Effect of ascorbic acid on arylsulfatase activities and sulfated proteoglycan metabolism in chondrocyte cultures.
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A correlation between increased arylsulfatase activities and decreased sulfated proteoglycan content in human osteoarthritic articular cartilage suggested a possible interrelationship between these parameters. Since we had previously shown that ascorbate caused a decrease in levels of arylsulfatase A and B activities in normal chondrocyte cultures, the validity of the above relationship was examined by measuring the effect of vitamin C on the biosynthesis and distribution of 35S-labeled proteoglycans and arylsulfatase A and B activities in cell extracts of chondrocytes derived from normal and osteoarthritic tissue. Arylsulfatase A and B activities were found to be reduced in the presence of ascorbic acid in all normal and osteoarthritic cell lines examined when measured 3, 6, 10, and 13 days after the introduction of the vitamin in the culture medium. Acid phosphatase activity, on the other hand, was found to be elevated in the presence of ascorbate. The inhibitory effect by ascorbic acid on arylsulfatase activities could be reversed by withdrawing the vitamin from the nutrient medium. Addition of EDTA to the cell extracts before assay also reversed the inhibiton. Sulfated proteoglycan biosynthesis as reflected in 35S-sulfate uptake per milligram of DNA was significantly increased in the presence of ascorbic acid. The distribution of the newly synthesized molecules between the cell layer and medium fractions was altered. In the presence of ascorbate, more deposition into the cell layer of newly synthesized macromolecules occurred. These data suggest an inverse relationship between arylsulfatase activities and the stability of the newly synthesized sulfated proteoglycans in the extracellular matrix. (+info)
Normal MPS excretion, but dermatan sulphaturia, combined with a mild Maroteaux-Lamy phenotype.
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A mildly affected Maroteaux-Lamy patient is described. Electrophoretic separation of acid mucopolysaccharides (MPS) in the urine showed an increased excretion of dermatan sulphate in spite of a normal total excretion of MPS. (+info)
Purification and some properties of arylsulphatases A and B from rabbit kidney cortex.
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Arylsulphatases A and B (EC 3.1.6.1) of rabbit kidney cortex were purified 5250- and 7720-fold respectively by a multiple-column-chromatography method. The specific activity toward 4-nitrocatechol sulphate was 42mumol/min per mg for arylsulphatase A and 62 mumol/min per mg for arylsulphatase B. Each enzyme migrated as a single band on polyacrylamide-gel electrophoresis, and the enzyme activity corresponded to the band of protein on the gel. The rate of hydrolysis of ascorbic acid 2-sulphate by arylsulphatase A was three times that for cerebroside 3-sulphate. Arylsulphatase B hydrolysed UDP-N--acetylgalactosamine 4-sulphate and glucosamine 4,6-disulphate, but not galactosamine 6-sulphate. (+info)
Analysis of N-acetylgalactosamine-4-sulfatase protein and kinetics in mucopolysaccharidosis type VI patients.
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A sensitive and specific, monoclonal antibody-based immunoquantification assay has facilitated determination of the N-acetylgalactosamine-4-sulfatase (4-sulfatase) protein content in cultured fibroblasts from normal controls and mucopolysaccharidosis type VI (MPS VI) patients. The assay enabled the quantification of 4-sulfatase protein by using a panel of seven monoclonal antibodies and has shown that fibroblasts from 16 MPS VI patients contained less than or equal to 5% of the level determined for normal controls. Fibroblasts from the most severely affected patients contained the lowest levels of 4-sulfatase protein, usually with few epitopes detected, while fibroblasts from mildly affected patients had higher levels of 4-sulfatase protein, with all seven epitopes detected. The pattern of epitope expression is proposed to reflect the conformational changes in the 4-sulfatase protein that arise from different mutations in the 4-sulfatase gene. Immunoquantification in combination with a specific and highly sensitive 4-sulfated trisaccharide-based assay of enzyme activity in these MPS VI patient fibroblasts enabled the determination of residual 4-sulfatase catalytic efficiency (kcat/Km). The capacity of fibroblasts to degrade substrate (catalytic capacity) was calculated as the product of 4-sulfatase catalytic efficiency and the content of 4-sulfatase in fibroblasts. One patient, 2357, with no clinical signs of MPS VI but with reduced 4-sulfatase activity and protein (both 5% of normal) and dermatansulfaturia, had 5% of normal catalytic capacity. The other 15 MPS VI patient fibroblasts had 0%-1.4% of the catalytic capacity of fibroblasts from normal controls and were representative of the spectrum of MPS VI clinical phenotypes, from severe to mild.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Restoration of arylsulphatase B activity in human mucopolysaccharidosis-type-VI fibroblasts by retroviral-vector-mediated gene transfer.
