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)

Decorin was isolated from 7 M urea extract of bovine placental cotyledons by ion-exchange and hydrophobic chromatography. Decorin and its core protein showed a broad band at about 115 kDa and a single band at 47 kDa, respectively by SDS-PAGE. Anti-decorin core protein antiserum from pig skin was reacted with placental decorin and its core protein in western blotting. The NH2-terminal amino acid sequence of core protein from placental cotyledons was not different from that of core protein from skin and bone. Glycosaminoglycan of decorin was identified as dermatan sulfate by electrophoresis on a cellulose-acetate membrane and chondroitinase digestivity. Decorin bound to collagen in the order for type III, I, and V.
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PMID:Affinity of placental decorin for collagen. 1119 21

Multiple proteoglycans (PGs) are present in all basement membranes (BM) and may contribute to their structure and function, but their effects on cell behavior are not well understood. Their postulated functions include: a structural role in maintaining tissue histoarchitecture, or aid in selective filtration processes; sequestration of growth factors; and regulation of cellular differentiation. Furthermore, expression PGs has been found to vary in several disease states. In order to elucidate the role of PGs in the BM, a well-characterized model of polarized epithelium, Madin-Darby canine kidney (MDCK) cells has been utilized. Proteoglycans were prepared from conditioned medium by DEAE anion exchange chromatography. The eluted PGs were treated with heparitinase or chondroitinase ABC (cABC), separately or combined, followed by SDS-PAGE. Western blot analysis, using antibodies specific for various PG core proteins or CS stubs generated by cABC treatment, revealed that both basement membrane and interstitial PGs are secreted by MDCK cells. HSPGs expressed by MDCK cells are perlecan, agrin, and collagen XVIII. Various CSPG core proteins are made by MDCK cells and have been identified as biglycan, bamacan, and versican (PG-M). These PGs are also associated with mammalian kidney tubules in vivo.
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PMID:Basement membrane and interstitial proteoglycans produced by MDCK cells correspond to those expressed in the kidney cortex. 1122 36

This report describes the isolation of guanidinium chloride extractable protein from demineralised bone extracts obtained from the 125-130 mya dinosaur Iguanodon. Protein products were isolated in the Mr. range 5,000-66,000 using SDS-PAGE and represent the first electrophoretically defined proteins isolated from dinosaur tissues. The levels of glycine, aspartate and serine tentatively suggest the presence of phosphoproteins. Hydroxylysine and hydroxyproline were not detected, confirming the presence of non-collagenous material. In addition the absence of ornithine confirmed lack of bacterial contamination. The relatively high level of leucine in the 2MNaCl NaCl fractions together with the abolition of alcian blue reactivity following protease-free chondroitinase digestion suggests the presence of proteoglycans. The study is of interest in describing the early proteins laid down in mineralised tissues for epitactic crystal growth and may provide evidence on evolutionary aspects of bone proteins.
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PMID:The isolation and detection of non-collagenous proteins from the compact bone of the dinosaur Iguanodon. 1126 72

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.
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PMID:The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels. 1154 22

Proteoglycans (PGs) were dissociatively extracted from human umbilical cord arteries (UCAs) with 4 M guanidine hydrochloride containing Triton X-100 and protease inhibitors, purified by Q-Sepharose anion exchange chromatography and lyophilized. They were analysed by gel filtration, SDS/PAGE and agarose gel electrophoresis before and after treatment with chondroitinase ABC. It was found that the PG preparation was especially enriched in chondroitin/dermatan sulphate PGs. The predominant PG fraction included small PGs that emerged from Sepharose CL-2B with Kav = 0.74. Their molecular mass, estimated by SDS/PAGE, was 160-200 kDa and 90-150 kDa, i.e. it was typical for biglycan and decorin, respectively. Treatment with chondroitinase ABC yielded the core proteins of 45 and 47 kDa, characteristic for both small PGs. Remarkable amounts of the 45 kDa protein were detected in non-treated PG samples, suggesting the presence of free core proteins of biglycan and decorin. Large PGs were present in lower amounts. In intact form they were eluted from Sepharose CL-2B with Kav = 0.17 and 0.43. Digestion with chondroitinase ABC yielded the core proteins with a molecular mass within the range of 180-360 kDa but predominant were the bands of 200, 250 and 360 kDa. The large PGs probably represent various forms of versican or perlecan bearing chondroitin sulphate chains.
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PMID:Proteoglycans of human umbilical cord arteries. 1199 98

