The human and murine arylsulfatase G - Biological function and deficiency

The mammalian sulfatases are a group of related enzymes, which catalyse the hydrolytic desulfation of steroid hormones, glycosaminoglycans and complex lipids. The biggest subgroup of these enzymes are the lysosomal sulfatases. The deficiency of each of these enzymes subsequently leads to a lysosomal storage disorder due to accumulation of the sulfatase substrates in the endolysosomal system. The physiological substrates of the lysosomal sulfatases with the exception of arylsulfatase A are sulfate groups attached to glycosaminoglycans. The storage of glycosaminoglycans is classified as a mucopolysaccharidosis and, consequently, almost all deficiencies of a lysosomal sulfatase lead to mucopolysaccharidoses. Arylsulfatase G was identified as the seventh lysosomal sulfatase by immunofluorescence studies. Additionally, arylsulfatase G exhibits an acidic pH optimum and the lysosomal sorting signal mannose 6-phosphate. Initial histological analyses of arylsulfatase G-knockout mice revealed slight pathological changes in visceral and central nervous system structures. The physiological substrate of the arylsulfatase G, however, has remained unknown until the present study, although activity against tyrosine sulfate as well as the sulfated thyroid hormone triiodothyronine sulfate was previously reported. In this study, a new arylsulfatase G-specific antibody was evaluated and successfully applied to study the tissue-specific and cell-type specific expression of the enzyme. It was found that arylsulfatase G is ubiquitously expressed in all tested tissues. Furthermore, immunofluorescence studies of mouse brain sections showed that arylsulfatase G expression is limited to particular cell types in the central nervous system. Furthermore, expression as well as subcellular fraction analyses showed that arylsulfatase G was processed from the 63 kDa precursor to several lower molecular weight forms in the lysosomes. This processing was strictly dependent on the lysosomal localisation of the enzyme. Moreover, the lysosomal proteases cathepsin B and cathepsin L were found to be involved in the processing of arylsulfatase G. Further studies led to the assignment of the particular arylsulfatase G subunits to different parts of the polypeptide and a lack of conservation in the amino acid residues present at the putative protease cleavage sites allowed the localisation of these sites. The sorting of arylsulfatase G into the lysosomes was found to be partially independent from the mannose 6-phosphate receptors in fibroblasts, although the sorting of most lysosomal hydrolases is strictly dependent on mannose 6-phosphate-mediated targeting. Moreover, CLN8-deficiency affected the sorting of arylsulfatase G. To elucidate the physiological function of arylsulfatase G, the previously generated arylsulfatase G knockout mouse model was thoroughly examined by histological, biochemical and mass spectrometric methods. The histological analyses showed severe pathological alterations in the cerebellum and also some changes in the visceral tissues of the arylsulfatase G knockout mice. The primary storage material was identified as heparan sulfate with 3-O-sulfate groups at the non-reducing end glucosamine by biochemical and mass spectrometric methods. The digest of a chemical synthesised standard of the non-reducing end glucosamine of the storage material showed arylsulfatase G activity against the 3-O-sulfate group of N-sulfoglucosamine 3-O-sulfate, identifying the sulfatase as the long-sought glucosamine 3-O-sulfatase in the degradation of heparan sulfate. Arylsulfatase G thus represents the fourth sulfatase found to be involved in this catabolic pathway. Furthermore, secondary pathological alterations were identified in the arylsulfatase G knockout mice, which comprise storage of lipid and carbohydrate moieties as well as accumulation of material with autofluorescent properties in various cell types of the central nervous system. The thorough examination of the arylsulfatase G-deficient mice allows an estimation of the symptoms putatively exhibited by human arylsulfatase G-deficient individuals and, hence, simplifies the search for these patients. The identification of the physiological substrate allowed the development of an arylsulfatase G-specific assay based on the physiological monosaccharide standard. The monosaccharide was labelled with 2-aminoacridone or 2-aminobenzoic acid and analysed by C18 reversed phase high-performance liquid chromatography. The assay revealed a specific activity of arylsulfatase G, which is comparable to activities found for arylsulfatase A and arylsulfatase B towards their corresponding physiological substrates. Furthermore, the substrate specificities of the heparan sulfate-degrading sulfatase glucosamine 6-sulfatase and sulfamidase as well as arylsulfatase G were examined using the established assay. It was found that arylsulfatase G-mediated 3-O-desulfation is the first step in the lysosomal degradation of a trissulfated glucosamine residues at the non-reducing end of a heparan sulfate chain. The other two sulfatases are acting in parallel, but only after the initial arylsulfatase G-mediated removal of the 3-O-sulfate. These results complete the understanding of the heparan sulfate degradation pathway elucidating enzyme actions at the glucosamine residue and demonstrating the influence of the rare 3-O-sulfate group on the heparan sulfate-degrading sulfatases.