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Mauro S G Pavao - One of the best experts on this subject based on the ideXlab platform.
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Dermatan sulfate in tunicate phylogeny order specific sulfation pattern and the effect of 4idoa 2 sulfate β 1 3galnac 4 sulfate β 1 motifs in Dermatan sulfate on heparin cofactor ii activity
BMC Biochemistry, 2011Co-Authors: Eliene O. Kozlowski, P C Lima, Tito Monteiro Da Cruz Lotufo, Cristina P Vicente, Kazuyuki Sugahara, Mauro S G PavaoAbstract:Previously, we have reported the presence of highly sulfated Dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated Dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian Dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to Dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Disaccharide analyses indicated a high content of disulfated disaccharide units in the Dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in Dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in Dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, Dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas Dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of Dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of Dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated Dermatan sulfates and 2,6-sulfated Dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of Dermatan sulfates and the presence of 2,4-sulfated units. Our results indicate that in ascidian Dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in Dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.
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Dermatan Sulfate In Tunicate Phylogeny: Order-specific Sulfation Pattern And The Effect Of [→4idoa(2-sulfate)β-1→3galnac(4-sulfate)β- 1→] Motifs In Dermatan Sulfate On Heparin Cofactor Ii Activity
Scopus, 2011Co-Authors: Eliene O. Kozlowski, P C Lima, Tito Monteiro Da Cruz Lotufo, Cristina P Vicente, Kazuyuki Sugahara, Mauro S G PavaoAbstract:Previously, we have reported the presence of highly sulfated Dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated Dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian Dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to Dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Disaccharide analyses indicated a high content of disulfated disaccharide units in the Dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in Dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in Dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, Dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas Dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of Dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate heparin cofactor II activity of Dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated Dermatan sulfates and 2,6-sulfated Dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of Dermatan sulfates and the presence of 2,4-sulfated units. Our results indicate that in ascidian Dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in Dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.
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antithrombotic activity of Dermatan sulfate in heparin cofactor ii deficient mice
Blood, 2004Co-Authors: Cristina P Vicente, Mauro S G Pavao, Li He, Douglas M TollefsenAbstract:Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of Dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of Dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin Dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII+/+ mice that was not observed in HCII-/- animals. Pharmacokinetic studies suggested that porcine skin Dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate Dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate Dermatan sulfates, which have higher charge densities than mammalian Dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII-/- mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin Dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged Dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004;104:3965-3970)
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Unbalanced effects of Dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding.
Thrombosis and haemostasis, 2001Co-Authors: Cristina P Vicente, P. Zancan, L. L. Peixoto, R. Alves-sa, F. S. Araujo, Paulo A.s. Mourão, Mauro S G PavaoAbstract:We compared the anticoagulant, antithrombotic and bleeding effects of highly sulfated Dermatan sulfates from invertebrates and their mammalian counterpart. An invertebrate Dermatan sulfate containing 2-O-sulfated α-L-iduronic acid and 4-O-sulfated N-acetyl-β-D-galactosamine residues is a potent anticoagulant due to a high heparin cofactor II activity. It inhibits thrombin due to the formation of a covalent complex with heparin cofactor II, as in the case of mammalian Dermatan sulfate, but the effect occurs at lower concentrations for the invertebrate polysaccharide. Surprisingly, the invertebrate Dermatan sulfate has a lower potency to prevent thrombus formation on an experimental model and a lower bleeding effect in rats than the mammalian Dermatan sulfate. In contrast, another invertebrate Dermatan sulfate, also enriched in 2-O-sulfated α-L-iduronic acid, but in this case sulfated at O-6 position of the N-acetyl-β-D-galactosamine units, has no in vitro or in vivo anticoagulant activity, does not prevent thrombus formation but shows a bleeding effect similar to the mammalian glycosaminoglycan. Overall, these results demonstrate unbalanced effects of Dermatan sulfates with different sulfation patterns on coagulation, thrombosis and bleeding, and raise interesting questions concerning the relationship among these three biological actions of sulfated polysaccharides.
