Muramoto T, Scott M, Cohen F E, Prusiner S B
Department of Neurology, University of California, San Francisco 94143, USA.
Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15457-62. doi: 10.1073/pnas.93.26.15457.
The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.
朊病毒蛋白（PrPSc）的瘙痒病异构体的N末端可以被截断而不丧失瘙痒病感染性，相应地，细胞异构体PrPC的N末端被截断后，仍可转化为PrPSc。为了评估PrP分子的其他片段是否可以被删除，我们之前去除了PrPC中假定的二级结构区域；在本研究中，我们发现删除四个预测螺旋中的每一个都阻止了PrPSc的形成，去除终止转移效应区域和C178A突变也有同样的效果。去除螺旋2和螺旋3之间的一个36个残基的环并不阻止蛋白酶抗性PrP的形成；产生的类似瘙痒病的蛋白，命名为PrPSc106，在N末端信号肽和用于添加糖脂锚的C末端序列被切割后含有106个残基。向细胞裂解物中添加去污剂十二烷基肌氨酸钠可溶解PrPSc106，其仍保留对蛋白酶K消化的抗性。这些结果表明，PrP中所有提议的二级结构区域对于PrPSc的形成都是必需的，稳定螺旋3和螺旋4的二硫键也是如此。PrPSc106的发现应有助于PrPSc的结构研究、PrPSc形成机制的研究以及PrPSc特异性抗体的产生。
