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

在人类中，硫酸酯酶组成了一个由17种酶组成的家族，这些酶负责水解各种底物如糖胺聚糖（GAG）中的硫酸酯。由于每种硫酸酯酶对其生理底物显示出严格的特异性，因此携带复杂硫酸化模式的GAG如硫酸乙酰肝素的溶酶体降解需要几种硫酸酯酶的活性-包括尚未表征的硫酸酯酶。因此，参与GAG连续降解途径的任何酶的缺乏导致称为粘多糖贮积症的溶酶体贮积病的一个亚类，其特征在于GAG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酯酶K（ARSK）的溶酶体定位，并证明了重组人ARSK对芳基硫酸酯作为假底物的抑制活性。在这个提议中，我们的目标是通过研究Arsk缺陷的组成型基因敲除小鼠模型来鉴定ARSK的生理底物。因此，我们已经产生了这样的小鼠模型，我们计划表征该模型的溶酶体贮积病的表现。我们将分析小鼠模型的行为，组织学，细胞和生化变化。作为本项目的一个重点，我们将分离溶酶体储存物质，并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，我们将在体外使用来自过表达CHO或昆虫细胞的重组ARSK来纯化分离的底物。

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.