Niemann-Pick type C disease: Subcellular location and functional characterization of NPC2 proteins with naturally occurring missense mutations
Niemann-Pick type C disease: Subcellular location and functional characterization of NPC2 proteins with naturally occurring missense mutations
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DOI：
10.1002/humu.20173
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发表时间：
2005-07-01
期刊：
影响因子：
3.9
通讯作者：
Millat, G
中科院分区：
文献类型：
--
作者：
Chikh, K;Rodriguez, C;Millat, G
C 型尼曼-匹克病 (NPC) 是一种严重的神经内脏溶酶体疾病，是由于 NPC1 基因突变或少数家族中的 NPC2 基因突变所致。针对 NPC2 蛋白的研究很少，仅描述了 13 种不同的致病突变（包括本报告中的三种新突变）。在目前已知的 NPC2 突变等位基因中，有 6 个导致提前终止密码子。仅鉴定出五种错义突变：c.115G > A (p.V39M)、c.140G > T (p.C47F)、c.199T > C (p.S67P)、c.278G > T (p.C93F) 和（本报告）c.295T > C (p.C99R)。在本研究中，我们生成了包含这些错义突变的 cDNA 构建体，并在具有无义 NPC2 突变的人成纤维细胞中过度表达后，通过免疫印迹、免疫细胞荧光显微镜和互补来表征突变蛋白。在两名患有成年神经系统疾病的患者中，p.V39M 突变以纯合状态被描述，导致合成了明显有功能的重组蛋白，正确靶向溶酶体。尽管在我们的过表达系统中可能会忽略轻微的功能影响，但与 NPCI 突变体的比较研究表明，轻微的突变可能不一定会影响蛋白质的定位或其在天然状态下的数量。相反，p.C47F、p.C93R、p.C99R 突变以及不太可预测的 p.S67P 突变导致了与内质网标记共定位的错误折叠重组蛋白的合成。后四种蛋白质通常是分泌的，但无法纠正 NPC2(-/-) 细胞中的胆固醇储存。在有临床病史的三例病例中，突变蛋白的功能特征显示出良好的基因型-表型相关性。 Hum Mutat 26(1), 20-28, 2005。(c) 2005 Wiley-Liss, Inc.
Niemann-Pick disease type C (NPC), a severe neurovisceral lysosomat disorder, is due to mutations on the NPC1 gene or, in a minority of families, the NPC2 gene. Few investigations have been devoted to the NPC2 protein, for which only 13 different disease-causing mutations (including three novel ones in this report) have been described. Among the currently known NPC2 mutant alleles, six resulted in a premature stop codon. Only five missense mutations, c.115G > A (p.V39M), c.140G > T (p.C47F), c.199T > C (p.S67P), c.278G > T (p.C93F), and (this report) c.295T > C (p.C99R) were identified. In the present study, we generated cDNA constructs harboring each of these missense mutations and, upon overexpression in human fibroblasts with a nonsense NPC2 mutation, characterized the mutated proteins by immunoblotting, immunocytofluorescence microscopy, and complementation. Mutation p.V39M, described in the homozygous state in two patients with an adult,onset neurological disease, resulted in the synthesis of apparently functional recombinant proteins correctly targeted to lysosomes. Although a mild functional impact could possibly be overlooked in our overexpression system, comparative studies with NPCI mutants indicated that mild mutations might not necessarily affect localization of the protein or its quantity in the native state. Conversely, mutations p.C47F, p.C93R, p.C99R but also, less predictably, p.S67P, led to the synthesis of misfolded recombinant proteins that colocalized with an endoplasmic reticulum marker. The four latter proteins were normally secreted but were unable to correct cholesterol storage in NPC2(-/-) cells. Functional characterization of the mutant proteins showed an excellent genotype-phenotype correlation in the three cases for whom a clinical history was available. Hum Mutat 26(1), 20-28, 2005. (c) 2005 Wiley-Liss, Inc.