Takahashi Nozomu, Onozuka Wataru, Watanabe Seiji, Wakasugi Keisuke
Department of Life Sciences Graduate School of Arts and Sciences The University of Tokyo Japan.
Present address: Department of Neuroscience and Pathobiology Research Institute of Environmental Medicine Nagoya University Nagoya Aichi Japan.
FEBS Open Bio. 2017 Aug 14;7(9):1338-1349. doi: 10.1002/2211-5463.12271. eCollection 2017 Sep.
Neuroglobin (Ngb) is a heme protein expressed in the vertebrate brain. We previously engineered a chimeric Ngb protein, in which module M1 of human Ngb is replaced by that of zebrafish Ngb, and showed that the chimeric ZHHH Ngb has a cell membrane-penetrating activity similar to that of zebrafish Ngb and also rescues cells from death caused by hypoxia/reoxygenation as does human Ngb. Recently, it was reported that overexpression of mammalian Ngb in neuronal cells induces neurite outgrowth. In this study, we performed neurite outgrowth assays of chimeric Ngb using rat pheochromocytoma PC12 cells. Addition of chimeric Ngb, but not human or zebrafish Ngb, exogenously to the cell medium induces neurite outgrowth. On the other hand, the K7A/K9Q chimeric Ngb double mutant, which cannot translocate into cells, did not induce neurite outgrowth, suggesting that the cell membrane-penetrating activity of the chimeric Ngb is crucial for its neurite outgrowth-promoting activity. We also prepared several site-directed chimeric Ngb mutants and demonstrated that residues crucial for neurite outgrowth-inducing activity of the chimeric Ngb are not exactly the same as those for its neuroprotective activity.
神经球蛋白（Ngb）是一种在脊椎动物大脑中表达的血红素蛋白。我们之前构建了一种嵌合Ngb蛋白，其中人Ngb的M1模块被斑马鱼Ngb的M1模块所取代，并且表明嵌合的ZHHH Ngb具有与斑马鱼Ngb相似的细胞膜穿透活性，而且与人Ngb一样能够挽救细胞免受缺氧/复氧引起的死亡。最近，有报道称在神经元细胞中过表达哺乳动物Ngb会诱导神经突生长。在本研究中，我们使用大鼠嗜铬细胞瘤PC12细胞对嵌合Ngb进行了神经突生长测定。向细胞培养基中外源添加嵌合Ngb而非人或斑马鱼Ngb可诱导神经突生长。另一方面，不能转运到细胞内的K7A/K9Q嵌合Ngb双突变体不会诱导神经突生长，这表明嵌合Ngb的细胞膜穿透活性对其促进神经突生长的活性至关重要。我们还制备了几种定点嵌合Ngb突变体，并证明嵌合Ngb诱导神经突生长活性的关键残基与神经保护活性的关键残基并不完全相同。
