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)

1. Human embryonic lung and skin fibroblasts were allowed to incorporate 32SO42- or 35SO42- and D-[1-3H]glucosamine. After removal of the medium the monolayer was subjected to sequential extractions by using EDTA, brief trypsin digestion, extraction with dithiothreitol ofllowed by freeze--thawing and extraction with trichloroacetic acid. The heparan sulphate and galactosaminoglycan contents of the various extracts were estimated after deaminative cleavage of the former component. Heparan sulphate was the major component of the trypsin digest, whereas galactosaminoglycans were the dominant component of other fractions. 2. Galactosaminoglycans of the various fractions were subjected to chemical (periodate oxidation/alkaline elimination) and enzymic (chondroitinase-AC and -ABC, as well as testicular hyaluronidase) degradations. Galactosaminoglycans from the insoluble cell fraction and the dithiothreitol extract contained larger amounts of L-iduronic acid than did those of other fractions. 3. Pulse-chase experiments were performed with and without replating of the cells at the start of the chase period. Radioactive glycans were isolated from the various extracts during the chase period. The half-lives of glycans of the insoluble cell fraction and the dithioreitol extract were shorter (5--8h) than were those of the trypsin digest and the EDTA extract (22h and 11h respectively). After replating of the cells in chase medium, radioactive cell-associated glycans were secreted from the cells and could be recovered in the trypsin digest, the EDTA extract and the medium. Furthermore, 35S/3H ratios of glycans from all these fractions decreased during the chase period. The following conclusions were reached. The insoluble cell fraction contains the synthesis pool and some structural material, whereas the soluble cell fraction is the storage and degradation pool. The dithiothreitol extract appears to contain the immediate precursors of secreted material. The trypsin-released glycans comprise structural components as well as material destined for pinocytosis or secretion into the medium. The EDTA extract is considered to consist of glycans en route to the medium. 4. The two presumptive precursor pools were preferentially depleted of L-iduronic acid-rich galactosaminoglycans during the chase. Glycans recovered from the trypsin digest, the EDTA extract and the medium during the chase contained larger amounts of periodate-resistant uronic acid residues (D-glucuronic acid and/or L-iduronic acid O-sulphate) than did their precursors. It is proposed that polymer-level modifications of secreted glycans are partly responsible for the results.
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PMID:Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments. 22 Sep 58

Dermatan sulfate-chondroitin sulfate copolymers have been isolated from human umbilical cord as a major galactosaminoglycan component of this tissue. The galactosaminoglycan fraction was obtained from this tissue by papain [EC 3.4.22.2] digestion followed by precipitation with cetylpyridinium chloride in a yield of 700 mg per 100 g of dry tissue. Ethanol fractionation resolved 4-5 subfractions differing in relative content of L-iduronic acid and D-glucuronic acid. No galactosaminoglycan containing either solely L-iduronic acid or D-glucuronic acid was obtained. The copolymeric structure of the material in each subfraction was demonstrated by analysis of oligosaccharide fragments obtained by chondroitinase-AC [EC 4.2.2.5] digestion. All the polymers contained repeating disaccharide units, D-glucuronosyl-N-acetylgalactosamine, D-glucuronosyl-N-acetylgalactosamine 4-sulfate, D-glucuronosyl-N-acetyl-galactosamine 6-sulfate, and L-iduronosyl-N-acetylgalactosamine 4-sulfate, of which D-glucuronosyl-N-acetylgalactosamine 6-sulfate and L-iduronosyl-N-acetylgalactosamine 4-sulfate were predominant. Both iduronic acid- and glucuronic acid-containing units were arranged in clusters. The presence of a considerable amount of nonsulfated disaccharide units was noted. The copolymers show extensive polydispersity in electrophoresis on cellulose acetate and gel chromatography on Sephadex G-200.
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PMID:Dermatan sulfate-chondroitin sulfate copolymers from ambilical cord. Isolation and characterization. 97 51

