Genotype-Phenotype Correlation in PEX5-Deficient Peroxisome Biogenesis Defective Cell Lines
Genotype-Phenotype Correlation in PEX5-Deficient Peroxisome Biogenesis Defective Cell Lines
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DOI：
10.1002/humu.20833
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发表时间：
2009-01-01
期刊：
影响因子：
3.9
通讯作者：
Waterham, Hans R.
中科院分区：
文献类型：
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作者：
Ebberink, Merel S.;Mooyer, Petra A. W.;Waterham, Hans R.
通过过氧化物酶体靶向序列1型（PTS 1）或2型（PTS 2）靶向预定用于过氧化物酶体基质的蛋白质。在人类中，任一类蛋白质的靶向依赖于由PEX 5基因编码的胞质受体蛋白。PEX 5的选择性剪接产生两种蛋白变体，PEX 5S和PEX 5L。PEX 5S专门参与PTS 1蛋白的输入，而PEX 5L介导PTS 1和PTS 2蛋白的输入。对来自被诊断患有过氧化物酶体生物发生障碍（PBD）的患者的500多种不同成纤维细胞系进行的遗传互补测试鉴定了11种在PEX 5中具有缺陷的细胞系。本研究的目的是在生化和遗传水平上表征这些细胞系。为此，分析培养的成纤维细胞的极长链脂肪酸（VLCFA）浓度，过氧化物酶体β-和α-氧化，二羟丙酮-磷酸酰基转移酶（DHAPAT）活性，过氧化物酶体硫解酶，和过氧化氢酶免疫荧光。对PEX 5基因的突变分析揭示了11种不同的突变，其中8种是新的。通过用标记有PTS 1或PTS 2的绿色荧光蛋白（GFP）转染细胞来评估PTS 1和PTS 2蛋白输入能力。6个细胞系显示PTS 1和PTS 2蛋白输入缺陷，而4个细胞系仅显示PTS 1蛋白输入缺陷。PEX 5氨基酸序列内不同突变的位置与在细胞系中观察到的过氧化物酶体蛋白输入缺陷相当好地相关。
proteins destined for the peroxisomal matrix are targeted by virtue of a peroxisomal targeting sequence type 1 (PTS1) or type 2 (PTS2). In humans, targeting of either class of proteins relies oil a cytosolic receptor protein encoded by the PEX5 gene. Alternative splicing of PEX5 results in two protein variants, PEX5S and PEX5L. PEX5S is exclusively involved in PTS1 protein import, whereas PEX5L mediates the import of both PTS1 and PTS2 proteins. Genetic complementation testing with over 500 different fibroblast cell lines from patients diagnosed with a peroxisome biogenesis disorder (PBD) identified 11 cell lines with it defect in PEX5. The aim of this study was to characterize these cell lines at a biochemical and genetic level. To this end, the cultured fibroblasts were analyzed for very long chain fatty acid (VLCFA) concentrations, peroxisomal beta-and alpha-oxidation, dihydroxyacetone-phosphate acyltransferase (DHAPAT) activity, peroxisomal thiolase, and catalase immunofluorescence. Mutation analysis of the PEX5 gene revealed 11 different mutations, eight of which are novel. PTS1- and PTS2-protein import capacity was assessed by transfection of the cells with green fluorescent protein (GFP) tagged with either PTS1 or PTS2. Six cell lines showed a defect in both PTS1 and PTS2 protein import, whereas four cell lines only showed a defect in PTS1 protein import. The location of the different mutations within the PEX5 amino acid sequence correlates rather well with the peroxisomal protein import defect observed in the cell lines.