Exposure to AG73, a synthetic peptide (LQVQLSIR) from the COOH-terminal region of the laminin alpha1 chain, induces a malignant phenotype in B16F10 melanoma cells. Coinjection of this peptide with the cells results in an increase of lung tumors and also the formation of liver tumors in approximately 50% of the mice (W. H. Kim et al., Int. J. Cancer, 77: 632-639, 1998). Here we have characterized the cell surface receptor and its functional groups on B16F10 cells. Peptide affinity chromatography identified a cell surface protein eluting with 1 M NaCl, which ran in SDS gels as a broad band of M(r) approximately 150,000-200,000. Digestion with heparitinase and chondroitinase produced a core protein of lower molecular weight (M(r) approximately 90,000). Involvement of the glycosaminoglycan (GAG) side chains was demonstrated by inhibition of cell binding to the peptide by heparin, heparan sulfate, and chondroitin sulfate B, but not by chondroitin sulfates A or C, or hyaluronic acid. The IC(50) for heparin was the lowest, followed by heparan sulfate, then chondroitin sulfate B, suggesting that the overall sulfation of the GAG side chain is critical. This was confirmed by inhibition of attachment with chemically modified heparin and heparan sulfate, which also showed that N or O linkages were not important for function. Using sized heparin fragments to inhibit cell binding to the peptide demonstrated that 16-mer is the minimum length required. B16F10 cells form a network when grown on Matrigel, and this is prevented by addition of the AG73 peptide. The GAGs alone did not affect network formation, but heparin, heparan sulfate, and chondroitin sulfate B reversed the inhibitory effect of the peptide, whereas other GAGs were inactive. Furthermore, removal of cell surface GAGs inhibited cell attachment to the peptide. Cells treated with glycosidases and coinjected with the peptide formed liver tumors equal to the control group receiving no peptide, suggesting that the GAGs play an early role in peptide-mediated tumor metastasis. These data indicate that the B16F10 cell receptor for a laminin metastasis-promoting sequence is a heparan sulfate/chondroitin sulfate-containing proteoglycan, and these GAG side chains are functionally important in the cell-peptide interaction.
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PMID:The B16F10 cell receptor for a metastasis-promoting site on laminin-1 is a heparan sulfate/chondroitin sulfate-containing proteoglycan. 1206 3

We developed a method to purify decorin core protein from tissue with the goal of preserving its native structure and biological function. Currently, most procedures rely on the use of denaturing reagents potentially altering the biological activity. Decorin was purified from corneal stromas without the use of detergents or chaotropic reagents. Proteoglycans isolated using anion exchange chromatography on Q-Sepharose were treated with chondroitinase ABC. Decorin was isolated by a second Q-Sepharose chromatography with affinity chromatographies on heparin-Sepharose and concanavalin A-Sepharose. SDS-PAGE revealed a 98.4% pure 44kDa protein identified as decorin with a yield of 35mg per 100 bovine corneas. Identification was confirmed by NanoESI and MALDI qTOF. The novel inclusion of 20% propylene glycol in extraction and column buffers resulted in recoveries of proteoglycans comparable with those observed with detergents and urea. Purified decorin did alter the rate of fibrillogenesis of type I collagen and inhibited the lateral fusion of collagen fibrils. It also bound to [125I]TGF-beta1 with an apparent K(d) of 40nM. Circular dichroism spectroscopy of decorin displayed the spectra of alpha-helices and beta-pleated sheets consistent with those obtained from recombinant decorin. Urea-induced unfolding was cooperative and reversible while thermal denaturation caused irreversible unfolding. Native decorin can be purified from tissue in quantity and quality for biophysical, biochemical, and biological assays.
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PMID:Extraction and purification of decorin from corneal stroma retain structure and biological activity. 1218 18

