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)

Sequencing of cDNA clones has shown that the carboxy terminal domain of the core protein of large proteoglycans (aggrecans) from human cartilage contains an epidermal growth factor-like (EGF-like) domain which is alternatively spliced. In a previous study it was found that the domain of the translated protein can be recognized by polyclonal antibodies to mouse EGF. A competitive enzyme-linked immunoabsorbent (ELISA) assay has been developed to evaluate the EGF-like domain content of aggrecans at various ages and in osteoarthritis. Fetal aggrecans digested with protease free chondroitinase ABC were adsorbed on polyvinyl chloride microtiter plates followed by blocking with bovine serum albumin and goat serum. Mixtures of known amounts of protein of digested aggrecans and constant amounts of anti-mouse EGF antibodies were incubated and added to plates. The second antibody was peroxidase-conjugate F(ab')2. Fetal, newborn and child aggrecan proteins have a higher content of EGF-like domain than aggrecan proteins from cartilage of older humans. Three areas of cartilages from osteoarthritic joints were separated: cartilages with normal macroscopic appearance, erosion border cartilage and osteophytic cartilage. Values derived from these samples were compared with values derived from nonosteoarthritic aged humans. The content of aggrecans from osteoarthritic cartilage with normal macroscopic appearance was similar to or slightly lower than the latter. The aggrecans from osteophytes had a higher EGF-like domain content. The aggrecans from the erosion border had a variable content, close to noneroded cartilages, to osteophytes or in between the values obtained for noneroded cartilages and for osteophytes. Variations in the amount of newly synthesized aggrecans, in the proteolysis of the carboxy terminal domain of aggrecans and in the alternating splicing of the EGF-like domain might explain the results shown here.
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PMID:The epidermal growth factor-like domain of the large proteoglycans from articular cartilage (aggrecans). Estimate of content at different ages and in osteoarthritis. 1544 24

Sequential extraction was applied to investigate the proteoglycan (PG) organization in healthy laryngeal cartilage (HLC) and laryngeal cartilage squamous cell carcinoma (LCSCC). Highly stable aggrecan aggregates, extracted from both HLC and LCSCC with strong dissociative reagents, i.e., 4 M guanidine HCl (GdnHCl), represented 53% and 7%, respectively, of total extracted macromolecules. Less stable complexes/aggregates, extracted with mild dissociative reagents (1 and 2 M GdnHCl), represented 40% and 61% of total extracted PGs from healthy and cancerous cartilage, respectively. Interestingly, a relative high proportion (32%) of uronic acid (UA)-containing macromolecules were removed from the cancerous cartilage using associative extracting solutions (PBS and 0.5 M GdnHCl), which obviously represented molecules freely extractable from the tissue. In contrast, the corresponding proportion in HLC was impressively low (about 7%). The major proportion of these molecules was chondroitin sulfate-containing PGs (CSPGs), which identified mainly as aggrecan. Differential digestion of the sequential extracts with chondroitinase ABC and chondroitinase AC II demonstrated the presence of dermatan sulfate-containing PGs (DSPGs) in both HLC and LCSCC, being mainly present in the 1 M GdnHCl extract, and identified as decorin. All cancerous extracts were found to be rich in 4-sulfated disaccharides, mostly participating in DS structures. In conclusion, the applied procedure permitted the elucidation of the changes in the cartilage status, regarding the stability and identity of its proteoglycan aggregates/complexes, in both HLC and LCSCC.
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PMID:The extractability of extracellular matrix components as a marker of cartilage remodeling in laryngeal squamous cell carcinoma. 1565 82