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highly sulfated Dermatan sulfates from ascidians structure versus anticoagulant activity of these glycosaminoglycans
Journal of Biological Chemistry, 1998Co-Authors: Mauro S G Pavao, Barbara Mulloy, Ana Paula Valente, Karin R M Aiello, Claudio C Werneck, L C F Silva, Niall S Colwell, Douglas M TollefsenAbstract:Abstract Dermatan sulfates with the same backbone structure [4-α-l-IdceA-1→3-β-d-GalNAc-1]nbut with different patterns of sulfation substitutions have been isolated from the ascidian body. All the ascidian Dermatan sulfates have a high content of 2-O-sulfated α-l-iduronic acid residues but differ in the pattern of sulfation of the N-acetyl-β-d-galactosamine units. Styela plicata and Halocynthia pyriformis have 4-O-sulfated units, but inAscidian nigra they are 6-O-sulfated. This collection of ascidian Dermatan sulfates (together with native and oversulfated mammalian Dermatan sulfate), where the extent and position of sulfate substitution have been fully characterized, were tested in anticoagulant assays. Dermatan sulfate from A. nigra has no discernible anticoagulant activity, which indicates that 4-O-sulfation of theN-acetyl-β-d-galactosamine is essential for the anticoagulant activity of this glycosaminoglycan. In contrast Dermatan sulfates from S. plicata and H. pyriformis are potent anticoagulants due to potentiation of thrombin inhibition by heparin cofactor II. These ascidian Dermatan sulfates have ∼10-fold and ∼6-fold higher activity with heparin cofactor II than native and an oversulfated mammalian Dermatan sulfate, respectively. They have no effect on thrombin or factor Xa inhibition by antithrombin. These naturally oversulfated ascidian Dermatan sulfates are sulfated at selected sites required for interaction with heparin cofactor II and thus have specific and potent anticoagulant activity.
Anders Malmstrom - One of the best experts on this subject based on the ideXlab platform.
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biological functions of iduronic acid in chondroitin Dermatan sulfate
FEBS Journal, 2013Co-Authors: Martin A. Thelin, Marco Maccarana, Ake Oldberg, Barbara Bartolini, Jakob B Axelsson, Renata Gustafsson, Emil Tykesson, Edgar M. Pera, Anders MalmstromAbstract:The presence of iduronic acid in chondroitin/Dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/Dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse regulatory functions. Iduronic acid is formed by two epimerases (i.e. Dermatan sulfate epimerase 1 and 2) that have different tissue distribution and properties. The role of iduronic acid in chondroitin/Dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler–Danlos syndrome: adducted thumb-clubfoot syndrome. Future research aims to understand the roles of the two epimerases and their interplay with the sulfotransferases involved in chondroitin sulfate/Dermatan sulfate biosynthesis. Furthermore, a better definition of chondroitin/Dermatan sulfate functions using different knockout models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation, as well as the role of iduronic acid in health and disease.
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Biological functions of iduronic acid in chondroitin/Dermatan sulfate
The FEBS journal, 2013Co-Authors: Martin A. Thelin, Marco Maccarana, Ake Oldberg, Barbara Bartolini, Jakob B Axelsson, Renata Gustafsson, Emil Tykesson, Edgar M. Pera, Anders MalmstromAbstract:The presence of iduronic acid in chondroitin/Dermatan sulfate changes the properties of the polysaccharides because it generates a more flexible chain with increased binding potentials. Iduronic acid in chondroitin/Dermatan sulfate influences multiple cellular properties, such as migration, proliferation, differentiation, angiogenesis and the regulation of cytokine/growth factor activities. Under pathological conditions such as wound healing, inflammation and cancer, iduronic acid has diverse regulatory functions. Iduronic acid is formed by two epimerases (i.e. Dermatan sulfate epimerase 1 and 2) that have different tissue distribution and properties. The role of iduronic acid in chondroitin/Dermatan sulfate is highlighted by the vast changes in connective tissue features in patients with a new type of Ehler–Danlos syndrome: adducted thumb-clubfoot syndrome. Future research aims to understand the roles of the two epimerases and their interplay with the sulfotransferases involved in chondroitin sulfate/Dermatan sulfate biosynthesis. Furthermore, a better definition of chondroitin/Dermatan sulfate functions using different knockout models is needed. In this review, we focus on the two enzymes responsible for iduronic acid formation, as well as the role of iduronic acid in health and disease.
