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)

The production and distribution of basement membrane-type heparan sulfate proteoglycans (BM HSPG) were investigated in a mouse glomerular epithelial cell line. Confluent cell monolayers were radiolabeled with [35S]sulfate or [35S]cysteine. Proteoglycans were isolated from the medium and cell layers by ion exchange chromatography and their nature determined by enzyme digestion (chondroitinase ABC) or degradative treatment (nitrous acid). It was found that more than 80% of the proteoglycans in both the cell layer and medium were heparan sulfate proteoglycans (HSPG) based on their susceptibility to nitrous acid degradation. More than half of the HSPG in the cell layer could be precipitated with an antiserum that specifically recognizes BM HSPG; only 10% of those released into the medium were precipitated with this antiserum. When immunoprecipitates of [35S] sulfate-labeled proteoglycans were analyzed by SDS-PAGE, the mature proteoglycans ran as a broad band at the top of the gel. When immunoprecipitates of [35S]cysteine-labeled proteoglycans were similarly analyzed, a 250 kd precursor core protein band was seen in addition to the mature proteoglycan. When BM HSPG were localized by immunofluorescence and immunoelectron microscopy (immunoperoxidase), they were found intracellularly in biosynthetic compartments (ER and Golgi cisternae) and extracellularly in deposits of basement membrane-like matrix located beneath and between the cells. These results indicate that l) BM HSPG are the predominant type of proteoglycans made by glomerular epithelial cells in culture; 2) these HSPG are assembled into a loosely organized matrix that is deposited beneath and between the cells; and 3) this cell type produces a higher proportion of BM HSPG than other cultured epithelial cells studied previously.
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PMID:Basement membrane heparan sulfate proteoglycan is the main proteoglycan synthesized by glomerular epithelial cells in culture. 252 72

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.
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PMID:Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone. 312 76

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.
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PMID:A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage. 679 63

The N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene, which is responsible for autosomal recessive mucopolysaccharidosis IVA (MPSIVA), has been assigned to the long arm of chromosome 16, subregion 24.3, an area where the adenine phosophoribosyltransferase (APRT) gene and renal dipeptidase (DPEP I) gene are also localized. Molecular genetic studies on a severely affected patient with MPSIVA (Morquio disease), without karyotypic abnormality, revealed a partial submicroscopic deletion of 16q24.3 and a single point mutation on the other allele, with no functional GALNS activity. The patient, his mother, and siblings were hemizygous for GALNS and APRT loci, evidenced by informative RFLP and gene dosage analyses combined with a fluorescence in situ hybridization, utilizing a partial genomic clone of GALNS, but heterozygosity was retained at the DPEP I locus and proximal D16S7. Haplotyping of the family members revealed recombinational events between DPEP I locus and three other polymorphic loci on the paternal chromosome, localizing GALNS gene on the proximal side to DPEP I gene. As estimated from the genetic distance between two flanking markers of proximal D16S7 and distal DPEP I locus, size of the deletion was less than 3Mb. Mother of the boy and two older siblings were asymptomatic, despite this interstitial deletion of the Giemsa-light G band. The remaining paternal allele had no gene rearrangement but GALNS activity was not encoded as Arginine at 386 was replaced with Cysteine (R386C), suggesting this alteration accounts for the severe phenotype. Allelic loss of APRT is frequently observed in cancer tissues, thereby suggesting that the tumor suppressor gene locates near the APRT locus. No family member has evidence of any malignant disease. This study is apparently the first documentation of interstitial deletion of 16q24.3, involving GALNS and APRT genes.
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PMID:Mucopolysaccharidosis IVA: submicroscopic deletion of 16q24.3 and a novel R386C mutation of N-acetylgalactosamine-6-sulfate sulfatase gene in a classical Morquio disease. 882 29