L-[14C]Iduronic acid-containing sulfated galactosaminoglycans were formed by incubation of a fibroblast particulate fraction with UDP-D[14C]glucuronic acid, UDP-N-acetylgalactosamine, and sulfate donor (3'-phosphoadenylylsulfate). The formation of L-iduronic acid was strongly promoted by concomitant sulfation of the polymer. In the absence of sulfate donor 5 to 10% of the [14C]uronic acid residues were L-iduronic acid. However, when 3'-phosphoadenylylsulfate was included in the incubation mixture the amount of L-iduronic acid in the product increased 3 to 5-fold. Furthermore, approximately the same quantity of L-[14C]iduronic acid was recovered from the product formed in a pulse-chase experiment where incorporation of 14C-isotope preceded sulfation. It was therefore concluded that C-5 inversion of D-glucuronic acid to L-iduronic acid occurred on the polymer level as shown previously for the biosynthesis of heparin (Hook, M., Lindahl, U., Backstrom, G., Malmstrom, A., AND Fransson, L-A., J. Biol. Chem. (1974) 249, 3908). This conclusion was supported by the finding that no L[14C]iduronic acid could be detected in the UDP-hexuronic acid pool during this experiment. Nonsulfated and sulfated [14C]galactosaminoglycan products were degraded separately with chondroitinase-AC. The non-sulfated products afforded primarily disaccharide and a small amount of tetrasaccharide, while the sulfated products yielded, in addition, a considerable amount of larger oligosaccharides. Tetrasaccharides from nonsulfated products contained L-iduronic acid indicating that C-5 inversion at solitary sites can occur in the absence of sulfation of adjacent hexosamine moieties. The larger oligosaccharides obtained after chondroitinase-AC digestion of sulfated products yielded L-iduronic acid upon acid hydrolysis and were susceptible to chondroitinase-ABC digestion. The split products were almost exclusively 4-sulfated disaccharides. These results demonstrate that formation of blocks of L-iduronic acid-containing repeat periods is associated with 4-sulfation of adjacent hexosamine moieties.
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PMID:Biosynthesis of dermatan sulfate. I. Formation of L-iduronic acid residues. 112 48

1. Pig skin dermatan sulphate was degraded by periodate oxidation followed by alkaline elimination or by chondroitinase-ABC to quantify irregular repeating units, i.e. those containing D-GlcUA (D-glucuronic acid) and L-IdUA-SO4 (sulphated iduronic acid). 2. Previous results of periodate oxidation (Fransson, 1974) indicated repeating sequences in pig skin dermatan sulphate containing, on average, 3D-GlcUA, 9 L-IdUA-SO4 or 28 L-IdUA units in addition to N-acetylgalactosamine sulphate. However, complete digestion with chondroitinase-ABC yielded, at the most, 3-4 disulphated disaccharides/chain. Consequently, more than one-half of the L-IdUA-SO4 residues were present in monosulphated periods, i.e. IdUA-(SO4)-GalNAc. 3. To determine the location of L-IdUA-SO4 residues along the copolymeric chain dermatan sulphate was digested with testicular hyaluronidase. (This enzyme cleaves GalNAc-GlcUA bonds within block regions containing D-GlcUA.) By NaB3H4 reduction GalNAc residues located in the reducing end of the fragments were converted into [3H]GalNAcOH (N-acetylgalactosaminitol). Finally, the radioactive product was fragmented by periodate oxidation followed by alkaline elimination. The bulk of the radioactivity was associated with periodate-resistant oligosaccharides indicating that clusters of GlcUA-GalNAc-SO4 periods are often adjacent to a varying number of (n = 1-4) of L-IdUA-SO4-containing periods. 4. To study the distribution of L-IdUA-SO4-containing periods in relation to blocks of IdUA-GalNAc-SO4 periods different fractions of hyaluronidase-degraded dermatan sulphate were degraded separately. In all types of fragments (mol. wts. 1,500-10,000) L-IdUA-SO4-containing periods were demonstrated. In short fragments reducing terminal GalNAc-6-SO4 (6-sulphated N-acetylgalactosamine) was found confirming that these sequences were joined to relatively long D-GlcUA-containing block sequences via GalNAc-6-SO4. Moreover, low-molecular-weight oligosaccharides composed of alternating sequences were encountered. An octasaccharide derived from the carbohydrate sequence -GalNAc---GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc-IdUA-GalNAc---GlcUA-GalNAc (--- indicates the position of cleavage by hyaluronidase) was identified.
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PMID:The co-polymeric structure of pig skin dermatan sulphate. Distribution of L-iduronic acid sulphate residues in co-polymeric chains. 115 66

