Stewart Richard S, Harris David A
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2003 Nov 14;278(46):45960-8. doi: 10.1074/jbc.M307833200. Epub 2003 Aug 21.
The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.
朊病毒蛋白（PrP）可呈现多种膜拓扑结构，包括完全转运形式（SecPrP）、两种跨膜形式（NtmPrP和CtmPrP）以及胞质形式。了解影响这些形式产生的因素很重要，因为其中两种形式，即CtmPrP和胞质PrP，被认为是某些朊病毒疾病中的关键神经毒性中间体。在本文中，我们对使用体外翻译系统合成的PrP进行了突变分析，以进一步确定影响CtmPrP形成的序列元件。我们发现，信号肽疏水核心中带电残基的取代增加了CtmPrP的合成，同时也降低了转运到微粒体中的效率。将这些突变与跨膜结构域中的取代相结合，会导致蛋白质仅以CtmPrP拓扑结构合成。减少信号肽与跨膜结构域之间的间距也会增加CtmPrP。相反，阻止信号肽切割的突变或删除糖基磷脂酰肌醇锚定添加的C末端信号不会改变拓扑结构。去除信号肽会完全阻断转运。综上所述，我们的结果与一个模型一致，即信号肽和跨膜结构域以不同方式作为PrP拓扑结构的决定因素发挥作用。我们还展示了一种抗体的特性，该抗体凭借其未切割的信号肽选择性识别CtmPrP和胞质PrP。通过使用这种抗体以及CtmPrP和胞质PrP独特的凝胶迁移率，我们表明培养细胞和啮齿动物脑中这两种形式的量不会因感染羊瘙痒病朊病毒而改变。我们得出结论，在家族性或传染性获得性朊病毒疾病中，CtmPrP和胞质PrP不太可能是必需的神经毒性中间体。
