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)

In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated [3H]glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight (approximately 370,000). Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors (apparent Mr approximately 360,000) suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions (the chondroitin sulfate rich regions of the proteoglycan) were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose acceptor activity comparable to the intact core protein.
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PMID:Deglycosylation of chondroitin sulfate proteoglycan and derived peptides. 234 Feb 82

Two major proteoglycans, which appear to be structurally closely related, were isolated from bovine chromaffin granule matrix proteins by ion-exchange chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis they have apparent average molecular sizes of 35-40 kDa (range of 23-75 kDa) and generate a 14-kDa core glycoprotein after chondroitinase treatment. Previous studies demonstrated that these two major chromaffin granule proteoglycans are very similar in terms of their peptide mapping patterns and carbohydrate composition (having a high proportion of tri- and tetraantennary N-glycosidic oligosaccharides, and O-glycosidic oligosaccharides consisting predominantly of disialyl derivatives of galactosyl(beta 1-3)N-acetylgalactosamine), and that they differed in these respects from the chromogranins. By using antisera to five synthetic peptide fragments of chromogranin A to stain immunoblots of purified chromaffin granule proteoglycans before and after chondroitinase treatment, we have now shown that these major proteoglycans are not immunochemically related to chromogranin A. However, it has recently been reported that some chromogranin A-immunoreactive material disappears after chondroitinase treatment, and our studies demonstrate that approximately 1-2% of the chromogranin A occurs in the form of a 110-kDa proteoglycan, which is converted to a 95-kDa core glycoprotein after chondroitinase treatment. Similar chromogranin A proteoglycans could be detected in rat PC12 pheochromocytoma cells, where they have a molecular size of 115-145 kDa and yield a 105-kDa core protein after chondroitinase treatment. Studies using antibodies to synthetic peptide fragments of chromogranin B (secretogranin I) did not provide any evidence that this related protein occurs in a proteoglycan form.
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PMID:Chromaffin granule and PC12 cell chondroitin sulfate proteoglycans and their relation to chromogranin A. 239 98

Corneal buttons were obtained from patients with types 1 and 2 macular corneal dystrophy (MCD) and from control patients with Fuchs' dystrophy or keratoconus. Buttons were incubated for 20 h in the presence of [3H]glucosamine or [2-3H]mannose. Radiolabeled proteoglycans and lactosaminoglycan-glycoproteins (L-GPs) were purified using chromatography on Q-Sepharose, Superose 6, and octyl-Sepharose. They were identified using chondroitinase ABC, keratanase or endo-beta-galactosidase digestion, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Superose 6 chromatography. This study confirms previous reports that type 1 MCD corneas synthesize a normal dermatan sulfate-proteoglycan (DS-PG) and an abnormal keratan sulfate-proteoglycan (KS-PG). The data indicate that typ 1 MCD corneas synthesize L-GP instead of KS-PG. This L-GP has a core protein of similar hydrophobicity (elution from octyl-Sepharose) and nearly similar mass (42 kDa) as the core protein of the KS-PG. It has identical glycoconjugates as those of the KS-PG except that they lack sulfate. Thus, type 1 MCD fails to synthesize keratan sulfate as a result of a defect in a sulfotransferase specific for sulfating lactosaminoglycans. Further, proteoglycans synthesized by a cornea from a patient with type 2 MCD were studied. This cornea synthesized a normal ratio of KS-PG to DS-PG although net synthesis of proteoglycans was approximately 30% below normal. The KS-PG appeared normal whereas the DS-PG had dermatan sulfate chains that were approximately 40% shorter than normal.
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PMID:Proteoglycan biosynthesis by human corneas from patients with types 1 and 2 macular corneal dystrophy. 239 54

Accumulation of glomerular extracellular matrix is a prominent feature of most forms of progressive glomerular disease. Since some growth factors may play a role in extracellular matrix production, we examined the effects of transforming growth factor-beta (TGF-beta), interleukin 1, platelet derived growth factor, and tumor necrosis factor on the production of extracellular matrix components by cultured rat mesangial cells. In control experiments we found that mesangial cells produced two distinct proteoglycans identified as the small chondroitin/dermatan sulfate proteoglycans biglycan (PG I) and decorin (PG II) by showing that their mobility on SDS-PAGE changed upon digestion by chondroitinase ABC, and that they reacted with antibodies raised against synthetic peptides from the core protein sequence of human biglycan and decorin. Exposure to TGF-beta for 48 hours stimulated an 8- to 10-fold increase in the biglycan and decorin bands, and induced a structural change detected as a shift in electrophoretic mobility. TGF-beta did not demonstrably affect the production of other matrix proteins by the mesangial cells. The other growth factors tested had no comparable effect on the production of proteoglycans or other extracellular matrix components by these cells. Our results show that TGF-beta is unique among growth factors in its regulatory effects on mesangial cell proteoglycan production. The release or activation of TGF-beta during glomerular injury could mediate the accumulation of proteoglycans in the extracellular matrix and predispose the kidney to development of glomerulosclerosis.
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PMID:Transforming growth factor-beta regulates production of proteoglycans by mesangial cells. 240 84

