Sillence Dan J, Puri Vishwajeet, Marks David L, Butters Terry D, Dwek Raymond A, Pagano Richard E, Platt Frances M
Glycobiology Institute, Department of Biochemistry, South Parks Road, University of Oxford, Oxford OX1 3QU, UK.
J Lipid Res. 2002 Nov;43(11):1837-45. doi: 10.1194/jlr.m200232-jlr200.
Glycosphingolipids are endocytosed and targeted to the Golgi apparatus, but are mistargeted to lysosomes in numerous sphingolipidoses. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Gaucher disease is a hereditary deficiency in glucocerebrosidase leading to glucosylceramide accumulation; however, Gaucher fibroblasts exhibited normal Golgi transport of lactosylceramide. To better understand the effects of glycosphingolipid accumulation on intracellular trafficking and the use of imino sugar inhibitors, we studied sphingolipid endocytosis in fibroblast and macrophage models for Gaucher disease. Treatment of fibroblasts or RAW macrophages with conduritol B epoxide, an inhibitor of lysosomal glucocerebrosidase, resulted in a change in the endocytic targeting of lactosylceramide from the Golgi to the lysosomes. Co-treatment of macrophages with conduritol B-epoxide and 12-25 microM N-butyldeoxygalactonojirimycin, an inhibitor of glycosphingolipid biosynthesis, prevented the mistargeting of lactosylceramide to the lysosomes and restored trafficking to the Golgi. Surprisingly, higher doses (>25 microM) of NB-DGJ induced targeting of lactosylceramide to the lysosomes, even in the absence of conduritol B-epoxide. These data demonstrate that both increases and decreases in glucosylceramide levels can dramatically alter the endocytic targeting of lactosylceramide and suggest a role for glucosylceramide in regulation of membrane transport.
糖鞘脂被内吞并靶向高尔基体，但在许多鞘脂贮积病中被错误靶向至溶酶体。底物减少疗法利用亚氨基糖抑制葡萄糖神经酰胺合酶，并可能消除贮积的影响。戈谢病是一种葡糖脑苷脂酶的遗传性缺乏症，导致葡萄糖神经酰胺积累；然而，戈谢病成纤维细胞对乳糖神经酰胺的高尔基体转运表现正常。为了更好地理解糖鞘脂积累对细胞内运输的影响以及亚氨基糖抑制剂的作用，我们在戈谢病的成纤维细胞和巨噬细胞模型中研究了鞘脂的内吞作用。用溶酶体葡糖脑苷脂酶抑制剂环氧化物处理成纤维细胞或RAW巨噬细胞，导致乳糖神经酰胺的内吞靶向从高尔基体转变为溶酶体。用环氧化物和12 - 25微摩尔N - 丁基脱氧半乳糖野黑霉素（一种糖鞘脂生物合成抑制剂）共同处理巨噬细胞，可防止乳糖神经酰胺错误靶向至溶酶体，并恢复向高尔基体的运输。令人惊讶的是，即使在没有环氧化物的情况下，更高剂量（>25微摩尔）的NB - DGJ也会诱导乳糖神经酰胺靶向至溶酶体。这些数据表明，葡萄糖神经酰胺水平的升高和降低都可显著改变乳糖神经酰胺的内吞靶向，并提示葡萄糖神经酰胺在膜运输调节中的作用。
