfate groups to the growing chain, and a C5-epimerase, which converts selected glucuronic acid residues to iduronic acid. All these modifications are believed to depend on initial glucosamine N-sulfation carried out by the enzyme glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here we report that heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 completely lacks N-sulfation but still contains 6-O-sulfate groups, demonstrating that 6-O-sulfation can occur without prior N-sulfation. Reverse transcriptase-PCR analysis indicates that all three identified 6-O-sulfotransferases are expressed by the cells, 6-O-sulfotransferase-1 being the dominating form. The 6-O-sulfated polysaccharide lacking N-sulfate groups also contains N-unsubstituted glucosamine units, raising questions about how these units are generated.
Asunto(s)
Amidohidrolasas/genética , Heparitina Sulfato/biosíntesis , Sulfotransferasas/genética , Azufre/metabolismo , Amidohidrolasas/fisiología , Animales , Blastocisto/metabolismo , Carbohidrato Epimerasas/química , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , ADN Complementario/metabolismo , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Glucosamina/química , Ácido Glucurónico/metabolismo , Glicosaminoglicanos , Ácido Idurónico/metabolismo , Ratones , Ratones Transgénicos , Ácido Nitroso/metabolismo , Polisacáridos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Sulfatos/química , Sulfotransferasas/fisiologíaRESUMEN
Sulfated glycosaminoglycans including heparin/heparan sulfate and chondroitin/dermatan sulfate have been implicated in numerous pathophysiological phenomena in vertebrates and invertebrates. The critical roles of glycosaminoglycans, especially heparan sulfate, in developmental processes involving the signaling of morphogens such as Wingless and Hedgehog proteins, as well as of fibroblast growth factor, in Drosophila have recently become evident. In biosynthesis, the tetrasaccharide sequence (GlcA-Gal-Gal-Xyl-), designated the protein linkage region, is first built on a specific Ser residue at the glycosaminoglycan attachment site of a core protein. A heparin/heparan sulfate chain is then polymerized on this fragment by alternate additions of N-acetylglucosamine and glucuronic acid (GlcA) through the actions of glycosyltransferases with overlapping specificity encoded by the tumor suppressor EXT family genes. In contrast, a chondroitin/dermatan sulfate chain is synthesized on the linkage region by alternate additions of N-acetylgalactosamine and GlcA through the actions of glycosyltransferases, designated chondroitin synthases. Recent studies have achieved purification of a few and molecular cloning of all of the glycosyltransferases responsible for these reactions and have revealed the bifunctional nature of a few of these enzymes. The availability of the cDNA probes has provided several important clues to help solve the molecular mechanisms of the biosynthetic sorting of heparin/heparan sulfate and chondroitin/dermatan sulfate chains, as well as of the chain elongation and polymerization of these glycosaminoglycans.
Asunto(s)
Glucuronosiltransferasa , Glicosaminoglicanos/biosíntesis , N-Acetilgalactosaminiltransferasas , Animales , Sulfatos de Condroitina/biosíntesis , Clonación Molecular , ADN Complementario , Regulación Enzimológica de la Expresión Génica , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/fisiología , Glicosaminoglicanos/fisiología , Glicosiltransferasas/genética , Glicosiltransferasas/fisiología , Heparitina Sulfato/biosíntesis , Humanos , Enzimas Multifuncionales , Especificidad por SustratoRESUMEN
Silicon plays a physiologically essential but mechanistically obscure role in promoting the synthesis of mucopolysaccharides and collagen. In light of reports that increased silicon ingestion impedes cholesterol-induced atherogenesis in rabbits and may be associated epidemiologically with reduced cardiovascular risk, it is reasonable to speculate that supplemental silicon may stimulate endothelial production of heparan sulfate proteoglycans that inhibit intimal hyperplasia.
