The human and murine arylsulfatase G - Biological function and deficiency

Kowalewski B. The human and murine arylsulfatase G - Biological function and deficiency. Bielefeld: Universität Bielefeld; 2017.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

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG)

Kowalewski B, Lange H, Galle S, Dierks T, Lübke T, Damme M. Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG). Biochemical Journal. 2021.The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wildtype and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate

Nature's Polyoxometalate Chemistry: X-ray Structure of the Mo Storage Protein Loaded with Discrete Polynuclear Mo-O Clusters

Kowalewski B, Poppe J, Demmer U, et al. Nature's Polyoxometalate Chemistry: X-ray Structure of the Mo Storage Protein Loaded with Discrete Polynuclear Mo-O Clusters. Journal of the American Chemical Society. 2012;134(23):9768-9774.Some N-2-fixing bacteria prolong the functionality of nitrogenase in molybdenum starvation by a special Mo storage protein (MoSto) that can store more than 100 Mo atoms. The presented 1.6 angstrom X-ray structure of MoSto from Azotobacter vinelandii reveals various discrete polyoxomolybdate clusters, three covalently and three noncovalently bound Mo-8, three Mo5-7, and one Mo-3 clusters, and several low occupied, so far undefinable clusters, which are embedded in specific pockets inside a locked cage-shaped (alpha beta)(3) protein complex. The structurally identical Mo-8 clusters (three layers of two, four, and two MoOn octahedra) are distinguishable from the [Mo8O26](4-) cluster formed in acidic solutions by two displaced MoOn octahedra implicating three kinetically labile terminal ligands. Stabilization in the covalent Mo-8 cluster is achieved by Mo bonding to His alpha 156-N-epsilon 2 and Glu alpha 129-O-epsilon 1. The absence of covalent protein interactions in the noncovalent Mo-8 cluster is compensated by a more extended hydrogen-bond network involving three pronounced histidines. One displaced MoOn octahedron might serve as nucleation site for an inhomogeneous Mo5-7 cluster largely surrounded by bulk solvent. In the Mo-3 cluster located on the 3-fold axis, the three accurately positioned His140-N-epsilon 2 atoms of the alpha subunits coordinate to the Mo atoms. The formed polyoxomolybdate clusters of MoSto, not detectable in bulk solvent, are the result of an interplay between self- and protein-driven assembly processes that unite inorganic supramolecular and protein chemistry in a host-guest system. Template, nucleation/protection, and catalyst functions of the polypeptide as well as perspectives for designing new clusters are discussed

Molecular Characterization of Arylsulfatase G: EXPRESSION, PROCESSING, GLYCOSYLATION, TRANSPORT, AND ACTIVITY

Kowalewski B, Lübke T, Kollmann K, et al. Molecular Characterization of Arylsulfatase G: EXPRESSION, PROCESSING, GLYCOSYLATION, TRANSPORT, AND ACTIVITY. The Journal of biological chemistry. 2014;289(40):27992-28005.Arylsulfatase G (ARSG) is a recently identified lysosomal sulfatase that was shown to be responsible for the degradation of 3-O-sulfated N-sulfoglucosamine residues of heparan sulfate glycosaminoglycans. Deficiency of ARSG leads to a new type of mucopolysaccharidosis, as described in a mouse model. Here, we provide a detailed molecular characterization of the endogenous murine enzyme. ARSG is expressed and proteolytically processed in a tissue-specific manner. The 63-kDa single-chain precursor protein localizes to pre-lysosomal compartments and tightly associates with organelle membranes, most likely the endoplasmic reticulum. In contrast, proteolytically processed ARSG fragments of 34-, 18-, and 10-kDa were found in lysosomal fractions and lost their membrane association. The processing sites and a disulfide bridge between the 18- and 10-kDa chains could be roughly mapped. Proteases participating in the processing were identified as cathepsins B and L. Proteolytic processing is dispensable for hydrolytic sulfatase activity in vitro. Lysosomal transport of ARSG in the liver is independent of mannose 6-phosphate, sortilin, and Limp2. However, mutation of glycosylation site N-497 abrogates transport of ARSG to lysosomes in human fibrosarcoma cells, due to impaired mannose 6-phosphate modification

Structural diversity of polyoxomolybdate clusters along the three-fold axis of the molybdenum storage protein

Poppe J, Warkentin E, Demmer U, et al. Structural diversity of polyoxomolybdate clusters along the three-fold axis of the molybdenum storage protein. Journal of Inorganic Biochemistry. 2014;138:122-128.The molybdenum storage protein (MoSto) can store more than 100 Mo or W atoms as discrete polyoxometalate (POM) clusters. Here, we describe the three POM cluster sites along the threefold axis of the protein complex based on four X-ray structures with slightly different polyoxomolybdate compositions between 1.35 and 2 angstrom resolution. In contrast to the Mo alpha-out binding site occupied by an Mo-3 cluster, the Mo alpha-in and Mo-beta binding sites contain rather weak and non-uniform electron density for the Mo atoms (but clearly identifiable by anomalous data), suggesting the presence of POM cluster ensembles and/or degradation products of larger aggregates. The "Mo alpha-in cluster ensemble" was interpreted as an antiprism-like Mo-6 species superimposed with an Mo-7 pyramide and the "Mo-beta cluster ensemble" as an Mo-13 cluster (present mostly ins degraded form) composed of a pyramidal Mo-7 and a Mo-3 building block linked by three spatially separated MoOx units. Inside the ball-shaped Mo-13 cluster sits an occluded central atom, perhaps a metal ion. POM cluster formation at the Mo alpha-in, and Mo-beta sites appears to be driven by filtering out and binding/protecting self-assembled transient species complementary to the protein template. (C) 2014 Elsevier Inc. All rights reserved

