RESUMEN
Insulin resistance and type 2 diabetes are associated with elevated circulating levels of insulin, nonesterified fatty acids (NEFAs), and lipoprotein remnants. Extracellular matrix proteoglycan (PG) alterations are also common in macro- and microvascular complications of type 2 diabetes. In liver, extracellular heparan sulfate (HS) PGs contribute to the uptake of triglyceride-rich lipoprotein remnants. We found that HepG2 cells cultured with 10 or 50 nmol/l insulin or 300 micromol/l albumin-bound linoleic acid changed their PG secretion. The glycosaminoglycans (GAGs) of the secreted PGs from insulin-treated HepG2 cells were enriched in chondroitin sulfate (CS) PGs. In contrast, cells exposed to linoleic acid secreted PGs with decreased content of CS. Insulin caused a moderate increase in mRNA for versican (secreted CS PG), whereas linoleic acid markedly decreased mRNA for versican in HepG2 cells, as did the peroxisomal proliferator-activated receptor-alpha agonist bezafibrate. The effects of insulin or linoleic acid on syndecan 1, a cell surface HS PG, were similar to those on versican, but less pronounced. The livers of obese Zucker fa/fa rats, which are insulin-resistant and have high levels of insulin, NEFAs, and triglyceride-rich remnants, showed increased expression of CS PGs when compared with lean littermates. These changes in PG composition decreased the affinity of remnant beta-VLDL particles to PGs isolated from insulin-treated HepG2 cells and obese rat livers. The results indicated that insulin and NEFAs modulate the expression of PGs in hepatic cells. We speculate that in vivo this exchange of CS for HS may reduce the clearance of remnant beta-VLDLs and contribute to the dyslipidemia of insulin resistance.
Asunto(s)
Ácidos Grasos/farmacología , Hiperlipidemias/etiología , Resistencia a la Insulina/fisiología , Insulina/farmacología , Hígado/metabolismo , Proteoglicanos/fisiología , Animales , Línea Celular , Matriz Extracelular/metabolismo , Humanos , Ácido Linoleico/farmacología , Lipoproteínas VLDL/sangre , Hígado/citología , Obesidad/genética , Obesidad/metabolismo , Proteoglicanos/metabolismo , Ratas , Ratas Zucker/metabolismo , DelgadezRESUMEN
Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (> or =0.5 m NaCl required for elution) contained an internal IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3))-IdoUA(2-OSO(3))-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1. FGF-2 was shown to bind to a mono-O-sulfated HS 6-mer carrying a single internal IdoUA(2-OSO(3))-unit. However, a di-O-sulfated -IdoUA(2-OSO(3))-GlcNSO(3)-IdoUA(2-OSO(3))-trisaccharide sequence within a HS 8-mer gave stronger binding. These findings show that not only the number but also the positions of individual sulfate groups determine affinity of HS for FGFs. Our findings support the notion that FGF-dependent processes can be modulated in vivo by regulated expression of distinct HS sequences.
Asunto(s)
Epítopos/química , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/inmunología , Secuencia de Carbohidratos , Factor 1 de Crecimiento de Fibroblastos , Heparitina Sulfato/químicaRESUMEN
Fibroblast growth factors (FGFs) are heparin-binding polypeptides that affect the growth, differentiation, and migration of many cell types. FGFs signal by binding and activating cell surface FGF receptors (FGFRs) with intracellular tyrosine kinase domains. The signaling involves ligand-induced receptor dimerization and autophosphorylation, followed by downstream transfer of the signal. The sulfated glycosaminoglycans heparin and heparan sulfate bind both FGFs and FGFRs and enhance FGF signaling by mediating complex formation between the growth factor and receptor components. Whereas the heparin/heparan sulfate structures involved in FGF binding have been studied in some detail, little information has been available on saccharide structures mediating binding to FGFRs. We have performed structural characterization of heparin/heparan sulfate oligosaccharides with affinity toward FGFR4. The binding of heparin oligosaccharides to FGFR4 increased with increasing fragment length, the minimal binding domains being contained within eight monosaccharide units. The FGFR4-binding saccharide domains contained both 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfoglucosamine residues, as shown by experiments with selectively desulfated heparin, compositional disaccharide analysis, and a novel exoenzyme-based sequence analysis of heparan sulfate oligosaccharides. Structurally distinct heparan sulfate octasaccharides differed in binding to FGFR4. Sequence analysis suggested that the affinity of the interaction depended on the number of 6-O-sulfate groups but not on their precise location.