Asunto(s)
Arteriosclerosis/prevención & control , Endotelio Vascular/metabolismo , Heparitina Sulfato/biosíntesis , Proteoglicanos/biosíntesis , Silicio/farmacología , Animales , Arteriosclerosis/epidemiología , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/prevención & control , Colesterol en la Dieta , Dieta Aterogénica , Endotelio Vascular/efectos de los fármacos , Proteoglicanos de Heparán Sulfato , Humanos , Modelos Cardiovasculares , Conejos , Factores de Riesgo , Silicio/uso terapéuticoRESUMEN
The presence of proteoglycans bearing galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.
Asunto(s)
Membrana Basal/química , Proteoglicanos Tipo Condroitín Sulfato/análisis , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/análisis , Neoplasias Experimentales/química , Proteoglicanos/análisis , Animales , Anticuerpos , Anticuerpos Monoclonales , Western Blotting , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Clonación Molecular , ADN Complementario , Técnica del Anticuerpo Fluorescente Indirecta , Heparitina Sulfato/biosíntesis , Humanos , Inmunohistoquímica , Corteza Renal/citología , Ratones , Proteoglicanos/biosíntesis , Conejos , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Piel/citologíaRESUMEN
Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Mesangio Glomerular/metabolismo , Glomérulos Renales/metabolismo , Proteoglicanos/biosíntesis , Adolescente , Adulto , Anciano , Secuencia de Bases , Células Cultivadas , Niño , Preescolar , Proteoglicanos Tipo Condroitín Sulfato/análisis , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Cromatografía Líquida de Alta Presión , ADN Complementario/biosíntesis , ADN Complementario/genética , Ensayo de Inmunoadsorción Enzimática , Epitelio/metabolismo , Glucosamina/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/genética , Humanos , Lactante , Glomérulos Renales/citología , Leucina/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteoglicanos/genética , ARN/genética , ARN/aislamiento & purificación , ARN/metabolismo , ARN Mensajero/análisis , ARN Mensajero/genética , Sulfatos/metabolismo , Radioisótopos de Azufre , Transcripción Genética , TritioRESUMEN
Proliferating cultures of bovine vascular smooth muscle cells synthesized a variety of proteoglycans corresponding closely to those reported previously for monkey smooth muscle cells. These included a chondroitin sulfate proteoglycan (CSPG) (47%), a dermatan sulfate proteoglycan (DSPG) (22%), and a heparan sulfate proteoglycan (HSPG) (6%) which were secreted into the medium. Heparan sulfate proteoglycan (6%) and a second dermatan sulfate proteoglycan (14%) were also present in the cell layer. Confluent cultures synthesized a similar spectrum of proteoglycans although the medium CSPG and DSPG were of smaller hydrodynamic size. The cell layer HSPG was much reduced relative to DSPG in early proliferating cultures. Previous reports have shown that heparin inhibits vascular smooth muscle cell proliferation. Heparin had two effects on proteoglycan synthesis. In control cultures, 35S-Labeled proteoglycan synthesis doubled during the first 12 h after releasing cells from growth arrest, decreasing during the following 12 h during which time cell division occurred. Treatment with heparin delayed the onset of proliferation by 24 h and this was accompanied by a corresponding delay in the increase in 35S-labeled proteoglycan synthesis associated with the early phase of the cell cycle. Secondly, heparin treatment resulted in an increase in the anionic properties of heparan sulfate proteoglycan synthesized by the cells. This was independent of the proliferative state of the cultures. Pentosan polysulfate, semi-synthetic heparin, and a highly sulfated heparan sulfate modulated both cell proliferation and heparan sulfate proteoglycan synthesis in the same way as heparin.
