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)

Proteoglycans have been implicated in the clustering of acetylcholine receptors (AChRs) on cultured myotubes and at the neuromuscular junction. We report that the presence of chondroitin sulfate is associated with the ability of cultured myotubes to form spontaneous clusters of AChRs. Three experimental manipulations of wild type C2 cells in culture were found to affect both glycosaminoglycans (GAGs) and AChR clustering in concert. Chlorate was found to have dose-dependent negative effects both on GAG sulfation and on the frequency of AChR clusters. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild-type C2 myotubes, increases were observed both in the level of cell layer-associated chondroitin sulfate and in the frequency of AChR clusters. Culture of wild-type C2 myotubes in the presence of chondroitinase ABC eliminated cell layer-associated chondroitin sulfate while leaving heparan sulfate intact and simultaneously prevented the formation of AChR clusters. Treatment with either chlorate or chondroitinase inhibited AChR clustering only if begun prior to the spontaneous formation of clusters. We propose that chondroitin sulfate plays an essential role in the initiation of AChR clustering and in the early events of synapse formation on muscle.
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PMID:Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate. 859 8

Uronic-acid-rich protein (UAP) is a urinary glycoprotein that inhibits calcium oxalate crystallization in vitro. It shows a structural similarity to bikunin, a component of inter-alpha-inhibitor (IalphaI) known for its inhibition of the action of many serine proteinases like trypsin and chymotrypsin. To clarify the relationship between these macromolecules, UAP, IalphaI, urinary bikunin, and plasma bikunin were purified and studied. Their calcium oxalate crystallization inhibitory activity was assayed before and after treatment with chondroitinase AC and pronase. Their molecular mass was determined by using SDS/PAGE before and after these treatments. Polyclonal bikunin antibody was used on Western blots for immunological identification. The partial amino acid sequence of UAP before and after chondroitinase treatment was determined. Also, the antitryptic activity of UAP was measured and compared to that of bikunin, which is responsible for the antiprotease activity of IalphaI. UAP exhibited a strong calcium oxalate crystallization inhibitory activity. IalphaI and both bikunins were less inhibitory. Chondroitinase AC had no effect on inhibitory activity of these proteins even when their molecular mass changed. However, after pronase treatment, the inhibitory activity of both bikunins and UAP was completely destroyed. The antitryptic activity of UAP was found to be 0.78 U/mg which is lower than that of bikunin which is about 1.9 U/mg. On Western blotting, bikunin antibody immunoreacted with UAP and both urinary and plasma bikunins. Partial amino acid sequence confirmed the identity of UAP as urinary bikunin.
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PMID:Identification of uronic-acid-rich protein as urinary bikunin, the light chain of inter-alpha-inhibitor. 866 22

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, enhanced the antithrombin activity of heparin cofactor II (HC II) more than 10000-fold. The apparent second-order rate constant of thrombin inhibition by HC II was calculated to be 4.2 x 10(4) M-1 min-1 in the absence of Ca-SP, and it increased in the presence of 50 micrograms/ml Ca-SP to 4.5 x 10(8) M-1 min-1. Ca-SP effectively induced the formation of a thrombin-HC II complex in plasma. In the presence of Ca-SP, both the recombinant HC II variants Lys173-->Leu and Arg 189-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHC II. This result indicates that the binding site of HC II for Ca-SP is different from the heparin- or dermatan sulfate-binding site. When we removed the calcium from the Ca-SP, the compound did not exert any antithrombin activity. Furthermore, Na-SP, which was prepared by replacement of the calcium in Ca-SP with sodium, accelerated the antithrombin activity of HC II as Ca-SP did. We therefore suggest that the molecular conformation maintained by Ca or Na is indispensable to the antithrombin activity of Ca-SP. The HC II-dependent antithrombin activity of Ca-SP was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, but not by treatment with chondroitinase ABC and chondroitinase AC II, suggesting that a heparin- or dermatan sulfate-like structure is not responsible for the activation of HC II by Ca-SP. Ca-SP is therefore thought to be a unique sulfated polysaccharide which shows a strong antithrombin effect in an exclusively HC II-dependent manner.
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PMID:Heparin cofactor II-dependent antithrombin activity of calcium spirulan. 887 66