Asunto(s)
Heparina/metabolismo , Heparitina Sulfato/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Sitios de Unión , Secuencia de Carbohidratos , Cromatografía de Afinidad , Disacáridos/química , Disacáridos/metabolismo , Heparina/química , Heparitina Sulfato/química , Humanos , Cinética , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The androgen-induced proliferation of S115 mouse mammary tumor cells has been suggested to involve autocrinic fibroblast growth factor signaling. Heparan sulfate proteoglycans are required for fibroblast growth factor signaling, presumably due to their ability to alter binding of fibroblast growth factors to their receptors. We have investigated the role of heparan sulfate proteoglycans in the testosterone-induced proliferation of S115 cells. We demonstrate that when the cells are treated with sodium chlorate, which inhibits the sulfation of endogenous heparan sulfate proteoglycans, cell growth becomes dependent on exogenous heparin. The shortest heparin oligosaccharides supporting cell growth were octasaccharides, whereas dodecasaccharides were almost as effective as native heparin. The N-, 2-O-, and 6-O-sulfate groups of heparin were all required for full testosterone response. Treatment of S115 cells with chlorate or testosterone did not alter the expression of fibroblast growth factor receptors 1 or 3, whereas the expression of fibroblast growth factor receptor 2 was down-regulated. We have previously shown that overexpression of syndecan-1 heparan sulfate proteoglycan renders S115 cells insensitive to testosterone and now demonstrate that this effect can be overcome by sodium chlorate treatment in combination with exogenous heparin. Our results suggest that heparin-like molecules are intimately involved in the androgen-mediated proliferation of S115 cells.
Asunto(s)
División Celular , Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas Tirosina Quinasas , Testosterona/metabolismo , Animales , Expresión Génica , Proteoglicanos de Heparán Sulfato/fisiología , Heparina/metabolismo , Neoplasias Mamarias Animales , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Ratones , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Relación Estructura-Actividad , Sindecano-1 , Sindecanos , Testosterona/farmacología , Células Tumorales CultivadasRESUMEN
The N-sulfated regions (NS domains) represent the modified sequences of heparan sulfate chains and mediate interactions of the polysaccharide with proteins. We have investigated the relationship between the type/extent of polymer modification and the length of NS domains in heparan sulfate species from human aorta, bovine kidney, and cultured NMuMG and MDCK cells. C5 epimerization of D-glucuronic acid to L-iduronic acid was found to be extensive and essentially similar in all heparan sulfate species studied, regardless of domain size, whereas the subsequent 2-O-sulfation of the formed iduronic acid residues varies appreciably. In aorta heparan sulfate, up to 90% of the formed iduronate residues were 2-O-sulfated, whereas in kidney heparan sulfate 2-O-sulfation occurred only in =50% of the iduronate residues. The degree of 2-O-sulfation was consistently increased with increasing NS domain length, suggesting a correlation between 2-O-sulfation efficiency and length of the polymeric substrate during heparan sulfate biosynthesis. By contrast, 6-O-sulfation of glucosamine units did not correlate to domain size. 6-O-Sulfation exceeded 2-O-sulfation in NS domains from kidney heparan sulfate, but was very low in aorta heparan sulfate. Remarkably, total O-sulfation of NS domains, i.e., the sum of 2-O- and 6-O-sulfate groups, was highly similar in all heparan sulfate samples investigated. The results reveal marked tissue-specific variation in the sulfation patterns of NS domains and indicate previously unrecognized distinctions in the coordination of the three polymer modification reactions during heparan sulfate biosynthesis.
