Katayama Hideki, Yokota Haruo, Akao Tetsuyuki, Nakamura Osamu, Ohba Michio, Mekada Eisuke, Mizuki Eiichi
Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume, Fukuoka 839-0861, Japan.
J Biochem. 2005 Jan;137(1):17-25. doi: 10.1093/jb/mvi003.
Pro-parasporin-1 is a parasporal inclusion protein of the non-insecticidal Bacillus thuringiensis strain A1190. Cytotoxic fragments, named parasporin-1, were generated from pro-parasporin-1 by trypsin digestion. Parasporin-1 was purified by a combination of chromatography procedures based on the cytotoxic activity to HeLa cells. Two different fragments of 15-kDa and 56-kDa were detected in the purified parasporin-1 fraction. These fragments were tightly associated with each other and could not be separated by chromatography under conditions that preserve cytotoxic activity, indicating that the active form of parasporin-1 is a heterodimer of the 15- and 56-kDa fragments. Amino acid sequencing and MALDI-TOF mass spectrometric analysis revealed that parasporin-1 is generated from pro-parasporin-1 by trypsin digestion at Arg 93 and Arg 231. Of 12 human cell lines tested, parasporin-1 showed strong cytotoxicity to four cell lines derived from cancer tissues, but low to no cytotoxicity to the other cell lines. The time-courses of cytotoxicity indicated that the mode of action of parasporin-1 to sensitive cells differs from that shown for previously isolated cytotoxic proteins from Bacillus thuringiensis, Cyt proteins, and other bacterial pore-forming toxins. Thus, parasporin-1 is a novel cytotoxic protein to human cancer cells produced by B. thuringiensis, and may be useful as a tool to recognize and destroy specific cancer cells.
前芽孢杆菌蛋白-1是无杀虫活性的苏云金芽孢杆菌菌株A1190的伴孢晶体蛋白。通过胰蛋白酶消化从前芽孢杆菌蛋白-1产生了名为芽孢杆菌蛋白-1的细胞毒性片段。基于对HeLa细胞的细胞毒性活性，通过多种色谱方法组合对芽孢杆菌蛋白-1进行了纯化。在纯化的芽孢杆菌蛋白-1组分中检测到15 kDa和56 kDa的两种不同片段。这些片段彼此紧密结合，在保留细胞毒性活性的条件下不能通过色谱分离，这表明芽孢杆菌蛋白-1的活性形式是15 kDa和56 kDa片段的异二聚体。氨基酸测序和基质辅助激光解吸电离飞行时间质谱分析表明，芽孢杆菌蛋白-1是通过胰蛋白酶在精氨酸93和精氨酸231处消化从前芽孢杆菌蛋白-1产生的。在测试的12种人类细胞系中，芽孢杆菌蛋白-1对四种源自癌组织的细胞系表现出强细胞毒性，但对其他细胞系的细胞毒性低或无细胞毒性。细胞毒性的时间进程表明，芽孢杆菌蛋白-1对敏感细胞的作用模式不同于先前从苏云金芽孢杆菌分离的细胞毒性蛋白、Cyt蛋白和其他细菌成孔毒素所显示的模式。因此，芽孢杆菌蛋白-1是苏云金芽孢杆菌产生的一种对人类癌细胞具有新型细胞毒性的蛋白，可能作为识别和破坏特定癌细胞的工具。
