Phosphorylation on protein and carbohydrate moieties of a lysosomal arylsulfatase B variant in human lung cancer transplanted into athymic mice.
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Human lung cancer transplanted into athymic mice contains predominantly an acidic variant (designated B1) of lysosomal arylsulfatase B. B1 enzyme was suggested to be phosphorylated and sialylated (Gasa, S., Makita, A., Kameya, T., Kodama, T., Koide, T., Tsumuraya, M., and Komai, T. (1981) Eur. J. Biochem. 116, 497-503). In order to determine the localization of phosphate in B1 enzyme, we labeled in vivo the transplanted tumor with [32P]H3PO4 or [3H]glucosamine and purified B1 enzyme by immunoprecipitation. Bio-Gel chromatography of the labeled B1 enzyme treated with endoglycosidase H demonstrated that both the excluded and included materials were labeled with 32P and 3H. From acid hydrolysate of the excluded materials, phosphorylated serine and threonine were detected. Protein phosphorylation of arylsulfatase was confirmed by in vitro labeling experiments with [gamma-32P]ATP. By incubation of the tumor homogenate with ATP followed by isolation of the enzymes, B1 enzyme had a significant amount of radioactivity, whereas the B enzyme had little; by exogenous protein kinase, partially purified B enzyme was phosphorylated 35 times more than B1 enzyme. Acid hydrolysate of the included materials in the Bio-Gel column demonstrated mannose 6-phosphate and an unknown phosphorylated compound which migrates more than Man-6-P on electrophoresis and chromatography. (+info)
Biosynthesis and maturation of arylsulfatase B in normal and mutant cultured human fibroblasts.
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The biosynthesis of arylsulfatase B in normal and mutant human skin fibroblasts was studied by metabolic labeling with radioactive amino acids, monosaccharides, or 32Pi and by specific immunoprecipitation followed by polyacrylamide gel electrophoresis and fluorography. Three major polypeptides with apparent molecular weights of 47,000, 40,000, and 31,000 were found intracellularly and one of 64,000 in the medium. Pulse-chase labeling and uptake experiments showed that arylsulfatase B synthesized and secreted as a 64,000 precursor was intracellularly processed within less than 24 h via short lived intermediates to two different forms. Form I (chains of 47,000 and 11,500) was labeled earlier and was about twice as stable as form II (chains of 40,000 and 31,000). The secreted 64,000 precursor and the 40,000 chain of form II contained oligosaccharides resistant to endo-beta-N-acetylglucosaminidase H. In the other chains mainly cleavable and phosphorylated oligosaccharides were found. Arylsulfatase B activity was associated with the 64,000 precursor and with form I, but not with form II. Fibroblasts of four patients with the severe form of mucopolysaccharidosis type VI, which were deficient in arylsulfatase B activity, synthesized and secreted the 64,000 precursor at a normal rate. This precursor, however, had little if any catalytic activity and one of its mature forms (I) was rapidly degraded. (+info)
A comparative study of brain arylsulfatases B1 and B2: the difference between the two forms in rates of uptake by multiple sulfatase deficient (MSD) disorder fibroblasts.
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Arylsulfatases B1 and B2 isolated from human brain were introduced into neonatal type MSD fibroblast culture. Arylsulfatase B2 was linearly taken up into MSD fibroblasts with respect to time and the dose of enzyme, whereas arylsulfatase B1 enzyme was practically not taken up into cells. Uptake of arylsulfatase B2 was inhibited in the presence of mannose 6-phosphate. These data suggest that the biological functional difference between arylsulfatases B1 and B2 is due to the presence of a phosphorylated residue acting as a recognition marker on one of the enzymes. (+info)
The pathology of the feline model of mucopolysaccharidosis VI.
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Three cats with feline arylsulfatase-B--deficient mucopolysaccharidosis were studied by light and transmission electron microscopy. Membrane-bound cytoplasmic inclusions were present in hepatocytes, bone marrow granulocytes, vascular smooth muscle cells, and fibroblasts in skin, cornea, and cardiac valves. Central nervous system lesions were restricted to mild ventricular dilatation, perithelial cell vacuolation, and, in one animal, cord compression by vertebral exostoses. The lesions in these cats closely resembled those described in human patients with mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). (+info)
Cellular location of enzymes involved in chondroitin sulfate breakdown by Bacteroides thetaiotaomicron.
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Bacteroides thetaiotaomicron, a gram-negative anaerobe found in human colons, could utilize chondroitin sulfate, a tissue mucopolysaccharide, as its sole source of carbohydrate. The enzymes responsible for the breakdown of chondroitin sulfate by B. thetaiotaomicron were similar to those produced by Proteus vulgaris and Flavobacterium heparinum and included a lyase (EC 4.2.2.4), which degraded chondroitin sulfate into sulfated disaccharides, sulfatases (EC 3.1.6.4), which removed the sulfate residues, and a glucuronidase, which broke the unsulfated disaccharides into monosaccharide components. Chondroitin sulfate lyase, the first enzyme in the breakdown sequence, was not extracellular. It appeared to be located in the periplasmic space since lyase activity was released by treatment with ethylenediaminetetraacetate and lysozyme. Moreover, sodium polyanethole sulfonate, a high-molecular-weight inhibitor of chondroitin lyase, did not inhibit breakdown of chondroitin sulfate by intact bacteria. The sulfatase and glucuronidase appeared to be intracellular. None of these enzymes was strongly bound to membranes, and none of the steps in the breakdown of chondroitin sulfate was sensitive to oxygen. (+info)
Feline mucopolysaccharidosis VI: purification and characterization of the resident arylsulfatase B activity.
