Lysosomal Storage Causes Cellular Dysfunction in Mucolipidosis II Skin Fibroblasts
Lysosomal Storage Causes Cellular Dysfunction in Mucolipidosis II Skin Fibroblasts
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DOI：
10.1074/jbc.m111.267930
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发表时间：
2011-10-07
影响因子：
4.8
通讯作者：
Sakai, Norio
中科院分区：
文献类型：
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作者：
Otomo, Takanobu;Higaki, Katsumi;Sakai, Norio
粘脂沉积症II（ML-II）是一种致命的遗传性代谢疾病，其由GlcNAc-磷酸转移酶缺乏引起，GlcNAc-磷酸转移酶在溶酶体酶上产生甘露糖6-磷酸识别标记物中起作用。在ML-II中，许多溶酶体酸水解酶被错误地从细胞中排出，溶酶体充满未消化的底物，这解释了包涵体细胞病作为这种疾病的替代名称。在这项研究中，我们揭示了ML-II皮肤成纤维细胞的各种细胞表型。我们定量细胞内的磷脂和胆固醇，并显示出类似的2倍积累在ML-II相比，正常细胞。ML-II细胞的溶酶体pH高于正常细胞（5.29 +/- 0.08对4.79 +/- 0.10，p < 0.001）。与正常细胞相比，ML-II细胞中增殖的溶酶体在量上积累接近3倍。细胞内物流，包括内吞作用和甘露糖6-磷酸受体再循环受损的ML-II细胞。为了确认这些ML-II细胞表型是否来源于溶酶体内的溶酶体酸水解酶缺陷，我们使用部分纯化的总酶混合物补充溶酶体酶，该混合物来源于NH 4Cl处理后的正常皮肤成纤维细胞的条件培养基。这种补充纠正了所有先前描述的ML-II表型。此外，自噬和线粒体损伤，我们以前报道的改善，和包涵体消失后，总溶酶体酶补充电子显微镜。我们的研究结果表明，ML-II中的各种细胞表型是由许多溶酶体酶的缺乏和大量积累的未消化的底物引起的。
Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed similar to 2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 +/- 0.08 versus 4.79 +/- 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated similar to 3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH4Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates.