TY  - THES
AB  - Sulfatases belong to an enzyme class involved in the hydrolytic cleavage of sulfate esters in steroid hormones, glycolipids, and above all in the degradation of glycosaminoglycans (GAGs). Thus, sulfatases play an essential physiological role in the degradation of macromolecules, signal transduction and hormone regulation. The loss of lysosomal sulfatase activity is associated with the accumulation of sulfated storage material, which leads to the manifestation of a progressive and often fatal lysosomal storage disease.<br />
Human arylsulfatase K (ARSK) was identified 2002 in a genome-wide screen searching for the highly conserved sulfatase signature sequence CXPSR. Based on its lysosomal sorting signal mannose-6-phosphate, the acidic pH-optimum as well as co-localization studies, the human ARSK was classified as a member of the lysosomal sulfatases. It was not until 2017 that the ARSK was assigned a glucuronate-2-O-sulfatase activity by demonstrating the turnover of synthetic 2-O-sulfated substrates and thus an involvement in lysosomal heparan sulfate (HS) and chondroitin sulfate (CS) degradation was suggested.<br />
In the present study, the physiological significance in the HS and CS catabolism of Arsk was investigated by the means of an Arsk-deficient mouse model. A variety of histological, biochemical and mass spectrometric analyzes were performed on knockout mouse-derived material. The validation of Arsk deficiency was performed on mRNA level as well as by the expression of the knockout lacZ-reporter cassette. At the cellular level, the knockout mouse displayed a very mild phenotype. Histological analyzes revealed moderately enriched electron-dense material in the Henle's loop of the kidney, which did not match the typically expected storage pathology observed in many other mucopolysaccharidoses. Furthermore, no striking influence on the regulation of the endo-lysosomal system of Arsk-deficient animals was observed. Neurologically, the knockout mouse is largely unremarkable and did not exhibit detectable degenerative or inflammatory processes. However, in behavioral tests on twelve-month-old knockout mice, reduced anxiety and delayed socializing were observed. Although an altered osteogenesis with an increased bone mineralization and a reduced number of osteoclasts was found experimentally, these results were not statistically significant due to large variances. Bone mineral density, however, was significantly decreased in 26 and 52 week old Arsk-deficient mice.<br />
Glucuronate-2-O-sulfatase activity of Arsk is critical for the degradation of glycosaminoglycans. With a DMMB-based assay for determining the amount of GAGs, increased GAG storage in mouse kidney and an increased urinary GAG excretion in the Arsk-deficient mice were diagnosed, which correspond to the characteristics of many mucopolysaccharidoses. The previously postulated ARSK-specific 2-O-sulfoglucuronate substrate was confirmed after GAG isolation from mouse tissue by GRIL-LC/MS analyzes and the digestion of isolated 2-O-sulfate-containing storage material by human recombinant ARSK. This result verifies the glucuronate-2-sulfatase activity of ARSK and the involvement in HS and CS/DS degradation.<br />
Finally an in vitro enzyme assay based on 2-aminoacridone-labeled disaccharides was established confirming not only the 2-O-glucuronate sulfate substrate but also the loss of glucuronate-2-O-sulfatase activity in Arsk-deficient mice. This assay demonstrated the sequential degradation of GAG-derived sulfated substrates by digestion with enriched lysosomal fractions (tritosomes) from wild-type mice. Tritosomes derived from Arsk-deficient mice were missing an GDS-activity and thus no substrate digestion was detectable.<br />
The Arsk-deficient mouse model represents an essential contribution to the understanding of the GAG degradation process in general. In addition, the phenotypic and molecular findings should help to screen patients with lysosomal storage and unclear molecular etiology for ARSK-deficiency.
DA  - 2018
LA  - ger
PY  - 2018
TI  - Funktionelle Studien zur Arylsulfatase K (Arsk) anhand eines Arsk-defizienten Mausmodells
UR  - https://nbn-resolving.org/urn:nbn:de:0070-pub-29214084
Y2  - 2024-11-25T00:38:45
ER  -