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)

This work, directed to characterization of proteoglycans present in normal human synovial fluid by Western blotting techniques, revealed an intimate relationship of these proteoglycans to those of articular cartilage. Analyses were performed on samples subjected to digestion with chondroitinase ABC, in the presence or absence of keratanase, yielding products containing core proteins with glycosaminoglycan side chain stubs. The proteoglycan core proteins contained epitopes reactive with monoclonal antibodies that distinguish between chondroitin sulfate-4 and chondroitin sulfate-6. Additionally, these products reacted with monoclonal antibody to keratan sulfate when keratanase was omitted from the digestion. The analysis of synovial fluid revealed that the proteoglycan core proteins expressed predominantly the chondroitin sulfate-6 epitope, with expression of the chondroitin sulfate-4 epitope demonstrable only in prepubertal individuals. There was coexpression of both chondroitin sulfate epitopes in all proteoglycan core proteins of prepubertal individuals. Coexpression of chondroitin sulfate and keratan sulfate epitopes occurred in all proteoglycan core proteins. Proteoglycan core proteins had M(r) similar to those obtained from articular cartilage. Hence, in individuals free of joint disease, most proteoglycans seem to be transferred from articular cartilage to the synovial fluid without major alteration in the apparent size of the proteoglycan core protein. Only a minor set of proteoglycan core proteins had no direct articular cartilage equivalent. As this set also contained keratan sulfate, it is likely to be of articular cartilage origin, but probably modified by proteolysis.
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PMID:Normal human synovial fluid and articular cartilage contain similar intact proteoglycans. 137 75

Two distinct classes of cell surface FGF-binding proteins have been identified. These receptors differ in both mode of interaction and in affinity for the FGFs. cDNAs that encode the low-affinity receptor were isolated from a hamster kidney cell line cDNA library by expression cloning. Transfected cells that contained these heparan sulfate proteoglycan FGF receptor cDNAs were enriched for by panning on basic FGF-coated plates. The analogous human cDNA was isolated from a hepatoma cell line cDNA library. The homology of our hamster cDNAs to the previously described murine integral membrane proteoglycan syndecan, together with an exact amino acid sequence match of our human-cDNA-encoded product to human syndecan, clearly indicates the identity of these independently isolated proteoglycans. Further confirmation that the expressed molecule serves as a proteoglycan core protein was achieved by immunoprecipitation of 35SO4-labeled material from solubilized transfected cells. Nitrous acid treatment and chondroitinase digestion revealed that 77% of the label was associated with heparan sulfate chains and 22% with chondroitin sulfate chains. These heparan sulfate chains contributed to the fivefold increase in the total heparan sulfate found to be present on the surface of the transfected cells compared with cells transfected with a vector lacking the cDNA insert.
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PMID:The molecular biology of heparan sulfate fibroblast growth factor receptors. 166 83

Monoclonal antibodies have been raised against determinants present in cartilage proteoglycan. Characterization of the specificity of these antibodies indicated that they recognize determinants present in the keratan sulfate glycosaminoglycan chain and on chondroitin sulfate oligosaccharide stubs attached to the proteoglycan core protein after chondroitinase digestion of the proteoglycan (i.e., delta-unsaturated 4- and 6-sulfated and unsulfated chondroitin sulfate on the proteoglycan core). The antibody recognizing keratan sulfate has been used to demonstrate the presence of a keratan sulfate-rich proteoglycan subpopulation that increases with increasing age of animal compared with chondroitin sulfate-rich proteoglycans. Monoclonal antibodies recognizing determinants on chondroitinase-treated proteoglycan have been used in immunohistochemical localization studies determining the differential distribution of 4- and 6-sulfated and unsulfated proteoglycans in tissue sections of cartilage and other noncartilaginous tissues. Digestion with chondroitinase ABC or ACII can be used to differentiate between chondroitin sulfate and dermatan sulfate proteoglycan in different connective tissues. In addition, the presence of a 6-sulfated chondroitin sulfate proteoglycan that is associated with membranes surrounding nerve and muscle fiber bundles is described. Monoclonal antibodies were also raised against the link protein(s) of cartilage proteoglycan aggregate. They have been used in peptide map analyses of link protein and in demonstrating the presence of a high-mannose oligosaccharide chain of the link proteins. The presence of high-mannose oligosaccharide structures on the link protein(s) accounts for the microheterogeneity of the link proteins (link proteins 1, 2, or 3) that is observed on sodium dodecyl sulfate-polyacrylamide gels.
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PMID:Production and characterization of monoclonal antibodies directed against connective tissue proteoglycans. 257 17

