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)

Chondroitin sulfate proteoglycans, cell surface and extracellular matrix molecules in both neural and non-neural tissues, are highly regulated during normal development. Entire proteoglycan molecules may be either up-regulated or down-regulated, or only the chondroitin sulfate glycosaminoglycan portions of these molecules may be modified. Subtle changes in the chemistries of chondroitin sulfate chains can now be identified through the use of a panel of anti-chondroitin sulfate monoclonal antibodies. Each of these antibodies recognizes specific chemical structures which are non-randomly dispersed along the lengths of chondroitin sulfate chains. The location of individual epitopes within defined domains in these chains is demonstrated through controlled treatments of aggrecan with chondroitinase ABC, whereby portions of these chains are removed from the non-reducing terminal ends and where the remainder of the chains remains covalently attached to the core protein. In these situations, some epitopes, such as those recognized by antibodies CS-56 and 6C3, can be removed without loss of other epitopes, such as that recognized by antibody 4C3. The independent expression of individual epitopes is demonstrated by immunocytochemical analyses of developing skin appendages in embryonic chicks and fetal humans. These are sites where highly patterned morphogenetic movements result from epithelial-mesenchymal interactions. In both chicks and humans, some epitopes are constitutively expressed while others are strictly regulated in the mesenchymal portions of the developing skin appendages. These data strongly suggest that chondroitin sulfate proteoglycans, including their chondroitin sulfate chains, have important roles in regulating these epithelial mesenchymal interactions. Furthermore, these data underscore the significance of the aforementioned observation that individual epitopes are located in specific domains within chondroitin sulfate chains. The highly organized expression of chondroitin sulfate proteoglycans in the development of the central nervous system strongly argues for a similar role for these molecules in the organs that comprise this system.
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PMID:Regulated expression of chondroitin sulfates at sites of epithelial-mesenchymal interaction: spatio-temporal patterning identified with anti-chondroitin sulfate monoclonal antibodies. 884 1

Sulfated glycosaminoglycans such as chondroitin sulfate are composed of three structural domains, a linkage oligosaccharide, connecting the chain to the core protein, a variably sulfated disaccharide repeat structure within the chain and a nonreducing terminal, and these domains may confer specific functions on particular chain populations. We report here a new and highly sensitive method for the detection and quantitation of all nonreducing terminal residues and internal disaccharides obtained by chondroitinase ABC or ACII digestion of aggrecan chondroitin sulfate. The procedure involves a quantitative reductive animation of the reducing ends of sulfated mono- and disaccharide chondroitinase products with 2-aminopyridine and boranedimethylamine. All derivatized saccharides can be separated and quantitated by fluorescence in a single chromatographic step on an AS4A anion exchange column, eluted with a gradient (0-500 nM) of sodium trifluoroacetate. The reproducibility and stability of the derivatisation, together with the sensitivity of the chromatography system, allowed for routine quantitation in the range of 3-500 pmol of reducing group (corresponding to about 1.5-250 ng of disaccharide or 0.75-125 ng of monosaccharide). Moreover, the fluorescence yield (fluorescence area units per pmol of reducing group) was virtually identical for all saccharides analyzed. Application of this method to an analysis of aggrecan purified from calf epiphyseal cartilage and from rat chondrosarcoma chondrocyte cultures allowed a precise identification and quantitation of the internal disaccharides and the nonreducing terminal structures, together with an estimation of the number average molecular weight of CS chains in these aggrecan preparations.
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PMID:Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine. 902 44