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Hepatic arylsulfatase B (ASB) from normal and mucopolysaccharidosis VI (MPS VI) cats was purified over 2,800- and 1,800-fold, respectively, and their physical and kinetic properties were characterized. In contrast to the normal feline enzyme, the partially purified MPS VI residual activity had a 100-fold greater Km value and was markedly less stable to thermal, cryo-, and pH-inactivation. In addition, the MPS VI enzyme had a more negative charge as determined by its migration on polyacrylamide gel electrophoresis and its elution profile on cation exchange chromatography. Finally, the MPS VI activity had approximately half the apparent molecular weight of the normal feline enzyme, which was a homodimer, suggesting that the genetic mutation in feline MPS VI altered the subunit association as well as the kinetic and stability properties of the mutant protein. (+info)
Enhancement of residual arylsulfatase B activity in feline mucopolysaccharidosis VI by thiol-induced subunit association.
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The molecular pathology of the deficient arylsulfatase B activity in feline mucopolysaccharidosis (MPS) VI was investigated. Compared with the highly purified normal feline hepatic enzyme, the purified MPS VI residual activity had a 100-fold higher Michaelis constant (K(m)), an altered electrophoretic mobility, half the apparent native molecular weight, and markedly decreased thermo-, cryo-, and pH stabilities. Molecular weight and alkylation studies were consistent with the normal enzyme being a homodimer and the residual MPS VI enzyme a monomer. When incubated with various sulfhydryl reagents, the residual specific activity was enhanced several-fold, whereas the activity of the purified normal enzyme was un-affected or slightly inhibited. In the presence of dithiothreitol (DTT) and cysteamine, a lysosomotropic aminothiol, the residual activity had an electrophoretic mobility and native molecular weight similar to those of the normal feline enzyme. These findings suggested that the monomeric residual enzyme was dimerized in the presence of thiol-reducing agents. To determine if thiol-induced subunit association could therapeutically increase the residual activity and degrade the accumulated dermatan sulfate, in vitro and in vivo experiments were undertaken. When 2 mM DTT or cysteamine was incubated with heparinized whole blood from an MPS VI cat, the leukocyte residual arylsulfatase B activity increased 11- and 20-fold, respectively, and the accumulated dermatan sulfate was degraded in the presence of both thiol reagents. Intravenous administration of DTT (50 mg/kg) effected an immediate, but transient, increase in leukocyte residual activity; however, the substrate levels were not significantly decreased. In contrast, intravenous administration of cysteamine (15 mg/kg) increased leukocyte residual activity more than sixfold 30 min postinfusion; concomitantly, the leukocyte substrate was decreased to 35% of the initial level immediately after infusion and to about 45% of preinfusion values during the 120-min period studied. These results suggest that the defective residual activity in feline MPS VI can be therapeutically manipulated by thiol-induced subunit association. Furthermore, this animal analog provides a prototype for the investigation of human inborn errors of metabolism resulting from enzymatic defects that might be amenable to enzyme manipulation therapy. (+info)
Human eosinophil arylsulfatase B. Structure and activity of the purified tetrameric lysosomal hydrolase.
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Arylsulfatase B from human eosinophils was purified free of contaminating proteins by gel filtration and sequential affinity chromatography on Affi-Gel Blue and zinc chelate Sepharose. 50 micrograms of the purified enzyme presented as a single stained band on alkaline disc gel electrophoresis. In both goats and rabbits, the purified enzyme elicited monospecific antisera that yielded single precipitation arcs on Ouchterlony analysis with a human eosinophil extract and the purified enzyme; the immunoprecipitation lines fused in a pattern of identity, providing immunochemical evidence for the homogeneity of the purified enzyme. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a dominant lower molecular weight protein and three other bands with molecular weights approximately two, three, and four times that of the major protein band were resolved. The prominence of the less rapidly migrating protein bands increased relative to the major band if the enzyme was maintained under acidic conditions or was reacted with the cross-linking agent dimethyl suberimidate under alkaline conditions before SDS-polyacrylamide gel electrophoresis, supporting the conclusion that the enzyme consists of four subunits. Two stained bands were present on acid disc gel electrophoresis; they were composed of oligomeric forms of enzyme on analysis by SDS-polyacrylamide gel electrophoresis in a second dimension. A minimum molecular weight of 70,190 was determined from amino acid composition analysis for the tetrameric form of the enzyme. The specific functional activity of the purified arylsulfatase B was concentration and time dependent, compatible with its association or dissociation into subunit forms with differing specific activities. Factors that govern subunit interactions of arylsulfatase B, including local enzyme concentration and pH, provide mechanisms for regulating the enzymatic activity of this lysosomal hydrolase. (+info)