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)

Antithrombin inhibits chemokine-induced migration of neutrophils by activating heparan sulfate proteoglycan-dependent signaling. Whether antithrombin affects migration of other types of leukocytes is not known. We investigated the effects of antithrombin on spontaneous and chemokine-triggered migration of lymphocytes and monocytes from human peripheral blood in modified Boyden chamber micropore filter assays. Lymphocyte and monocyte populations from human peripheral blood were purified using magnetic antibody cell sorting. The signaling mechanisms required for antithrombin-dependent migration were studied using signaling enzyme blockers. Expression of heparan sulfate proteoglycan core protein was studied by RT-PCR and flow cytometry. The antithrombins used were Kybernin P from human plasma and a monoclonal-antibody-purified preparation from this plasma. Pretreatment of lymphocytes and monocytes with antithrombin inhibited chemotaxis toward optimal concentrations of interleukin-8 or Rantes (regulated upon activation normal T-cell expressed and activated) at concentrations of antithrombin as low as 10 nU/ml. In the absence of the chemokines, direct exposure of cells to gradients of antithrombin stimulated migration. Effects of antithrombin were abolished by pretreating cells with heparinase-1, chondroitinase, sodium chlorate and anti-syndecan-4 antibodies. Expression of syndecan-4 mRNA and protein in monocytes and lymphocytes was demonstrated in RT-PCR and anti-syndecan-4 immunoreactivity assays, respectively. In the presence of pentasaccharide, antithrombin lost its effect on cells. Data indicate that antithrombin directly inhibits chemokine-stimulated migration of monocytes and lymphocytes via the effects of its heparin-binding site on cell surface syndecan-4 by activation of protein kinase C and Rho signaling.
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PMID:Syndecan-4 mediates antithrombin-induced chemotaxis of human peripheral blood lymphocytes and monocytes. 1180 40

Cell adhesion to fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with alpha5beta1 integrin and the heparin-binding domain II (Hep II) domain with syndecan-4. Within Hep II, this activity was mapped to repeat III13 and to the peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic heparin-binding peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds heparin and mediates cell adhesion via chondroitin sulphate proteoglycans. We have now studied whether HBP/III5 co-operates with alpha5beta1 and drives a full cytoskeletal response in melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7-FNIII10 (FNIII7-10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7-10 or adding soluble HBP/III5 to cells prespread on FNIII7-10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the small GTPase family, abolished the HBP/III5 effect. Both chondroitinase and heparitinase diminished focal adhesions, indicating that both types of proteoglycans bound HBP/III5 in melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in fibronectin with focal-adhesion-promoting activity. The last two arginine (R) residues of this sequence are required for activity, since their replacement by alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large fibronectin fragments. Our results establish that the Hep III region provides co-operative signals to alpha5beta1 for the progression of the cytoskeletal response and that these include activation of RhoA.
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PMID:A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells. 1251 80

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

Opioid receptors are expressed in cells of the immune system, and potent immunomodulatory effects of their natural and synthetic ligands have been reported. In some studies, the opiate receptor antagonist naloxone itself displayed immunomodulatory actions. We investigated effects of naloxone on leukocyte chemotaxis. Cell migration was tested in micropore filter assays using modified Boyden chambers, and receptor expression was investigated using radiolabel binding assays. Naloxone induced peripheral blood nonadherent mononuclear cell and neutrophil chemotaxis at nanomolar concentrations and deactivated their migration toward beta-endorphin, angiotensin II, somatostatin, or interleukin-8 but not toward RANTES, vasoactive intestinal peptide, or substance P. Ligand binding studies showed no alteration in the binding of interleukin-8 to neutrophils by naloxone. Cleavage of heparan sulfate from proteoglycans on the cells' surface completely inhibited chemotactic and deactivating properties of naloxone but not other attractants. Chemotactic properties were abolished by pretreating cells with heparinase, chondroitinase, sodium chlorate, and anti-syndecan-4 antibodies, indicating the involvement of syndecan-4. The extent of migration toward naloxone was diminished by pretreatment with dimethylsphingosine, a specific sphingosine kinase inhibitor. As syndecan-4 signaling in leukocyte chemotaxis involves activation of sphingosine kinase, results indicate that naloxone interacts with syndecan-4 function in cell migration and suggest a role for heparan sulfate proteoglycans as coreceptors to members of the delta-opiate receptor family.
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PMID:Heparan sulfate proteoglycans are involved in opiate receptor-mediated cell migration. 1470 51