Myofibroblasts play an important role in fibrogenesis. Myofibroblasts secrete several components of the extracellular matrix, including decorin. To clarify the properties of decorin synthesized by myofibroblasts, we have purified and characterized decorin secreted into culture medium by the myofibroblast cell line MRC-5. Decorin was purified by successive chromatography steps using Hitrap Q and Superdex 200. Purified decorin showed a broad band on SDS-polyacrylamide gel electrophoresis, which was resolved into two smaller molecular weight bands after digestion with chondroitinase ABC. Further digestion with N-glycanase resolved these two bands into a single band, indicating that the N-glycation pattern of decorin is heterogeneous. The N-terminal amino acid sequence analysis of the purified protein and its reactivity towards an antibody raised against a C-terminal peptide of decorin indicate that MRC-5 cells secrete full-length decorin into the culture medium. To characterize the glycosaminoglycan chains attached to decorin, glycosaminoglycans from the purified protein were treated with chondroitinase ACI, chondroitinase ACII, chondroitinase ABC and chondroitinase B. The resulting disaccharides were analyzed by chromatography, which indicated that decorin secreted by MRC-5 cells is a dermatan sulfate proteoglycan. In conclusion, the decorin secreted by MRC-5 cells has similar characteristics to the decorin expressed in several tissues. Thus, culturing MRC-5 cells may be highly useful for studying the role of decorin and myofibroblasts in fibrosis.
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PMID:Purification and characterization of decorin from the culture media of MRC-5 cells. 1514 41

Dentin sialoprotein (DSP) is a glycoprotein that is critical for proper tooth dentin formation, but little is known about the nature of its carbohydrate attachments and other post-translational modifications. We have isolated DSP from pig dentin and demonstrate that it is a proteoglycan. Polyclonal antibodies were raised in chicken against recombinant pig DSP, and used to identify native DSP in fractions of tooth dentin proteins extracted from developing pig molars. Amino acid analyses and characterization of lysylendopeptidase cleavage products confirmed that the purified protein was DSP, and that Arg391 is at the DSP C terminus. On SDS-PAGE and on urea gels, DSP appeared as a smear extending from 280 to 100 kDa, but in the presence of beta-mercaptoethanol the top of the DSP smear disappeared. The high molecular weight material was likely comprised of covalent DSP dimers connected by a disulfide bridge at Cys205. Oligosaccharides were released from DSP following N- and O-linked glycosidase digestions, but these digestions had little effect on the apparent molecular weight of DSP on SDS-PAGE, when compared with the significant reduction following chondroitinase ABC digestion. Glycosaminoglycanases with assorted glycosaminoglycan (GAG) cleavage specificities coupled with Western analyses of the cleaved GAG "stubs" demonstrated that the DSP GAG attachments contain chondroitin 6-sulfate, but not keratan sulfate, heparan sulfate, chondroitin, or chondroitin 4-sulfate. DSP binds biotin-labeled hyaluronic acid, and such binding is inhibited by the addition of unlabeled hyaluronic acid. We conclude that DSP is a proteoglycan and that GAG attachments are the predominant structural feature of porcine DSP.
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PMID:Porcine dentin sialoprotein is a proteoglycan with glycosaminoglycan chains containing chondroitin 6-sulfate. 1553 41

A biglycan was isolated from bovine aorta intima media by 4M guanidine HCl extraction of the tissue; the material was fractionated and purified by using isopycnic ultracentrifugation and DEAE Sephacel ion-exchange chromatography. Core proteins, resulting from digestion of the proteoglycan preparation with chondroitinase ABC, were resolved by SDS-polyacrylamide gel electrophoresis into three bands. The apparent molecular weight of the fast migrating major protein band was 47 kDa and the other slow-moving minor protein bands were 90 and 105 kDa. These proteins were recognized by a monoclonal anti-proteoglycan deltaDi-6S (MAb 3-B-3/Cl). The amino acid composition of 47 kDa core protein revealed a high content of aspartic acid, glutamic acid and leucine, similar to those found for biglycans isolated from bovine cartilage, rat vascular smooth muscle cell culture and human bone. The N-terminal sequence of 47 kDa core protein was determined as Asp-Glu-Glu-Ala-X-Gly-Ala-Glu-Thr-Thr-X-Gly-Ile-Pro-Asp which is identical to the sequence of bovine articular cartilage biglycan. The proteoglycan had two glycosaminoglycan chains.
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PMID:N-terminal sequence of a core protein from a biglycan isolated from bovine aorta. 1561 28


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