Asunto(s)
Heparina/farmacología , Músculo Liso Vascular/metabolismo , Proteoglicanos/biosíntesis , Animales , Aorta , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Dermatán Sulfato/biosíntesis , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Poliéster Pentosan Sulfúrico/farmacología , Polisacáridos/farmacologíaRESUMEN
Selenate, a sulfation inhibitor, blocks the synthesis of heparan sulfate and chondroitin sulfate by cultured endothelial cells. In contrast, selenate does not affect the production of hyaluronic acid, a nonsulfated glycosaminoglycan. No differences in molecular weight, [3H]glucosamine/[35S]sulfuric acid ratios, or disaccharide composition were observed when the heparan sulfate synthesized by selenate-treated cells was compared with that of control cells. The absence of undersulfated chains in preparations from cultures exposed to selenate supports the concept that, in the intact cell, the polymerization of heparan sulfate might be dependent on the sulfation of the saccharide units added to the growing glycosaminoglycan chain.
Asunto(s)
Endotelio Vascular/metabolismo , Glicosaminoglicanos/biosíntesis , Heparitina Sulfato/biosíntesis , Selenio/farmacología , Animales , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Heparitina Sulfato/antagonistas & inhibidores , Cinética , ConejosRESUMEN
We have isolated five Chinese hamster ovary cell mutants defective in galactosyltransferase I (UDP-D-galactose:xylose beta-1,4-D-galactosyltransferase) and studied the effect of p-nitrophenyl-beta-D-xyloside supplementation on glycosaminoglycan biosynthesis in the mutant cells. Assays of galactosyltransferase I showed that the mutants contained less than 2% of the enzyme activity present in wild-type cells, and enzyme activity was additive in mixtures of mutant and wild-type cell extracts, suggesting that the mutations most likely defined the structural gene encoding the enzyme. Cell hybridization studies showed that the mutations in all five strains were recessive and that the mutants belonged to the same complementation group. The mutants contained wild-type levels of xylosyltransferase (UDP-D-xylose:core protein (serine) beta-D-xylosyltransferase), lactose synthase (UDP-D-galactose:N-acetyl-glucosaminide beta-1,4-D-galactosyltransferase), and lactosylceramide synthase (UDP-D-galactose:glucosylceramide beta-1,4-D-galactosyltransferase). Their sensitivity to lectin-mediated cytotoxicity was virtually identical to that of the wild-type, indicating that there were no gross alterations in glycoprotein or glycolipid compositions. Anion-exchange high performance liquid chromatography of 35S-glycosaminoglycans from one of the galactosyltransferase I-deficient mutants showed a dramatic reduction in both heparan sulfate and chondroitin sulfate, demonstrating that galactosyltransferase I is responsible for the formation of both glycosaminoglycans in intact cells. Surprisingly, the addition of 1 mM-p-nitrophenyl-beta-D-xyloside, a substrate for galactosyltransferase I, restored glycosaminoglycan synthesis in mutant cells. This finding suggested that another galactosyltransferase, possibly lactose synthase, can transfer galactose to xylose in intact cells.
Asunto(s)
Sulfatos de Condroitina/biosíntesis , Condroitín/análogos & derivados , Glicosaminoglicanos/biosíntesis , Heparitina Sulfato/biosíntesis , Pentosiltransferasa/genética , Animales , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , Femenino , Prueba de Complementación Genética , Glicosaminoglicanos/metabolismo , Mutación , Ovario/enzimología , Ovario/metabolismo , Pentosiltransferasa/deficiencia , UDP Xilosa Proteína XilosiltransferasaRESUMEN
The insect Rhodnius prolixus synthesizes heparan sulfate and chondroitin sulfate after a blood meal containing [35S]-inorganic sulfate. A 40 to 80% inhibition of heparan sulfate synthesis was obtained when the meal was supplemented with 10(-5) and 10(-4) M sodium selenate respectively. Likewise an inhibition of the molting in the order of 30 to 60% was observed when the insects were fed with blood containing 10(-5) and 10(-4) M selenate respectively. The insects after a subsequent meal without selenate molted normally. Except for the inhibition of the ecdysis no gross physiological or morphological changes could be observed in the insects. Based on these and other findings the possible role of sulfated glycosaminoglycans in the control of cell growth is discussed.