艾杜糖2-硫酸酯酶的Man6P非依赖转运机制的研究

Lysosomal enzymes have long been known to be responsible for the degradation of different substrates in the cell. They are delivered to lysosomal compartments, where they finally become active due to the acidic PH characteristic of the lysosomal compartment. The majority of the enzymes are transported to the lysosomal compartment in the Man6P dependent transporting pathway. Only beta-glucocerebrosidase, acid sphingomyelinase and few other lysosomal enzymes have been suggested to be targeted to lysosome in Man6P independent transporting pathway. Mucolipidosis is in the rare case of lysosome storage diseases. Deficiency of the GlcNac-1-phosphotransferase leads to a global defect of lysosome biogenesis. Newly synthesized Man6P dependent lysosomal enzymes are not equipped with the critical lysosomal trafficking marker Man6P, thus escape from lysosomal sorting and are released outside the cell. Here, Mucolipidosis was used as a model to find Man6P independent lysosomal enzymes since they should not be affected by GlcNac-1-phosphotransferase deficiency. Our previous quantitative analysis by mass spectrometry shows that the amount of iduronate 2-sulfatase in leukocytes from Mucolipidosis patients is 1.4 times more than in leukocytes from normal control, which indicates that iduronate 2-sulfatase might have Man6P independent transporting pathway. In this research, western blotting and enzyme activity assay would be used to verify the increase in the amount of iduronate 2-sulfatase from Mucolipidosis patients compared with normal control. Furthermore, possible interactions of iduronate 2-sulfatase with two known Man6P independent receptors LIMP-2 and Sortilin would be tested in leukocytes from Mucolipidosis patients by immunoprecipitation based on cross-linking. At the same time, other possible receptors would be identified using mass spectrometry after immunoprecipitation in order to explore the potential Man6P independent transporting mechanism.

溶酶体酶在细胞内负责物质的降解，它们被转运到酸性环境的溶酶体发挥功能。大部分溶酶体酶经甘露糖6-磷酸(Man6P)依赖途径进入溶酶体，仅β-葡糖脑苷脂酶、鞘磷脂酶等少数溶酶体酶被单独报道存在Man6P非依赖途径。黏脂贮积症是N-乙酰氨基葡萄糖-1-磷酸转移酶缺陷的罕见溶酶体贮积症，该酶的缺陷导致Man6P依赖的溶酶体酶不能被转运到溶酶体而分泌到细胞外从而致病。本研究前期以黏脂贮积症为模型，质谱定量发现艾杜糖2-硫酸酯酶在黏脂贮积症病人白细胞中的含量是正常人的1.4倍，推测该酶存在Man6P非依赖途径。本研究拟运用免疫印迹和酶活性测定的方法验证艾杜糖2-硫酸酯酶在黏脂贮积症病人白细胞中含量的升高。运用交联结合免疫沉淀的方法在病人白细胞中试验该酶是否与已知Man6P非依赖途径受体LIMP-2和sortilin有相互作用，同时进行质谱分析寻找其他可能的受体，以探索该酶的MAn6P非依赖转运机制。

溶酶体酶在细胞内负责物质的降解，它们被转运到溶酶体发挥功能。大部分溶酶体酶经甘露糖6-磷酸（Man6P）依赖途径进入溶酶体，仅β-葡糖脑苷脂酶等少数溶酶体酶被单独报道存在Man6P非依赖途径。黏脂贮积症是N-乙酰氨基葡萄糖-1-磷酸转移酶缺陷的溶酶体贮积症，该酶的缺陷导致Man6P依赖的溶酶体酶不能被转运到溶酶体而分泌到细胞外从而致病。本研究运用酶活性测定的方法验证艾杜糖2-硫酸酯酶在黏脂贮积症病人白细胞中含量不降低，推测该酶存在Man6P依赖转运机制的同时还可能存在Man6P非依赖转运机制。本研究验证了该酶与已知Man6P非依赖途径受体LIMP-2和sortilin的相互作用。在正常人白细胞中以艾杜糖2-硫酸酯酶为靶蛋白进行免疫沉淀试验后以LIMP-2和sortilin的抗体进行免疫印迹试验，结果提示sortilin可能与艾杜糖2-硫酸酯酶发生相互作用。同时运用免疫沉淀、质谱鉴定的方法在正常人白细胞中鉴定了艾杜糖2-硫酸酯酶可能的受体。通过定量分析，蛋白质功能分析，结合已有的相互作用数据库中的数据，挑选其可能的受体AP3M1进行验证。在过表达艾杜糖2-硫酸酯酶的293T细胞中通过免疫沉淀-免疫印迹的方法验证了AP3M1可能与艾杜糖2-硫酸酯酶发生相互作用。本研究推测，艾杜糖2-硫酸酯酶可能通过AP complex转运至溶酶体，其Man6P非依赖转运的机制可能是与sortilin受体发生相互作用。