Asunto(s)
Glucosamina/metabolismo , Ácido Glucurónico/metabolismo , Proteoglicanos de Heparán Sulfato/química , Proteoglicanos de Heparán Sulfato/metabolismo , Ácido Idurónico/metabolismo , Acetilglucosamina/metabolismo , Anciano , Animales , Conformación de Carbohidratos , Carbohidrato Epimerasas/metabolismo , Bovinos , Línea Celular , Células Cultivadas , Disacáridos/aislamiento & purificación , Disacáridos/metabolismo , Perros , Proteoglicanos de Heparán Sulfato/aislamiento & purificación , Ácidos Hexurónicos/metabolismo , Humanos , Masculino , Ratones , Relación Estructura-Actividad , Sulfatos/metabolismoRESUMEN
Fibroblast growth factors (FGFs) are known to induce formation of new blood vessels, angiogenesis. We show that FGF-induced angiogenesis can be modulated using selectively desulfated heparin. Chinese hamster ovary cells (CHO677) deficient in heparan sulfate biosynthesis were employed to assess the function of heparin/heparan sulfate in FGF receptor-1 (FGFR-1) signal transduction and biological responses. In the presence of FGF-2, FGFR-1 kinase and subsequent mitogen-activated protein kinase Erk2 activities were augmented in a dose-dependent manner, whereas high concentrations of heparin resulted in decreased activity. The length of the heparin oligomer, minimally an 8/10-mer, was critical for the ability to enhance FGFR-1 kinase activity. The N- and 2-O-sulfate groups of heparin were essential for binding to FGF-2, whereas stimulation of FGFR-1 and Erk2 kinases by FGF-2 also required the presence of 6-O-sulfate groups. Sulfation at 2-O- and 6-O-positions was moreover a prerequisite for binding of heparin to a lysine-rich peptide corresponding to amino acids 160-177 in the extracellular domain of FGFR-1. Selectively 6-O-desulfated heparin, which binds to FGF-2 but fails to bind the receptor, decreased FGF-2-induced proliferation of CHO677 cells, presumably by displacing intact heparin. Furthermore, FGF-2-induced angiogenesis in chick embryos was inhibited by 6-O-desulfated heparin. Thus, formation of a ternary complex of FGF-2, heparin, and FGFR-1 appears critical for the activation of FGFR-1 kinase and downstream signal transduction. Preventing complex formation by modified heparin preparations may allow regulation of FGF-2 functions, such as induction of angiogenesis.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/fisiología , Heparina/farmacología , Neovascularización Fisiológica/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Alantoides/irrigación sanguínea , Secuencia de Aminoácidos , Animales , Células CHO , División Celular/efectos de los fármacos , Embrión de Pollo , Corion/irrigación sanguínea , Cricetinae , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparina/química , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Datos de Secuencia Molecular , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Relación Estructura-Actividad , TransfecciónRESUMEN
We have analyzed the effect of sodium chlorate treatment of Madin-Darby canine kidney cells on the structure of heparan sulfate (HS), to assess how the various sulfation reactions during HS biosynthesis are affected by decreased availability of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate. Metabolically [(3)H]glucosamine-labeled HS was isolated from chlorate-treated and untreated Madin-Darby canine kidney cells and subjected to low pH nitrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternating N-acetylated and N-sulfated disaccharide units, and (iii) the N-acetylated domains were recovered and subjected to compositional disaccharide analysis. Upon treatment with 50 mM chlorate, overall O-sulfation of HS was inhibited by approximately 70%, whereas N-sulfation remained essentially unchanged. Low chlorate concentrations (5 or 20 mM) selectively reduced the 6-O-sulfation of HS, whereas treatment with 50 mM chlorate reduced both 2-O- and 6-O-sulfation. Analysis of saccharides representing the different domain types indicated that 6-O-sulfation was preferentially inhibited in the alternating domains. These data suggest that reduced 3'-phosphoadenosine 5'-phosphosulfate availability has distinct effects on the N- and O-sulfation of HS and that O-sulfation is affected in a domain-specific fashion.
