Oh-hashi Kentaro, Ito Masatoshi, Tanaka Tatsuhide, Hirata Yoko, Kiuchi Kazutoshi
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Mol Cell Biochem. 2009 Mar;323(1-2):1-7. doi: 10.1007/s11010-008-9958-3. Epub 2008 Nov 13.
Glial cell line-derived neurotrophic factor (GDNF) is synthesized as a precursor, proGDNF. However, the molecular mechanisms for the processing and secretion of GDNF are not fully characterized, since the amount of its biosynthesis and secretion in glial cells are below the detection limit of western blotting. We established stably GDNF-overexpressing C6 cells, and this enabled us to monitor its spontaneous secretion, as well as its processed forms in the cells. GDNF secretion was augmented by stimulation with high potassium, while it was inhibited by treatment with either tunicamycin, an inhibitor of protein glycosylation, or brefeldin A, a disturbing factor of ER-Golgi transport. Wild-type GDNF transfected cells secreted three forms of processed GDNF. After deglycosylation, the highest molecular weight of secreted GDNF showed the same mobility on electrophoresis as recombinant human GDNF without a whole pro-domain. Mutations in the pro-domain and two cysteines at the C-terminal of GDNF markedly diminished the secretion of resultant proteins into the culture medium. GDNF proteins having mutations in the putative furin-consensus sequence were secreted partly as unprocessed forms, and forms with lower molecular weights than a mature form were secreted from the C6 cells. Taking these observations together, we conclude that GDNF is likely secreted both with and without processing by furin-like proteases, and that the pro-domain and C-terminal cysteines of GDNF play important roles in its processing and secretion in cultured astrocytes and C6 cells.
胶质细胞系源性神经营养因子（GDNF）最初是以一种前体形式，即前GDNF合成的。然而，由于胶质细胞中GDNF的生物合成量和分泌量低于蛋白质印迹法的检测限，其加工和分泌的分子机制尚未完全明确。我们建立了稳定过表达GDNF的C6细胞系，这使我们能够监测其自发分泌情况以及细胞内的加工形式。高钾刺激可增强GDNF的分泌，而蛋白质糖基化抑制剂衣霉素或内质网-高尔基体转运干扰因子布雷菲德菌素A的处理则会抑制其分泌。转染野生型GDNF的细胞分泌三种加工形式的GDNF。去糖基化后，分泌的GDNF的最高分子量在电泳上显示出与不含整个前结构域的重组人GDNF相同的迁移率。GDNF前结构域和C末端的两个半胱氨酸发生突变，会显著减少所得蛋白质向培养基中的分泌。在假定的弗林蛋白酶切割位点序列发生突变的GDNF蛋白部分以未加工形式分泌，且分子量低于成熟形式的GDNF蛋白从C6细胞中分泌出来。综合这些观察结果，我们得出结论，GDNF可能在经过和未经过弗林蛋白酶样蛋白酶加工的情况下都被分泌，并且GDNF的前结构域和C末端半胱氨酸在其在培养的星形胶质细胞和C6细胞中的加工和分泌过程中发挥重要作用。
