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Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG)
Biochemical Journal ( IF 4.4 ) Pub Date : 2021-09-17 , DOI: 10.1042/bcj20210415
Björn Kowalewski 1 , Heike Lange 1 , Sabrina Galle 1 , Thomas Dierks 1 , Torben Lübke 2 , Markus Damme 3
Affiliation  

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 wild-type 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.

中文翻译:

用芳基硫酸酯酶 G (ARSG) 解码含有硫酸乙酰肝素的 3-O-硫酸盐的连续溶酶体降解

硫酸乙酰肝素的溶酶体降解由九种不同酶的协同作用介导。在此降解途径中,芳基硫酸酯酶 G (ARSG) 对从氨基葡萄糖中去除 3-O-硫酸盐至关重要,而 ARSG 中的突变是导致 IV 型亚瑟综合征的原因。我们使用硫酸化单糖底物开发了一种特定的 ARSG 酶测定,这反映了其天然底物的衍生物。这些硫酸化化合物与 ARSG 一起温育,在分别化学添加荧光染料 2-氨基吖啶酮或 2-氨基苯甲酸后,通过反相 HPLC 分析所得产物。我们应用该测定进一步表征 ARSG 对含有额外硫酸基团和 N-乙酰化的 3-O-硫酸化单糖的水解特异性。重组 ARSG 和过表达 ARSG 的细胞以及来自野生型和 Arsg 敲除小鼠的分离溶酶体的应用验证了我们测定的效用。我们进一步利用该测定来确定参与硫酸乙酰肝素的 3-O-硫酸化葡糖胺残基溶酶体分解代谢的不同硫酸酯酶的顺序作用。我们的结果证实并扩展了 ARSG 底物特异性的表征,并有助于确定 (3-O-) 硫酸化硫酸乙酰肝素的溶酶体分解代谢分解的顺序。我们进一步利用该测定来确定参与硫酸乙酰肝素的 3-O-硫酸化葡糖胺残基溶酶体分解代谢的不同硫酸酯酶的顺序作用。我们的结果证实并扩展了 ARSG 底物特异性的表征,并有助于确定 (3-O-) 硫酸化硫酸乙酰肝素的溶酶体分解代谢分解的顺序。我们进一步利用该测定来确定参与硫酸乙酰肝素的 3-O-硫酸化葡糖胺残基溶酶体分解代谢的不同硫酸酯酶的顺序作用。我们的结果证实并扩展了 ARSG 底物特异性的表征,并有助于确定 (3-O-) 硫酸化硫酸乙酰肝素的溶酶体分解代谢分解的顺序。
更新日期:2021-09-08
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