Asunto(s)
Heparitina Sulfato/química , Cloruro de Sodio/química , Animales , Línea Celular , Perros , Concentración de Iones de Hidrógeno , Cloruro de Sodio/farmacología , SulfatosRESUMEN
Recombinant mouse endostatin produced by mammalian cells was shown to bind to heparin with a K(d) of 0.3 microM, suggesting that this interaction may play a role in its anti-angiogenic activity. Alanine mutagenesis demonstrated that a major site of four clustered arginines (positions 155, 158, 184 and 270) and a second site (R193, R194) are essential for binding. The same epitopes also participate in endostatin binding to heparan sulfate and sulfatides but not in its binding to the extracellular protein ligands fibulin-1 and fibulin-2. Analyses with various heparin fragments demonstrated a minimum size (12mer) for efficient binding to endostatin and a crucial role of 2-O- and 6-O-sulfation. Furthermore, a substantial proportion (10-50%) of heparan sulfate chains obtained from various tissues showed a distinct binding to endostatin, indicating its potential to interact with extracellular and/or membrane-bound proteoglycans. Angiogenesis induced by basic fibroblast growth factor-2 (FGF-2), but not by vascular endothelial growth factor (VEGF), in a chick chorioallantoic membrane assay could be inhibited by endostatin in a dose-dependent manner. The mutational block of heparin binding decreased endostatin inhibition to low levels but elimination of zinc binding had no effect.
Asunto(s)
Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/metabolismo , Colágeno/química , Colágeno/metabolismo , Heparina/química , Heparina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Alantoides/irrigación sanguínea , Sustitución de Aminoácidos , Inhibidores de la Angiogénesis/farmacología , Animales , Sitios de Unión , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Embrión de Pollo , Corion/irrigación sanguínea , Colágeno/farmacología , Endostatinas , Factores de Crecimiento Endotelial/farmacología , Epítopos/química , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Cinética , Linfocinas/farmacología , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Neovascularización Fisiológica/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Mutación Puntual , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Porcinos , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Heparan sulfate found in the cerebral plaques of Alzheimer's disease binds to beta-amyloid (Abeta) fibrils. This interaction has been proposed to enhance fibril deposition and mediate Abeta-induced glia activation and neurotoxicity. On the other hand, heparan sulfate augments signaling of fibroblast growth factor-2 (FGF-2), a neuroprotective factor that antagonizes the neurotoxic effects of Abeta. We defined structures in heparan sulfate from human cerebral cortex that bind Abeta fibrils. The minimal binding site is found in N-sulfated hexasaccharide domains and contains critical 2-O-sulfated iduronic acid residues. By contrast, binding of Abeta monomers requires, in addition, 6-O-sulfate groups on glucosamine residues. The binding specificity of fibrillar Abeta is shared by FGF-2, and we here show that cerebral heparan sulfate domains selected for binding to Abeta-(1-40) fibrils bind also to FGF-2. These data suggest that neurotoxic and neuroprotective signals may converge by competing for the same binding sites on the heparan sulfate chain.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Corteza Cerebral/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/química , Sitios de Unión , Disacáridos/química , Factor 2 de Crecimiento de Fibroblastos/química , Glucosamina/análogos & derivados , Glucosamina/química , Glucosamina/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/aislamiento & purificación , Humanos , Ácido Idurónico/análogos & derivados , Ácido Idurónico/química , Ácido Idurónico/metabolismo , Oligosacáridos/química , Fragmentos de Péptidos/químicaRESUMEN
It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Ácido Glucurónico/química , Heparitina Sulfato/metabolismo , Ácido Idurónico/química , Aorta/citología , Aorta/efectos de los fármacos , Aorta/metabolismo , Sitios de Unión , Secuencia de Carbohidratos , División Celular/efectos de los fármacos , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heparitina Sulfato/síntesis química , Heparitina Sulfato/química , Humanos , Isomerismo , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Oligosacáridos/síntesis química , Oligosacáridos/químicaRESUMEN