Aggrecan (PG) was isolated from Swarm rat chondrosarcoma and the chondroitin 4-sulfate removed with chondroitinase ABC (ABC) or ACII (AC), leaving a 4-deoxy-beta-d-gluc-4-enuronosyl (DeltaGlcA) residue on the nonreducing terminus of the attached chondroitin sulfate chains. Mercuric acetate (as low as 5 mm) removed the DeltaGlcA from the PG-ABC within 10 min at 25 degrees C at pH 5.0, and the rate was pH independent between pH 3.0 and 5.0. The reaction was readily monitored by following the loss of reactivity to the monoclonal antibodies specific for 4-sulfated and nonsulfated unsaturated disaccharides in ELISA. After mercury treatment, there was a loss of carbazole-positive material and a decrease in the size of the linkage region oligosaccharides consistent with DeltaGlcA being removed. Aside from the loss of DeltaGlcA, neutral sugar composition and sialic acid content remained unchanged. After electrophoresis in a 4% polyacrylamide gel, Hg-treated PG-ABC and PG-AC migrated as single major bands, but with reduced mobilities, which is consistent with a loss of charge. There was a loss of reactivity to specific monoclonal antibodies. Treated aggrecan did not bind hyaluronic acid. This loss was not completely prevented by being present in a complex with link protein and hyaluronic acid. However, link protein could partially restore the hyaluronic acid interaction, so the effect of mercuric acetate on biological function will have to be assessed on an individual basis. Treatment with mercuric acetate is an effective, rapid, reproducible way of removing DeltaGlcA from both chondroitinase ABC and ACII-digested proteoglycan.
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PMID:Mercuric salt-catalyzed removal of unsaturated glucuronic acid from chondroitinase-treated proteochondroitin sulfate. 902 74

Proteoglycans influence axonal outgrowth in several experimental paradigms, and their distribution during development suggests a role in axon guidance. We have used a monoclonal antibody, 5D4, that recognizes an epitope on sulfated keratans (KS), to define the distribution of keratan sulfate proteoglycans (KSPGs) in the developing thalamus and cortex of the rat. During development, 5D4 immunolabeling is present on thalamic axons as they grow through the internal capsule and subplate but is not present in the adjacent pathway for cortical efferent axons. Individual thalamic nuclei differ markedly in their expression of KSPGs; these distinctions persist throughout the period of developmentally regulated expression. Major cortical domains also differ in their expression of KSPGs, which are expressed throughout medial (cingulate and retrosplenial) cortex well before neocortex. Immunolabeling for KSPGs diminishes 2 weeks after birth; in the adult it is associated with small glia. The 5D4 epitope is present on several KSPGs (320, 220, and 160 kD) on Western blots during development but only in a broad 200-kD band in adult brain. Immunolabeling is degraded on sections and Western blots by keratanase II but not by keratanase I or chondroitinase ABC, confirming that the antibody recognizes KS. Bands identified by 5D4 on Western blots differ from those identified by antibodies to known KSPGs (aggrecan, claustrin, SV2, ABAKAN, phosphacan-KS), indicating that 5D4 is labeling KSPGs not previously described in the brain. The selective expression of KSPGs during development suggests that they may be a part of the molecular identity of thalamic nuclei and cortical domains that defines their connectivity.
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PMID:Developmental expression of keratan sulfate-like immunoreactivity distinguishes thalamic nuclei and cortical domains. 908 31

Proteoglycans are among the major extracellular matrix components of the central nervous system. In the cerebral cortex and many subcortical regions, chondroitin sulphate proteoglycans, which are related to the aggrecan-versican-neurocan family, have been detected immunocytochemically in perineuronal nets that surround various types of neurons. This indicates that, in the brain, there is a nonhomogeneous but defined distribution of extracellular matrix components. The present study is a further attempt to characterize the perineuronal nets in the cerebral cortex. Sections obtained from fixed and unfixed rat brains were subjected to different enzymatic treatments prior to the visualization of perineuronal nets using N-acetylgalactosamine-binding Wisteria floribunda agglutinin, antibodies against chondroitin sulphate proteoglycans or hyaluronectin, and biotinylated hyaluronectin which detects hyaluronan. In all perineuronal nets the binding of the Wisteria floribunda agglutinin was abolished after the incubation of sections with chondroitinase ABC. The protein components of the proteoglycan complexes became easier to digest after removal of chondroitin sulphate chains or hyaluronan. Since only quantitative, and not qualitative, differences in the labelling properties and the structural appearance of cortical perineuronal nets were observed after the various treatments, it is concluded that, with regard to their proteoglycan composition, these structures have common basic properties.
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PMID:Characterization of proteoglycan-containing perineuronal nets by enzymatic treatments of rat brain sections. 908 41

