Role of Arylsulfatase K (ARSK) in lysosomal function in health and disease

在人类中，硫酸酯酶由17种酶组成，负责水解各种底物中的硫酸酯，如糖胺聚糖（GAGs）。由于每种磺化酶对其生理底物都表现出严格的特异性，因此像硫酸肝素这样携带复杂磺化模式的GAGs的溶酶体降解需要几种磺化酶的活性，包括尚未表征的磺化酶。因此，任何参与GAGs序列降解途径的酶的缺乏都会导致溶酶体储存疾病的一个亚类，称为粘多糖病，其特征是GAGs作为储存物质的积累。最近，我们已经验证了新发现的芳基硫酸盐酶K （ARSK）的溶酶体定位，并证明了重组人ARSK对作为假底物的芳基硫酸盐的脱硫活性。在本研究中，我们的目标是通过研究ARSK缺失的组成敲除小鼠模型来确定ARSK的生理基础。因此，我们已经建立了这样一个小鼠模型，我们计划在溶酶体贮积病的表现方面对这个模型进行表征。我们将分析小鼠模型的行为、组织学、细胞和生化变化。作为本项目的重点，我们将分离溶酶体储存物质，确定其性质和分子结构，以确定ARSK的生理底物。作为最后的证明，我们将使用来自过表达CHO或昆虫细胞的重组ARSK在体外脱硫分离的底物。

In human, sulfatases constitute a family of 17 enzymes that are responsible for the hydrolysis of sulfate esters in a variety of substrates like glycosaminoglycans (GAGs). As each sulfatase shows a strict specificity towards its physiological substrate, the lysosomal degradation of GAGs like heparan sulfate, carrying complex sulfation patterns, needs the activity of several sulfatases - including as yet uncharacterized sulfatases. As a consequence, deficiency of any enzyme involved in the sequential degradation pathway of GAGs results in a subclass of lysosomal storage diseases called mucopolysaccharidoses which is characterized by the accumulation of GAGs as storage material. Recently, we have validated the lysosomal localization of the newly discovered arylsulfatase K (ARSK) and demonstrated desulfation activity of recombinant human ARSK towards arylsulfates serving as pseudosubstrates. In this proposal we aim to identify the physiological substrate of ARSK by studying a constitutive knock out mouse model for Arsk-deficiency. Therefore, we have already generated such a mouse model and we plan to characterize this model regarding the manifestation of a lysosomal storage disease. We will analyze the mouse model regarding behavioral, histological, cellular and biochemical alterations. As a focus of this project, we will isolate the lysosomal storage material and determine its nature and molecular structure in order to identify the physiological substrate of ARSK. As a final proof, we will desulfate the isolated substrate in vitro using recombinant ARSK derived from overexpressing CHO or insect cells.