Fibroblast growth factors FGF-1 and FGF-2 mediate their biological effects via heparan sulfate-dependent interactions with cell surface FGF receptors. While the specific heparan sulfate domain binding to FGF-2 has been elucidated in some detail, limited information has been available concerning heparan sulfate structures involved in the recognition of FGF-1. In the current study we present evidence that the minimal FGF-1 binding heparan sulfate sequence comprises 5-7 monosaccharide units and contains a critical trisulfated IdoA(2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. N-Sulfated heparan sulfate decasaccharides depleted of FGF-1 binding domains showed dose-dependent and saturable binding to FGF-2. These data indicate that the FGF-1 binding domain is distinct from the minimal FGF-2 binding site, previously shown to contain an IdoA(2-OSO3) residue but no 6-O-sulfate groups. We further show that the FGF-1 binding heparan sulfate domain is expressed in human aorta heparan sulfate in an age-related manner in contrast to the constitutively expressed FGF-2 binding domain. Reduction of heparan sulfate O-sulfation by chlorate treatment of cells selectively impedes binding to FGF-1. The present data implicate the 6-O-sulfation of IdoA(2-OSO3)-GlcNSO3 units in cellular heparan sulfate in the control of the biological activity of FGF-1.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Animales , Sitios de Unión , Secuencia de Carbohidratos , Bovinos , Factor 1 de Crecimiento de Fibroblastos , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , PorcinosRESUMEN
The biological activity of heparan sulphate (HS) and heparin largely depends on internal oligosaccharide sequences that provide specific binding sites for an extensive range of proteins. Identification of such structures is crucial for the complete understanding of glycosaminoglycan (GAG)-protein interactions. We describe here a simple method of sequence analysis relying on the specific tagging of the sugar reducing end by 3H radiolabelling, the combination of chemical scission and specific enzymic digestion to generate intermediate fragments, and the analysis of the generated products by strong-anion-exchange HPLC. We present full sequence data on microgram quantities of four unknown oligosaccharides (three HS-derived hexasaccharides and one heparin-derived octasaccharide) which illustrate the utility and relative simplicity of the technique. The results clearly show that it is also possible to read sequences of inhomogeneous preparations. Application of this technique to biologically active oligosaccharides should accelerate progress in the understanding of HS and heparin structure-function relationships and provide new insights into the primary structure of these polysaccharides.
Asunto(s)
Heparina/química , Heparitina Sulfato/química , Oligosacáridos/análisis , Análisis de Secuencia/métodos , Animales , Sitios de Unión , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Disacáridos/análisis , Disacáridos/química , Disacáridos/aislamiento & purificación , Disacáridos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Iduronato Sulfatasa/metabolismo , Iduronidasa/metabolismo , Lisosomas/enzimología , Datos de Secuencia Molecular , Membrana Mucosa , Ácido Nitroso/metabolismo , Oligosacáridos/química , Oligosacáridos/aislamiento & purificación , Oligosacáridos/metabolismo , Unión Proteica , Relación Estructura-Actividad , Sulfatasas/metabolismo , Porcinos , Factores de TiempoRESUMEN
Cell surface heparan sulfate proteoglycans are involved in several aspects of the lipoprotein metabolism. Most of the biological activities of these proteoglycans are mediated via interactions of their heparan sulfate moieties with various protein ligands, including lipoproteins and lipases. The binding of lipoproteins to heparan sulfate is largely determined by their apoprotein composition, and apoproteins B and E display the highest affinity for heparan sulfate. Interactions of lipoproteins with heparan sulfate are important for the cellular uptake and turnover of lipoproteins, in part by enhancing the accessibility of lipoproteins to lipoprotein receptors and lipases. Apoprotein B may interact with receptors without involving heparan sulfate. Heparan sulfate has been further implicated in presentation and stabilization of lipoprotein lipase and hepatic lipase on cell surfaces and in the transport of lipoprotein lipase from extravascular cells to the luminal surface of the endothelia. In atherosclerosis, heparan sulfate is intimately involved in several events important to the pathophysiology of the disease. Heparan sulfate thus binds and regulates the activity of growth factors, cytokines, superoxide dismutase and antithrombin, which contribute to aberrant cell proliferation, migration and matrix production, scavenging of reactive oxygen radicals and thrombosis. In this review we discuss the various roles of heparan sulfate proteoglycans in vascular biology, with emphasis on interactions of heparan sulfate with lipoproteins and lipases and the molecular basis of such interactions.