The structure of dermatan [35S]sulphate-chondroitin [35S]sulphate copolymers synthesized and secreted by fibroblasts in culture was studied. 35S-labelled glycosaminoglycans were isolated from the medium, a trypsin digest of the cells and the cell residue after 72h of 35SO42-incorporation. The galactosaminoglycan component (dermatan sulphatechondroitin sulphate copolymers) was isolated and subjected to various degradation procedures including digestion with testicular hyaluronidase, chondroitinase-AC and-ABC and periodate oxidation followed by alkaline elimination. The galactosaminoglycans from the various sources displayed significant structural differences with regard to the distribution of various repeating units, i.e. IdUA-GalNAc-SO4 (L-iduronic acid-N-acetyl-galactosamine sulphate), GlcUA-GalNAc-SO4 (D-glucuronic acid-N-acetylgalactosamine-sulphate) and IdUA(-SO4)-GalNAc (L-iduronosulphate-N-acetylgalactosamine). The galactosaminoglycans of the cell residue contained larger amounts of IdUA-GalNAc-SO4 than did those isolated from the medium or those released by trypsin. In contrast, the glycans from the latter 2 sources contained large proportions of periodate-resistant repeat periods [GlcUA-GalNAc-SO4 and IdUA(-SO4)-GalNAc]. Periods containing L-iduronic acid sulphate were particularly prominent in copolymers found in the medium. Kinetic studies indicated that the 35S-labelled glycosaminoglycan of the cell residue accumulated radioactivity more slowly than did the glycans of other fractions, indicating that the material remaining with the cells was not exclusively a precursor of the secreted polymers. The presence of copolymers rich in glucuronic acid or iduronic acid sulphate residues in the soluble fractions may be the result of selective secretion from the cells. Alternatively, extracellular, polymer-level modifications such as C-5 inversion of L-iduronic acid to D-glucuronic acid, or sulphate rearrangements, would yield similar results.
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PMID:The copolymeric structure of dermatan sulphate produced by cultured human fibroblasts. Different distribution of iduronic acid and glucuronic acid-containing units in soluble and cell-associated glycans. 121 88

Chondroitin sulfate was examined in the extracellular matrix of the canine medial and lateral superior olivary nuclei by light and electron microscopic immunocytochemistry. The extracellular matrix around the large neurons was intensely stained with a monoclonal antibody recognizing D-glucuronic acid 2-sulfate----N-acetylgalactosamine 6-sulfate (D-unit) and this staining degree was remarkably reduced after chondroitinase ABC digestion. Neuronal cytoplasm, glial cells or capillaries in these nuclei were not stained with the monoclonal antibody. The results indicate the presence of disaccharide residue of D-glucuronic acid 2-sulfate----N-acetylgalactosamine 6-sulfate in the chondroitin sulfate proteoglycan of the extracellular matrix.
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PMID:Chondroitin sulfate in the extracellular matrix of the medial and lateral superior olivary nuclei in the dog. 138 Aug 72

N-Acetylgalactosamine-6-sulfate sulfatase from human placenta was purified 33,600-fold using beta-N-acetyl-D-galactosamine 6-sulfate-(1----4)-beta-D-glucuronic acid-(1----3)-N-acetyl-D-[3H]galactosaminitol 6-sulfate as the substrate. This enzyme is an oligomer with a molecular mass of 120 kDa and consists of polypeptides of 40 and 15 kDa. The 15 kDa polypeptide is a glycoprotein. This purified protein has activities of N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. Rabbit antiserum was raised against the purified protein. The antibody titrated N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. The size of the precursor of the enzyme is 60 kDa, as determined by cell-free translation. The optimal pH values of the N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase activities are pH 3.8-4.0, and the Kms are 8 and 13 microM, respectively. Sulfate and phosphate ions are potent competitive inhibitors for the enzyme and their inhibition constants are 35 and 200 microM, respectively. Cross-reactive materials of 40 and 15 kDa were detected by immunoblot analysis, in the placenta, liver, and normal fibroblasts, but not in fibroblasts from a patient with Morquio disease.
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PMID:N-acetylgalactosamine-6-sulfate sulfatase in human placenta: purification and characteristics. 179 86

