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通过抗体检测细胞表面PrP(C)的构象。

Conformation of PrP(C) on the cell surface as probed by antibodies.

作者信息

Leclerc Estelle, Peretz David, Ball Haydn, Solforosi Laura, Legname Giuseppe, Safar Jiri, Serban Ana, Prusiner Stanley B, Burton Dennis R, Williamson R Anthony

机构信息

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.

出版信息

J Mol Biol. 2003 Feb 14;326(2):475-83. doi: 10.1016/s0022-2836(02)01365-7.

Abstract

We have investigated the conformation of Syrian hamster PrP(C) 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 PrP(C) 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 PrP(C) revealed a clear heterogeneity in the accessibility of different specific epitopes. Fab D18, recognizing sequence incorporating the first alpha-helix of PrP(C), 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 PrP(C) molecules bound by Fab D18. The pattern of antibody reactivity we observed may, in part, result from N-terminal truncation of a proportion of PrP(C) 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 PrP(C)-PrP(C) interaction, interaction between PrP(C) and other constituents on the cell membrane, or the existence of PrP(C) subspecies with distinct conformations.

摘要

我们通过在一组针对蛋白质中特定表位的九个单克隆抗体片段(Fab)之间进行交叉竞争实验,研究了转染的CHO细胞表面叙利亚仓鼠PrP(C)的构象。在识别位于PrP(C)无结构N端部分的表位的抗体与识别位于分子有序C端一半内的表位的抗体之间未观察到竞争。然而,在识别重叠表位的抗体之间以及在PrP序列中彼此相邻的表位的抗体之间观察到了竞争。滴定每种Fab对细胞表面PrP(C)的反应性揭示了不同特定表位可及性的明显异质性。识别包含PrP(C)第一个α螺旋序列的Fab D18结合了细胞表面PrP群体的最大部分。相比之下,结合PrP极端N端表位的Fab E123和结合PrP中央区域表位的Fab 13A5能够识别的PrP(C)分子数量不到Fab D18结合数量的一半。我们观察到的抗体反应模式可能部分是由于在细胞表面发现的一部分PrP(C)分子的N端截短所致。然而,截短不能解释Fab D18和13A5表位暴露之间的明显差异,这两个表位在PrP序列中相邻。13A5表位相对不可及可能反映了PrP(C)-PrP(C)相互作用、PrP(C)与细胞膜上其他成分之间的相互作用,或具有不同构象的PrP(C)亚类的存在。

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