Xiong L W, Raymond L D, Hayes S F, Raymond G J, Caughey B
Laboratory of Persistent Viral Diseases and Microscopy Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana 59840, USA.
J Neurochem. 2001 Nov;79(3):669-78. doi: 10.1046/j.1471-4159.2001.00606.x.
The conversion of protease-sensitive prion protein (PrP-sen) to a high beta-sheet, protease-resistant and often fibrillar form (PrP-res) is a central event in transmissible spongiform encephalopathies (TSE) or prion diseases. This conversion can be induced by PrP-res itself in cell-free conversion reactions. The detergent sodium N-lauroyl sarkosinate (sarkosyl) is a detergent that is widely used in PrP-res purifications and is known to stimulate the PrP-res-induced conversion reaction. Here we report effects of sarkosyl and other detergents on recombinant hamster PrP-sen purified from mammalian cells under oxidizing conditions that maintain the single native disulfide bond. Low concentrations of sarkosyl (0.001-0.1%) induced aggregation of PrP-sen molecules, increased light scattering, altered fluorescence excitation and emission spectra, and enhanced the proportion of beta-sheet secondary structure according to circular dichroism and infrared spectroscopies. An enhancement of beta-sheet content was also seen with 0.001% sodium dodecyl sulfate (SDS) but not several other types of detergents. Electron microscopy revealed that sarkosyl induced the formation of both amorphous and fibrillar aggregates. The fibrils appeared to be constructed from spherical bead-like protofibrils. Neither TSE infectivity nor the characteristic partial proteinase K resistance of PrP-res was detected in the sarkosyl-induced PrP aggregates. We conclude that certain anionic detergents can disrupt the conformation of PrP-sen and induce high beta-sheet aggregates that are distinct from scrapie-associated PrP-res in terms of protease-resistance, infrared spectrum and infectivity. These results reinforce the idea that not all high-beta aggregates of PrP are equivalent to the pathologic form, PrP-res.
蛋白酶敏感型朊病毒蛋白（PrP-sen）转变为高β-折叠、蛋白酶抗性且通常为纤维状的形式（PrP-res）是传染性海绵状脑病（TSE）或朊病毒疾病的核心事件。在无细胞转化反应中，这种转变可由PrP-res自身诱导。去污剂月桂酰肌氨酸钠（ Sarkosyl）是一种广泛用于PrP-res纯化的去污剂，已知其能刺激PrP-res诱导的转化反应。在此，我们报告了Sarkosyl和其他去污剂对在维持单个天然二硫键的氧化条件下从哺乳动物细胞中纯化的重组仓鼠PrP-sen的影响。低浓度的Sarkosyl（0.001 - 0.1%）诱导PrP-sen分子聚集，增加光散射，改变荧光激发和发射光谱，并根据圆二色性和红外光谱增强β-折叠二级结构的比例。0.001%的十二烷基硫酸钠（SDS）也能增强β-折叠含量，但其他几种类型的去污剂则不能。电子显微镜显示，Sarkosyl诱导形成无定形和纤维状聚集体。这些纤维似乎由球形珠状原纤维构成。在Sarkosyl诱导的PrP聚集体中未检测到TSE感染性或PrP-res特有的部分蛋白酶K抗性。我们得出结论，某些阴离子去污剂可破坏PrP-sen的构象并诱导形成高β-折叠聚集体，这些聚集体在蛋白酶抗性、红外光谱和感染性方面与羊瘙痒病相关的PrP-res不同。这些结果强化了并非所有PrP的高β聚集体都等同于病理形式PrP-res的观点。
