Luangwattananun Piriya, Eiamphungporn Warawan, Songtawee Napat, Bülow Leif, Isarankura Na Ayudhya Chartchalerm, Prachayasittikul Virapong, Yainoy Sakda
Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
J Biotechnol. 2017 Apr 10;247:50-59. doi: 10.1016/j.jbiotec.2017.03.001. Epub 2017 Mar 6.
Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions.
主要抗氧化酶，如超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GPx）的协同作用比任何单一酶的作用更有效。最近，我们设计了一种具有SOD、CAT和细胞穿透活性的三功能酶，即6His-MnSOD-TAT/CAT-MnSOD（M-TAT/CM）。该蛋白能主动内化进入细胞，并且与融合了TAT的天然酶相比，在抵抗氧化应激诱导的细胞死亡方面表现出更好的保护作用。为了改善其分子大小、酶活性和稳定性，在本研究中，将工程蛋白的MnSOD部分替换为CuZnSOD，CuZnSOD是最小且最耐热的SOD同工型。新设计的蛋白，即CAT-CuZnSOD/6His-CuZnSOD-TAT（CS/S-TAT），分子大小减少了42%，SOD和CAT活性分别增加了22%和99%。在70°C孵育10分钟后，CS/S-TAT的残余SOD活性高达54%，而M-TAT/CM的SOD活性则完全丧失。此外，该蛋白在70°C下的半衰期提高了5倍。因此，这项工作为设计和合成一种更小但更稳定的多功能抗氧化酶提供了思路，该酶能够进入哺乳动物细胞，作为针对氧化应激相关病症的保护/治疗剂进一步应用。