Cytotactin is an extracellular matrix protein that is involved in neuron-glia adhesion and is found in both neural and nonneural sites. It is synthesized by glia but not by neurons. In this study, we have examined the binding of cytotactin to a variety of extracellular matrix components using uniform microscopic beads (Covaspheres) that could be labeled and then linked to purified molecules. Cytotactin-coated beads bound well to neurons, and this binding was strongly inhibited by anti-cytotactin antibodies but not by anti-neural cell adhesion molecule (anti-N-CAM) antibodies. In contrast, the binding of N-CAM-coated beads to neurons was inhibited by anti-N-CAM antibodies and not by anti-cytotactin antibodies. To identify a neuronal ligand for cytotactin, we tested several molecules for their ability to block the binding of cytotactin-coated beads to cells. A proteoglycan-containing fraction that copurified with cytotactin from brain extracts strongly inhibited binding, whereas neither a heparan sulfate proteoglycan from Engelbreth-Holm-Swarm tumor cells nor soluble cytotactin itself had a significant inhibitory effect. The neural proteoglycan also inhibited the binding of cytotactin-coated beads to fibroblasts. Digestion with chondroitinase, heparitinase, and hyaluronidase as well as immunological analyses suggested that the predominant species in the active fraction was a chondroitin sulfate proteoglycan with a Mr280,000 core protein bearing HNK-1 antigenic determinants and also indicated that hyaluronic acid was present in this fraction. In experiments on in vitro synthesis, it was found that the proteoglycan was synthesized in culture by embryonic chicken brain tissue but not by embryonic chicken glial cells. A series of binding experiments was performed on appropriately derivatized beads to confirm that the proteoglycan is a ligand for cytotactin and to check for the possibility that other extracellular matrix proteins might interact with one or the other member of this binding couple. Proteoglycan-coated beads and cytotactin-coated beads coaggregated readily. The aggregation was inhibitable by anti-cytotactin antibodies, soluble cytotactin, or soluble proteoglycan. Addition of laminin inhibited the binding of cytotactin-coated beads to proteoglycan-coated beads or to cells; this is consistent with data indicating that laminin interacts with a component of the proteoglycan-containing fraction. In contrast, fibronectin bound to cytotactin, but it did not bind to proteoglycan or interfere with the binding of cytotactin to proteoglycan. The results of this study are in accord with the idea that the functions of extracellular matrix components during neural and nonneural development may be modulated both by competition for shared cell surface receptors and by a network of molecular interactions among the matrix components themselves.
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PMID:A proteoglycan with HNK-1 antigenic determinants is a neuron-associated ligand for cytotactin. 243 34

Proteoglycan, one of the major non-collagenous protein in the connective tissue, is bound with fibronectin and other cell adhesion proteins, and has a role in the formation of the tissue and the organ. Although the glycosaminoglycan components in various tissue have been widely investigated, the molecular structure of periodontal ligament proteoglycan (PDL-PG) was rarely reported. In present study, proteoglycans of bovine periodontal ligament were purified by chromatography from material adsorbed by DEAE-Sephacel from a guanidium HCl extract. The sequential chromatographic steps consisted of ion-exchange chromatography on DEAE-Sephacel in 4M urea and gel filtration on Sepharose CL-4B in 4M guanidium HCl. The preparation contained a relatively small proteoglycan (Mr = 132,000 dalton) and a free glycosaminoglycan chain (Mr = 88,000 dalton). A Mr = 58,000 dalton core protein was shown by gradient SDS gel electrophoresis after chondroitinase ABC or chondroitinase AC II treatment. The glycosaminoglycan chains after chondroitinase AC II hydrolysis were seen on gel as polydispersed, broad alcian blue staining material (Mr = 20,000-60,000 dalton) while chains were totally hydrolyzed by chondroitinase ABC. These indicate a chondroitin sulfate/dermatan sulate (CS/DS) hybrid glycosaminoglycan chain. Papain digestion of the proteoglycan resulted in a single glycosaminoglycan chain after SDS gel electrophoresis with no protein band. These results suggest that the PDL-PG is slightly larger than that of bone and contains a single chondroitin sulphate/dermatan sulphate chain attached to a 58 K core protein. Antisera raised against PDL-PGs cross-reacted with PDL-PGs but not with other PDL proteins or bone PGs. It has been shown that during biosynthesis of dematan sulfate, L-iduronic acid is formed by epimerization of D-glucuronic acid, and sulfation. The degree of epimerization and sulfation may be related to the function of PDL in buffering the mechanical force applied to the tooth.
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PMID:[Isolation and characterization of proteoglycan in bovine periodontal ligament]. 248 42