We have isolated a chondroitin sulfate proteoglycan from mouse brain by affinity chromatography with a fragment of the extracellular matrix glycoprotein tenascin-R (TN-R) that comprises the amino-terminal cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats. The isolated chondroitin sulfate proteoglycan has a molecular mass of 500-600 kDa and carries the HNK-1 carbohydrate epitope. Treatment with chondroitinase ABC reveals a major band of approximately 400 kDa and two minor bands at 200 and 150 kDa. Immunoblot analysis relates the molecule to phosphacan but not to the chondroitin sulfate proteoglycans neurocan and versican. Binding of the phosphacan-related molecule to the epidermal growth factor-like repeats of TN-R is Ca2+-dependent. Co-localization of the molecule with TN-R in the retina and optic nerve by immunocytochemistry suggests a functional relationship between the two molecules in vivo. Inhibition of neurite outgrowth from hippocampal neurons by the phosphacan-related molecule in vitro is neutralized by TN-R when coated as a uniform substrate. Furthermore, the phosphacan-related molecule neutralizes growth cone repulsion induced by TN-R coated as a sharp substrate boundary with or without prior treatment with chondroitinase ABC. These observations indicate that TN-R can interact with a phosphacan-related molecule and thereby modulate its inhibitory influence on neuritogenesis.
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PMID:Isolation of a tenascin-R binding protein from mouse brain membranes. A phosphacan-related chondroitin sulfate proteoglycan. 940 6

Transforming growth factor-beta (TGF-beta) is normally secreted in a latent form, and plasmin-mediated proteolytic cleavage of latency-associated peptide (LAP), a component of latent TGF-beta complex that makes the complex inactive, activates latent TGF-beta. In the present study, we investigated the possible involvement of calpain, one of the cysteine proteases, in the activation of latent TGF-beta. When recombinant latent TGF-beta was incubated with calpain (1-10 u/ml) in a test tube, calpain cleaved LAP and released mature TGF-beta from the latent complex. When calpain was applied to cultured bovine capillary endothelial (BCE) cells, a low concentration of calpain (0.05-0.1 u/ml) inhibited the migration and proliferation of the cells, and these inhibitory effects were abrogated by anti-TGF-beta antibody as well as by calpain inhibitor peptide, but not by alpha2-antiplasmin, a specific inhibitor of plasmin. Active TGF-beta was detected in the conditioned medium of BCE cells collected in the presence of calpain. Chemical cross-linking of (125)I-calpain to BCE cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that calpain bound to the cell surface through chondroitinase ABC-sensitive proteoglycan. In addition, treatment of the BCE cells with chondroitinase ABC abrogated the inhibitory effect of calpain on the migration of these cells. Our data thus suggest that calpain is able to activate latent TGF-beta through a mechanism independent of plasmin. This activation is efficient in the presence of cells, and calpain binds to the cell surface via proteoglycan and activates latent TGF-beta, which is targeted to the same surface.
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PMID:Cell-associated activation of latent transforming growth factor-beta by calpain. 942 5

Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been associated with a family of chondrodysplasias that includes diastrophic dysplasia (DTD), atelosteogenesis type 2 (AO2) and the lethal condition achondrogenesis type 1B (ACG1B). There is a correlation between the nature of the mutations and the clinical phenotype, but our understanding of the pathophysiology of the disorder, which involves defective sulfation of cartilage proteoglycans, is far from complete. To evaluate the degree of proteoglycan undersulfation in vivo, we have extracted chondroitin sulfate proteoglycans from cartilage of twelve patients with sulfate transporter chondrodysplasias and analyzed their disaccharide composition by HPLC after digestion with chondroitinase ABC. The amount of non-sulfated disaccharide was elevated in patients' samples (controls, 5.5%+/-2.8 (n=10); patients, 11% to 77%), the highest amount being present in ACG1B patients, indicating that undersulfation of chondroitin sulfate proteoglycans occurs in cartilage in vivo and is correlated with the clinical severity. To investigate further the biochemical mechanisms responsible for the translation of genotype to phenotype, we have studied fibroblast cultures of patients with DTD, AO2 and ACG1B, and controls, by double-labelling with [35S]sulfate and [3H]glucosamine. The incorporation of extracellular sulfate, estimated by the 35S/3H ratio in proteoglycans, was reduced in all patients' cells, with ACG1B cells showing the lowest values. However, disaccharide analysis of chondroitin sulfate proteoglycans showed that these were normally sul fated or only moderately undersulfated; marked undersulfation was observed only after addition of the artificial glycosaminoglycan-chain initiator, beta-D-xyloside, to the culture medium. These results suggest that, while utilization of extracellular sulfate is impaired, fibroblasts can replenish their intracellular sulfate pool by oxidizing sulfur-containing compounds (such as cysteine) and thus partially rescue PG sulfation under basal conditions. This rescue pathway becomes insufficient when GAG synthesis rate is stimulated by beta-D-xyloside. These findings may explain why phenotypic consequences of DTDST mutations are restricted to cartilage, a tissue with high GAG synthesis rate and poor vascular supply, and imply that pharmacological therapy aimed at restoring the intracellular sulfate pool might improve PG sulfation in DTD and related disorders.
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PMID:Proteoglycan sulfation in cartilage and cell cultures from patients with sulfate transporter chondrodysplasias: relationship to clinical severity and indications on the role of intracellular sulfate production. 982 2

Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.
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PMID:Plasmodium falciparum: cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A. 1091 Jul 12

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), required for degradation of keratan sulfate and chondroitin-6-sulfate. In order to study the effects of a missense mutation in the active site cysteine in the GALNS gene that is conserved in all mammalian sulfatases, we produced a p.C76S (an active site replacement) knock-in mouse by replacing the Cys76 with Ser in the endogenous murine Galns by targeted mutagenesis. Homozygous Galns(tm(C76S)slu) mice had no detectable GALNS enzyme activity. At age of 2-4 months, lysosomal storage was present primarily within reticuloendothelial cells such as Kupffer cells and spleen sinusoidal lining cells. Vacuolar change was present in glomerular visceral epithelial cells and was not present in hepatocytes or renal tubular cells. In the brain, hippocampal and neocortical neurons and meningeal cells showed lysosomal storage. Radiographs revealed no change in the skeletal bones of mice up to 12 months old. Thus, the Galns(tm(C76S)slu) mice had visceral storage of GAGs in organs but lacked the skeletal features of human MPS IVA. In contrast to a previously reported transgenic model (Galns(tm(hC79S.mC76S)slu)), in which the inactive human GALNS transgene was overexpressed, no reduction in other sulfatases was observed. In addition, the Galns(tm(C76S)slu) mice displayed milder storage. We conclude that the milder phenotype is characteristic of isolated GALNS deficiency while the more severe phenotype reflected in the Galns(tm(hC79S.mC76S)slu) mice was due to deficiency of other sulfatases caused by oversaturation of the sulfate modifying enzyme by the inactive human gene product.
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PMID:Murine model (Galns(tm(C76S)slu)) of MPS IVA with missense mutation at the active site cysteine conserved among sulfatase proteins. 1749 92

Fibroblast growth factor-18 (FGF-18) has been shown to regulate the growth plate chondrocyte proliferation, hypertrophy and cartilage vascularization necessary for endochondral ossification. The heparan sulfate proteoglycan perlecan is also critical for growth plate chondrocyte proliferation. FGF-18 null mice exhibit a skeletal dwarfism similar to that of perlecan null mice. Growth plate perlecan contains chondroitin sulfate (CS) and heparan sulfate (HS) chains and FGF-18 is known to bind to heparin and to heparan sulfate from some sources. We used cationic filtration and immunoprecipitation assays to investigate the binding of FGF-18 to perlecan purified from the growth plate and to recombinant perlecan domains expressed in COS-7 cells. FGF-18 bound to perlecan with a K(d) of 145 nM. Near saturation, approximately 103 molecules of FGF-18 bound per molecule of perlecan. At the lower concentrations used, FGF-18 bound with a K(d) of 27.8 nM. This binding was not significantly altered by chondroitinase nor heparitinase digestion of perlecan, but was substantially and significantly reduced by reduction and alkylation of the perlecan core protein. This indicates that the perlecan core protein (and not the CS nor HS chains) is involved in FGF-18 binding. FGF-18 bound equally to full-length perlecan purified from the growth plate and to recombinant domains I-III and III of perlecan. These data indicate that low affinity binding sites for FGF-18 are present in cysteine-rich regions of domain III of perlecan. FGF-18 stimulated 3H-thymidine incorporation in growth plate chondrocyte cultures derived from the lower and upper proliferating zones by 9- and 14-fold, respectively. The addition of perlecan reversed this increased incorporation in the lower proliferating chondrocytes by 74% and in the upper proliferating cells by 37%. These results suggest that perlecan can bind FGF-18 and alter the mitogenic effect of FGF-18 on growth plate chondrocytes.
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PMID:The core protein of growth plate perlecan binds FGF-18 and alters its mitogenic effect on chondrocytes. 1797 Dec 91


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