From the Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, Japan.
J Biol Chem. 2014 May 2;289(18):12922-30. doi: 10.1074/jbc.M113.536326. Epub 2014 Mar 18.
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.
Reelin 是一种分泌性糖蛋白，在大脑中发挥着重要作用。Reelin 可在两个不同的位点被特异性切割，称为 N-t 和 C-t，前者为主。N-t 切割既可以发生在细胞外空间，也可以发生在内涵体中，尽管内涵体中 N-t 切割的生理意义尚未得到研究。在本研究中，我们首先确定了由大脑皮质神经元分泌的蛋白酶催化的精确 N-t 切割位点。切割发生在 Reelin 重复 3 内的 Pro-1244 和 Ala-1245 之间。将 Pro-1244 替换为天冬氨酸的 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 保持更长时间的活性。有趣的是，Reelin-PD 比野生型 Reelin 更长时间地留在细胞内隔间中，并持续激活下游信号。因此，Reelin 的 N-t 切割对于在细胞外空间以及靶神经元的内涵体中停止信号机制都是必需的。我们建立了一种针对未切割 Reelin 蛋白的单克隆抗体，并发现它定位于产生 Reelin 的细胞附近，而 N 端片段扩散或被运输到远处的区域。这些数据表明，Reelin 的 N-t 切割在调节 Reelin 功能的持续时间和范围方面在细胞外环境和细胞内隔室中都发挥着关键作用。