Fibrocartilaginous regions of bovine deep flexor tendon were treated with chondroitinase-ABC and trypsin in order to extract proteoglycans from the extracellular matrix and thereby investigate the contribution of proteoglycan and collagen organization to tissue material properties. Chondroitinase-ABC digestion of tendon specimens for 24 h resulted in extraction of 60% of tissue glycosaminoglycan and leaching of the degraded large proteoglycan from the tissue residue. The totally degraded core protein of the small dermatan sulfate proteoglycan remained with the tissue residue, indicating that it is specifically associated with the tissue residue and that this association is not dependent on the glycosaminoglycan chains. Treatment of residues with trypsin after chondroitinase-ABC digestion depleted the specimens of proteoglycan. Bulk swelling tests on enzyme-extracted specimens showed that the distinct swelling properties of the fibrocartilaginous regions of the distal flexor tendon could be partially accounted for by elevated levels of proteoglycan. Swelling tests also showed that the distinct collagen organization of this region contributes significantly to the tissue's material properties. These results suggest that the fibrocartilaginous organization and composition of the articulating layer of distal tendon are adapted for mechanical requirements unique to this site, which receives compressive and frictional loads in addition to tensile loads.
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PMID:Effects of chondroitinase-ABC on proteoglycans and swelling properties of fibrocartilage in bovine flexor tendon. 249 83

Radioisotopically labeled proteoglycans were isolated from a 4 M guanidine HCl, 2% Triton X-100 extract of corneal stroma from day 18 chicken embryos by anion-exchange chromatography. Two predominant proteoglycans in the sample were separated by octyl-Sepharose chromatography using a gradient elution of detergent in 4 M guanidine HCl. One proteoglycan had an overall mass of approximately 125 kDa, a single dermatan sulfate chain (approximately 85-90% chondroitin 4-sulfate, low iduronate content) of approximately 65 kDa, and a core protein after chondroitinase ABC digestion of approximately 45 kDa which also contained one to three N-linked oligosaccharides and one O-linked oligosaccharide. The other proteoglycan had an overall size of approximately 100 kDa, two to three keratan sulfate chains of approximately 15 kDa each, and a core protein following keratanase digestion of approximately 51 kDa which included two to three N-linked but no O-linked oligosaccharides. A larger size, a greater overall hydrophobicity (as measured by its interaction with octyl-Sepharose) and an absence of O-linked oligosaccharides argue that this core protein is a distinct gene product from the core protein of the dermatan sulfate proteoglycan.
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PMID:Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. II. Structural characterization of the keratan sulfate and dermatan sulfate proteoglycans from corneal stroma. 252 80

Purified NMuMG mouse mammary epithelial cell surface proteoglycan (PG), a membrane-intercalated core protein bearing both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains, binds to a thrombospondin (TSP) affinity column and is eluted by a salt gradient. Double immunofluorescence microscopy demonstrates extensive co-localization of bound exogenous TSP and cells bearing exposed cell surface PG at their apical surface. The binding, as assayed by both methods, is heparitinase-sensitive, but not chondroitinase-sensitive. Alkali-released heparan sulfate chains bind to a TSP affinity column, similarly to native PG, whereas the chrondroitin sulfate chains do not. Core protein does not bind to TSP. These results indicate that NMuMG cells bind TSP via their surface PG and that the binding is mediated by the heparan sulfate chains.
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PMID:Heparan sulfate-mediated binding of epithelial cell surface proteoglycan to thrombospondin. 252 31

The production and distribution of basement membrane-type heparan sulfate proteoglycans (BM HSPG) were investigated in a mouse glomerular epithelial cell line. Confluent cell monolayers were radiolabeled with [35S]sulfate or [35S]cysteine. Proteoglycans were isolated from the medium and cell layers by ion exchange chromatography and their nature determined by enzyme digestion (chondroitinase ABC) or degradative treatment (nitrous acid). It was found that more than 80% of the proteoglycans in both the cell layer and medium were heparan sulfate proteoglycans (HSPG) based on their susceptibility to nitrous acid degradation. More than half of the HSPG in the cell layer could be precipitated with an antiserum that specifically recognizes BM HSPG; only 10% of those released into the medium were precipitated with this antiserum. When immunoprecipitates of [35S] sulfate-labeled proteoglycans were analyzed by SDS-PAGE, the mature proteoglycans ran as a broad band at the top of the gel. When immunoprecipitates of [35S]cysteine-labeled proteoglycans were similarly analyzed, a 250 kd precursor core protein band was seen in addition to the mature proteoglycan. When BM HSPG were localized by immunofluorescence and immunoelectron microscopy (immunoperoxidase), they were found intracellularly in biosynthetic compartments (ER and Golgi cisternae) and extracellularly in deposits of basement membrane-like matrix located beneath and between the cells. These results indicate that l) BM HSPG are the predominant type of proteoglycans made by glomerular epithelial cells in culture; 2) these HSPG are assembled into a loosely organized matrix that is deposited beneath and between the cells; and 3) this cell type produces a higher proportion of BM HSPG than other cultured epithelial cells studied previously.
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PMID:Basement membrane heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture. 252 72


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