Niemann-Pick type C disease: importance of N-glycosylation sites for function and cellular location of the NPC2 protein
Niemann-Pick type C disease: importance of N-glycosylation sites for function and cellular location of the NPC2 protein
复制标题
DOI：
10.1016/j.ymgme.2004.06.013
复制
发表时间：
2004-11-01
影响因子：
3.8
通讯作者：
Millat, G
中科院分区：
文献类型：
--
作者：
Chikh, K;Vey, S;Millat, G
C 型尼曼-匹克病 (NPC) 是一种神经内脏疾病，其特征是溶酶体/晚期内体系统中胆固醇和糖脂的积累，是由于 NPC1 或 NPC2 基因突变所致。尽管 NPC1 和 NPC2 蛋白似乎对于正常的细胞胆固醇运输至关重要，但它们的确切功能和关系仍然难以捉摸。 NPC2 患者的突变鉴定并未提供结构与功能关系的见解，但最近的研究提供了有关 NPC2 蛋白胆固醇结合位点的重要信息。目前的工作重点是 NPC2 的定位和 N-糖基化，因为糖基化通常对于蛋白质的靶向、稳定性和生物功能至关重要。在培养的人成纤维细胞中使用免疫细胞荧光，我们发现天然 NPC2 蛋白本质上是溶酶体的，与 NPC1 的晚期内体定位不同。影响 NPC2(-/-) 成纤维细胞中三个潜在 NPC2 N-糖基化位点中每一个的 cDNA 突变体的表达显示仅使用了两个位点。细胞内人 NPC2 蛋白以两种 N-糖基化形式存在，其中一条寡糖链连接到 Asn 58，或者两条寡糖连接到 Asn 58 和 135。寡糖链是杂合和/或高甘露糖类型，没有复杂的链。对Asn 58和Asn 135突变蛋白的细胞定位及其各自对恢复NPC2(-/-)细胞中正常胆固醇运输的影响的进一步研究得出这样的结论：只有Asn 58携带的寡糖链负责将NPC2正确靶向溶酶体，并且对于NPC2功能至关重要。 (C) 2004 Elsevier Inc. 保留所有权利。
Niemann-Pick disease type C (NPC), a neurovisceral disorder characterized by accumulation of cholesterol and glycolipids in the lysosomal/late endosomal system, is due to mutations on either the NPC1 or the NPC2 genes. Although NPC1 and NPC2 proteins appear essential for proper cellular cholesterol trafficking, their precise functions and relationship have remained elusive. Mutation identification in NPC2 patients did not provide insights into structure-function relationships, but recent studies brought important information on the cholesterol-binding site of the NPC2 protein. The present work was focused on localization and N-glycosylation of NPC2, considering that glycosylation is often essential for targeting, stability and biological function of proteins. Using immunocytolluorescence in cultured human fibroblasts, we found that the native NPC2 protein is essentially lysosomal, at variance with the late endosomal location of NPC1 Expression of cDNA mutants affecting each of the three potential NPC2 N-glycosylation sites in NPC2(-/-) fibroblasts showed that only two sites are used. The intracellular human NPC2 protein occurred as two N-glycosylated forms, with either one single oligosaccharide chain attached to Asn 58 or two oligosaccharides attached to Asn 58 and 135. The oligosaccharidic chains were of the hybrid and/or high mannose type, with no complex chains. Further studies on the cellular location of Asn 58 and Asn 135 mutant proteins and their respective effect on restoration of normal cholesterol traficking in NPC2(-/-) cells led to the conclusion that only the oligosaccharide chain carried by Asn 58 is responsible for proper targeting of NPC2 to lysosomes, and is crucial for NPC2 function. (C) 2004 Elsevier Inc. All rights reserved.