We found that neurocan, a major brain chondroitin sulphate proteoglycan, interacts with HSPGs (heparan sulphate proteoglycans) such as syndecan-3 and glypican-1. Binding of these HSPGs to neurocan was prevented by treatment of the HSPGs with heparitinases I and II, but not by treatment of neurocan with chondroitinase ABC. Scatchard plot analysis indicated that neurocan has two binding sites for these HSPGs with different affinities. It is known that neurocan in the rodent brain is proteolytically processed with aging into N- and C-terminal fragments. When a mixture of whole neurocan and N- and C-terminal fragments prepared from neonatal mouse brains or recombinant N- and C-terminal fragments was applied to a heparin column, the whole molecule and both the N- and C-terminal fragments bound to heparin. A centrifugation cell adhesion assay indicated that both the N- and C-terminal neurocan fragments could interact with these HSPGs expressed on the cell surface. To examine the biological significance of the HSPG-neurocan interaction, cerebellar granule cells expressing these HSPGs were cultured on the recombinant neurocan substrate. A significant increase in the rate of neurite outgrowth was observed on the wells coated with the C-terminal neurocan fragment, but not with the N-terminal one. Neurite outgrowth-promoting activity was inhibited by pretreatment of neurocan substrate with heparin or the addition of heparitinase I to culture medium. These results suggest that HSPGs such as syndecan-3 and glypican-1 serve as the cell-surface receptor of neurocan, and that the interaction of these HSPGs with neurocan through its C-terminal domain is involved in the promotion of neurite outgrowth.
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PMID:Heparan sulphate proteoglycans interact with neurocan and promote neurite outgrowth from cerebellar granule cells. 1519 37

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

Chronic inflammation is characterized by tissue infiltration with monocytes/macrophages, which possess broad proinflammatory, destructive, and remodeling capacities. Elevated levels of osteoprotegerin, an important regulator of differentiation and activation of osteoclasts that also affects different cells of the immune system, were found in the serum of patients with chronic inflammatory diseases. The study of whether osteoprotegerin affects monocyte locomotion in vitro and the possible mechanisms and pathways involved was investigated using Boyden microchemotaxis chambers and Western blot analyses. Osteoprotegerin significantly stimulated monocyte chemotaxis, whereas preincubation of monocytes with osteoprotegerin inhibited monocyte migration toward optimal concentrations of regulated upon activation normal T cell expressed and secreted, monocyte chemotactic protein -1, and procalcitonin. The effects of osteoprotegerin were abolished by pretreating cells with heparinase I and chondroitinase or antibodies against the ectodomain of syndecan-1. Osteoprotegerin signaling was shown to involve protein kinase C, phosphatidylinositol 3-kinase/Akt, and tyrosine kinase. Data suggest that osteoprotegerin affects monocyte mi-gration and protein kinase C and phosphatidylinositol 3-kinase/Akt activation via syndecan-1. Osteoprotegerin-induced deactivation of monocyte chemotaxis toward different chemokines is due to interaction of osteoprotegerin with heparan sulfate and chondroitin sulfate.
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PMID:Syndecan-1 is involved in osteoprotegerin-induced chemotaxis in human peripheral blood monocytes. 1572 9