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Dermatan sulfate epimerase 1 deficient mice have reduced content and changed distribution of iduronic acids in Dermatan sulfate and an altered collagen structure in skin
Molecular and Cellular Biology, 2009Co-Authors: Marco Maccarana, Sebastian Kalamajski, M Kongsgaard, Peter S Magnusson, Ake Oldberg, Anders MalmstromAbstract: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.
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Biosynthesis of Dermatan Sulphate in Cultured Fibroblasts
European journal of biochemistry, 2005Co-Authors: Ingrid Sjöberg, Anders MalmstromAbstract:Fibroblasts in culture were incubated with 35SO4 and [3H]glucosamine for periods ranging from 5 min to 24 h. 35S radioactivity was incorporated linearily into intracellular sulphated glycosaminoglycans for 10 min. At this time the rate of incorporation levelled off and radioactive glycans started to emerge in the pericellular pool. 20 min later radioactive glycans could be isolated from the medium. Incorporation of [3H]glucosamine into corresponding glycans was delayed by about 2–3 h. Dermatan sulphate isolated from the cells after half an hour of incubation contained much less iduronic acid than did chains obtained at a later times. The uronate composition of Dermatan sulphate from the pericellular and medium fractions did not change appreciably during the incubation period. To obtain further information about the structure of newly synthesized glycosaminoglycans as well as intermediates thereof, microsomes were incubated with UDP-[14C]glucuronic acid, 3′-phosphoadenosine 5′-phospho-[35S] sulphate and UDP-N-acetylgalactosamine. Polymeric acceptors for the synthesis of hyaluronate, heparan sulphate and Dermatan sulphate were present. Chondroitin, an intermediate in Dermatan sulphate synthesis could also be demonstrated. The copolymeric structure of newly synthesized, microsomal, Dermatan sulphate was similar to that of Dermatan sulphate obtained after 0.5–1 h of synthesis in cultured cells.
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The glucuronyl C5-epimerase activity is the limiting factor in the Dermatan sulfate biosynthesis.
Archives of biochemistry and biophysics, 2001Co-Authors: Kerstin Tiedemann, Thomas Larsson, Dick Heinegård, Anders MalmstromAbstract:Abstract An early step in the biosynthesis of Dermatan sulfate is polymerization to chondroitin, which then is modified by the d -glucuronyl C5-epimerase and mainly 4-O-sulfotransferase. The final structure of the Dermatan sulfate side chains varies and our aim was to identify, which of the two enzymes that are crucial to generate Dermatan sulfate copolymeric structures in tissues. Dermatan sulfate side chains of biglycan and decorin were prepared from fibroblasts and nasal and articular chondrocytes and characterized regarding detailed structure. Microsomes were prepared from these cells and the activities of d -glucuronyl C5-epimerase and 4-O-sulfotransferase were determined. Chondrocytes from nasal cartilage synthesized biglycan and decorin containing 10%, articular chondrocytes 20–30%, and fibroblast 80% of the uronosyl residues in the l -iduronyl configuration. All three tissues contained high amount of 4-O-sulfotransferase activity. The activity of d -glucuronyl C5-epimerase showed different relationships. Fibroblasts contained a high level of the epimerase activity, articular chondrocytes intermediary activity, and in nasal cartilage it was barely detectable. The data indicate that the activity of the d -glucuronyl C5-epimerase is the main factor for formation of Dermatan sulfate in tissues.
Douglas M Tollefsen - One of the best experts on this subject based on the ideXlab platform.