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Lipoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Antitrombinas/metabolismo , Apoproteínas/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Glicosaminoglicanos/metabolismo , Proteoglicanos de Heparán Sulfato/química , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Lipasa/metabolismo , Lipoproteína Lipasa/metabolismo , Hígado/enzimología , Macrófagos/inmunología , Macrófagos/metabolismo , Glicoproteínas de Membrana/química , Unión Proteica , Proteoglicanos/metabolismo , Superóxido Dismutasa/metabolismo , SindecanosRESUMEN
Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O-sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.
Asunto(s)
Diferenciación Celular , Neoplasias del Colon/química , Heparitina Sulfato/química , Acetilación , Células CACO-2 , Conformación de Carbohidratos , Neoplasias del Colon/patología , Sustancias de Crecimiento/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Unión ProteicaRESUMEN
Heparan sulfate is thought to regulate the biological activities of several proteins implicated in the pathogenesis of atherosclerosis. While the interactions of heparan sulfate with lipoprotein lipase and various growth factors have been actively studied, little is known of the cellular regulation of heparan sulfate biosynthesis in response to lipid accumulation. We have investigated heparan sulfate biosynthesis during conversion of murine J774 macrophages into lipid-laden foam cells. Such conversion is shown to accelerate the rate of glycosaminoglycan synthesis and the transport of newly synthesized proteoglycans into the medium. Moreover, the structure of heparan sulfate is specifically altered due to an approximately 30% increase in the 6-O-sulfation of glucosamine residues within the N-sulfated heparan sulfate domains, whereas the sulfation of chondroitin sulfate remains unaffected. These results suggest a selective effect of foam cell conversion on the biosynthesis of heparan sulfate.
Asunto(s)
Células Espumosas/metabolismo , Heparitina Sulfato/biosíntesis , Macrófagos/metabolismo , Animales , Arteriosclerosis/fisiopatología , Línea Celular , Sulfatos de Condroitina/metabolismo , Disacáridos/química , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/química , Humanos , Lipoproteínas LDL/metabolismo , Ratones , Ácido Nitroso/metabolismo , Oligosacáridos/químicaRESUMEN
Heparan sulfate interacts with growth factors, matrix components, effectors and modulators of enzymatic catalysis as well as with microbial proteins via sulfated oligosaccharide domains. Although a number of such domains have been characterized, little is known about the regulation of their formation in vivo. Here we show that the structure of human aorta heparan sulfate is gradually modulated during aging in a manner that gives rise to markedly enhanced binding to isoforms of platelet-derived growth factor A and B chains containing polybasic cell retention sequences. By contrast, the binding to fibroblast growth factor 2 is affected to a much lesser extent. The enhanced binding of aorta heparan sulfate to platelet-derived growth factor is suggested to be due to an age-dependent increase of GlcN 6-O-sulfation, resulting in increased abundance of the trisulfated L-iduronic acid (2-OSO3)-GlcNSO3(6-OSO3) disaccharide unit. Such units have been shown to hallmark the platelet-derived growth factor A chain-binding site in heparan sulfate.
Asunto(s)
Envejecimiento/metabolismo , Aorta Abdominal/metabolismo , Heparitina Sulfato/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Conformación de Carbohidratos , Femenino , Sustancias de Crecimiento/metabolismo , Heparitina Sulfato/química , Humanos , Masculino , Persona de Mediana Edad , Unión ProteicaRESUMEN
Heparan sulfate at cell surfaces and in the extracellular matrix regulates cell proliferation and adhesion by binding to growth factors and matrix proteins via structurally specific oligosaccharide domains. We have used the hormonally regulated mouse mammary carcinoma cell line S115 as a model to elucidate the effect of malignant transformation on the structure of heparan sulfate. When cultured in the presence of testosterone, S115 cells form tumor cell colonies in soft agar and exhibit fibroblast-like morphology; withdrawal of testosterone results in a loss of the tumorigenic capacity and a switch towards epithelial morphology. Metabolically 35SO4-labeled heparan sulfate was isolated from testosterone-treated and non-treated S115 cells and subjected to structural analysis. We found that the testosterone-dependent malignant transformation was associated with reduced sulfation of heparan sulfate due to a approximately 40% decrease in the amount of GlcN6S units. By contrast, no significant differences were observed in the amounts of 2-O-sulfate or N-sulfate groups. The reduced 6-O-sulfation of GlcN units in heparan sulfate from transformed S115 cells led to a marked decrease in the amount of trisulfated IdoA2S-GlcNS6S units (IdoA, L-iduronic acid), implicated in many heparan sulfate-protein interactions.
Asunto(s)
Transformación Celular Neoplásica , Heparitina Sulfato/metabolismo , Sulfatos/metabolismo , Testosterona/farmacología , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Disacáridos/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Heparitina Sulfato/química , Heparitina Sulfato/aislamiento & purificación , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Glicoproteínas de Membrana/biosíntesis , Ratones , Modelos Biológicos , Proteoglicanos/biosíntesis , Proteínas Recombinantes/biosíntesis , Radioisótopos de Azufre , Sindecanos , Transfección , Células Tumorales CultivadasRESUMEN
Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.
Asunto(s)
Heparina/metabolismo , Heparitina Sulfato/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Sitios de Unión , Conformación de Carbohidratos , Cromatografía de Afinidad , Disacáridos/química , Glicosaminoglicanos/metabolismo , Oligosacáridos/química , Factor de Crecimiento Derivado de Plaquetas/genética , PorcinosRESUMEN
The sulfated glycosaminoglycans, heparan sulfate and heparin, are increasingly implicated in cell-biological processes such as cytokine action, cell adhesion, and regulation of enzymic catalysis. These activities generally depend on interactions of the polysaccharides with proteins, mediated by distinct saccharide sequences, and expressed at various levels of specificity, selectivity, and molecular organization. The formation of heparin/ heparan sulfate in the cell requires an elaborate biosynthetic machinery, that is conceived in terms of a novel model of glycosaminoglycan assembly and processive modification. Recent advances in the identification and molecular analysis of the enzymes and other proteins involved in the biosynthesis provide novel tools to study the regulation of the process, presently poorly understood, at the subcellular and cellular levels. The potential medical importance of heparin-related compounds is likely to promote the biotechnological exploitation of components of the biosynthetic machinery.
Asunto(s)
Heparitina Sulfato , Animales , Secuencia de Carbohidratos , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas/químicaRESUMEN
Syndecans are a family of integral membrane proteoglycans with conserved membrane-spanning and intracellular domains but with structurally distinct extracellular domains (ectodomains). They are known to function as heparan sulphate co-receptors in fibroblast growth factor signalling as well as to link cells directly to the extracellular matrix. These and other biological activities of syndecans involve specific interactions of the heparan sulphate side chains of syndecans with cytokines and extracellular matrix proteins. Four different vertebrate syndecans, designated as syndecans 1-4 (or syndecan, fibroglycan, N-syndecan and amphiglycan, respectively), are known. During embryonic development, syndecans have specific and highly regulated expression patterns that are distinct from the expression in adult tissue, suggesting an active role in morphogenetic processes. The developmental expression of syndecans is particularly intense in mesenchymal condensates and at epithelium mesenchyme interfaces, where a number of heparan sulphate-binding cytokines and matrix components are also expressed in a regulated manner, often spatially and temporally co-ordinated with the syndecan expression. Recent evidence indicates that the regulation of heparan sulphate fine structure (mainly the number and arrangement of sulphate groups along the polymer) provides a mechanism for the cellular control of syndecan-protein interactions. Furthermore, morphogenetically active cytokines such as fibroblast growth factor-2 and transforming growth factor-beta participate in the regulation of syndecan expression and glycosaminoglycan structure. This review discusses the developmental expression and binding functions of syndecans as well as the molecular regulation of specific heparan sulphate-protein interactions.