Cell surface proteoglycans play an important part in the functional and metabolic behaviour of leucocytes. We studied the expression of cell surface proteoglycans in human monocytes, in monocyte-derived immature and mature dendritic cells and in macrophages by metabolic labelling with [(35)S]-sulphate, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Immature dendritic cells had the highest metabolic activity for the synthesis of cell surface proteoglycans. The major part of these proteoglycans was in phosphatidylinositol-anchored form and was released after treatment with phospholipase C. A minor part was released by trypsin. Digestion with chondroitinase ABC and mild HNO(2) treatment showed that cell surface proteoglycans had a higher proportion of chondroitin sulphate, both in the phospholipase C and trypsin fractions, suggesting that at least some glypicans contained chondroitin sulphate chains. RT-PCR detected the transcripts of glypicans 1, 3, 4 and 5 and all syndecans. Immature dendritic cells expressed a most complex spectrum of glypicans and syndecans, glypican-1 and syndecan-1 being expressed preferentially by this type of cells. Mature dendritic cells expressed glypican-3, which was not present in other lineages. These results suggest that different mononuclear cells synthesize cell surface proteoglycans actively with characteristic expression of different syndecans and glypicans genes, depending on the degree of cell differentiation and/or maturation.
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PMID:Cell surface proteoglycan expression during maturation of human monocytes-derived dendritic cells and macrophages. 1673 18

Cell surface heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans have been implicated in a multitude of biological processes, including embryonic implantation, tissue morphogenesis, wound repair, and neovascularization through their ability to regulate growth factor activity and morphogenic gradients. However, the direct role of the glycosaminoglycan (GAG) sugar-side chains in the control of human mesenchymal stem cell (hMSC) differentiation into the osteoblast lineage is poorly understood. Here, we show that the abundant cell surface GAGs, HS and CS, are secreted in proteoglycan complexes that directly regulate the bone morphogenetic protein (BMP)-mediated differentiation of hMSCs into osteoblasts. Enzymatic depletion of the HS and CS chains by heparinase and chondroitinase treatment decreased HS and CS expression but did not alter the expression of the HS core proteins perlecan and syndecan. When digested separately, depletion of HS and CS chains did not effect hMSC proliferation but rather increased BMP bioactivity through SMAD1/5/8 intracellular signaling at the same time as increasing canonical Wnt signaling through LEF1 activation. Long-term culturing of cells in HS- and CS-degrading enzymes also increased bone nodule formation, calcium accumulation, and the expression of such osteoblast markers as alkaline phosphatase, RUNX2, and osteocalcin. Thus, the enzymatic disruption of HS and CS chains on cell surface proteoglycans alters BMP and Wnt activity so as to enhance the lineage commitment and osteogenic differentiation of hMSCs.
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PMID:Disruption of heparan and chondroitin sulfate signaling enhances mesenchymal stem cell-derived osteogenic differentiation via bone morphogenetic protein signaling pathways. 2609 86

The sulfatase enzymes, N-acetylgalactosamine-4-sulfatase (arylsulfatase B (ASB)) and galactose-6-sulfatase (GALNS) hydrolyze sulfate groups of CS. Deficiencies of ASB and GALNS are associated with the mucopolysaccharidoses. To determine if expression of ASB and GALNS impacts on glycosaminoglycans (GAGs) and proteoglycans beyond their association with the mucopolysaccharidoses, we modified the expression of ASB and GALNS by overexpression and by silencing with small interference RNA in MCF-7 cells. Content of total sulfated GAG (sGAG), chondroitin 4-sulfate (C4S), and total chondroitin sulfates (CSs) was measured following immunoprecipitation with C4S and CS antibodies and treatment with chondroitinase ABC. Following silencing of ASB or GALNS, total sGAG, C4S, and CS increased significantly. Following overexpression of ASB or GALNS, total sGAG, C4S, and CS declined significantly. Measurements following chondroitinase ABC treatment of the cell lysates demonstrated no change in the content of the other sGAG, including heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Following overexpression of ASB and immunoprecipitation with C4S antibody, virtually no sGAG was detectable. Total sGAG content increased to 23.39 (+/-1.06) microg/mg of protein from baseline of 12.47 (+/-0.68) microg/mg of protein following ASB silencing. mRNA expression of core proteins of the CS-containing proteoglycans, syndecan-1 and decorin, was significantly up-regulated following overexpression of ASB and GALNS. Soluble syndecan-1 protein increased following increases in ASB and GALNS and reduced following silencing, inversely to changes in CS. These findings demonstrate that modification of expression of the lysosomal sulfatases ASB and GALNS regulates the content of CSs.
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PMID:Distinct effects of N-acetylgalactosamine-4-sulfatase and galactose-6-sulfatase expression on chondroitin sulfates. 1828 41


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