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antithrombotic activity of Dermatan sulfate in heparin cofactor ii deficient mice
Blood, 2004Co-Authors: Cristina P Vicente, Mauro S G Pavao, Li He, Douglas M TollefsenAbstract:Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of Dermatan sulfate or heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of Dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin Dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII+/+ mice that was not observed in HCII-/- animals. Pharmacokinetic studies suggested that porcine skin Dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate Dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate Dermatan sulfates, which have higher charge densities than mammalian Dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII-/- mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin Dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged Dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004;104:3965-3970)
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amino acid residues of heparin cofactor ii required for stimulation of thrombin inhibition by sulphated polyanions
Biochimica et Biophysica Acta, 1999Co-Authors: Niall S Colwell, Michael J Grupe, Douglas M TollefsenAbstract:Abstract A variety of sulphated polyanions in addition to heparin and Dermatan sulphate stimulate the inhibition of thrombin by heparin cofactor II (HCII). Previous investigations indicated that the binding sites on HCII for heparin and Dermatan sulphate overlap but are not identical. In this study we determined the concentrations (IC50) of various polyanions required to stimulate thrombin inhibition by native recombinant HCII in comparison with three recombinant HCII variants having decreased affinity for heparin (Lys-173→Gln), Dermatan sulphate (Arg-189→His), or both heparin and Dermatan sulphate (Lys-185→Asn). Pentosan polysulphate, sulphated bis-lactobionic acid amide, and sulphated bis-maltobionic acid amide resembled Dermatan sulphate, since their IC50 values were increased to a much greater degree (≥8-fold) by the mutations Arg-189→His and Lys-185→Asn than by Lys-173→Gln (≤1.5-fold). By contrast, the IC50 values for fucosylated chondroitin sulphate, chondroitin sulphate E, dextran sulphate, and fucoidan were minimally affected. Only in the case of heparin was the IC50 increased to a greater degree by both Lys-173→Gln and Lys-185→Asn (≥6-fold) than by Arg-189→His (≤1.5-fold). None of the polyanions significantly stimulated inhibition of thrombin by an N-terminal deletion mutant of HCII (Δ1–74). These results suggest that, like Dermatan sulphate and heparin, other polyanions stimulate HCII primarily by an allosteric mechanism requiring the N-terminal acidic domain.
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highly sulfated Dermatan sulfates from ascidians structure versus anticoagulant activity of these glycosaminoglycans
Journal of Biological Chemistry, 1998Co-Authors: Mauro S G Pavao, Barbara Mulloy, Ana Paula Valente, Karin R M Aiello, Claudio C Werneck, L C F Silva, Niall S Colwell, Douglas M TollefsenAbstract:Abstract Dermatan sulfates with the same backbone structure [4-α-l-IdceA-1→3-β-d-GalNAc-1]nbut with different patterns of sulfation substitutions have been isolated from the ascidian body. All the ascidian Dermatan sulfates have a high content of 2-O-sulfated α-l-iduronic acid residues but differ in the pattern of sulfation of the N-acetyl-β-d-galactosamine units. Styela plicata and Halocynthia pyriformis have 4-O-sulfated units, but inAscidian nigra they are 6-O-sulfated. This collection of ascidian Dermatan sulfates (together with native and oversulfated mammalian Dermatan sulfate), where the extent and position of sulfate substitution have been fully characterized, were tested in anticoagulant assays. Dermatan sulfate from A. nigra has no discernible anticoagulant activity, which indicates that 4-O-sulfation of theN-acetyl-β-d-galactosamine is essential for the anticoagulant activity of this glycosaminoglycan. In contrast Dermatan sulfates from S. plicata and H. pyriformis are potent anticoagulants due to potentiation of thrombin inhibition by heparin cofactor II. These ascidian Dermatan sulfates have ∼10-fold and ∼6-fold higher activity with heparin cofactor II than native and an oversulfated mammalian Dermatan sulfate, respectively. They have no effect on thrombin or factor Xa inhibition by antithrombin. These naturally oversulfated ascidian Dermatan sulfates are sulfated at selected sites required for interaction with heparin cofactor II and thus have specific and potent anticoagulant activity.