High performance capillary electrophoresis was used to determine impurities in glycosaminoglycans. The counterion of glycosaminoglycans was analyzed with indirect UV-detection using a 40 mM 4-aminopyridine buffer. Calcium, lithium, potassium and sodium could be resolved. A linear correlation between the area under the curve and the concentration of sodium (r2 = 0.98) and calcium (r2 = 0.99) was found. Using enzymatic depolymerization, chondroitin sulfates were cleaved to disaccharides. The resulting disaccharides, with the structure 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid (delta UA) 2 x (1-->3)-D-GalNY6X (X = H, sulfate and Y = acetyl, sulfate) for dermatan sulfate, were detected selectively at 230 nm using capillary electrophoresis. Dermatan sulfate disaccharides were analyzed using a 50 cm long fused silica capillary (75 microns ID). The buffer used was 10 mM sodium tetraborate and 50 mM SDS, pH 8.8. The detection was at 230 nm. Using the main peak delta UA (1-->3)-D-GalNAc4S as standard, between 1 and 80% dermatan sulfate in heparin preparations were analyzed. The disaccharide showed a linear correlation of the peak area versus the concentration with a correlation coefficient r2 = 0.98. The methods are useful in characterizing the identity and concentration of the counterion of glycosaminoglycans after chondroitinase degradation.
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PMID:Purity of glycosaminoglycan-related compounds using capillary electrophoresis. 890 Sep 50

Morquio syndrome (mucopolysaccharidosis IV) presents with multiple bone dysplasia and is characterized by the inability to degrade keratan sulfate due to deficient N-acetylgalactosamine-6-sulfate sulfatase in Morquio A syndrome and deficient beta-D-galactosidase in Morquio B syndrome. The aim of our study was to investigate into the pathogenetic mechanism as it is not clear whether the accumulation of keratan sulfate is toxic for osteoblasts or inhibits osteoblast activity as e.g. bone resorption. The glycosaminoglycans keratan sulfate, heparan sulfate, dermatan sulfate, chondroitin-4,6-sulfate and hyaluronic acid were tested in rat neonatal calvarian cultures for their effects on bone resorption, osteoblast activity and toxicity. Bone resorption was evaluated by calcium release into the medium, osteoblast activity by the determination of alkaline phosphatase and toxicity by measuring lactate dehydrogenase in the culture media. Keratan sulfate had no effect on bone resorption but inhibited osteoblast activity at the low, nontoxic concentration of 10 ng per ml organ culture supernatant significantly (p<0.05). At a concentration of 100 ng per ml keratan sulfate revealed toxic effects as reflected by significantly (p<0.05) elevated lactate dehydrogenase activity. None of the other glycosaminoglycans inhibited osteoblast activities. Heparan sulfate showed at toxic levels (10 microg per ml supernatant) significantly increased bone resorption (p<0.05) accompanied by increased alkaline phosphatase activity. The specific keratan sulfate effects of inhibiting osteoblast activity and toxicity towards bone, which were never tested before, suggest a role for this glycosaminoglycan in the pathogenesis of bone dysplasia in Morquio syndrome.
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PMID:The effects of acid glycosaminoglycans on neonatal calvarian cultures--a role of keratan sulfate in Morquio syndrome? 927 6