The structure of a unique focose-branched chondroitin sulfate isolated from the body wall of a sea cucumber was examined in detail. This glycosaminoglycan contains side chain disaccharide units of sulfated fucopyranosyl units linked to approximately one-half of the glucuronic acid moieties through the O-3 position of the acid. The intact polysaccharide is totally resistant to chondroitinase degradation, whereas, after defucosylation, it is partially degraded by the enzyme. However, only after an additional step of desulfation, the chondroitin from sea cucumber is almost totally degraded by chondroitinase AC or ABC. This result, together with the methylation and NMR studies of the native and chemically modified polysaccharide, suggest that besides the fucose branches, the sea cucumber chondroitin sulfate contains sulfate esters at position O-3 of the beta-D-glucuronic acid units. Furthermore, the proteoglycan from the sea cucumber chondroitin sulfate is recognized by anti-Leu-7 monoclonal antibody, which specifically recognizes 3-sulfoglucuronic acid residues. In analogy with the fucose branched units, the 3-O-sulfo-beta-D-glucuronosyl residues are resistant to chondroitinase degradation. Regarding the position of the glycosidic linkage and site of sulfation in the fucose branches, our results suggest high heterogeneity. Tentatively, it is possible to suggest the preponderance of disaccharide units formed by 3,4-di-O-sulfo-alpha-L-fucopyranosyl units glycosidically linked through position 1----2 to 4-O-sulfo-alpha-L-fucopyranose. Finally, the presence of unusual 4/6-disulfated disaccharide units, together with the common 6-sulfated and non-sulfated units, was detected in the chondroitin sulfate core of this polysaccharide.
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PMID:Structure of a fucose-branched chondroitin sulfate from sea cucumber. Evidence for the presence of 3-O-sulfo-beta-D-glucuronosyl residues. 190 78

A D-glucuronic acid rich, copolymeric chondroitin sulfate (CS)-dermatan sulfate (DS) proteoglycan (PG) from post-burn hypertrophic scar tissue (HSc) was obtained by DEAE-cellulose chromatography and differential ethanol fractionation, and further purified on a Sepharose CL-6B column. CS-DS-PG protein content was 14% (w/w). The amino-terminal amino acid sequence of the first ten residues was as follows: NH2-Asp-Glu-Ala-B-Gly-Ile-Gly-Pro-Glu-Val. This sequence is identical to that of human embryonic fibroblast cell (IMR-90) CS-DS-PG, as well as to human HSc-DS-PG. After chondroitinase ABC treatment, two peptides (Mr 22,000 and 16,000 daltons) were detected by sodium dodecyl sulfate-(polyacryl)amide gel electrophoresis (SDS-PAGE). ELISA analysis using rabbit antiserum raised against a synthetic peptide that contained 15 amino acids in the same sequence as the amino terminus of human fetal membrane PG showed significant reactivity with HSc CS-DS-PG. HSc CS-DS-PG had an apparent Mr of approximately 78,000 daltons, as determined by Sepharose CL-6B chromatography and SDS-PAGE. Alkaline borohydride treatment of CS-DS-PG liberated CS-DS glycosaminoglycan (GAG) chains having an Mr of 29,000 daltons. The conversion of xylose to xylitol indicated that the GAG chains are attached to the PG protein core at O-3 through a xylosyl-seryl linkage. CS-DS-PG also contained both N and O-linked oligosaccharides and did not aggregate with hyaluronic acid. These results, together with those reported previously, showed that HSc CS-DS-PG and DS-PG have the same A1-A15 amino acid sequence at the amino terminus but different protein cores. HSc CS-DS-PG was completely digested with chondroitinase AC and is, therefore, distinctly different from HSc DS-PG.
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PMID:Isolation and some structure analyses of a copolymeric chondroitin sulfate-dermatan sulfate proteoglycan from post-burn, human hypertrophic scar. 234 48

Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains. One of these monoclonal antibodies (IgM; designated MO-225) reacts with PG-M, chick embryo cartilage proteoglycans (PG-H, PG-Lb, and PG-Lt), and bovine nasal cartilage proteoglycan, but not with Swarm rat chondrosarcoma proteoglycan. The reactivity of PG-H to MO-225 is not affected by keratanase digestion but is completely abolished after chondroitinase digestion. Competitive binding analyses with various glycosaminoglycan samples indicate that the determinant recognized by MO-225 resides in a D-glucuronic acid 2-sulfate(beta 1----3)N-acetylgalactosamine 6-sulfate disaccharide unit (D-unit) common to antigenic chondroitin sulfates. A tetrasaccharide trisulfate containing D-unit at the reducing end is the smallest chondroitin sulfate fragment that can inhibit the binding of the antibody to PG-H. Decreasing the size of a D-unit-rich chondroitin sulfate by hyaluronidase digestion results in progressive reduction in its inhibitory activity. The results suggest that the epitope has a requirement for a long stretch of a disaccharide-repeating structure for a better fit to the antibody.
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PMID:A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain. 243 33


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