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a unique Dermatan sulfate like glycosaminoglycan from ascidian its structure and the effect of its unusual sulfation pattern on anticoagulant activity
Journal of Biological Chemistry, 1995Co-Authors: Mauro S G Pavao, Barbara Mulloy, Paulo A.s. Mourão, Douglas M TollefsenAbstract:A Dermatan sulfate, similar to the mammalian glycosaminoglycans but not identical with any of them, has been isolated from the body of the ascidian Ascidia nigra. Degradation with chondroitin ABC lyase, analysis of the disaccharide products by digestion with chondro-4- and −6-sulfatases, and 1H and 13C NMR data confirm that the predominant structure is [4-α-L-IdoA-(2SO4)-1→3-β-D-GalNAc(6SO4)-1]n. Mammalian Dermatan sulfate is an anticoagulant due to its ability to potentiate inhibition of thrombin by heparin cofactor II. The structure in Dermatan sulfate which binds to heparin cofactor II is [4-α-L-IdoA-(2SO4)-1→3-β-D-GalNAc(4SO4)-1]n, where n ≥ 3. We have compared the ascidian Dermatan sulfate with mammalian Dermatan sulfate and with chemically oversulfated mammalian Dermatan sulfate for anticoagulant activity as measured by the activated partial thromboplastin time assay and for its ability to potentiate heparin cofactor II. In spite of its high content of 2-O-sulfated α-L-iduronic acid residues, the ascidian compound had no discernible anticoagulant activity and had low ability to potentiate heparin cofactor II. These results suggest that 4-O-sulfation of the N-acetyl-β-D-galactosamine residues is essential for the anticoagulant activity of Dermatan sulfate.
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role of lysine 173 in heparin binding to heparin cofactor ii
Journal of Biological Chemistry, 1991Co-Authors: Herbert C. Whinna, Morey A Blinder, Mark Szewczyk, Douglas M Tollefsen, Frank C ChurchAbstract:Abstract Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits alpha-thrombin in a reaction that is dramatically enhanced by heparin and other glycosaminoglycans/polyanions. We investigated the glycosaminoglycan binding site in HC by: (i) chemical modification with pyridoxal 5'-phosphate (PLP) in the absence and presence of heparin and Dermatan sulfate; (ii) molecular modeling; and (iii) site-directed oligonucleotide mutagenesis. Four lysyl residues (173, 252, 343, and 348) were protected from modification by heparin and to a lesser extent by Dermatan sulfate. Heparin-protected PLPHC retained both heparin cofactor and Dermatan sulfate cofactor activity while Dermatan sulfate-protected PLPHC retained some Dermatan sulfate cofactor activity and little heparin cofactor activity. Molecular modeling studies revealed that Lys173 and Lys252 are within a region previously shown to contain residues involved in glycosaminoglycan binding. Lys343 and Lys348 are distant from this region, but protection by heparin and Dermatan sulfate might result from a conformational change following glycosaminoglycan binding to the inhibitor. Site-directed mutagenesis of Lys173 and Lys343 was performed to further dissect the role of these two regions during HC-heparin and HC-Dermatan sulfate interactions. The Lys343----Asn or Thr mutants had normal or only slightly reduced heparin or Dermatan sulfate cofactor activity and eluted from heparin-Sepharose at the same ionic strength as native recombinant HC. However, the Lys173----Gln or Leu mutants had greatly reduced heparin cofactor activity and eluted from heparin-Sepharose at a significantly lower ionic strength than native recombinant HC but retained normal Dermatan sulfate cofactor activity. Our results demonstrate that Lys173 is involved in the interaction of HC with heparin but not with Dermatan sulfate, whereas Lys343 is not critical for HC binding to either glycosaminoglycan. These data provide further evidence for the determinants required for glycosaminoglycan binding to HC.
Richard L Gallo - One of the best experts on this subject based on the ideXlab platform.
