Ogino Himari, Yamakage Yuko, Yamashita Mihoshi B, Kohno Takao, Hattori Mitsuharu
Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan.
Methods Mol Biol. 2020;2043:105-111. doi: 10.1007/978-1-4939-9698-8_9.
Proteolytic cleavage of the secreted signaling protein Reelin has been suggested to play causative roles in many neuropsychiatric and neurodegenerative disorders. Therefore, characterization of the proteolytic activity against Reelin is important not only for understanding how the brain works but also for the development of novel therapy for these disorders. Notably, ADAMTS family proteases are the primary suspects of Reelin-cleaving proteases under many, though not all, circumstances. Here we describe how to measure the Reelin-cleaving activity of ADAMTS (or of any other protease that may cleave Reelin), how to purify the Reelin-cleaving protease ADAMTS-3 from the culture supernatant of cortical neurons, and how to detect endogenous Reelin protein and its fragments in the brain.
分泌型信号蛋白Reelin的蛋白水解切割被认为在许多神经精神疾病和神经退行性疾病中起致病作用。因此，表征针对Reelin的蛋白水解活性不仅对于理解大脑的工作方式很重要，而且对于开发这些疾病的新疗法也很重要。值得注意的是，在许多（尽管不是所有）情况下，ADAMTS家族蛋白酶是切割Reelin的蛋白酶的主要嫌疑对象。在这里，我们描述了如何测量ADAMTS（或任何其他可能切割Reelin的蛋白酶）的Reelin切割活性，如何从皮质神经元的培养上清液中纯化切割Reelin的蛋白酶ADAMTS-3，以及如何检测大脑中的内源性Reelin蛋白及其片段。