The major proteoglycan in bovine parietal pericardium is a low molecular weight dermatan-sulfate proteoglycan. It possesses structural and immunologic characteristics similar to those of the small proteoglycan found in tendon. We demonstrate that digestion of purified pericardial proteoglycan with low levels of V8 protease results in the liberation of the glycosaminoglycan chain and of a 40 kDa resistant fragment. A similar 40 kDa fragment can be obtained by V8 protease digestion of the proteoglycan deglycosylated by chondroitinase ABC. Although the protein core size of the pericardial proteoglycan is similar to that of tendon PG II, the size of the glycosaminoglycan chain liberated from the former is smaller. The pericardial proteoglycan and its V8 protease products reacted with an anti-PG II antiserum by immunoblotting. The anti-PG-II antibody localized in the pericardial tissue by the immunoperoxidase technique. The presence of intrachain disulfide bonds in the structure of pericardial proteoglycan core protein and V8 resistant fragment was demonstrated by their decreased electrophoretic mobility after disulfide reduction. Digestion of pericardial proteoglycan with Cathepsin C resulted in a rapid liberation of the glycosaminoglycan chain from the core protein, indicating that its attachment site was very close to the amino terminus. Ultrastructural examination of pericardial tissue utilizing Cuprolinic Blue revealed a periodic association of the proteoglycan with the d/e band on the collagen fibrils. Electron microscopic immunohistochemical studies confirmed the perifibrillar association of pericardial proteoglycan. The present data demonstrate that, although the pericardial proteoglycan possesses some unique structural features, it shares structural and immunological characteristics to place it in the category of the small PG II family.
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PMID:Bovine pericardial proteoglycan: biochemical, immunochemical and ultrastructural studies. 267 26

We have isolated cDNA clones that code for a proteoglycan-related polypeptide with unique properties. A lambda gt11 expression library made from human fibroblast mRNA was screened with an antiserum made against a proteoglycan fraction from human fetal membranes. One group of positive clones revealed an open reading frame coding for 685 amino acids from the COOH terminus of a polypeptide. This amino acid sequence contains a domain that is strongly homologous with the COOH-terminal core protein domain of the large aggregating cartilage proteoglycan. This domain also contains sequences that are homologous with vertebrate lectins that bind terminal galactosyl, N-acetyl-glucosaminyl or mannosyl residues. On the NH2-terminal side of the lectin-like domain the cDNA-derived amino acid sequence contains two epidermal growth factor-related segments. The cDNA clones were shown to belong to a chondroitin sulfate proteoglycan by using antisera made against two peptides predicted from the cDNA sequence. These antisera were reactive with a proteoglycan fraction from fibroblasts after chondroitinase treatment of the fraction but not after treatment with heparinase or no treatment. Among the several polypeptides reactive with the anti-peptide antibodies the largest one, corresponding to a molecular weight of about 400,000, is likely to be the intact core protein, whereas the smaller polypeptides may be processing products or products of artifactual proteolysis. These results show that the amino acid sequence belongs to a proteoglycan core protein, and the sequence, therefore, provides a molecular definition to this proteoglycan. The lectin-related and growth factor-like sequences in the core protein of this proteoglycan suggest that it may play a role in intercellular signaling.
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PMID:A fibroblast chondroitin sulfate proteoglycan core protein contains lectin-like and growth factor-like sequences. 282 Sep 64