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structural and sequence motifs in Dermatan sulfate for promoting fibroblast growth factor 2 fgf 2 and fgf 7 activity
Journal of Biological Chemistry, 2005Co-Authors: Kristen R Taylor, Jennifer A Rudisill, Richard L GalloAbstract:Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although Dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of Dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active Dermatan sulfate oligosaccharides, Dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated Dermatan sulfate preparations. This indicated a preference for specific Dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.
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Dermatan sulfate proteoglycan and glycosaminoglycan synthesis is induced in fibroblasts by transfer to a three dimensional extracellular environment
Journal of Biological Chemistry, 2004Co-Authors: Phillip Lee, Janet M Trowbridge, Kristen R Taylor, Vera B Morhenn, Richard L GalloAbstract:Composition and architecture of the extracellular matrix dictate cell behavior. Proteoglycans bind multiple components of the extracellular matrix by serving as important regulators of cell behavior. Given the influence of culture architecture on cell function, we investigated whether switching NIH3T3 fibroblasts from growth on type 1 collagen in monolayer to a collagen gel might influence Dermatan sulfate expression. Immunofluorescent staining, immunoblot, and Western blot demonstrated an induction in decorin expression in cells switched to collagen gels. This induction was associated with a 40-fold increase in decorin transcript expression determined by quantitative real time PCR. Disaccharide analysis of extracted glycosaminoglycans from collagen gels showed an increase in total glycosaminoglycan and in the ratio of chondroitin sulfate to heparan sulfate compared with monolayer culture. The ratio of chondroitin sulfate to heparan sulfate likewise increased on syndecan-1 from gel culture. Digestion with chondroitinase B showed that this induced chondroitin sulfate was Dermatan sulfate. Syndecan-1 extracted from wounded mouse skin also displayed an increase in Dermatan sulfate synthesis compared with unwounded skin. Furthermore, glycosaminoglycans from collagen gel culture activated keratinocyte growth factor, whereas glycosaminoglycans from monolayer culture lacked this ability. These findings suggest that regulation of Dermatan sulfate and Dermatan sulfate proteoglycan is dependent on extracellular matrix architecture. The ability of collagen gel culture to mimic better the in vivo dermal environment may be due in part to this influence on Dermatan sulfate and Dermatan sulfate proteoglycan synthesis.
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Dermatan sulfate binds and potentiates activity of keratinocyte growth factor fgf 7
Journal of Biological Chemistry, 2002Co-Authors: Janet M Trowbridge, Jennifer A Rudisill, Richard L GalloAbstract:Abstract FGF-7 is induced after injury and induces the proliferation of keratinocytes. Like most members of the FGF family, the activity of FGF-7 is strongly influenced by binding to heparin, but this glycosaminoglycan is absent on keratinocyte cell surfaces and minimally present in the wound environment. In this investigation we compared the relative activity of heparan sulfate and chondroitin sulfate B (Dermatan sulfate), glycosaminoglycans that are present in wounds. A lymphoid cell line (BaF/KGFR) containing the FGF-7 receptor (FGFR2 IIIb) was treated with FGF-7 and with various glycosaminoglycans. FGF-7 did not support cell proliferation in the absence of glycosaminoglycan or with addition of heparan sulfate or chondroitin sulfate A/C but did stimulate BaF/KGFR division in the presence of Dermatan sulfate or highly sulfated low molecular weight fractions of Dermatan. Dermatan sulfate also enabled FGF-7-dependent phosphorylation of mitogen-activated protein kinase and promoted binding of radiolabeled FGF-7 to FGFR2 IIIb. In addition, Dermatan sulfate and FGF-7 stimulated growth of normal keratinocytes in culture. Thus, Dermatan sulfate, the predominant glycosaminoglycan in skin, is the principle cofactor for FGF-7.
Theodore Tsegenidis - One of the best experts on this subject based on the ideXlab platform.
