C-Terminal Region-Dependent Change of Antibody-Binding to the Eighth Reelin Repeat Reflects the Signaling Activity of Reelin
C-Terminal Region-Dependent Change of Antibody-Binding to the Eighth Reelin Repeat Reflects the Signaling Activity of Reelin
复制标题
DOI：
10.1002/jnr.22143
复制
发表时间：
2009-11-01
影响因子：
4.2
通讯作者：
Hattori, Mitsuharu
中科院分区：
文献类型：
--
作者：
Kohno, Takao;Nakano, Yoshimi;Hattori, Mitsuharu
Reelin是一种分泌型糖蛋白，在大脑的发育和功能中起着关键作用，但它如何激活下游细胞内信号转导尚不完全清楚。我们最近报道，高度保守的C-末端区域（CTR）的Reelin是必需的，其完整的信号传导活动，虽然潜在的机制仍然未知。在Reelin的生物化学研究期间，我们偶然发现，在Western印迹上，一种市售的抗Reelin抗体G20可以与缺乏CTR的突变体Reelin蛋白结合，但不能与野生型Reelin结合。G20表位位于Reelin-repeat 8（RR 8）的最后19个残基，翻译后修饰和蛋白水解都不能解释这种效应。此外，当在RR 8和CTR之间插入不相关的序列（例如FLAG标签）时，相应抗体的反应性大大降低。这些结果表明，RR 8和CTR形成紧密结构，使得周围序列无法接近抗体。利用这一现象，我们首次在体内发现了缺乏CTR的Reelin同种型，并估计了其对分泌的Reelin总量的贡献。重要的是，Reelin突变体与G20反应的程度与它们的信号传导活性负相关，表明CTR诱导的RR 8结构变化是下游信号传导激活的先决条件，推测是通过与某种神经元膜分子结合。(C)2009 Wiley-Liss，Inc.
Reelin is a secreted glycoprotein that plays pivotal roles in the development and function of the brain, but how it activates downstream intracellular signaling is not fully understood. We have recently reported that the highly conserved C-terminal region (CTR) of Reelin is required for its full signaling activity, although the underlying mechanism remains unknown. During biochemical study of Reelin, we serendipitously found that one commercially available anti-Reelin antibody G20 can bind to CTR-lacking mutant Reelin proteins, but not wild-type Reelin, on Western blotting. The G20 epitope resides in the last 19 residues of Reelin-repeat 8 (RR8), and neither posttranslational modification nor proteolysis can explain this effect. Furthermore, when an unrelated sequence, such as FLAG-tag, is inserted between RR8 and CTR, the reactivity of the corresponding antibody greatly decreases. These results suggest that RR8 and CTR form a tight structure that makes the surrounding sequence inaccessible to an antibody. Taking advantage of this phenomenon, we show the existence of CTR-lacking Reelin isoform in vivo for the first time and estimate its contribution to the total amount of secreted Reelin. Importantly, the extent to which Reelin mutants react with G20 is inversely correlated with their signaling activity, indicating that the CTR-induced structural change of RR8 is a prerequisite for downstream signaling activation, presumably via binding to a certain neuronal membrane molecule(s). (C) 2009 Wiley-Liss, Inc.