Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.6.4 (chondroitinase)
2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Thrombomodulin (TM) is an anticoagulant glycoprotein on the surface of endothelial cell that directly inhibits the procoagulant activities of thrombin, and the TM-thrombin complex accelerates thrombin-catalyzed activation of protein C. Soluble TM in urine has no glycosaminoglycan (GAG) chain which accelerates the anticoagulant activities. Therefore, we expressed recombinant GAG-modified urinary thrombomodulin (GAG-UTM) in C127 cells. The glycosylation sites were determined by amino acid sequence analysis of peptides digested with trypsin after S-carboxymethylation. The structures of N-linked oligosaccharides were estimated by two-dimensional sugar mapping of pyridylaminated oligosaccharides that were treated with exoglycosidase. The disaccharide composition analysis of the GAG chain was performed by HPLC using digestion with chondroitinase ABC, ACII and B. Consequently, it was revealed that the N-linked oligosaccharides were assigned to Asn29, Asn98, Asn364, Asn391; those structures were estimated biantennary, 2-6 branched triantennary and 2-4 branched triantennary complex type oligosaccharides that were linked by fucose at the ratio of 1.0:0.5:0.1, respectively. Moreover, the attachment site of the GAG chain was assigned to Ser472. It was then estimated that the GAG chain contained chondroitin-4-sulfate and dermatan sulfate, which were repeated approximately 30 times. In this paper, the GAG attachment site and structural characteristics of GAG-UTM, were confirmed. Moreover, structures of the N-linked oligosaccharides of GAG-UTM are described for the first time.
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PMID:The glycosylation sites and structural characteristics of oligosaccharides on recombinant human thrombomodulin. 959 55

Circulating endotoxin is elevated in sepsis and plays a role in endothelial dysfunction whereas antithrombin is decreased by virtue of its consumption during complex formation with clotting factors and by proteolytic degradation by granulocyte elastase. Dysfunction of endothelium results in enhanced leukocyte rolling and diapedesis into tissues leading to edema formation and injury. Antithrombin exerts beneficial effects on endothelial function in sepsis. A direct anti-inflammatory action of anti-thrombin in inflammatory cells is exerted via heparan sulfate proteoglycans. In this study, we investigated whether antithrombin affects endotoxin-induced adhesion of neutrophils to human endothelial cells in vitro and whether glycosaminoglycans are involved in its signaling. Adhesion of human neutrophils to monolayers of umbilical vein endothelial cells was tested under static conditions. Endothelial cells were pretreated with endotoxin, interleukin-1, heparinase-I, chondroitinase-ABC or anti-syndecan-4-antibody. Endotoxin and interleukin-1 increased neutrophil adherence to human umbilical vein endothelial cells which was inhibited by antithrombin. Concomitant incubation with pentasaccharide abolished this effect of antithrombin. Treatment of endothelial cells with heparinase or chondroitinase led to higher adhesion and prevented effects of antithrombin. With antibodies to syndecan-4, enhanced adhesion of neutrophils was observed. As studied by Western blotting, endotoxin-induced signaling was diminished by antithrombin and the effect was reversible by chondroitinase or heparinase. From our results, we can conclude that endotoxin-induced adhesion of leukocytes to endothelium can be reversed by ligation of syndecan-4 with antithrombin's heparin-binding site and interferences with stress response signaling events in endothelium.
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PMID:Syndecan-4-dependent signaling in the inhibition of endotoxin-induced endothelial adherence of neutrophils by antithrombin. 1465 50

