Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme
Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme
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DOI：
10.1016/j.jmb.2009.01.049
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发表时间：
2009-03-20
影响因子：
5.6
通讯作者：
Fontana, Angelo
中科院分区：
文献类型：
--
作者：
Frare, Erica;Mossuto, Maria F.;Fontana, Angelo
野生型人溶菌酶在pH3.0和60℃条件下的聚集过程分析了在聚集途径上形成的一系列不同的物种，特别是淀粉样蛋白单体前体蛋白、低聚可溶性原纤维聚集体和成熟原纤维。由于最近的研究表明，溶菌酶在成熟淀粉样原纤维出现之前形成的低聚物对细胞是有毒的，因此对低聚物种类的结构特性的分析一直受到特别关注。在这里，通过一系列技术分析了人类溶菌酶的可溶性低聚物，包括与荧光探针（如硫黄素T和1-苯胺-萘-8-磺酸盐）的结合，傅里叶变换红外光谱和控制蛋白质水解。蛋白孵育5天后分离出低聚物，透射电镜观察为直径为8-17 nm的球形颗粒。与单体蛋白不同，低聚物具有溶剂暴露的疏水性斑块，能够结合荧光探针1-苯胺-萘-8-磺酸盐。与单体溶菌酶相比，低聚物的傅里叶变换红外光谱显示了错误折叠的物种，具有随机结构的普遍存在，但具有成熟原纤维特征的β -片结构的重要元素。此外，寡聚溶菌酶聚集体被发现比单体蛋白和成熟原纤维更容易被胃蛋白酶水解，进一步表明它们的组织结构较差。综上所述，本研究表明可溶性溶菌酶低聚物是局部未折叠的物种，在聚集的初始阶段以低浓度存在。它们所组成的溶菌酶分子的非天然构象特征可能是赋予它们与包括膜在内的各种细胞成分不适当相互作用能力的因素。2009爱思唯尔有限公司版权所有。
The aggregation process of wild-type human lysozyme at pH3.0 and 60 degrees C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infra red spectroscopy, and con trol led proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the beta-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes. (C) 2009 Elsevier Ltd. All rights reserved.