We have isolated a chondroitin sulfate proteoglycan from mouse brain by affinity chromatography with a fragment of the extracellular matrix glycoprotein tenascin-R (TN-R) that comprises the amino-terminal cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats. The isolated chondroitin sulfate proteoglycan has a molecular mass of 500-600 kDa and carries the HNK-1 carbohydrate epitope. Treatment with chondroitinase ABC reveals a major band of approximately 400 kDa and two minor bands at 200 and 150 kDa. Immunoblot analysis relates the molecule to phosphacan but not to the chondroitin sulfate proteoglycans neurocan and versican. Binding of the phosphacan-related molecule to the epidermal growth factor-like repeats of TN-R is Ca2+-dependent. Co-localization of the molecule with TN-R in the retina and optic nerve by immunocytochemistry suggests a functional relationship between the two molecules in vivo. Inhibition of neurite outgrowth from hippocampal neurons by the phosphacan-related molecule in vitro is neutralized by TN-R when coated as a uniform substrate. Furthermore, the phosphacan-related molecule neutralizes growth cone repulsion induced by TN-R coated as a sharp substrate boundary with or without prior treatment with chondroitinase ABC. These observations indicate that TN-R can interact with a phosphacan-related molecule and thereby modulate its inhibitory influence on neuritogenesis.
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PMID:Isolation of a tenascin-R binding protein from mouse brain membranes. A phosphacan-related chondroitin sulfate proteoglycan. 940 6

We have studied the interactions of the nervous tissue-specific chondroitin sulfate proteoglycans neurocan and phosphacan with the extracellular matrix protein tenascin-R and two heparin-binding proteins, amphoterin and the heparin-binding growth-associated molecule (HB-GAM), using a radioligand binding assay. Both proteoglycans show saturable, high affinity binding to tenascin-R with apparent dissociation constants in the 2-7 nM range. Binding is reversible, inhibited in the presence of unlabeled proteoglycan, and increased by approximately 60% following chondroitinase treatment of the proteoglycans, indicating that the interactions are mediated via the core (glyco)proteins rather than by the glycosaminoglycan chains, which may in fact partially shield the binding sites. In contrast to their interactions with tenascin-C, in which binding was decreased by approximately 75% in the absence of calcium, binding of phosphacan to tenascin-R was not affected by the absence of divalent cations in the binding buffer, although there was a small but significant decrease in the binding of neurocan. Neurocan and phosphacan are also high affinity ligands of amphoterin and HB-GAM (Kd = 0.3-8 nM), two heparin-binding proteins that are developmentally regulated in brain and functionally involved in neurite outgrowth. The chondroitin sulfate chains on neurocan and phosphacan account for at least 80% of their binding to amphoterin and HB-GAM. The presence of amphoterin also produces a 5-fold increase in phosphacan binding to the neural cell adhesion molecule contactin. Immunocytochemical studies showed an overlapping localization of the proteoglycans and their ligands in the embryonic and postnatal brain, retina, and spinal cord. These studies have therefore revealed differences in the interactions of neurocan and phosphacan with the two major members of the tenascin family of extracellular matrix proteins, and also suggest that chondroitin sulfate proteoglycans play an important role in the binding and/or presentation of differentiation factors in the developing central nervous system.
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PMID:High affinity binding and overlapping localization of neurocan and phosphacan/protein-tyrosine phosphatase-zeta/beta with tenascin-R, amphoterin, and the heparin-binding growth-associated molecule. 950 7