The present studies were undertaken to confirm the presence and identity of a putative proteoglycan associated with laminin in neurite-promoting factor complexes isolated from rat schwannoma cell conditioned medium. Sucrose density gradient centrifugation of the complex resolved two laminin-associated Na2[35S]O4-labeled peaks which were termed Pools A and B. Both pools had nearly all their [35S] cpms associated with glycosaminoglycan, contained heparan sulfate-proteoglycan core protein antigen and displayed a similarly high neurite promoting potency relative to their laminin contents. However, Pool A contained about twice as many [35S] cpms and twice as much proteoglycan core protein per laminin than Pool B. Seventy percent of Pool A cpms was associated with heparan sulfate and 30% with chondroitin sulfate whereas the inverse was true for Pool B. Treatment with heparitinase and/or chondroitinase ABC caused laminin in either pool to elute at lower salt concentrations from DEAE cellulose. In SDS-PAGE the [35S] cpms of both pools ran with the same mobility as laminin but could be separated from laminin under reducing conditions. The Pool A cpms remained at 900 KD and the Pool B cpms spread over the 200-900 KD range. By rotary shadowing electron microscopy, Pool B fractions contained primarily cross-shaped laminin images, often associated with proteoglycan-like images. Pool A fractions contained i) dense, aggregated images including intact laminin from which emanated proteoglycan-like strands, ii) circular images bearing globular domains and less commonly, iii) distorted cross-shaped laminin-like images. These studies support the existence of at least two forms of laminin-proteoglycan complexes which differ in biochemical, immunochemical and ultrastructural characteristics.
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PMID:Association of laminin with heparan and chondroitin sulfate-bearing proteoglycans in neurite-promoting factor complexes from rat schwannoma cells. 296 Sep 8

The high-buoyant-density proteoglycans of the nucleus pulposus and annulus fibrosus of the beagle intervertebral disc have been isolated by CsCl density gradient ultracentrifugation. The sulphated proteoglycans were labelled in vivo with 35SO4, 24 h and 60 days prior to killing. The hydrodynamic size and aggregation of the 24 h, 60 day and resident (from hexuronic acid and hexosamine analysis) proteoglycan subunit populations were determined by Sepharose CL-2B chromatography in the presence or absence of excess hyaluronic acid. The hydrodynamic size of the keratan sulphate-proteoglycan core protein complexes were also determined by Sepharose CL-2B chromatography after chondroitinase ABC digestion of proteoglycans. When initially synthesised (24 h) or after 60 days, the percentage aggregation and hydrodynamic size of the proteoglycans derived from the annulus fibrosus were larger than those present in the nucleus pulposus. Hexosamine, hexuronic and protein determination of the high-buoyant-density fractions showed that the proteoglycans of the nucleus pulposus were richer in chondroitin sulphate than those in the annulus. However there was no difference in Mr of the chondroitin sulphate and keratan sulphate attached to the proteoglycans of the two disc regions, nor were differences detected by HPLC between the proportions of chondroitin 4-sulphate and chondroitin 6-sulphate present in these high-density fractions. In contrast, the low-buoyant-density (1.54 greater than p greater than 1.45) proteoglycan fractions and tissue residues remaining after 4 M GuHCl extraction were found to contain dermatan sulphate, suggesting the presence of a third proteoglycan species possibly associated with the collagen of the fibrocartilagenous matrix.
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PMID:The proteoglycans of the canine intervertebral disc. 392 Oct 57

Using enzyme-linked immunosorbent assays and radioimmunoassays employing chondroitinase ABC-treated rabbit cartilage proteoglycan, we have shown that approximately one-third of the outbred New Zealand white rabbits we have examined possess naturally occurring antibodies which react with oligosaccharides of hyaluronic acid (independently of chain length) bearing saturated and 4,5-unsaturated glucuronosyl residues at the nonreducing ends. Such antibodies were also found in a similar proportion of rabbits with an experimental inflammatory arthritis. There was a preferential reactions in the majority of sera with unsaturated oligosaccharides of hyaluronic acid. One serum (R64) reacted only with unsaturated oligosaccharides of hyaluronic acid. Sera reacted also with unsaturated (never saturated) oligosaccharides of chondroitin 4-sulfate and with chondroitin 6-sulfate, particularly when chondroitin sulfate oligosaccharides remained bound to a proteoglycan core protein. Reactions were also observed to both unsaturated and saturated oligosaccharides of chondroitin. Some of these sera also reacted with intact hyaluronic acid and chondroitin but never with intact chondroitin sulfate. The antibodies were present in the IgG fraction of four sera studied and in the IgM fraction of one of these sera: they bound through the F(ab')2 region of the molecule. These observations suggest that, in some rabbits, humoral immunity to hyaluronic acid and/or chondroitin sulfate bound to core protein can develop after these reactive glycosaminoglycans have been degraded by eliminases or hydrolases produced by naturally occurring bacteria and rabbit cells, respectively. Immunological studies of proteoglycans and hyaluronic acid treated with eliminases and hydrolases employing rabbit antisera, and possibly those from other species, should be evaluated in the light of these observations.
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PMID:Rabbit antibodies to degraded and intact glycosaminoglycans which are naturally occurring and present in arthritic rabbits. 399 11

