Biochemical characterization and intracellular localization of the Menkes disease protein
Biochemical characterization and intracellular localization of the Menkes disease protein
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DOI：
10.1073/pnas.93.24.14030
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发表时间：
1996-11-26
影响因子：
11.1
通讯作者：
Gitlin, JD
中科院分区：
文献类型：
--
作者：
Yamaguchi, Y;Heiny, ME;Gitlin, JD
Menkes病是一种致命的儿童神经退行性疾病，由于缺乏或功能障碍的推定铜转运P型ATP酶编码的X染色体上。为了阐明该蛋白的生物合成和亚细胞定位，针对编码人Menkes蛋白氨基末端的第4至第6个铜结合结构域的细菌融合蛋白产生多克隆抗血清。RNA印迹分析显示，Menkes基因在几种细胞系中表达丰富，利用这种抗血清的免疫印迹研究很容易在这些细胞的裂解物中检测到178-kDa的蛋白质。脉冲追踪研究表明，这种蛋白质合成为单链多肽，其通过N-连接的糖基化修饰为成熟的糖苷内切酶H-抗性形式，HeLa细胞裂解物的蔗糖梯度分级分离，然后用已知细胞内位置的蛋白质的抗体对单个级分进行免疫印迹，鉴定出与含有阳离子非依赖性甘露糖-6-磷酸受体的级分相似的Menkes ATP酶。与这一观察结果一致，这些相同的细胞的共聚焦免疫荧光研究定位这种蛋白质的trans-Golgi网络和囊泡隔室，在细胞核或质膜上没有表达。两者合计，这些数据提供了一个独特的模型铜运输到哺乳动物细胞的分泌途径，这是兼容的临床观察受影响的患者和同源蛋白在原核生物和酵母中鉴定的最新数据。
Menkes disease is a fatal neurodegenerative disorder of childhood due to the absence or dysfunction of a putative copper-transporting P-type ATPase encoded on the X chromosome. To elucidate the biosynthesis and subcellular localization of this protein, polyclonal antisera were generated against a bacterial fusion protein encoding the 4th to 6th copper-binding domains in the amino terminus of the human Menkes protein. RNA blot analysis revealed abundant Menkes gene expression in several cell lines, and immunoblotting studies utilizing this antiserum readily detected a 178-kDa protein in lysates from these cells. Pulse-chase studies indicate that this protein is synthesized as a single-chain polypeptide which is modified by N-linked glycosylation to a mature endoglycosidase H-resistant form, Sucrose gradient fractionation of HeLa cell lysates followed by immunoblotting of individual fractions with antibodies to proteins of known intracellular location identified the Menkes ATPase in fractions similar to those containing the cation-independent mannose-6-phosphate receptor. Consistent with this observation, confocal immunofluorescence studies of these same cells localized this protein to the trans-Golgi network and a vesicular compartment with no expression in the nucleus or on the plasma membrane. Taken together, these data provide a unique model of copper transport into the secretory pathway of mammalian cells which is compatible with clinical observations in affected patients and with recent data on homologous proteins identified in prokaryotes and yeast.