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The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI; MPS VI) is a lysosomal storage disease caused by deficiency of the enzyme arylsulphatase B (ASB). A human ASB cDNA has been subcloned into the retroviral vector pXT1 containing the bacterial neomycin-resistance gene and an internal thymidine kinase promoter for transcription of the inserted gene. Replication defective retrovirus was generated by transfecting the construct into the amphotropic packaging cell line PA317. Human MPS VI fibroblasts infected with recombinant retrovirus integrated the provirus into their genome and expressed retrovirus-encoded ASB mRNAs. In infected fibroblasts the level of ASB was up to 36-fold higher than in normal fibroblasts. Biosynthesis and processing of ASB in infected MPS VI fibroblasts was accomplished as in normal fibroblasts, and mature, enzymically active, ASB accumulated in dense lysosomes, indicating that the ASB deficiency in MPS VI fibroblasts was corrected by the retroviral gene transfer. (+info)
Glycosaminoglycan and collagen metabolism in arylsulfatase B-deficient retinal pigment epithelium in vitro.
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Regional differences in retinal pigment epithelial (RPE) cell glycosaminoglycan (GAG) and collagen metabolism were studied using cells obtained from normal cats and those with deficient activity of arylsulfatase B (ASB), a lysosomal enzyme involved in GAG catabolism. Control and ASB-deficient RPE cultures initiated from superior equatorial (superior) and inferior equatorial (inferior) regions of the eye were radiolabeled for 72 hr with 35SO4, and GAGs from the media and cell layers were analyzed separately. In ASB-deficient RPE, there was an accumulation of dermatan/chondroitin sulfate in the cell layer of cultures initiated from the superior region of the eye but not in those initiated from the inferior region. This agrees with previous in situ and in vitro morphologic observations that accumulation of inclusions in ASB-deficient RPE was greater in the superior region of the eye than in the inferior region. By contrast, media from ASB-deficient cultures initiated from the inferior region of the eye contained much higher levels of radiolabeled dermatan/chondroitin sulfate than ASB-deficient cultures from the superior region or normal cultures. Increased GAG content in the media may result from increased secretion of proteoglycans, increased turnover of cell surface or extracellular matrix components, or extrusion of lysosomal contents. These results indicate that one or more of these mechanisms vary regionally throughout the eye in the RPE of ASB-deficient animals. Collagen production was determined in normal and ASB-deficient RPE cultures. In normal RPE, no differences in collagen synthesis were noted between the inferior and superior regions.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Phylogenetic conservation of arylsulfatases. cDNA cloning and expression of human arylsulfatase B.
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A 2.2-kilobase cDNA clone for human arylsulfatase B (ASB) and several genomic clones were isolated and sequenced. The deduced amino acid sequence of 533 amino acids contains a 41-amino acid N-terminal signal peptide and a mature polypeptide of 492 amino acid residues. Overexpression of ASB in transfected baby hamster kidney (BHK) cells resulted in up to 68-fold higher ASB activity than in untransfected BHK cells. Pulse-chase labeling showed that ASB was synthesized and secreted as a 64-kDa precursor and processed to a 47-kDa mature form in BHK cells. The 47-kDa ASB form was located in dense lysosomes. Transport of ASB to the lysosomes was accomplished in a mannose 6-phosphate receptor-dependent manner. The ASB cDNA clone hybridizes to 4.8-, 2.5-, and 1.8-kilobase species of RNA from human fibroblasts. The same pattern was observed in RNA from fibroblasts of three Maroteaux-Lamy patients who were deficient in ASB activity, as well as in RNA from fibroblasts of three patients with multiple sulfatase deficiency, in which all known sulfatases were markedly diminished. Deduced amino acid sequences of human arylsulfatase A, human ASB, human steroid sulfatase, human glucosamine-6-sulfatase, and an arylsulfatase from sea urchin showed a substantial degree of similarity suggesting that they arose from a common ancestral gene and are members of an arylsulfatase gene family. (+info)
Phosphorylation of human lysosomal arylsulfatase B by cAMP-dependent protein kinase. Different sites of phosphorylation between normal and cancer tissues.
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We previously demonstrated that an acidic variant (B1) of lysosomal arylsulfatase B from transplanted human lung cancer is phosphorylated on its protein and carbohydrate moieties (Gasa, S., and Makita, A. (1983) J. Biol. Chem. 258, 5034-5039). The present study identifies that a cAMP-dependent protein kinase is responsible for phosphorylation of arylsulfatase B. The protein kinase activity toward the sulfatase was considerably higher in the transplanted lung cancer than in normal lung in the presence of cAMP. B enzyme purified from normal human liver was found to contain 0.6 mol/mol B enzyme, and protein kinase treatment added further 1.3 mol of Pi to give a single phosphopeptide (X). On the other hand, B1 enzyme purified from the transplanted human lung cancer which had been labeled in vivo with 32Pi revealed at least two phosphopeptides (X and Y). Assuming that the sulfatase from normal liver and lung cancer possesses the same number of available phosphorylation sites, phosphorylation of site X which was available only by deliberate phosphorylation of the native, ordinary B enzyme appears to be cancer-associated. Increasing phosphorylation of the sulfatase resulted in a maximum 50% elevation in arylsulfatase activity, followed by a decrease of the activity upon overphosphorylation, using an artificial substrate. (+info)