Cleavage within Reelin Repeat 3 Regulates the Duration and Range of the Signaling Activity of Reelin Protein
Cleavage within Reelin Repeat 3 Regulates the Duration and Range of the Signaling Activity of Reelin Protein
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DOI：
10.1074/jbc.m113.536326
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发表时间：
2014-05-02
影响因子：
4.8
通讯作者：
Hattori, Mitsuharu
中科院分区：
文献类型：
--
作者：
Koie, Mari;Okumura, Kyoko;Hattori, Mitsuharu
背景：Reelin蛋白水解的生理作用在很大程度上是未知的。结果：一个不可切割的Reelin突变体在很长一段时间内保持活性，并且被切割的Reelin片段与全长的Reelin片段定位不同。结论：细胞外和细胞内的Reelin裂解是阻断裂解产物的下游信号传导和运输所必需的。意义：抑制Reelin裂解有利于神经精神疾病的治疗。Reelin是一种分泌糖蛋白，在大脑中起着至关重要的作用。Reelin在两个不同的位点被切割，称为N-t和C-t，前者是主要的。N-t切割可以发生在细胞外空间和核内体中，尽管尚未研究核内体N-t切割的生理重要性。在这项研究中，我们首先确定了由大脑皮质神经元分泌的蛋白酶催化的确切N-t切割位点。在Reelin repeat 3中，Pro-1244和Ala-1245发生了切割。用天冬氨酸取代Pro-1244的Reelin突变体（Reelin- pd）对培养的大脑皮质神经元分泌的蛋白酶具有抗性，其生物活性比野生型Reelin保持活性的时间更长。有趣的是，与野生型Reelin相比，Reelin- pd在细胞内的时间更长，并持续激活下游信号。因此，需要Reelin的N-t切割来停止细胞外空间和靶神经元核内体内的信号机制。我们建立了一种针对未切割的Reelin蛋白的单克隆抗体，发现它定位于产生Reelin的细胞附近，而n端片段则扩散或被运输到遥远的区域。这些数据表明，在细胞外环境和细胞内区室中，N-t切割对调节Reelin功能的持续时间和范围起着关键作用。
Background: The physiological role of Reelin proteolysis is largely uncharacterized. Results: An uncleavable Reelin mutant remains active for a long time, and the cleaved Reelin fragment localizes differently than full-length Reelin. Conclusion: Extracellular and intracellular Reelin cleavage is required for halting downstream signaling and transport of the cleaved product. Significance: Inhibition of Reelin cleavage will be beneficial for treating neuropsychiatric diseases.Reelin is a secreted glycoprotein that plays essential roles in the brain. Reelin is specifically cleaved at two distinct sites, called N-t and C-t, with the former being the major one. N-t cleavage can occur both in the extracellular space and in the endosomes, although the physiological importance of endosomal N-t cleavage has not been investigated. In this study, we first determined the exact N-t cleavage site catalyzed by a protease secreted by cerebral cortical neurons. Cleavage occurred between Pro-1244 and Ala-1245 within Reelin repeat 3. A Reelin mutant in which Pro-1244 was replaced with aspartate (Reelin-PD) was resistant to a protease secreted by cultured cerebral cortical neurons, and its biological activity stayed active longer than that of wild-type Reelin. Interestingly, Reelin-PD remained in the intracellular compartments longer than wild-type Reelin and persistently activated downstream signaling. Therefore, N-t cleavage of Reelin is required for halting the signaling machinery in the extracellular space as well as within endosomes of target neurons. We established a monoclonal antibody specific to uncleaved Reelin protein and found that it is localized in the vicinity of Reelin-producing cells, whereas the N-terminal fragment diffuses, or is transported, to distant regions. These data demonstrate that N-t cleavage of Reelin plays critical roles in regulating the duration and range of Reelin functions both in the extracellular milieu and in the intracellular compartments.