Structure of a human lysosomal sulfatase.

BACKGROUND: . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase. RESULTS: . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme. CONCLUSIONS: . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.

Inhibition of arylsulfatase B by ascorbic acid.

One hypothesis for atherosclerotic lesion formation is the response to injury hypothesis. If catabolism of the ground substance of the intima of blood vessels is viewed as an injury, then inhibition of this catabolism could lead to a decrease in atherosclerotic lesions. Arylsulfatase B catalyses the desulfation of glycosaminoglycans in the catabolism of the intimal ground substance. We have found that ascorbic acid inhibits arylsulfatase B (Km = 2.06 mM, KI = 4.89 mM), thus providing a possible mechanism by which ascorbic acid may decrease atherosclerosis.

Inability of thiol compounds to restore CNS arylsulfatases inhibited by methyl mercury.

Arylsulfatase A and B activities have been analysed in different areas of the CNS (olfactory bulbs, cerebral hemispheres, cerebellum, medulla oblongata and spinal cord) during methyl mercuric chloride (MMC), N-acetyl-DL-homocysteine thiolactone (NAHT) and glutathione (GSH) treatment. Male albino rats were administered with two different doses of MMC (1 mg/kg and 10 mg/kg), NAHT (40 mg/kg and 80 mg/kg) and GSH (100 mg/kg and 150 mg/kg) for 2, 7 and 15 days, and sacrificed on the 3rd, 8th and 16th day, respectively. Likewise, eight groups of animals, after treatment for 7 days with MMC, were kept for another 7 days and sacrificed on the 15th day (normal withdrawal). Sixteen groups of 7 days MMC pretreated animals were given either NAHT or GSH for another seven days before sacrifice on the 15th day. The study revealed 1) a dose and duration dependent inhibition of the enzymes in all CNS areas, 2) a maximum inhibition of the enzymes among various hydrolases reported so far, and 3) none of the antagonists restored the enzymes significantly except for one group with NAHT. It is, therefore, concluded that arylsulfatases are more sensitive to MMC than other lysosomal enzymes, and the antagonists used here have not helped their restoration.

Comparative biochemistry of mammalian arylsulfatases A and B.

1. Arylsulfatases A and B occurred as a major anionic and cationic isozyme, respectively, among eleven eutherian mammalian species. 2. Minor anionic arylsulfatase B isozymes were observed in rodents, dog, whale and pig, and were either monomeric (vole, Mr = 67 +/- 2 kDa), an apparent aggregate (dog, whale, pig; Mr = 192 +/- 10 kDa), or both (rat, mouse; monomeric Mr = 57 +/- 2 kDa; apparent dimeric Mr = 114 +/- 3 kDa). 3. Minor cationic arylsulfatase A isozymes were isolated from the deer, whale and pig. 4. Opossum arylsulfatases A and B were both anionic, had similar relative molecular weights, were not inhibited by silver, and were not precipitated by anti-murine arylsulfatase B nor anti-bovine arylsulfatase A IgG preparations.

Inhibition of homogeneous human eosinophil arylsulfatase B by sulfidopeptide leukotrienes.

Arylsulfatase B, purified to homogeneity from human eosinophils, is a tetrameric enzyme whose activity varied in accordance with the state of association of its monomeric subunits. The rate of dissociation of oligomeric forms was slow relative to the rate of the enzymatic reaction so that the kinetic properties of the enzyme depended on the concentration of the enzyme before assay. For concentrated enzyme solutions (14 micrograms/ml), Lineweaver-Burk analysis demonstrated substrate inhibition at greater than or equal to 20 mM substrate and revealed two distinct regions of activity at low and intermediate substrate concentrations. The addition of bovine serum albumin (60 micrograms/ml) or sucrose (0.25 M), which prevent subunit dissociation, yielded a linear relationship on Lineweaver-Burk analysis at non-inhibitory substrate concentrations. For dilute enzyme concentrations (4.7 micrograms/ml), inhibition occurred at greater than or equal to 2 mM substrate. Nanomolar amounts of leukotriene C4 (LTC4), relative to millimolar concentrations of substrate, inhibited eosinophil arylsulfatase B. On Lineweaver-Burk analysis, the pattern of inhibition of LTC4 with concentrated enzyme was compatible with competitive inhibition of only one oligomeric form of the enzyme, whereas at low enzyme concentrations the pattern of inhibition was apparently competitive. These findings suggest that LTC4 is a potent competitive inhibitor of a dissociated, possibly dimeric, form of the enzyme. Nanomolar concentrations of LTC4, leukotriene D4, and leukotriene E4 were equally inhibitory, whereas leukotriene B4 and isomeric 5,12-dihydroxyeicosatetraenoic acids had no inhibitory activity, indicating a requirement for a thiopeptide at C-6. Thiopeptide leukotriene analogs without an intact triene structure also lacked inhibitory activity. Sulfoxide analogs of LTC4 and leukotriene D4 were potent inhibitors, although two sulfone analogs of leukotriene D4 were not inhibitory. Arylsulfatase B did not inactivate the spasmogenic activity of sulfidopeptide leukotrienes. These findings indicate that sulfidopeptide leukotrienes and their sulfoxide derivatives may regulate by competitive inhibition the activity of oligomeric forms of the eosinophil lysosomal hydrolase, arylsulfatase B.

Inhibition of rabbit liver arylsulfatase B by phosphate esters.

Arylsulfatase B (aryl-sulfate sulfohydrolase, EC 3.1.6.1) purified from rabbit liver is competitively inhibited at modest concentrations by a variety of phosphate esters derived from amino acids, amines and simple sugars. Phospho-L-serine coupled to Sepharose 4B could be used as an affinity column to enhance the purity of a crude preparation of the enzyme. It is suggested that phosphate esters containing functional groups can be used to obtain affinity reagents to purify arylsulfatases and also to probe their active sites.

Purification and properties of arylsulphatase B of human liver.

1. A purification scheme for an arylsulphatase B from human liver is described. Specificity of purification was achieved by the use of the affinity chromatography on an agrose-4-hydroxy-2-nitrophenyl sulphate derivative. The scheme provides a rapid and convenient method for preparation of a highly purified enzyme. 2. The purified enzyme was examined by isoelectric focusing electrophoresis on polyacrylamide gel and by ultracentrifugation and was found to be catalytically homogenous, with an apparent molecular weight of 50000 and a specific activity of 93.3 units/mg of protein. 3. The kinetic properties of the purified preparation and the effect of various amino acid group-specific reagents on the catalysis of the enzyme are described. The involvement of histidine residues in the active site of the enzyme is suggested. 4. The purified enzyme lost activity rapidly on freezing. The implication of this observation is discussed in terms of a possible dissociation-reaggregation phenomenon induced by cold treatment.