Immunization of mice with a plasma membrane-enriched fraction from human malignant melanoma cells and subsequent generation of hybridomas resulted in the isolation of an IgG1 monoclonal antibody, 155.8, that recognizes chondroitin sulfate proteoglycans. By cell binding analysis, 155.8 was shown to react with seven of eight cultured melanoma cell lines, but not with a variety of lymphoblastoid cell lines or cultured tumor cells derived from other solid tumor types. Indirect immunoprecipitation of the 155.8 antigen from intrinsically labeled melanoma cells revealed a glycoprotein of Mr = 250,000 and a sulfated molecule of Mr greater than 400,000. The antigen was identified as a chondroitin sulfate type A/C proteoglycan synthesized by melanoma cells on the basis of its sensitivity to chondroitinase ABC digestion and the identification of sulfated glycosaminoglycans released from the antigen immunoprecipitated by 155.8. The determinants recognized by antibodies 155.8 and 9.2.27, another anti-chondroitin sulfate proteoglycan, immunoprecipitate only a proteoglycan from high density cesium chloride gradient fractions, (1.487 g/liter); however, they immunoprecipitate a free glycoprotein of Mr = 250,000 from low density fractions (1.317 g/liter). This demonstrated that the 155.8 and 9.2.27 determinants, both of which reside on the glycoprotein of Mr = 250,000, are also present in the proteoglycan, suggesting that this glycoprotein is the proteoglycan core protein. Monoclonal antibody 155.8 reacts with a determinant on the core protein distinct from that recognized by 9.2.27. Proteoglycans bearing 155.8 determinants are distributed on the surface of cultured melanoma cells in a punctated fashion that apparently resolves to short, filamentous structures at high magnification. Immunohistochemical analysis demonstrated that 155.8-defined proteoglycans are found in freshly biopsied melanoma tissue, suggesting that these antigens are also synthesized in vivo by melanoma cells.
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PMID:Characterization of monoclonal antibody 155.8 and partial characterization of its proteoglycan antigen on human melanoma cells. 619 23

Several monoclonal antibodies which recognize different antigenic determinants of chondroitin sulfate proteoglycan were used to study chondroitin sulfate proteoglycan biosynthesis in chicken chondrocyte cultures. The intracellular sites of synthesis and processing and extracellular deposition in matrix were localized by double immunofluorescence reactions. One rat monoclonal antibody, S103L , which recognizes an antigenic determinant of the core protein of the chicken cartilage chondroitin sulfate proteoglycan monomer, was used to identify both extracellular chondroitin sulfate proteoglycan and intracellular compartments containing chondroitin sulfate proteoglycan precursors. Intracellular staining with S103L was localized to perinuclear regions, and, in some chondrocytes, to a few other cytoplasmic vesicles as well. When chondrocytes were not fed for several days, intracellular chondroitin sulfate proteoglycan precursors were accumulated in larger compartments distributed throughout the cytoplasm. Polyclonal chondroitin sulfate proteoglycan antibodies displayed similar staining characteristics. In contrast, several of the monoclonal antibodies, including the rat monoclonals S11D and P100D , and the mouse monoclonals 1-B-5, 3-B-3 and 9-A-2, did not recognize native chondroitin sulfate proteoglycan, but reacted only with chondroitinase ABC-digested (and/or hyaluronidase-digested) chondroitin sulfate proteoglycan. These antibodies were particularly useful in the demonstration of the extracellular codistribution of chondroitin sulfate proteoglycan with either type II collagen or fibronectin. In other experiments, the monoclonal antibodies to chondroitin sulfate proteoglycan served to demonstrate that the perinuclear subset of intracellular compartments is uniquely involved in the addition of chondroitin sulfate oligosaccharides to the chondroitin sulfate proteoglycan core protein. Lastly, using the mouse monoclonal 5-D-4, which recognizes keratan sulfate determinants, the perinuclear region was identified as the site for keratan sulfate addition. Results suggest heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan.
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PMID:Immunofluorescence studies of chondroitin sulfate proteoglycan biosynthesis: the use of monoclonal antibodies. 620 57


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