The eggshell of the chicken is a useful model to study matrix components which affect biomineralization. As an extension of our previous immunohistochemical work which suggested the presence of dermatan sulfate proteoglycans in the mineralized region of the eggshell, a study was undertaken to characterize these molecules biochemically. After demineralization with HCl and extraction with 4 M guanidinium chloride containing protease inhibitors, the extract was partitioned by anion exchange chromatography. Step elution with 0.25 M and 1.0 M sodium chloride resulted in the generation of two fractions, both of which contain chondroitinase-sensitive proteoglycans with molecular weights estimated at 200,000 by gel electrophoresis. The proteoglycans in each fraction have core proteins with molecular weights of approximately 120,000 and glycosaminoglycans with average molecular weights of 22,000. Based on differential sensitivity to chondroitinase ABC and AC II, these glycosaminoglycans contain a small proportion of dermatan sulfate. The disaccharide compositions of these glycosaminoglycans differ for the proteoglycans eluted with 0.25 M and 1.0 M sodium chloride. Those eluted with lower sodium chloride are enriched in unsulfated chondroitin and have much more 4-sulfated than 6-sulfated disaccharides; those eluted with 1.0 M sodium chloride contain primarily 4-sulfated disaccharides, a small amount of 6-sulfated disaccharides, and less unsulfated disaccharides than the proteoglycans eluted with 0.25 M sodium chloride. The large difference in the proportions of unsulfated chondroitin may be the reason for the elution at different sodium chloride concentrations. Both of the anion exchange column fractions contain other proteins in addition to the proteoglycans. These proteins are not separated from the proteoglycans by a second anion exchange column or by molecular sieve chromatography under dissociative conditions. Of particular interest is the observation that the eggshell proteoglycans and their core proteins are recognized by a monoclonal antibody which recognizes an epitope on the core protein of avian versican. This suggests that, in spite of the large differences in the sizes of the core proteins of versican and the eggshell proteoglycans, these core proteins share some homology. Because anionic molecules are thought to be important regulators of biomineralization, and because preparations like those analyzed in this study have been shown to influence in vitro calcium carbonate crystallization, the eggshell proteoglycans may play a role in eggshell mineralization.
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PMID:Dermatan sulfate proteoglycans from the mineralized matrix of the avian eggshell. 951 87

Platelet factor 4 (PF-4), a member of the alpha-chemokine subfamily of cytokines, activates human neutrophils independently of intracellular free calcium mobilization or binding to IL-8R. In the present study, we have identified and partially characterized a receptor for PF-4 on human neutrophils, which displays weak cross-reactivity with the IFN-gamma-inducible protein 10, but not with other alpha-chemokines such as IL-8, neutrophil-activating peptide 2, or melanoma growth-stimulatory activity (GRO alpha). Binding studies revealed that human neutrophils express a high number of receptors (Bmax approximately 7.6 x 10(6) sites/cell) of moderate affinity (Kd approximately 650 nM). The kinetics of PF-4-binding correlates with the proportion of PF-4 tetramers in solution and with the activation of neutrophils for exocytosis. Reduction of PF-4 binding and PF-4-induced exocytosis in the presence of various glycosaminoglycans or following treatment of cells with chondroitinase ABC (but not other glycosaminoglycan-degrading enzymes) altogether demonstrates that the PF-4 receptor is a proteoglycan of the chondroitin sulfate class. Cross-linking experiments with radiolabeled PF-4 revealed a receptor-ligand complex of approximately 250 kDa. Taken together, our data show that a distinct chondroitin sulfate proteoglycan represents specific receptors for tetrameric PF-4 on human neutrophils.
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PMID:A chondroitin sulfate proteoglycan on human neutrophils specifically binds platelet factor 4 and is involved in cell activation. 978 Feb 12

Lp(a) is a major inherited risk factor for premature atherosclerosis. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to interfere with plasminogen activation and its atherogenic potential as a lipoprotein particle after receptor-mediated uptake. We demonstrate that Lp(a) stimulates production of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin in cultured human coronary artery endothelial cells (HCAEC). This effect resulted from a rise in intracellular free calcium induced by Lp(a) and could be inhibited by the intracellular calcium chelator, BAPTA/AM. The involvement of the LDL and VLDL receptors in Lp(a) activation of HCAEC were ruled out since Lp(a) induction of adhesion molecules was not prevented by an antibody (IgGC7) to the LDL receptor or by receptor-activating protein, an antagonist of ligand binding to the VLDL receptor. Addition of alpha2-macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM-1 and E-selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor-related protein nor cell-surface proteoglycans are involved in Lp(a)-induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM-1 and E-selectin were not significantly decreased by the addition of glu-plasminogen, the lysine analog epsilon-aminocaproic acid, or by trans-4-(aminomethyl)-cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r-apo(a)] competed with Lp(a) and attenuated the expression of VCAM-1 and E-selectin. In summary, we have identified a calcium-dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM-1 and E-selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell-activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.
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PMID:Expression of adhesion molecules by lp(a): a potential novel mechanism for its atherogenicity. 983 67


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