Nanobodies Raised against Monomeric α-Synuclein Distinguish between Fibrils at Different Maturation Stages
Nanobodies Raised against Monomeric α-Synuclein Distinguish between Fibrils at Different Maturation Stages
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DOI：
10.1016/j.jmb.2013.01.040
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发表时间：
2013-07-24
影响因子：
5.6
通讯作者：
De Genst, Erwin
中科院分区：
文献类型：
--
作者：
Guilliams, Tim;El-Turk, Farah;De Genst, Erwin
纳米抗体是骆驼抗体的单结构域片段，正在成为生物技术中的通用工具。我们在这里描述了一种特定的纳米抗体，NbSyn 87，与单体和纤维形式的α-突触核蛋白（α-Syn）的相互作用。一种140个残基的蛋白质，其聚集与帕金森病有关。我们已经使用一系列的生物物理技术，包括核磁共振和圆二色谱，等温滴定量热法和石英晶体微量天平测量这些相互作用的特点。此外，我们将结果与我们先前报道的针对不同纳米抗体NbSyn 2的结果进行了比较，NbSyn 2也针对单体aSyn产生。该比较表明，NbSyn 87和NbSyn 2以纳摩尔亲和力结合可溶性aSyn的C-末端结构域内的独特表位，所述C-末端结构域分别包含大约氨基酸118-131和137-140。量热和石英晶体微量天平数据表明，当aSyn转化成其纤维状结构时，两种纳米抗体的表位仍然是可接近的。然而，定义纳米抗体和原纤维之间结合的表观亲和力和其他热力学参数随着原纤维形成过程进行的时间长度和形成发生的条件而显著变化，表明α-Syn的C-末端结构域的环境随着原纤维组装的发生而变化。这些结果表明，纳米抗体能够靶向在其结构的相对次要细节上彼此不同的潜在致病性聚集体的形式，例如与原纤维成熟相关的那些。(C)2013爱思唯尔有限公司保留所有权利。
Nanobodies are single-domain fragments of camelid antibodies that are emerging as versatile tools in biotechnology. We describe here the interactions of a specific nanobody, NbSyn87, with the monomeric and fibrillar forms of alpha-synuclein (alpha Syn),. a 140-residue protein whose aggregation is associated with Parkinson's disease. We have characterized these interactions using a range of biophysical techniques, including nuclear magnetic resonance and circular dichroism spectroscopy, isothermal titration calorimetry and quartz crystal microbalance measurements. In addition, we have compared the results with those that we have reported previously for a different nanobody, NbSyn2, also raised against monomeric aSyn. This comparison indicates that NbSyn87 and NbSyn2 bind with nanomolar affinity to distinctive epitopes within the C-terminal domain of soluble aSyn, comprising approximately amino acids 118-131 and 137-140, respectively. The calorimetric and quartz crystal microbalance data indicate that the epitopes of both nanobodies are still accessible when aSyn converts into its fibrillar structure. The apparent affinities and other thermodynamic parameters defining the binding between the nanobody and the fibrils, however, vary significantly with the length of time that the process of fibril formation has been allowed to progress and with the conditions under which formation occurs, indicating that the environment of the C-terminal domain of alpha Syn changes as fibril assembly takes place. These results demonstrate that nanobodies are able to target forms of potentially pathogenic aggregates that differ from each other in relatively minor details of their structure, such as those associated with fibril maturation. (C) 2013 Elsevier Ltd. All rights reserved.