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:3.1.6.12 (chondroitinase)
2,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human brain proteins were partially purified by using arginine-Sepharose 4B affinity chromatography, which traps proteins having an affinity to certain groups of arginine residue, such as serine proteases and zymogens. Bound proteins were analyzed for binding and cleavage related to the brain beta-amyloid precursor protein (APP). They were then further separated and isolated using a preparative gel system having a liquid-phase collection apparatus, using a non-denaturing gel system. Each fractionated protein was also analyzed for the above activity using natural APP. Among these, we found several fractions that bind preferentially to APP treated with chondroitinase ABC but not to intact APP, and that also generate particular beta-amyloid containing C-terminal peptides of APP via proteolysis. Our results suggest that sulfated glycoconjugates attached to APP play a role in the substrate specificity of APP for proteases, and also that the nature of natural APP processing mechanisms in vivo is very complex.
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PMID:A human brain proteolytic activity capable of cleaving natural beta-amyloid precursor protein is affected by its substrate glycoconjugates. 953 6

This study characterized proteoglycan metabolites present in gingival crevicular fluid (GCF) collected from sites with clinical evidence of advanced periodontal disease. The metabolites were purified by anion-exchange chromatography from which a chondroitin sulphate rich fraction was identified by cellulose acetate electrophoresis. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of this fraction revealed a broad silver-staining band with mol. wt 55-65 k and Western blotting suggested that this band was immunoreactive with CS-56, a monoclonal antibody for chondroitin sulphate. Digestion of the metabolite with chondroitinase ABC (protease-free) led to the loss of the silver-staining band. Dot-blot analysis identified components in this fraction that were immunoreactive for the monoclonal/polyclonal antibodies against the C-termino of decorin and biglycan. Amino acid analysis revealed the composition of the proteoglycan metabolite to be rich in glycine, serine and glutamic acid. Immunochemical and biochemical analyses were compared with those of proteoglycan purified from human alveolar bone. Changes in the amino acid composition were noted, suggesting the proteoglycan metabolite has undergone extensive modification and fragmentation to the protein core. The results suggest that the proteoglycan metabolite from GCF represented a degradation product originating from the active destruction of the alveolar bone. They provide further support for the proposal that the appearance of proteoglycan metabolites in GCF is a biomarker for active destruction of alveolar bone, the biochemical analysis of which provides important information on mechanisms involved in the pathology of periodontal diseases.
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PMID:Immunochemical detection of the proteoglycans decorin and biglycan in human gingival crevicular fluid from sites of advanced periodontitis. 983 4

Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region. In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.
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PMID:Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region. Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride. 1023 73

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.
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PMID:Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastin-binding protein. 1071 2

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

The media layer of the arterial cryo-cross sections, is defective for vWf-dependent platelet adhesion. Exposure of the same layer by stripping off the most inner portions of the vessel wall results in a highly thrombogenic surface. Stripping or balloon dilation was applied to porcine arteries prior to functional assays. Cryosections of treated or untreated arteries were perfused with porcine blood at 3,350 s(-1) and platelet deposition was detected by indirect immunofluorescence. Following balloon dilation, vWf-dependent platelet deposition increased; covering 9.08 +/- 1.36% of the total media surface area, this value for untreated vessels was 0.88 +/- 0.14%. A 10-fold increase was also found in the binding of vWf-coated fluorescent beads to the media. In addition to mechanical procedures, treatment by serine-proteases like trypsin, chymotrypsin and proteinase 3, or by chondroitinase ABC, but not by heparitinase also resulted in a 7-10-fold increase in platelet coverage over the media. Collagen in the media may be complexed with another vessel wall component shielding the vWf-binding sites. Mechanical or biochemical processes unmask these sites, and increase the thrombogenicity of the vessel wall.
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PMID:Physical and enzymatic perturbation of the architecture of the Tunica media destroys its inherent thromboresistance. 1242 2

CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.
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PMID:Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding. 1600 44

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

Human leukocyte elastase (HLE) and cathepsin G (CG) are expressed at high levels on the surface of activated human neutrophils (PMN) in catalytically active but inhibitor-resistant forms having the potential to contribute to tissue injury. Herein we have investigated the mechanisms by which HLE and CG bind to PMN plasma membranes. (125)I-Labeled HLE and CG bind to PMN at 0 degrees C in a saturable and reversible manner (K(D) = 5.38 and 4.36 x 10(-7) m and 11.5 and 8.1 x 10(6) binding sites/cell, respectively). Incubation of PMN with radiolabeled HLE and CG in the presence of a 200-fold molar excess of unlabeled HLE, CG, myeloperoxidase, lactoferrin, proteinase 3, phenylmethylsulfonyl fluoride (PMSF)-inactivated HLE, or PMSF-inactivated CG inhibited binding of radiolabeled ligands. This indicates that these PMN granule proteins share binding sites on PMN and that functional active sites of HLE and CG are not required for their binding to PMN. The sulfate groups of heparan sulfate- and chondroitin sulfate-containing proteoglycans are the PMN binding sites for HLE and CG since binding of HLE and CG to PMN was inhibited by incubating PMN with 1) trypsin, chondroitinase ABC, and heparitinases, but not other glycanases, and 2) purified chondroitin sulfates, heparan sulfate, and other sulfated molecules, but not with non-sulfated glycans. Thus, heparan sulfate- and chondroitin sulfate-containing proteoglycans are low affinity, high volume PMN surface binding sites for HLE and CG, which are well suited to bind high concentrations of active serine proteinases released from degranulating PMN.
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PMID:The sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans in neutrophil plasma membranes are novel binding sites for human leukocyte elastase and cathepsin G. 1738 12

Disruptions in neuronal dendrite development alter brain circuitry and are associated with debilitating neurological disorders. Nascent apical dendrites of cortical excitatory neurons project into the marginal zone (MZ), a cell-sparse layer characterized by intense chondroitin sulfate proteoglycan (CSPG) expression. Paradoxically, CSPGs are known to broadly inhibit neurite growth and regeneration. This raises the possibility that the growing apical dendrite is somehow insensitive to CSPG-mediated neurite growth inhibition. To test this, developing cortical neurons were challenged with both soluble CSPGs and CSPG-positive stripe substrates in vitro Soluble CSPGs inhibited dendritic growth and cortical dendrites respected CSPG stripe boundaries, effects that could be counteracted by prior CSPG inactivation by chondroitinase. Importantly, addition of Reelin, an extracellular signaling protein highly expressed in the MZ, partially rescued dendritic growth in the presence of CSPGs. High-resolution confocal imaging revealed that the CSPG-enriched areas of the MZ spatially correspond with the areas of reduced dendritic density in the Reelin null (reeler) cortex compared with controls. Chondroitinase injections into reeler explants resulted in increased dendritic growth into the MZ, recovering to near wild-type levels. Activation of the serine threonine kinase Akt is required for Reelin-dependent dendritic growth and we find that CSPGs induce Akt dephosphorylation, an effect that can be counteracted by Reelin addition. In contrast, CSPG application had no effect on the cytoplasmic adaptor Dab1, which is rapidly phosphorylated in response to Reelin and is upstream of Akt. These findings suggest CSPGs do inhibit cortical dendritic growth, but this effect can be counteracted by Reelin signaling.
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PMID:Reelin Counteracts Chondroitin Sulfate Proteoglycan-Mediated Cortical Dendrite Growth Inhibition. 3264 98


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