Ataxia is the major neuropathological finding in Arylsulfatase G deficient mice: Similarities and dissimilarities to Sanfilippo disease (Mucopolysaccharidosis type III)

Kowalewski B, Heimann P, Ortkras T, et al. Ataxia is the major neuropathological finding in Arylsulfatase G deficient mice: Similarities and dissimilarities to Sanfilippo disease (Mucopolysaccharidosis type III). Human Molecular Genetics. 2015;24(7):1856-1868.: Deficiency of Arylsulfatase G (ARSG) leads to a lysosomal storage disease in mice resembling biochemical and pathological features of the mucopolysaccharidoses and particularly features of mucopolysaccharidosis type III (Sanfilippo syndrome). Here we show that Arsg KO mice share common neuropathological findings with other Sanfilippo syndrome models and patients, but can be clearly distinguished by the limitation of most phenotypic alterations to the cerebellum, presenting with ataxia as the major neurological finding. We determined in detail the expression of ARSG in the central nervous system and observed highest expression in perivascular macrophages (which are characterized by abundant vacuolization in Arsg KO mice) and oligodendrocytes. To gain insight into possible mechanisms leading to ataxia, the pathology in older adult mice (> 12 months) was investigated in detail. This study revealed massive loss of Purkinje cells and gliosis in the cerebellum, and secondary accumulation of glycolipids like GM2 and GM3 gangliosides and unesterified cholesterol in surviving Purkinje cells, as well as neurons of some other brain regions. The abundant presence of ubiquitin and p62-positive aggregates in degenerating Purkinje cells coupled with the absence of significant defects in macroautophagy is consistent with lysosomal membrane permeabilization playing a role in the pathogenesis of Arsg deficient mice and presumably Sanfilippo disease in general. Our data delineating the phenotype of mucopolysaccharidosis IIIE in a mouse KO model should help in the identification of possible human cases of this disease

Tyrosine hydroxylase (TH) immunoreactive axons are more abundant 9 weeks after enzyme treatment than in control mice.

<p>One µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal of this site in mice with severe compression injury. After 9 weeks, the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence. Double immunostaining for TH and neurofilament-M (NF-M) shows higher immunoreactivities caudal to the injury site in the ChaseABC (<b>D,E,F</b>) and ARSB (<b>G,H,I</b>) treated mice versus the buffer treated control mice (<b>A,B,C</b>). (<b>A,D,G</b>) Immunostainings for TH and (<b>B,E,H</b>) NF-M, and (<b>C,F,I</b>) merged for TH with NF-M. Immunoreactive areas were quantified above threshold using Image J software (<b>J</b>). Mean fluorescence intensity of the area between the injury site and 1 mm caudal to it shows significantly higher immunofluorescence intensity in ARSB versus buffer treated control and ChaseABC treated mice. Asterisks indicate significant differences between the groups *p<0.05 and **p<0.01 as assessed by one-way ANOVA followed by Tukey’s <i>post-hoc</i> analysis. Data represent means ± SEM, (n = 4 mice).</p

Thermostability of ARSB and ChaseABC.

<p>Stock solutions of each enzyme were incubated at 37°C/pH 6.8 for up to 125 hours. At the indicated time points aliquots were diluted into pre-warmed assay buffer (pH 8.0 for ChaseABC, pH 5.6 for ARSB) and subjected to activity determination (see Methods). Asterisks indicate significant differences between the groups *p<0.001 as assessed by t-Test. Data represent means ± SEM (n = 4).</p

C4S and C6S immunoreactivities are reduced at 6 and 9 weeks after ARSB and ChaseABC treatment.

<p>Immediately after moderate (<b>A,B</b>) and severe (<b>C–E</b>) spinal cord injury, one µl ChaseABC (10 U/ml), ARSB (10 U/ml) or buffer was injected at the injury site and 0.5 mm rostral and caudal to this site. After 6 (<b>A,B</b>) and 9 weeks (<b>C–E</b>), the mice were perfused, and sagittal spinal cord sections were analyzed by immunofluorescence using the CS56 antibody reacting with C4S and C6S. Immunoreactivity is more intense at the injury site in the buffer treated control mice (<b>A,C</b>) versus the ChaseABC (<b>D</b>) and ARSB (<b>B,E</b>) treated mice. Immunoreactivities of the entire images were quantified above threshold using Image J software (<b>F</b>). Mean fluorescence intensities of the area at 0.4 mm equidistant rostral and caudal to the center of the injury site show significantly less expression of CSs in ChaseABC and ARSB treated mice versus buffer treated control mice. Reduction of immunoreactivity is not significantly different between applications of ChaseABC versus ARSB. Arrows indicate the injury site. Asterisks indicate significant differences between the groups *p<0.05 (ARSB) and **p<0.01 (ChaseABC) as assessed by one-way ANOVA followed by Tukey’s <i>post-hoc</i> analysis. Data represent means ± SEM, (n = 3 mice).</p