Mesenchymal cell condensation is an essential step for cartilage development. Versican/PG-M, a large chondroitin sulfate proteoglycan, is one of the major molecules expressed in the extracellular matrix during condensation. However, its role, especially as an environment for cells being condensed, has not been elucidated. Here we showed several lines of evidence for essential roles of versican/PG-M in chondrogenic condensation using a new chondrocytic cell line, N1511. Chondrogenic stimuli (treatment with parathyroid hormone, dexamethasone, 10% serum) induced a marked increase in the transcription and protein synthesis of versican/PG-M. Stable antisense clones for versican/PG-M, depending on suppression of the expression of versican/PG-M, showed different capacities for chondrogenesis, as indicated by the expression and deposition of aggrecan, a major chondrocytic cell product. The cells in the early stages of the culture only expressed V0 and V1 forms, having more chondroitin sulfate chains among the four variants of versican/PG-M, and treatment of those cells with chondroitinase ABC suppressed subsequent chondrogenesis. Furthermore, treatment with beta-xyloside, an artificial chain initiator of chondroitin sulfate synthesis to consequently inhibit the synthesis on the core proteins, suppressed chondrogenesis. In addition, forced expression of the variant V3, which has no chondroitin sulfate chain, disrupted the deposition and organization of native versican/PG-M (V0/V1) and other extracellular matrix molecules known to be expressed during the mesenchymal condensation and resulted in the inhibition of subsequent chondrogenesis. These results suggest that versican/PG-M is involved in positively regulating the formation of the mesenchymal matrix and the onset of chondrocyte differentiation through the attached chondroitin sulfate chains.
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PMID:Versican/PG-M regulates chondrogenesis as an extracellular matrix molecule crucial for mesenchymal condensation. 1625 55

A simple, rapid, and reproducible microtiter-based chondroitinase (CSase) assay is reported here, based on the competition of chondroitin sulfate (CS) with immobilized hyaluronan (HA) for the binding of TSG-6 protein, the product of TNF-inducible gene 6. Although the catabolic reaction of bacterial and other prokaryotic CSase enzymes, often referred to as the chondroitin lyases, can be followed by tracking the generation of unsaturated bonds by the spectrophotometrical determination of the absorbance at 232 nm, no rapid, sensitive, and simple assay has been devised to date for measuring the activity of the vertebrate enzymes that cleave their substrate exclusively by hydrolysis. We provide data demonstrating that the CSase assay described here is suitable for the determination of the activities of both classes of enzymes. For the bacterial enzyme CSase ABC, both the determination of the absorbance at 232 nm and the assay based on TSG-6 binding are suitable using the same range of enzyme activities. However, for testicular hyaluronidase, considerably higher enzyme activities were needed to cleave CS than to cleave HA. Using the HA-binding domain of aggrecan for a comparison, we determined that the interaction between TSG-6 and chondroitin sulfate is uniquely suited for this CSase assay.
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PMID:An assay for bacterial and eukaryotic chondroitinases using a chondroitin sulfate-binding protein. 1628 10

Aggrecan, a large chondroitin sulfate (CS) and keratan sulfate (KS) proteoglycan, has not previously been expressed as a full-length recombinant molecule. To facilitate structure/function analysis, we have characterized recombinant bovine aggrecan (rbAgg) and link protein expressed in COS-7 cells. We demonstrate that C-terminally truncated rbAgg was not secreted. Gel filtration chromatography of rbAgg and isolated glycosaminoglycan (GAG) chains, and their susceptibility to chondroitinase ABC digestion indicate that the GAG chains are predominantly CS, which likely occupy fewer serine residues than native aggrecan. To confirm functionality, we determined that rbAgg bound hyaluronan and recombinant link protein to form proteoglycan aggregates. In addition, cleavage of rbAgg by ADAMTS-4 revealed that the p68 form of ADAMTS-4 preferentially cleaves within the CS-2 domain, whereas the p40 form only effectively cleaves within the interglobular domain (IGD). MMP-13 cleaved rbAgg within the IGD, but cleaved more rapidly at a site within the CS domains, suggesting a role in C-terminal processing of aggrecan. Our results demonstrate that recombinant aggrecan can be used for in vitro analyses of matrix protease-dependent degradation of aggrecan in the IGD and CS domains, and both recombinant aggrecan and link protein can be used to study the assembly of proteoglycan aggregates with hyaluronan.
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PMID:Mammalian expression of full-length bovine aggrecan and link protein: formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13. 1642 4

We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.
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PMID:Composition of perineuronal net extracellular matrix in rat brain: a different disaccharide composition for the net-associated proteoglycans. 1664 27