The accumulation of extracellular matrix components such as proteoglycans is a hallmark of an atherosclerotic lesion. A large heparan sulfate proteoglycan, perlecan, dramatically increases in the advanced lesion, and vascular smooth muscle cells are the cell type responsible for the accumulation. In this study, we investigated the effects of thrombin on the proteoglycan synthesis in cultured human coronary smooth muscle cells to determine the interrelationship between the accumulation of proteoglycans and the procoagulant state of blood in atherosclerosis. The cells were metabolically labeled with [(35)S]sulfate or (35)S-labeled amino acids in the presence of thrombin, and the labeled proteoglycans were characterized by Sepharose CL-4B molecular sieve chromatography and DEAE-Sephacel ion-exchange chromatography. The glycosaminoglycan M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was determined by SDS-polyacrylamide gel electrophoresis before and after digestion with chondroitinase ABC or papain. The results indicate that thrombin increases the cell layer-associated heparan sulfate proteoglycan with a core protein size of approximately 400 kDa without any change in the length of the glycosaminoglycan chains when the cell density is high. The heparan sulfate proteoglycan was identified as perlecan by Western blot analysis. In addition, quantitative reverse transcription-polymerase chain reaction showed that thrombin elevated the steady-state level of perlecan mRNA but not that of versican, decorin, and syndecan-1 mRNAs, although that of biglycan mRNA was moderately elevated. Furthermore, the percentage of disaccharide units that compose perlecan heparan sulfate chains remained unaffected by thrombin. Therefore, it is suggested that thrombin induces the perlecan core protein synthesis without influencing the formation of the heparan sulfate chains in human coronary smooth muscle cells at a high cell density. The regulation of proteoglycan synthesis by thrombin may be involved in the accumulation of perlecan in advanced lesions of atherosclerosis.
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PMID:Induction of synthesis of a large heparan sulfate proteoglycan, perlecan, by thrombin in cultured human coronary smooth muscle cells. 1571 25

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.
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PMID:N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate. 1662 94

The endothelial cell membrane glycoprotein thrombomodulin (TM) plays a critical role in the regulation of coagulation. TM is an essential cofactor in protein C activation by thrombin, and a direct inhibitor of thrombin-induced platelet activation and fibrinogen clotting. Protease nexin-1 (PN-1) is a serpin synthesized and secreted by a variety of cells including endothelial cells. PN-1 bound to the cell surface through interactions with glycosaminoglycans, is an efficient inhibitor of thrombin and controls thrombin-induced cell responses. An investigation of the interaction of PN-1 with TM using purified proteins and cultured human aortic endothelial cells was performed. Purified PN-1 was observed to bind to purified TM in a concentration-dependent manner. Double immunofluorescence studies indicated that PN-1 and TM were colocalized at the endothelial cell surface from which they were coprecipitated. Pretreatment of the cells with chondroitinase ABC greatly decreased the amount of the PN-1 associated to TM at the cell surface demonstrating the involvement of the TM chondroitin-sulfate chain in the formation of complexes. The inhibitory activity of the PN-1/TM complexes on the catalytic activity of thrombin, and on thrombin-induced fibrinogen clotting, was markedly enhanced when compared with the inhibitory activity of each partner. PN-1-overexpressing human aortic endothelial cells and PN-1-underexpressing human aortic endothelial cells exhibited respectively a significantly reduced ability and enhanced capacity to activate protein C. Furthermore, PN-1 decreased the cofactor activity of TM on thrombin activable fibrinolysis inhibitor activation by thrombin. These data show for the first time that PN-1 forms complexes with TM and modulates its anticoagulant activity.
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PMID:Protease nexin-1 interacts with thrombomodulin and modulates its anticoagulant effect. 1737 30

A dermatan sulfate isolated from the shark Scyliorhinus canicula skin by enzymatic digestion followed by purification with anion exchange chromatography was identified by chondroitinase and nitrous acid treatment and partially characterized by Fourier-transform infrared spectroscopy. Dermatan sulfate was the major glycosaminoglycan and represented 75% of the polysaccharide fraction in the sharkskin. This dermatan sulfate had a 38.6 kDa average molecular weight and 23% sulfate content. The anticoagulant action of this dermatan sulfate was checked by several coagulometric and colorimetric assays such as the activated partial thromboplastin time, thrombin time, thrombin generation and heparin cofactor II and antithrombin-mediated inhibition of thrombin and compared with that of porcine intestinal mucosa dermatan sulfate. The effects on platelet activation and aggregation were investigated using flow cytometry and aggregometry, respectively. The dermatan sulfate prolonged activated partial thromboplastin time and thrombin time, delayed and inhibited thrombin generation in a concentration-dependent manner. The specific anticoagulant activity of the sharkskin dermatan sulfate was 43 UI/mg. The anticoagulant effect of sharkskin dermatan sulfate was higher than that of the porcine dermatan sulfate and was due to the potentiation of thrombin inhibition by heparin cofactor II. Moreover, it had no effect on platelet aggregation and activation induced by various agonists and thereby constitutes a potentially useful drug of interest in anticoagulant therapy.
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PMID:Anticoagulant activity of a dermatan sulfate from the skin of the shark Scyliorhinus canicula. 2058 62


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