The movement of neural crest cells is controlled in part by extracellular matrix. Aggrecan, the chondroitin sulfate proteoglycan from adult cartilage, curtails the ability of neural crest cells to adhere, spread, and move across otherwise favorable matrix substrates in vitro. Our aim was to isolate, characterize, and compare the structure and effect on neural crest cells of aggrecan and proteoglycans purified from the tissues through which neural crest cells migrate. We metabolically radiolabeled proteoglycans in E2.5 quail embryos and isolated and characterized proteoglycans from E3.3 quail trunk and limb bud. The major labeled proteoglycan was highly negatively charged, similar in hydrodynamic size to chick limb bud versican/PG-M, smaller than adult cartilage aggrecan but larger than reported for embryonic sternal cartilage aggrecan. The molecular weight of the iodinated core protein was about 400 kDa, which is more than reported for aggrecan but less than that of chick versican/PG-M. The proteoglycan bore chondroitin sulfate glycosaminoglycan chains of 45 kDa, which is larger than those of aggrecan. It lacked dermatan sulfate, heparan sulfate, or keratan sulfate chains. It bound to collagen type I, like aggrecan, but not to fibronectin (unlike versican/PG-M), collagen type IV, or laminin-1 in solid-phase assays and it bound to hyaluronate in gel-shift assays. When added at concentrations between 10 and 30 microg/ml to substrates of fibronectin, trunk proteoglycan inhibited neural crest cell spreading and migration. Attenuation of cell spreading was shown to be the most sensitive and titratable measure of the effect on neural crest cells. This effect was sensitive to digestion with chondroitinase ABC. Similar cell behavior was also produced by aggrecan and the small dermatan sulfate proteoglycan decorin; however, 30-fold more aggrecan was required to produce an effect of similar magnitude. When added in solution to neural crest cells which were already spread and migrating on fibronectin, the embryonic proteoglycan rapidly and reversibly caused complete rounding of the cells, being at least 30-fold more potent than aggrecan in this activity.
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PMID:Isolation and characterization of chondroitin sulfate proteoglycans from embryonic quail that influence neural crest cell behavior. 940 1

This study used biochemical and light and electron microscopic immunohistochemical methods to localize and characterize large hyaluronate-binding proteoglycans in the developing mandible of fetal rats at embryonic day 15 (Day 15) to Day 18 using a monoclonal antibody (MAb) 5D5. This antibody is derived from bovine sclera and specifically recognizes the core protein of large proteoglycan such as versican, neurocan and brevican, but not that of aggrecan. At the light microscopic level, MAb 5D5 moderately stained the extracellular matrices among osteoblasts at the centers of ossification in Day 15 mandible specimens. Weaker staining was observed in osteoblasts, whereas Meckel's cartilage lacked staining. Ultrastructural immunocytochemistry showed the presence of immunogold particles over unmineralized matrices among osteoblasts and their intracellular organelles. In Day 16 to 18 specimens, bone nodules were recognized in LR gold sections before immunostaining, but, after immunostaining, consistently appeared devoid of mineral crystals and were seen as a demineralized structure that had an electron dense periphery within which fine filamentous and granular material were present. The appearance of these structures was created by the demineralization of thin sections on grids during immunostaining. Specific immunogold staining was clearly seen over the demineralized structures corresponding to bone nodules. The majority of immunogold particles tended to localize inside of the structures. Bone proteins were extracted from fresh, Day 18 specimens with a three-step technique: 4 M guanidine HCl (GdnCl,-extract), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis after chondroitinase ABC digestion, showed that G1-extract gave a 5D5 reactive band greater than 400 kDa, whereas E-extract produced two major reactive populations of small molecular size with core proteins approximately 63 and 74 kDa. These results indicate that the large proteoglycan having smaller molecular weight is preferentially localized to bone nodules and may correlate with bone matrix mineralization.
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PMID:Immunocytochemical localization and biochemical characterization of large proteoglycans in developing rat bone. 968 Jul 65

