Hornemann Simone, Schorn Christian, Wüthrich Kurt
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, 8093 Zürich, Switzerland.
EMBO Rep. 2004 Dec;5(12):1159-64. doi: 10.1038/sj.embor.7400297. Epub 2004 Nov 26.
NMR structures of recombinant prion proteins from various species expressed in Escherichia coli have been solved during the past years, but the fundamental question of the relevancy of these data relative to the naturally occurring forms of the prion protein has not been directly addressed. Here, we present a comparison of the cellular form of the bovine prion protein isolated and purified from healthy calf brains without use of detergents, so that it contains the two carbohydrate moieties and the part of the GPI anchor that is maintained after enzymatic cleavage of the glycerolipid moiety, with the recombinant bovine prion protein expressed in E. coli. We show by circular dichroism and (1)H-NMR spectroscopy that the three-dimensional structure and the thermal stability of the natural glycoprotein and the recombinant polypeptide are essentially identical. This result indicates possible functional roles of the glycosylation of prion proteins in healthy organisms, and provides a platform and validation for future work on the structural biology of prion proteins, which will have to rely primarily on the use of recombinant polypeptides.
在过去几年里，已解析出在大肠杆菌中表达的来自各种物种的重组朊病毒蛋白的核磁共振结构，但这些数据与朊病毒蛋白天然存在形式的相关性这一基本问题尚未得到直接解决。在此，我们对从健康牛脑分离纯化的牛朊病毒蛋白的细胞形式与在大肠杆菌中表达的重组牛朊病毒蛋白进行了比较。前者在不使用去污剂的情况下进行分离纯化，因此含有两个碳水化合物部分以及甘油脂质部分经酶切后保留的糖基磷脂酰肌醇（GPI）锚定部分。我们通过圆二色光谱和（1）H - NMR光谱表明，天然糖蛋白和重组多肽的三维结构及热稳定性基本相同。这一结果表明了朊病毒蛋白糖基化在健康生物体中的可能功能作用，并为未来朊病毒蛋白结构生物学研究提供了一个平台及验证，而未来研究将主要依赖于重组多肽的使用。