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels. Here, we characterized cartilage versican during development and growth. Immunostaining showed that versican was mainly localized in the interterritorial zone of the articular surface at 2 weeks in mice, whereas aggrecan was in the pericellular zone of prehypertrophic and hypertrophic cells of the growth plate. Although its transcription level rapidly diminished during growth, versican remained in the articular cartilage. Biochemical analysis of normal articular cartilage and aggrecan-null cartilage from cmd (cartilage matrix deficiency)/cmd mice revealed that versican was present as a proteoglycan aggregate with both link protein and hyaluronan. Chondroitin sulfate chains of versican digested with chondroitinase ABC contained 71% nonsulfated and 28% 4-sulfated unsaturated disaccharides, whereas those of aggrecan contained 25% nonsulfated and 70% 4-sulfated. Link protein overexpression in chondrocytic N1511 cells at the early stage of differentiation, in which versican is expressed, enhanced versican deposition in the matrix and prevented subsequent aggrecan deposition. These results suggest that versican is present as an aggregate distinct from the aggrecan aggregate and may play specific roles in the articular surface.
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PMID:Identification and characterization of versican/PG-M aggregates in cartilage. 1664 31

Aggrecan is degraded by several aggrecanase-1 (ADAMTS-4) isoforms differing in the number of sulfated glycosaminoglycan (sGAG)-binding motifs. ADAMTS-4 and MMPs cleave aggrecan more efficiently within the chondroitin sulfate (CS)-rich region than the interglobular domain (IGD). We investigated the influence of CS on aggrecan core protein cleavage by ADAMTS-4 (p68) and (p40) as well as MMP-13, which has no recognizable GAG-binding sites. Chondroitinase ABC-treated cartilage aggrecan was cleaved with ADAMTS-4 (p68) less efficiently than CS-substituted aggrecan within the CS-2 domain. Keratanase-treated aggrecan exhibited reduced IGD cleavage, but when both CS and KS were removed, the IGD cleavage was restored. This result suggests that KS in the IGD may compete with CS for ADAMTS-4 (p68) binding. In the absence of KS, however, p68 binding was shifted to the CS-2 domain. CS-deficient full-length recombinant aggrecan (rbAgg) was produced by chondroitinase ABC treatment, or by expression in the xylosyltransferase-deficient CHO-pgsA745 cell line. When digested with the ADAMTS-4 (p68), each of these preparations exhibited reduced CS-2 domain cleavage compared to CS-substituted CHO-K1 cell-derived aggrecan. Additionally, CS-deficient rbAgg showed increased IGD scission prior to cleavage within the CS-2 domain. ADAMTS-4 (p40) readily cleaved both rbAggs within the IGD, but cleaved poorly within the CS-2 domain, indicating little CS dependence. MMP-13, in contrast, cleaved the CS region and the IGD of both CS-substituted and CS-deficient rbAgg equally well. These data indicate that covalently bound CS enhances ADAMTS-4-mediated cleavage within the CS-rich region. MMP-13 also cleaves preferentially within the CS-region, but by an apparently CS-independent mechanism.
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PMID:Effects of covalently attached chondroitin sulfate on aggrecan cleavage by ADAMTS-4 and MMP-13. 1694 13

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.
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PMID:Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans. 1725 Jul 96

A novel in-gel endoglycosidase technique to study oligosaccharides with graphitized carbon LC-MS has revealed differences in the sulfation profile between the linkage and repeat regions of chondroitin sulfate on aggrecan. Bovine articular cartilage aggrecan was isolated in a composite agarose PAGE gel or diluted in ammonium acetate buffer and was digested overnight with chondroitinase ABC. Including a chemical release/reduction protocol after digestion, we could separate and detect three differentially sulfated chondroitin sulfate disaccharides of the repeat region (DeltaUA1-3GalNAc0/4/6S-ol) from the three differentially sulfated linkage region hexasaccharides (DeltaUA1-3GalNAc0/4/6Sbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylitol). Graphitized carbon LC-MS in the negative ion mode was able to resolve isomeric disaccharides and linkage region hexasaccharides. Specific MS2 and MS3 enabled us to confirm the sulfate location on all oligosaccharides by comparing their fragmentation with sulfated disaccharide standards. The presence of unsulfated, 6-sulfated, and 4-sulfated linkage regions was correlated with positive Western blot staining with the respective CS linkage region neoepitope antibodies (1B5, 3B3, 2B6) on digested aggrecan. Our strategy of examining linkage region and repeat region profiles is applicable to screening GAGs from various biological samples in order to detect differences between normal and disease states.
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PMID:Graphitized carbon LC-MS characterization of the chondroitin sulfate oligosaccharides of aggrecan. 1741 Oct 12


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