We report here the isolation and sulphation isomer analyses of trisaccharides GalNAcS(beta1,4)GlcA(beta1,3)GalNAcS (in which S indicates sulphate) derived from the non-reducing termini of aggrecan chondroitin sulphate. Rat chondrosarcoma and human aggrecans were digested for 1 h at 37 degrees C with 30 micro-units of endo-chondroitinase ABC per microgram of chondroitin sulphate, and trisaccharides were isolated from the digests by ToyoPearl HW40S gel-filtration chromatography. Four trisaccharide species were identified; their sulphation isomer compositions, as determined by digestion with chondroitinase ACII and fluorescence-based ion-exchange HPLC, were GalNAc4Sbeta1,4GlcAbeta1,3GalNAc4S, GalNAc4Sbeta1,4GlcAbeta1,3GalNAc6S, GalNAc4,6Sbeta1,4GlcAbeta1, 3GalNAc4S and GalNAc4,6Sbeta1,4GlcAbeta1,3GalNAc6S. The abundances of such sequences in chondroitin sulphate on aggrecan from normal (foetal to 72 years of age) and from osteoarthritic human knee cartilages were also established. The results showed that non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphated disaccharide, suggesting that the sulphation of the last disaccharide might not have a direct effect on the specificity of chondroitin sulphate terminal GalNAc sulphotransferases. Furthermore, for each aggrecan preparation examined, the 4S-to-6S ratio of all chain interior disaccharides was equivalent to that in the last repeating disaccharides at the non-reducing terminus, suggesting that neither chondroitin 4-sulphotransferase nor chondroitin 6-sulphotransferase shows preferential activity near the chain terminus.
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PMID:Sulphation heterogeneity in the trisaccharide (GalNAcSbeta1, 4GlcAbeta1,3GalNAcS) isolated from the non-reducing terminal of human aggrecan chondroitin sulphate. 1043 20

A biotinylated complex of aggrecan G1-domain and link protein was used to characterize the distribution of hyaluronan in paraffin-embedded sections of adult human and canine intervertebral disc and cartilage endplate. Limited chondroitinase ABC and trypsin digestions of the sections before staining was utilized to expose hyaluronan potentially masked by aggrecan. Hyaluronan concentration and hyaluronan to uronic acid ratio in different parts of the discs were measured as a background for the histological analysis. Hyaluronan staining was strong in the nucleus pulposus and inner parts of annulus fibrosus of both species, corroborated by biochemical assays of the same compartments. Particularly in human samples, hyaluronan in the interterritorial matrix of nucleus pulposus and annulus fibrosus was readily accessible to the probe without enzyme treatments. In contrast, the cell-associated hyaluronan signal was enhanced after trypsin or limited chondroitinase ABC-treatment of the sections, suggesting that pericellular hyaluronan was more masked by aggrecan than in the distant matrix. A puzzling feature of canine cartilage endplate cells was their intensive cell-associated hyaluronan signal, part of which appeared intracellular. Hyaluronan was abundant between the collagenous lamellae in annulus fibrosus, perhaps important in the plasticity of this tissue.
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PMID:Hyaluronan distribution in the human and canine intervertebral disc and cartilage endplate. 1057 27

Pleiotrophin (PTN) is a secreted heparin-binding, developmentally regulated protein that is found in abundance in fetal, but not mature, cartilage. SDS-page and glycosaminoglycan (GAG) analysis of sulfate-radiolabeled proteoglycans isolated from the medium of mature cultured chondrocytes treated with PTN showed a threefold increase in the levels of proteoglycan synthesis. In contrast, in cultures of fetal chondrocytes, no changes in proteoglycan synthesis were observed. Thymidine incorporation experiments showed a dose-dependent decrease in proliferation of treated cells compared with control cultures, suggesting that pleiotrophin had an inhibitory effect on growth of chondrocytes. Neither FGF or heparin reversed the inhibitory effect of PTN. Capillary electrophoresis of chondroitinase ABC-digested proteoglycans isolated from mature chondrocytes showed 2-4-fold increases in the amounts of the 4S- and 6S-substituted GAG chains for the PTN-treated chondrocytes. Northern analysis showed a twofold upregulation in the mRNA levels of biglycan and collagen type II, but no difference in the message levels for decorin and aggrecan. These results establish that PTN inhibits cell proliferation, while stimulating the synthesis of proteoglycans in mature chondrocytes in vitro, suggesting that PTN may act directly or indirectly to regulate growth and proteoglycan synthesis in the developing matrix of fetal cartilage.
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PMID:Pleiotrophin inhibits chondrocyte proliferation and stimulates proteoglycan synthesis in mature bovine cartilage. 1060 16


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