Characterization of recombinant, membrane-attached full-length prion protein
Characterization of recombinant, membrane-attached full-length prion protein
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DOI：
10.1074/jbc.m400952200
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发表时间：
2004-06-11
影响因子：
4.8
通讯作者：
Glockshuber, R
中科院分区：
文献类型：
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作者：
Eberl, H;Tittmann, P;Glockshuber, R
正常细胞朊病毒蛋白 (PrPC) 的异常亚型 PrPSc 是朊病毒疾病病原体的主要成分。发现两种同工型具有相同的共价结构，包括 C 端糖基磷脂酰肌醇锚，但二级和三级结构不同。在这项研究中，在大肠杆菌中重组产生了 C 端具有未配对半胱氨酸的全长 PrP 变体，与硫醇反应性磷脂共价偶联，并掺入脂质体中，作为研究重组 PrP 在膜附着后结构和稳定性可能变化的模型。 PrP 与脂质体的共价偶联不会导致远紫外圆二色性可观察到的显着结构变化。此外，有限的蛋白水解实验未能检测到脂质体结合的 PrP 相对于可溶性 PrP 的稳定性变化。这些数据表明，先前在细胞培养模型中观察到的 PrPC 向 PrPSc 转化需要筏定位的要求，并不是因为筏脂质对膜结合 PrPC 的结构和稳定性的直接影响，而是由其他因素引起，例如。 g。增加局部 PrP 浓度或膜相关转换因子的高有效浓度。共价连接至脂质体的重组 PrP 的可用性为利用膜表面重组 PrP 进行系统性体外转化测定提供了基础。此外，我们的结果表明，膜中哺乳动物 PrPC 的三维结构与溶液中重组 PrP 的三维结构相同。
An abnormal isoform, PrPSc, of the normal cellular prion protein ( PrPC) is the major component of the causative agent of prion diseases. Both isoforms were found to possess the same covalent structures, including a C-terminal glycosylphosphatidylinositol anchor, but different secondary and tertiary structures. In this study, a variant of full-length PrP with an unpaired cysteine at the C terminus was recombinantly produced in Escherichia coli, covalently coupled to a thiol-reactive phospholipid, and incorporated into liposomes to serve as a model for studying possible changes in structure and stability of recombinant PrP upon membrane attachment. Covalent coupling of PrP to liposomes did not result in significant structural changes observable by far-UV circular dichroism. Moreover, limited proteolysis experiments failed to detect changes in the stability of liposome-bound PrP relative to soluble PrP. These data suggest that the requirement of raft localization for the PrPC to PrPSc conversion, observed previously in cell culture models, is not because of a direct influence of raft lipids on the structure and stability of membrane-bound PrPC but caused by other factors, e. g. increased local PrP concentrations or high effective concentrations of membrane-associated conversion factors. The availability of recombinant PrP covalently attached to liposomes provides the basis for systematic in vitro conversion assays with recombinant PrP on the surface of membranes. In addition, our results indicate that the three-dimensional structure of mammalian PrPC in membranes is identical to that of recombinant PrP in solution.