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isolation and characterisation of a small Dermatan sulphate proteoglycan from ray skin raja clavata
Comparative Biochemistry and Physiology B, 1999Co-Authors: Costas C Chatziioannidis, Nikos K. Karamanos, Theodore TsegenidisAbstract:The major proteoglycan of ray (Raja clavata) skin, which is a Dermatan sulphate proteoglycan, DSI-PG, was extracted, isolated and characterised. This proteoglycan has an average molecular size (Mr) of 110 kDa, and is composed of one Dermatan sulphate chain (Mr 55 kDa) and uronic acid containing oligosaccharides attached to the protein core. SDS-polyacrylamide gel electrophoresis of chondroitinase ABC deglycosylated PG revealed three protein cores with Mrs of about 57, 43 and 31 kDa. The amino acid composition of this preparation showed large amounts of phenylalanine, glutamic acid/glutamine, glycine, threonine and serine; cysteine was not detected. Dermatan sulphate is rich in iduronic acid (68% of total uronic acid) and composed mainly of mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (61%) or C-6 (4%), disulphated disaccharides with a peculiar sulphation pattern (32%) and a minor portion of non-sulphated ones (3%). The findings indicate that the major proteoglycan of ray skin is a small Dermatan sulphate proteoglycan and suggest that the Dermatan sulphate chain contains some sulphated disaccharide units with esterified sulphate groups possibly at the C-2 and/or C-3 position(s) of the uronic acid.
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PURIFICATION AND CHARACTERISATION OF A MINOR LOW-SULPHATED Dermatan SULPHATE-PROTEOGLYCAN FROM RAY SKIN
Biochimie, 1999Co-Authors: Costas C Chatziioannidis, Stavros T. Anagnostides, Nikos K. Karamanos, Theodore TsegenidisAbstract:Abstract A minor low-sulphated Dermatan sulphate proteoglycan was isolated from ray skin by extraction with 2% sodium dodecyl sulphate, followed with ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan with a relative molecular mass ( M r ) ranging from 70 to 120 kDa is composed of about two Dermatan sulphate chains ( M r 33 kDa) bound on a protein core of M r 27 kDa, and oligosaccharides consisting of uronic acids, hexosamines and neutral sugars. The major amino acids of the protein core were glycine (corresponding to about one-fourth of the total amino acids), serine, threonine, glutamic acid/glutamine, leucine and cysteine, together amounting to 56% of the total. The isolated proteoglycan does not interact with hyaluronic acid and does not form self-aggregates. Dermatan sulphate was rich in iduronic acid (62% of total uronic acid) and composed of non-sulphated (44%), and mono-sulphated disaccharides bearing esterified sulphate groups at positions C-4 (53%) or C-6 (3%) of the N-acetyl galactosamine. HPLC analysis of a pure preparation of Dermatan sulphate, showed the presence of galactose and glucose possibly as branches on the Dermatan sulphate chain.
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isolation and high performance liquid chromatographic analysis of ray raja clavata skin glycosaminoglycans
Comparative Biochemistry and Physiology B, 1991Co-Authors: Nikos K. Karamanos, A Manouras, D Politou, P Gritsoni, Theodore TsegenidisAbstract:Abstract 1. 1. Ray skin contains three glycosaminoglycans, the major one being Dermatan sulphate ( ca 80% of the tissue glycosaminoglycans), and the others low-sulphated Dermatan sulphate and hyaluronic acid. 2. 2. Dermatan sulphate of M r 6 × 10 4 was rich in iduronic acid and composed of 60.5% Δdi-4S,6% Δdi-6S, 3.5% Δdi-0S and 30% Δdi-diS. Disulphated Δ-disaccharides were of the following types: 19% Δdi-diS k , 12.6% Δdi-diS D . 62.5% of the units sulphated at C-2 and C-3 of the uronic acids, and 6% of the units sulphated at C-2 or C-3 of the uronic acids and at the galactosamine amino group. It also contained the neutral monosaccharides xylose, glucose and galactose. 3. 3. Low-sulphated Dermatan sulphate of M r 1.25 × 10 5 was rich in glucuronic acid and contained the neutral monosaccharides glucose and galactose. 4. 4. Hyaluronic acid ( ca 1% of the tissue glycosaminoglycans) was detected as Δdi-0S HA by HPLC.