Conformation of PrPc on the cell surface as probed by antibodies
Conformation of PrPc on the cell surface as probed by antibodies
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DOI：
10.1016/s0022-2836(02)01365-7
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发表时间：
2003-02-14
影响因子：
5.6
通讯作者：
Williamson, RA
中科院分区：
文献类型：
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作者：
Leclerc, E;Peretz, D;Williamson, RA
我们通过在一组九个单克隆抗体片段 (Fab) 之间进行交叉竞争实验，研究了叙利亚仓鼠 PrPC 在转染的 CHO 细胞表面上的构象，这些单克隆抗体片段针对整个蛋白质中的定义表位。在识别位于 PrPC 非结构化 N 端部分内的表位的抗体与识别位于分子有序 C 端部分内的表位的抗体之间没有观察到竞争。然而，在识别重叠表位的抗体之间以及识别PrP序列中彼此相邻的表位的抗体之间观察到竞争。滴定每个 Fab 对细胞表面 PrPC 的反应性揭示了不同特定表位的可及性存在明显的异质性。 Fab D18 识别包含 PrPC 第一个 α 螺旋的序列，结合细胞表面 PrP 群体的最大部分。相比之下，结合PrP最N末端的表位的Fab E123和结合Prp中心区域的表位的Fab 13A5能够识别少于Fab D18结合的PrPC分子数量的一半。我们观察到的抗体反应性模式可能部分是由于细胞表面发现的部分 PrPc 分子的 N 端截短造成的。然而，截短不能解释 Fab D18 和 13A5 表位暴露之间的显着差异，这两个表位在 PrP 序列中相邻。 13A5 表位相对难以接近可能反映了 PrPC-PrPC 相互作用、PrPC 与细胞膜上其他成分之间的相互作用，或者具有不同构象的 PrPC 亚种的存在。 (C) 2003 Elsevier Science Ltd. 保留所有权利。
We have investigated the conformation of Syrian hamster PrPC on the surface of transfected CHO cells by performing cross-competition experiments between a set of nine monoclonal antibody fragments (Fab) directed to defined epitopes throughout the protein. No competition was observed between antibodies recognizing epitopes located within the unstructured N-terminal portion of PrPC and those recognizing epitopes located within the ordered C-terminal half of the molecule. However, competition was observed between antibodies recognizing overlapping epitopes and between antibodies recognizing epitopes lying adjacent to one another in the PrP sequence. Titrating the reactivity of each Fab against cell-surface PrPC revealed a clear heterogeneity in the accessibility of different specific epitopes. Fab D18, recognizing sequence incorporating the first alpha-helix of PrPC, bound the largest fraction of the cell-surface PrP population. In contrast, Fab E123, binding an epitope at the extreme N terminus of PrP, and Fab 13A5, binding an epitope in the central region of Prp, were able to recognize fewer than half the number of PrPC molecules bound by Fab D18. The pattern of antibody reactivity we observed may, in part, result from N-terminal truncation of a proportion of PrPc molecules found at the cell surface. However, truncation cannot account for the marked disparity between exposure of the Fab D18 and 13A5 epitopes, which lie adjacent in the PrP sequence. The relative inaccessibility of the 13A5 epitope likely reflects either PrPC-PrPC interaction, interaction between PrPC and other constituents on the cell membrane, or the existence of PrPC subspecies with distinct conformations. (C) 2003 Elsevier Science Ltd. All rights reserved.